WO2015103235A1 - Detection of entamoeba nucleic acids - Google Patents

Detection of entamoeba nucleic acids Download PDF

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Publication number
WO2015103235A1
WO2015103235A1 PCT/US2014/072709 US2014072709W WO2015103235A1 WO 2015103235 A1 WO2015103235 A1 WO 2015103235A1 US 2014072709 W US2014072709 W US 2014072709W WO 2015103235 A1 WO2015103235 A1 WO 2015103235A1
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Prior art keywords
negative
positive
histolytica
seq
primer
Prior art date
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PCT/US2014/072709
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French (fr)
Inventor
Steven Knapp
Charlotte BROWN
Karen Lenz
Melissa Adams
Robert Swan
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Becton, Dickinson And Company
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Publication date
Application filed by Becton, Dickinson And Company filed Critical Becton, Dickinson And Company
Priority to JP2016544385A priority Critical patent/JP2017501733A/en
Priority to AU2014373826A priority patent/AU2014373826A1/en
Priority to US15/108,771 priority patent/US20160319374A1/en
Priority to BR112016015537A priority patent/BR112016015537A2/en
Priority to CA2934877A priority patent/CA2934877A1/en
Priority to EP14877335.1A priority patent/EP3090069A4/en
Priority to MX2016008778A priority patent/MX2016008778A/en
Publication of WO2015103235A1 publication Critical patent/WO2015103235A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Embodiments herein relate generally to methods and compositions that are useful for detecting the presence of Entamoeba nucleic acids.
  • Amebiasis is a disease that can be caused by infection with the protozoan Entamoeba histolytica.
  • E. histolytica infection is typically in the intestinal tract, and can cause colitis, and amoebic dysentery.
  • E. histolytica infection can also spread to other organs, including the liver, the lungs, or central nervous system.
  • E. dispar is a non-pathogenic species, and is morphologically indistinguishable from the pathogenic E. histolytica (Verweij et al, J. Clin. Microbiol. 42: 1220-23, 2004).
  • E. dispar and E. histolytica genomes have a high degree of nucleic acid sequence homology. It has been estimated that E.
  • Quantitative nucleic acid amplification reactions can be useful for quantifying the relative and/or absolute amount of target nucleic acid sequences present in a sample. Due to the highly sensitive nature of quantitative nucleic acid amplification reactions, in order to avoid false positives, false negatives, overestimation of target or product quantity, or underestimation of target or product quantity, extreme care must be taken when selecting reagents and methods for quantitative nucleic acid amplification. Ribosomal DNA (rDNA) genes are highly conserved. The high degree of conservation of rDNA sequences can result in little variability between different organisms of the same species, a feature that can make rDNA genes useful for nucleic-acid-based detection assays directed to the detection of a desired species.
  • rDNA Ribosomal DNA
  • E. histolytica and E. dispar rDNA genes can complicate quantitative nucleic acid amplification for the specific detection of the different species.
  • multi-template PCR amplification or rDNA genes can be subject to bias, and can produce various artifacts (Kanagawa, J. Bioscience and Bioengineering 96: 317-23, 2003; Wang et al., Microbiology 142: 1 107-14, 1996).
  • a method of detecting the presence of an E. histolytica polynucleotide sequence in a sample can comprise contacting the sample with a first primer consisting essentially of SEQ ID NO: 1 (GTACAAAATGGCCAATTCATTCAATG).
  • the method can comprise contacting the sample with a second primer consisting essentially of SEQ ID NO: 2 (ACTACCAACTGATTGATAGATCAG).
  • the method can comprise extending the first and second primer, thereby producing at least one amplicon if the E. histolytica polynucleotide sequence is present in the sample.
  • the method can comprise contacting the sample with an oligonucleotide probe comprising a polynucleotide consisting essentially of SEQ ID NO: 3 (ATTGTCGTGGCATCCTAACTCA) or its complement.
  • the probe provides detectable signal when it is bound to a substantially complementary nucleic acid, but does not provide detectable signal when it is single-stranded.
  • the method can comprise detecting the signal, if the amplicon is present.
  • the first primer and second primer amplify the E. histolytica polynucleotide sequence, but do not substantially amplify any E. dispar polynucleotide sequence.
  • the first primer hybridizes to the E. histolytica polynucleotide sequence if contacted with the E. histolytica polynucleotide sequence at a temperature of at least about 50°C in 5 mM MgC12, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA, but does not hybridize to any E. dispar polynucleotide sequence if contacted with any E.
  • the second primer hybridizes to the E. histolytica polynucleotide sequence if contacted with E.
  • histolytica polynucleotide sequence at a temperature of at least about 60°C in 5 mM MgC12, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSAl .96% Trehalose, 0.6 mg/ml BSA, and hybridizes to an E. dispar polynucleotide sequence if contacted with the E.
  • each of the first primer and second primer hybridizes to the E. histolytica polynucleotide sequence if contacted with the E.
  • histolytica polynucleotide sequence at a temperature of at least about 60°C in in 5 mM MgC12, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSAl.96% Trehalose, 0.6 mg/ml BSA, but the second primer does not hybridize to any E. dispar polynucleotide sequence if contacted with any E.
  • the sample comprises E. histolytica and E. dispar.
  • the sample comprises fecal material of a human.
  • the sample comprises fixed material.
  • the sample is non-fixed.
  • a 95% limit of detection for E. histolytica comprises no more than about 17 E. histolytica genomes per milliliter.
  • the primers and probes do not cross-react with any of the following organisms, if present in the sample: Abiotrophia defectiva, Acinetobacter baumannii, Acinetobacter Iwoffli, Aeromonas hydrophila, Alcaligenes faecalis subsp.
  • faecalis Anaerococcus tetradius, Arcobacter butzleri, Arcobacter cryaerophilus, Bacillus cereus, Bacteroides caccae, Bacteroides merdae, Bacteroides stercoris, Bifidobacterium adolescentis, Bifidobacterium longum, Camplylobacter coli, Campylobacter concisus, Campylobacter curvus, Campylobacter fetus subsp. fetus, Campylobacter fetus subsp.
  • the primers and probes do not cross-react with any of the following organisms, if present in the sample: Entamoeba coli, Entamoeba dispar, Entamoeba polecki, Entamoeba muris, Entamoeba nuttalli, Entamoeba hartmanni, and Entamoeba bovis.
  • the primers and probes produce fewer than 1 in 1600 false positives for samples that do not comprise E. histolytica.
  • E. dispar if present, does not inhibit production of the amplicon if the E. histolytica polynucleotide sequence is present in the sample. In some embodiments, E. dispar, if present, does not inhibit determining the presence or absence of E. histolytica.
  • a kit can comprise a first primer.
  • the kit can comprise a second primer.
  • the first primer and second primer amplify a E. histolytica polynucleotide sequence, thereby producing an amplicon, but do not substantially amplify any E. dispar polynucleotide sequence.
  • the kit can comprise a probe, wherein the probe comprises a polynucleotide consisting essentially of a sequence, wherein the sequence or its complement is present in each of the amplicon, a polynucleotide sequence of E. histolytica, and a polynucleotide sequence of E. dispar.
  • the probe comprises a fluorophore; and a quencher.
  • the primers and probes amplify an E. histolytica polynucleotide sequence with a 95% limit of detection of no more than about 17 E. histolytica organisms per mililiter.
  • the primers and probes do not cross-react with any of the following organisms, if present in the sample: Abiotrophia defectiva, Acinetobacter baumannii, Acinetobacter Iwoffii, Aeromonas hydrophila, Alcaligenes faecalis subsp.
  • faecalis Anaerococcus tetradius, Arcobacter butzleri, Arcobacter cryaerophilus, Bacillus cereus, Bacteroides caccae, Bacteroides merdae, Bacteroides stercoris, Bifidobacterium adolescentis, Bifidobacterium longum, Camplylobacter coli, Campylobacter concisus, Campylobacter curvus, Campylobacter fetus subsp. fetus, Campylobacter fetus subsp.
  • the first primer comprises a polynucleotide having at least about 90% identity to SEQ ID NO: 1 (GTACAAAATGGCCAATTCATTCAATG) or its complement. In some embodiments, the first primer consists essentially of SEQ ID NO: 1 (GTACAAAATGGCCAATTCATTCAATG) or its complement.
  • the second primer comprises a polynucleotide having at least about 90% identity to SEQ ID NO: 2 (ACTACCAACTGATTGATAGATCAG) or its complement. In some embodiments, the second primer comprises a polynucleotide having the sequence of SEQ ID NO: 2 (ACTACCAACTGATTGATAGATCAG) or its complement.
  • the probe comprises a polynucleotide having at least about 90% identity to SEQ ID NO: 3 (ATTGTCGTGGCATCCTAACTCA) or its complement. In some embodiments, the probe comprises a polynucleotide having the sequence of SEQ ID NO: 3 (ATTGTCGTGGCATCCTAACTCA) or its complement.
  • the primers and probes do not cross-react with any of the following organisms, if present in the sample: Entamoeba coli, Entamoeba dispar, Entamoeba poiecki, Entamoeba muris, Entamoeba nuttalli, Entamoeba hartmanni, and Entamoeba bovis.
  • the primers and probes produce fewer than 1 in 1600 false positives for samples that do not comprise E. histolytica.
  • E. dispar if present, does not inhibit production of the amplicon if the E. histolytica polynucleotide sequence is present in the sample.
  • E. dispar if present, does not inhibit determining the presence or absence of E. histolytica.
  • a kit can comprise a first primer comprising a polynucleotide having at least about 90% identity to SEQ ID NO: 1 (GTACAAAATGGCCAATTCATTCAATG).
  • the kit can comprise a second primer comprising polynucleotide having at least about 90% identity to SEQ ID NO: 2 (ACTACCAACTGATTGATAGATCAG).
  • the kit can comprise a probe comprising a polynucleotide having at least about 90% identity to SEQ ID NO: 3 (ATTGTCGTGGCATCCTAACTCA) or its complement; a flurophore; and a quencher.
  • the first primer consists essentially of SEQ ID NO: 1 (GTACAAAATGGCCAATTCATTCAATG).
  • the second primer consists essentially of SEQ ID NO: 2 (ACTACCAACTGATTGATAGATCAG).
  • the probe comprises a polynucleotide consisting essentially of SEQ ID NO: 3 (ATTGTCGTGGCATCCTAACTCA) or its complement.
  • the primers and probes do not cross-react with any of the following organisms, if present in the sample: Entamoeba coli, Entamoeba dispar, Entamoeba polecki, Entamoeba muris, Entamoeba nuttalli, Entamoeba hartmanni, and Entamoeba bovis.
  • the primers and probes produce fewer than 1 in 1600 false positives for samples that do not comprise E. histolytica.
  • E. dispar if present, does not inhibit production of the amplicon if the E. histolytica polynucleotide sequence is present in the sample. In some embodiments, E. dispar, if present, does not inhibit determining the presence or absence of E. histolytica.
  • a method of detecting the presence of an E. histolytica polynucleotide sequence in a sample can comprise contacting the sample with a first primer.
  • the method can comprise contacting the sample with a second primer.
  • the first primer and second primer amplify the E. histolytica polynucleotide sequence, but do not substantially amplify any E. dispar polynucleotide sequence.
  • the method can comprise extending the first and second primer, thereby producing at least one amplicon if the E. histolytica polynucleotide sequence is present in the sample.
  • the method can comprise contacting the sample with an oligonucleotide probe.
  • the probe provides detectable signal when it is bound to a substantially complementary nucleic acid, but does not provide detectable signal when it is single-stranded.
  • the probe comprises a polynucleotide consisting essentially of sequence that is a portion of the E. histolytica polynucleotide sequence, a polynucleotide sequence of E. dispar, and a sequence of the amplicon.
  • the method can comprise detecting the signal, if the amplicon is present.
  • the first primer hybridizes to the E. histolytica polynucleotide sequence if contacted with the E.
  • the second primer hybridizes to the E. histolytica polynucleotide sequence if contacted with E. histolytica polynucleotide sequence at a temperature of at least about 60°C in 5 mM MgC12, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA, and hybridizes to an E. dispar polynucleotide sequence if contacted with the E.
  • each of the first primer and second primer hybridizes to the E. histolytica polynucleotide sequence if contacted with the E.
  • the first primer comprises a polynucleotide having at least about 90% identity to SEQ ID NO: 1 (GTACAAAATGGCCAATTCATTCAATG) or its complement. In some embodiments, the first primer consists essentially of SEQ ID NO: 1 (GTACAAAATGGCCAATTCATTCAATG) or its complement.
  • the second primer comprises a polynucleotide having at least about 90% identity to SEQ ID NO: 2 (ACTACCAACTGATTGATAGATCAG) or its complement. In some embodiments, the second primer comprises a polynucleotide having the sequence of SEQ ID NO: 2 (ACTACCAACTGATTGATAGATCAG) or its complement.
  • the probe comprises a polynucleotide having at least about 90% identity to SEQ ID NO: 3 (ATTGTCGTGGCATCCTAACTCA) or its complement. In some embodiments, the probe comprises a polynucleotide having the sequence of SEQ ID NO: 3 (ATTGTCGTGGCATCCTAACTCA) or its complement. In some embodiments, the amplicon comprises a polynucleotide having at least about 95% identity to SEQ ID NO: 7 (GTACAAAATGGCCAATTCATTCAATGAATTGAGAAATGACATTCTAAGTGAG TTAGGATGCCACGACAATTGTAGAACACACAGTGTTTAACAAGTAACCAATG AGAATTTCTGATCTATCAATCAGTTGGTAGT).
  • the amplicon comprises a polynucleotide having the sequence of SEQ ID NO: 7 (GTACAAAATGGCCAATTCATTCAATGAATTGAGAAATGACATTCTAAGTGAG TTAGGATGCCACGACAATTGTAGAACACACAGTGTTTAACAAGTAACCAATG AGAATTTCTGATCTATCAATCAGTTGGTAGT).
  • the sample comprises E. histolytica and E. dispar.
  • the sample comprises fecal material of a human.
  • the sample comprises fixed material.
  • the sample is non-fixed.
  • a 95% limit of detection for E. histolytica comprises no more than about 17 E.
  • the primers and probes do not cross-react with any of the following organisms, if present in the sample: Abiotrophia defectiva, Acinetobacter baumannii, Acinetobacter Iwoffli, Aeromonas hydrophila, Alcaligenes faecalis subsp.
  • faecalis Anaerococcus tetradius, Arcobacter butzleri, Arcobacter cryaerophilus, Bacillus cereus, Bacteroides caccae, Bacteroides merdae, Bacteroides stercoris, Bifidobacterium adolescentis, Bifidobacterium longum, Camplylobacter coli, Campylobacter concisus, Campylobacter curvus, Campylobacter fetus subsp. fetus, Campylobacter fetus subsp.
  • the primers and probes do not cross-react with any of the following organisms, if present in the sample: Entamoeba coli, Entamoeba dispar, Entamoeba polecki, Entamoeba muris, Entamoeba nuttalli, Entamoeba hartmanni, and Entamoeba bovis.
  • the primers and probes produce fewer than 1 in 1600 false positives for samples that do not comprise E. histolytica.
  • E. dispar if present, does not inhibit production of the amplicon if the E. histolytica polynucleotide sequence is present in the sample. In some embodiments, E. dispar, if present, does not inhibit determining the presence or absence of E. histolytica.
  • a method of determining the presence or absence of an E. histolytica nucleic acid sequence in a sample can comprise performing a nucleic acid amplification reaction on the sample, the nucleic acid amplification comprising a first oligonucleotide primer and a second oligonucleotide primer, in which the first oligonucleotide primer has a length of 15-75 nucleotides and hybridizes under standard conditions to SEQ ID NO: 10 or its complement, if present,, but does not hybridize under standard conditions to SEQ ID NO: 1 1 or its complement, if present, and in which the second oligonucleotide primer has a length of 15-75 nucleotides and hybridizes under standard conditions to a SEQ ID NO: 10 or its complement, if present, and wherein the second oligonucleotide primer hybridizes under standard conditions to SEQ ID NO: 11 or its complement, if present.
  • the method can comprise detecting a signal, if present, from a detectably labeled probe that hybridizes to an amplicon of the the first and second oligonucleotide primers under standard hybridization conditions if the amplicon is present, in which the signal indicates the presence or absence of the amplicon, and in which the amplicon has a length of 75-350 nucleotides.
  • the first oligonucleotide primer comprises at least 10 consecutive nucleotides of SEQ ID NO: 1, and wherein the first oligonucleotide primer has at least 80% identity to a target sequence of SEQ ID NO: 10 or its complement.
  • the second oligonucleotide primer comprises at least 10 consecutive nucleotides of SEQ ID NO: 2, and wherein the second oligonucleotide primer has at least 80% identity to a target sequence of SEQ ID NO: 10 or its complement.
  • the first oligonucleotide primer comprises at least 12 consecutive nucleotides of SEQ ID NO: 1.
  • the first oligonucleotide primer comprises at least 15 consecutive nucleotides of SEQ ID NO: 1.
  • the first oligonucleotide primer comprises at least 20 consecutive nucleotides of SEQ ID NO: 1.
  • the first oligonucleotide primer has at least 85% identity to a target sequence of SEQ ID NO: 10 or its complement.
  • the first oligonucleotide primer has at least 90% identity to a target sequence of SEQ ID NO: 10 or its complement.
  • the first oligonucleotide primer has at least 95% identity to a target sequence of SEQ ID NO: 10 or its complement.
  • the first oligonucleotide primer has 100% identity to a target sequence of SEQ ID NO: 10 or its complement.
  • the second oligonucleotide primer comprises at least 12 consecutive nucleotides of SEQ ID NO: 2.
  • the second oligonucleotide primer comprises at least 15 consecutive nucleotides of SEQ ID NO: 2.
  • the second oligonucleotide primer comprises at least 20 consecutive nucleotides of SEQ ID NO: 2.
  • the second oligonucleotide primer has at least 85% identity to a target sequence of SEQ ID NO: 10 or its complement.
  • the second oligonucleotide primer has at least 90% identity to a target sequence of SEQ ID NO: 10 or its complement.
  • the second oligonucleotide primer has at least 95% identity to a target sequence of SEQ ID NO: 10 or its complement.
  • the second oligonucleotide primer has 100% identity to a target sequence of SEQ ID NO: 10 or its complement.
  • the probe comprises at least 10 consecutive nucleotides of SEQ ID NO: 3, and wherien the probe has at least 80% identity to a target sequence of SEQ ID NO: 10 or its complement.
  • the probe comprises at least 12 consecutive nucleotides of SEQ ID NO: 3.
  • the probe comprises at least 15 consecutive nucleotides of SEQ ID NO: 3.
  • the probe comprises at least 20 consecutive nucleotides of SEQ ID NO: 3.
  • the probe has at least 85% identity to a target sequence of SEQ ID NO: 10 or its complement.
  • the probe has at least 90% identity to a target sequence of SEQ ID NO: 10 or its complement.
  • the probe has at least 95% identity to a target sequence of SEQ ID NO: 10 or its complement.
  • the probe has 100% identity to a target sequence of SEQ ID NO: 10 or its complement.
  • the first oligonucleotide primer is about 20-50 nucleotides long.
  • the first oligonucleotide primer is about 23-45 nucleotides long.
  • the second oligonucleotide primer is about 20-50 nucleotides long.
  • the second oligonucleotide primer is about 23-45 nucleotides long.
  • the detectably labeled probe is about 15-75 nucleotides long.
  • the detectably labeled probe is about 20-45 nucleotides long.
  • the probe is capable of hybridizing to SEQ ID NO: 10 and toSEQ ID NO: 11 under standard hybridization conditions.
  • the probe is capable of hybridizing to SEQ ID NO: 10 but not toSEQ ID NO: 11 under standard hybridization conditions.
  • the probe comprises a fluorophore or a quencher.
  • the amplicon has a length of 100-150 nucleotides.
  • the amplicon comprises SEQ ID NO: 7.
  • a kit comprising any of the first oligonucleotide primer, the second oligonucleotide primer, and the detectably labeled probe as described above is provided.
  • E. dispar if present, does not inhibit production of the amplicon if the E. histolytica polynucleotide sequence is present in the sample. In some embodiments, E. dispar, if present, does not inhibit determining the presence or absence of E. histolytica.
  • Figure 1 is a diagram showing primers and probes as used in some of the embodiments disclosed herein.
  • Figure 2A is an alignment showing E. dispar and E. histolytica nucleic acids sequences.
  • Figure 2B is an annotated diagram of an E. histolytica gene encoding small subunit ribosomal RNA (GenBank: AB608092.1) (SEQ ID NO: 10)
  • Figure 3 is a graph showing quantitative PCR signal detection using previously-known primers and probes, for which the presence of E. dispar depresses amplification signal and can cause false negatives.
  • Figure 4 is a graph showing quantitative PCR signal detection using primers and probes in embodiments as described herein, for which the presence of E. dispar does not interfere with amplification signal.
  • Detection of E. histolytica, and quantification of relative levels of E. histolytica can be useful in guiding clinical decisions.
  • Quantitative nucleic acid amplification for example quantitative assays involving nucleic acid amplification, such as polymerase chain reaction (qPCR) can be highly sensitive, and useful for quantification of nucleic acid levels, and thus can be used to infer relative quantities of E. histolytica based on quantification of nucleic acid.
  • qPCR polymerase chain reaction
  • some embodiments herein provide methods and reagents for detecting and quantifying E. hisotolytica nucleic acids, without substantial interference from the presence of E. dispar. Some embodiments herein provide methods of detecting E. hisotolytica nucleic acids by qPCR. Some embodiments herein provide reagents and/or kits for detecting E. hisotolytica without substantial interference from the presence of E. dispar.
  • ranges are meant to include the starting value and the ending value and any value or value range there between unless otherwise specifically stated.
  • “from 0.2 to 0.5” means 0.2, 0.3, 0.4, 0.5; ranges there between such as 0.2-0.3, 0.3-0.4, 0.2-0.4; increments there between such as 0.25, 0.35, 0.225, 0.335, 0.49; increment ranges there between such as 0.26-0.39; and the like.
  • compositions, and kits, and methods of using the same for use in detecting and/or distinguishing or identifying Entamoeba nucleic acids. Accordingly, some embodiments provide nucleic acid sequences for use in nucleic acid detection assays, e.g., in amplification assays.
  • nucleic acid detection assays e.g., in amplification assays.
  • the reverse compliment can be readily obtained, and that a disclosure of a nucleic acid sequence also provides a disclosure of the reverse compliment of that sequence.
  • a person skilled in the art will appreciate that for any DNA sequence disclosed herein, a corresponding RNA sequence can be readily obtained, and that for any RNA sequence, a corresponding DNA can readily be obtained, for example by reverse transcription.
  • upstream refers one or more locations 5' of a position on a nucleic acid sequence
  • downstream refers to one or more locations 3' of a position on a nucleic acid sequence
  • nucleic acids provided herein can be in various forms.
  • the nucleic acids are dissolved (either alone or in combination with various other nucleic acids) in solution, for example buffer.
  • nucleic acids are provided, either alone or in combination with other isolated nucleic acids, as a salt.
  • nucleic acids are provided in a lyophilized form that can be reconstituted.
  • the isolated nucleic acids disclosed herein can be provided in a lyophilized pellet alone, or in a lyophilized pellet with other isolated nucleic acids.
  • nucleic acids are provided affixed to a solid substance, such as a bead, a membrane, or the like.
  • nucleic acids are provided in a host cell, for example a cell line carrying a plasmid, or a cell line carrying a stably integrated sequence.
  • nucleic acids are isolated from a host cell, for example one or more Entamoeba cells.
  • nucleic acids are synthesized, for example chemically or in a cell-free system.
  • nucleic acid amplification can include qualitative nucleic acid amplification, e.g. to determine whether a nucleic acid sequence is present or absent in a sample, for example, an E. histolytica-specific or E. dispar- specific nucleic acid sequence.
  • nucleic acid amplification can include quantitative nucleic acids amplification, e.g. to measure the relative or absolute amount of nucleic acid present in a sample.
  • nucleic acid amplification can include quantitative and qualitative nucleic acid amplification, e.g. to determine whether a nucleic acid sequence is present in a sample, and if present, to measure the relative or absolute amount of nucleic acid sequence present in the sample.
  • the method of amplification includes a multiplex assay for identifying the presence of two or more parasitic organisms from a sample, such as a human stool sample, for example at least two or more of E. histolytica, E. dispar, Giardia lamblia, Cryptosporidium parvum, Cryptosporidium hominis, and the like.
  • Methods of nucleic acid amplification can include, but are not limited to: polymerase chain reaction (PCR), strand displacement amplification (SDA), for example multiple displacement amplification (MDA), loop-mediated isothermal amplification (LAMP), ligase chain reaction (LCR), immuno-amplification, and a variety of transcription-based amplification procedures, including transcription-mediated amplification (TMA), nucleic acid sequence based amplification (NASBA), self- sustained sequence replication (3SR), and rolling circle amplification.
  • PCR polymerase chain reaction
  • SDA strand displacement amplification
  • MDA multiple displacement amplification
  • LAMP loop-mediated isothermal amplification
  • LCR loop-mediated isothermal amplification
  • TMA transcription-mediated amplification
  • NASBA nucleic acid sequence based amplification
  • SR self- sustained sequence replication
  • rolling circle amplification See, e.g., Mullis, "Process for Amplifying, Detecting, and/or Clon
  • RNA sequence is amplified.
  • the target RNA sequence is reverse-transcribed, and the reverse transcript includes a DNA that is amplified using a nucleic acid amplification method described herein.
  • the nucleic acid amplification is quantitative.
  • Quantitative nucleic acid amplification can include detection of the amount of amplicon produced.
  • the detection can be performed continuously or periodically. For example, detection can be performed at a certain point, e.g., at the end of every Nth cycle or fraction thereof, where N is one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 21, 23, 24, 25, 26, 27, 28, 29, 30, 21, 32, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 75, 80, 85, 95, 100 or the like.
  • detection can include measuring fluorescence, for example the intensity of electromagnetic radiation at the emission wavelength of a fluorophore tethered to a probe as described herein, or a wavelength range including the emission wavelength of the fluorophore tethered to the probe.
  • exemplary probes include molecular beacons, SCORPIONSTM probes (Sigma), TAQMANTM probes (Life Technologies), and the like.
  • detection can include detecting FRET.
  • detection can include detecting intensity of a non-specific detectable marker that binds to dsDNA, but does not bind to ssDNA.
  • substantially amplification refers to amplification that produces exponential yields of an amplicon or amplicons under standard amplification conditions.
  • PCR-derived forms of amplification and LAMP can produce discrete, double stranded amplicons, for which each strand can serve as a template in successive rounds of amplification, thus permitting exponential amplification.
  • a template can be substantially amplified and detected by polynucleotide that have less than 100% complementarity to the template, for example primers and/or probes having degenerate nucleotides, inosines, or the like at one or more positions.
  • compositions disclosed herein can be used in various types of nucleic acid amplification reactions, as disclosed herein.
  • the compositions disclosed herein can be used in polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • standard amplification conditions refer to 5 mM MgCl 2 , 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA with a denaturation temperature of 97°C, and an annealing temperature of 62°C.
  • amplification conditions include the conditions disclosed in the references cited herein, such as, for example, 5 mM MgCl 2 , 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA with an annealing temperature of 72°C; 5 mM MgCl 2 ; 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA with an annealing temperature of 62°C; 5 mM MgCi 2 , 100 mM Tris, 10 m
  • an annealing temperatures as described herein is modified, for example to at least about 50°C, for example 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, or 75°C.
  • At least one polymerase is provided.
  • the polymerase can be used for quantitative PCR.
  • Different nucleic acid polymerases are available for use, including but not limited to the FASTSTARTTM Taq DNA polymerase (Roche), the KlenTaq 1 (AB peptides Inc.), the HOTGOLDSTARTM DNA polymerase (Eurogentec), the KAPATAQTM HotStart DNA polymerase or the KAPA2GTM Fast HotStart DNA polymerase (Kapa Biosystemss), and the PHUSIONTM Hot Start (Finnzymes).
  • Thermal cycling conditions can vary in time as well as in temperature for each of the different steps, depending on the thermal cycler used as well as other variables that could modify the amplification's performance.
  • a 2- step protocol is performed, in which the protocol combines the annealing and elongation steps at a common temperature, optimal for both the annealing of the primers and probes as well as for the extension step.
  • a 3 -step protocol is performed, in which a denaturation step, an annealing step, and an elongation step are performed.
  • compositions disclosed herein can be used in connection with devices for real-time amplification reactions, e.g., the BD MAX® (Becton Dickinson and Co., Franklin Lakes, NJ), the VIPER® (Becton Dickinson and Co., Franklin Lakes, NJ), the VIPER LT® (Becton Dickinson and Co., Franklin Lakes, NJ), the SMARTCYLCER® (Cepheid, Sunnyvale, CA), ABI PRISM 7700® (Applied Biosystems, Foster City, CA), ROTOR-GENETM (Corbett Research, Sydney, Australia), LIGHTCYCLER® (Roche Diagnostics Corp, Indianapolis, IN), ICYCLER® (BioRad Laboratories, Hercules, CA), IMX4000® (Stratagene, La Jolla, CA), CFX96TM Real- Time PCR System (Bio-Rad Laboratories Inc), and the like.
  • BD MAX® Becton Dickinson and Co.,
  • compositions disclosed herein can be used in methods comprising isothermal amplification of nucleic acids.
  • Isothermal amplification conditions can vary in time as well as temperature, depending on variables such as the method, enzyme, template, and primer or primers used. Examples of amplification methods that can be performed under isothermal conditions include, but are not limited to, some versions of LAMP, SDA, and the like.
  • Isothermal amplification can include an optional denaturation step, followed by an isothermal incubation in which nucleic acid is amplified.
  • an isothermal incubation is performed without an initial denaturing step.
  • the isothermal incubation is performed at least about 25°C, for example about 25°C, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 °C, including ranges between any of the listed values.
  • the isothermal incubation is performed at about 37°C. In some embodiments, the isothermal incubation is performed at about 64°C. In some embodiments, the isothermal incubation is performed for 180 minutes or less, for example about 180, 165, 150, 135, 120, 105, 90, 75, 60, 45, 30, or 15 minutes, including ranges between any two of the listed values. Oligonucleotides
  • oligonucleotides are provided, for example primers and/or probes.
  • the terms "primer” and “probe” include, but are not limited to oligonucleotides.
  • the oligonucleotide primers and/or probes disclosed herein can be between 8 and 45 nucleotides in length.
  • the primers and or probes can be at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or more nucleotides in length.
  • Primers and/or probes can be provided in any suitable form, included bound to a solid support, liquid, and lyophilized, for example.
  • the primer and probe sequences disclosed herein can be modified to contain additional nucleotides at the 5' or the 3' terminus, or both. The skilled artisan will appreciate, however, that additional bases to the 3' terminus of amplification primers (not necessarily probes) are generally complementary to the template sequence.
  • the primer and probe sequences disclosed herein can also be modified to remove nucleotides at the 5' or the 3' terminus.
  • Oligonucleotide primers and probes can bind to their targets at an annealing temperature, which is a temperature less than the melting temperature (T m ).
  • T m melting temperature
  • melting temperature are interchangeable terms which refer to the temperature at which 50% of a population of double-stranded polynucleotide molecules becomes dissociated into single strands.
  • the Tm of a hybrid polynucleotide may also be estimated using a formula adopted from hybridization assays in 1 M salt, and commonly used for calculating Tm for PCR primers: [(number of A+T) x 2°C +(number of G+C) x 4°C]. See, e.g., C. R. Newton et al. PCR, 2nd Ed., Springer-Verlag (New York: 1997), p. 24. Other more sophisticated computations exist in the art, which take structural as well as sequence characteristics into account for the calculation of T m .
  • the melting temperature of an oligonucleotide can depend on complementarity between the oligonucleotide primer or probe and the binding sequence, and on salt conditions.
  • an oligonucleotide primer or probe provided herein has a T m of less than about 90°C in 50mM KC1, 10 mM Tris-HCl buffer, for example about 89°C, 88, 87, 86, 85, 84, 83, 82, 81, 80 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39°C, or less, including ranges between any two of the listed values.
  • an oligonucleotide primer or probe provided herein has a T m of less than about 90°C in 5 mM MgCi 2 , 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA, for example about 89°C, 88, 87, 86, 85, 84, 83, 82, 81, 80 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39°C, or less, including ranges between any two of the listed values.
  • the primers disclosed herein are provided as an amplification primer set, e.g., comprising a forward primer and a reverse primer.
  • the forward and reverse primers have Tm's that do not differ by more than 10°C, e.g., that differ by less than 10°C, less than 9°C, less than 8°C, less than 7°C, less than 6°C, less than 5°C, less than 4°C, less than 3°C, less than 2°C, or less than 1°C.
  • the primer and probe sequences may be modified by having nucleotide substitutions (relative to the target nucleic acid sequence) within the oligonucleotide sequence, provided that the oligonucleotide contains enough complementarity to hybridize specifically to the target nucleic acid sequence. In this manner, at least 1, 2, 3, 4, or up to about 5 nucleotides can be substituted.
  • the term "complementary" refers to sequence complementarity between regions of two polynucleotide strands or between two regions of the same polynucleotide strand.
  • a first region of a polynucleotide is complementary to a second region of the same or a different polynucleotide if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide of the first region is capable of base pairing with a base of the second region. Therefore, it is not required for two complementary polynucleotides to base pair at every nucleotide position.
  • “Fully complementary” refers to a first polynucleotide that is 100% or "fully” complementary to a second polynucleotide and thus forms a base pair at every nucleotide position.
  • Partially complementary also refers to a first polynucleotide that is not 100% complementary (e.g., 90%, or 80% or 70% complementary) and contains mismatched nucleotides at one or more nucleotide positions.
  • an oligonucleotide includes a universal base.
  • hybridization is used in reference to the pairing of complementary (including partially complementary) polynucleotide strands.
  • Hybridization and the strength of hybridization is impacted by many factors well known in the art including the degree of complementarity between the polynucleotides, stringency of the conditions involved affected by such conditions as the concentration of salts, the melting temperature of the formed hybrid, the presence of other components (e.g., the presence or absence of polyethylene glycol), the molarity of the hybridizing strands and the G:C content of the polynucleotide strands.
  • the primers are designed such that the Tm of one primer in the set is within 2°C of the T m of the other primer in the set.
  • the term “specific hybridization” or “specifically hybridizes” refers to the hybridization of a polynucleotide, e.g., an oligonucleotide primer or probe or the like to a target sequence, such as a sequence to be quantified in a sample, a positive control target nucleic acid sequence, or the like, and not to unrelated sequences, under conditions typically used for nucleic acid amplification.
  • the primers and/or probes include oligonucleotides that hybridize to a target nucleic acid sequence over the entire length of the oligonucleotide sequence.
  • Such sequences can be referred to as “fully complementary” with respect to each other.
  • an oligonucleotide is referred to as “substantially complementary” with respect to a nucleic acid sequence herein, the two sequences can be fully complementary, or they may form mismatches upon hybridization, but retain the ability to hybridize under stringent conditions or standard PCR conditions as discussed below.
  • the term “substantially complementary” refers to the complementarity between two nucleic acids, e.g., the complementary region of the oligonucleotide and the target sequence.
  • the complementarity need not be perfect; there may be any number of base pair mismatches that between the two nucleic acids. However, if the number of mismatches is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence is not a substantially complementary sequence.
  • substantially complementary it is meant that the sequences are sufficiently complementary to the each other to hybridize under the selected reaction conditions.
  • nucleic acid complementarity and stringency of hybridization sufficient to achieve specificity is well known in the art and described further below in reference to sequence identity, melting temperature and hybridization conditions. Therefore, substantially complementary sequences can be used in any of the detection methods disclosed herein.
  • probes can be, for example, perfectly complementary or can contain from 1 to many mismatches so long as the hybridization conditions are sufficient to allow, for example discrimination between a target sequence and a non-target sequence.
  • substantially complementary sequences can refer to sequences ranging in percent identity from 100%, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 75, 70% or less, or any number in between, compared to the reference sequence.
  • the oligonucleotides disclosed herein can contain 1, 2, 3, 4, 5, or more mismatches and/or degenerate bases (e.g.
  • variant oligonucleotides as compared to the target sequence to which the oligonucleotide hybridizes, with the proviso that the oligonucleotides are capable of specifically hybridizing to the target sequence under, for example, standard nucleic acid amplification conditions.
  • the primers described herein can be prepared using techniques known in the art, including, but not limited to, cloning and digestion of the appropriate sequences and direct chemical synthesis.
  • Chemical synthesis methods that can be used to make the primers of the described herein include, but are not limited to, the phosphotriester method described by Narang et al. (1979) Methods in Enzymology 68:90, the phosphodiester method disclosed by Brown et al. (1979) Methods in Enzymology 68: 109, the diethylphosphoramidate method disclosed by Beaucage et al. (1981) Tetrahedron Letters 22: 1859, and the solid support method described in U.S. Patent No. 4,458,066.
  • oligonucleotide synthesizer to prepare synthetic oligonucleotide primers described herein is also contemplated herein. Additionally, if desired, the primers can be labeled using techniques known in the art and described below.
  • a set of amplification primers is provided.
  • the set of amplification primers can include one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, or more primer pairs.
  • the term "primer pair" can refer to two amplification primers that individually hybridize to opposite strands of a target nucleic acid sequence (e.g., a sequence of E. histolytica, a sequence of E. dispar, or a sequence found in both E. histolytic and E. dispar), in which each primer can be extended at its 3' end to form a target amplification product, for example in PCR.
  • the target amplification product can include an amplicon.
  • a primer pair can include a forward primer and a reverse primer.
  • the primer set includes amplification primers that will anneal to, and amplify, a sequence of E. histolytica under standard amplification conditions, but will not anneal to a sequence of E. dispar, or will anneal to a sequence of E. dispar, but not substantially amplify this sequence of E. dispar under the same or similar amplification conditions. Accordingly, in some embodiments, the primer set is used to detect the presence of E. histolytica, but not E. dispar. Due to the high degree of homology between E. histolytica and E. dispar, an alternative approach for quantitative amplification of E.
  • histolytica sequences would be to select a primer set that amplifies a polynucleotide sequence found in both E. histolytica and E. dispar (e.g., a homologous sequence), and then use a probe that hybridizes only to the polynucleotide sequence of E. histolytica to detect amplification of E. histolytica product (see Verweij, et al, Clin. Microbiol. 42: 1220-23, 2004).
  • Verweij et al, Clin. Microbiol. 42: 1220-23, 2004.
  • Example 1 and Figure 3 when a primer set that amplifies both E. histolytica and E. dispar nucleic acid sequences was used, a known copy number of E. histolytica target nucleic acid sequence became nearly undetectable in the presence of a high copy number of E. dispar target nucleic acid sequences. Without being bound by any one theory, it is contemplated that homo-and hetero duplex formation between amplification products of E. histolytica and E. dispar can block available E. histolytica probe binding sequences as the proportion of E.
  • E. histolytica Performing quantitative nucleic acid amplification of E. histolytica according to some embodiments herein can minimize or eliminiate interfering effects of E. dispar nucleic acids.
  • depression of E. histolytica signal by E. dispar can be minimized.
  • depression of E. histolytica signal by E. dispar can be effectively eliminated.
  • primers are designed to substantially amplify a E. histolytica target nucleic acid sequence under standard amplification conditions, without substantially amplifying any E. dispar nucleic acid sequences.
  • the primers of the primer set will individually hybridize to opposite strands of a target nucleic acid of E. histolytica under standard amplification conditions, so as to define a target amplification product.
  • the primers when extended at their respective 3 ' ends, the primers will produce a target amplification product. Accordingly, in some embodiments, when extended, the primers will substantially amplify an E. histolytica target nucleic acid sequence.
  • neither primer of the primer pair will hybridize to a strand of E. dispar nucleic acid under standard amplification conditions, and thus will not substantially amplify any sequence of E. dispar.
  • only one primer of the primer pair will hybridize to a strand of E. dispar nucleic acid under standard amplification conditions, while the other primer will not hybridize to any E. dispar nucleic acid under these conditions, so that the primer set will fail to substantially amplify any E. dispar sequence.
  • each primer of the primer pair will hybridize to E. dispar nucleic acid under standard amplification conditions, but these primers will not hybridize in an orientation that will form an amplification product when each primer is extended at its 3 ' end (e.g.
  • the primers may hybridize to the same strand, or hybridize too far apart to form an amplification product when extended, or hybridize in an orientation so that when extended at its 3' end, at least one primer extends "away” from the other primer). Accordingly, in some embodiments, the primers of the primer pair will not substantially amplify any nucleic acid sequence of E. dispar.
  • primers that reliably amplify sequences of E. histolytica but not E. dispar it can be useful to select primers that amplify a conserved region of E. histolytica, so as to minimize false negatives due to strain-to-strain variation among E. histolytica, but do not amplify a conserved region of E. dispar, so as to minimize false positives that could otherwise be caused by the presence of E. dispar.
  • a highly conserved sequence with ancestral differences between E. dispar and E. histolytica can be a useful region from which to select a target nucleic acid (e.g. a "template") for a target amplification sequence.
  • the target amplification sequence includes an rDNA gene or portion thereof.
  • a gene product for example, an rRNA or portion thereof
  • the primer pair amplifies a polynucleotide sequence that includes at least a portion of the gene encoding the E. histolytica small subunit ribosomal RNA (GenBank Accession No: AB608092.1)(SEQ ID NO: 10).
  • An annotated diagram of the E. histolytica small subunit ribosomal RNA gene is illustrated in Figure 2B.
  • the target sequence includes a polynucleotide having SEQ ID NO: 7 (e.g. positions 191-325 of SEQ ID NO: 10)
  • the target amplification product includes at least about 30 continuous nucleotides of SEQ ID NO: 7, for example at least about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135 continuous nucleotides of SEQ ID NO: 7, including ranges between any two of the listed values.
  • the target amplification sequence includes at least about 30-135 continuous nucleotides of SEQ ID NO: 7, for example about 30-100, 30-110, SOUS, 30-120, 30-125, 30-130, 30-135, 40-100, 40-110, 40-115, 40-120, 40-125, 40-130, 40-135, 50-100, 50-110, 50-115, 50-120, 50-125, 50-130, 50-135, 60-100, 60-110, 60- 115, 60-120, 60-125, 60-130, 60-135, 70-100, 70-110, 70-115, 70-120, 70-125, 70-130, 70-135, 80-100, 80-110, 80-115, 80-120, 80-125, 80-130, 80-135, 90-100, 90-110, 90- 115, 90-120, 90-125, 90-130, 90-135, 100-110, 100-115, 100-120, 100-125, 100-130, 100-135, 110-115, 110-120, 110-125, 110-125, 110
  • the target amplification produce has at least 70% nt-nt identity to SEQ ID NO: 7, for example at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.2% nt- nt identity, including ranges between any two of the listed values.
  • the target amplification sequence includes a polynucleotide having the sequence of SEQ ID NO: 7, and at least one additional polynucleotide upstream and/or downstream of a 5' end or 3 ' end of SEQ ID NO: 7 (e.g. positions 191-325 of SEQ ID NO: 10).
  • the target amplification sequence includes at least about 1 nucleotide upstream of the 5' end of SEQ ID NO: 7 as shown in SEQ ID NO: 10 (see Figure 2B), for example at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides upstream.
  • the target amplification includes at least about 1 nucleotide downstream of the 3' end of SEQ ID NO: 7 as shown in SEQ ID NO: 10 (see Figure 2B), for example at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides upstream.
  • the target amplification sequence includes nucleotides both upstream and downstream of the ends of SEQ ID NO: 7 (as shown in SEQ ID NO: 10), as described herein.
  • the pimer pair does not amplify, under standard amplification conditions, the E. dispar small subunit rRNA.
  • a sequence of the E. dispar small subunit rRNA can be found Genbank accession umber AB282661, which is provided herein as SEQ ID NO: 11
  • the primer set includes a first primer that comprises SEQ ID NO: 1 (GTACAAAATGGCCAATTCATTCAATG).
  • the primer set includes a second primer that comprises SEQ ID NO: 2 (ACTACCAACTGATTGATAGATCAG).
  • a primer that comprises SEQ ID NO: 1 can have high (up to 100%) identity to a sequence encoding the E. histolytica small subunit rRNA, while having lower identity to a homologous region of the E. dispar genome.
  • a primer comprising SEQ ID NO: 1 hybridizes to E. histolytica genomic DNA, but does not hybridize to E. dispar genomic DNA under standard amplification conditions.
  • a primer that comprises SEQ ID NO: 2 can hybridize to genomic DNA in the region of the small subunit rRNA gene of both E. histolytica and E. dispar. Accordingly, in some embodiments, a primer set that includes a first primer comprising SEQ ID NO: 1, and a second primer comprising SEQ ID NO: 2, can amplify a target sequence of E. histolytica nucleic acid, but not E. dispar nucleic acids.
  • the nucleic acid includes genomic DNA.
  • the nucleic acid includes nucleic acid reverse-transcribed from a gene product, for example an mRNA or rRNA.
  • the first primer comprises at least about 10 consecutive nucleotides of SEQ ID NO: l, for example at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleotides of SEQ ID NO: 1 or its complement.
  • the first primer comprises a polynucleotide sequence that is at least about 38% identical of SEQ ID NO: 1, for example at least about 38%, 42, 46, 50, 53, 57, 61, 65, 69, 73, 76, 80, 84, 88, 92, or 96% identical to SEQ ID NO: 1.
  • the first primer comprises SEQ ID NO: 1, and at least 1 additional nucleotide 5' of the 5' terminus of SEQ ID NO: 1, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides 5' of the 5' terminus of SEQ ID NO: 1.
  • one or more of the nucleotides 5' of SEQ ID NO: 1 are complementary to the template strand of SEQ ID NO: 10 as shown in Figure 2B.
  • the first primer has a length of 15-50 nucleotides and comprises at least about 10 consecutive nucleotides of SEQ ID NO: l, for example at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleotides of SEQ ID NO: 1 or its complement, including ranges between any two of the listed values, for example 10-15, 10-20, 15-20, 10-26, 15-26, or 20-26 consecutive nucleotides.
  • the first primer can hybridize to a target sequence of SEQ ID NO: 10, and have at least about 80% identity to the target sequence.
  • the first primer has a length of 15-50 nucleotides and comprises at least about 10 consecutive nucleotides of SEQ ID NO: l, for example at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleotides of SEQ ID NO: 1 or its complement, including ranges between any two of the listed values, for example 10-15, 10-20, 15-20, 10-26, 15-26, or 20-26 consecutive nucleotides.
  • the first primer can hybridize to a target sequence of SEQ ID NO: 10, and have at least about 85% identity to the target sequence.
  • the first primer has a length of 15-50 nucleotides and comprises at least about 10 consecutive nucleotides of SEQ ID NO: l, for example at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleotides of SEQ ID NO: 1 or its complement, including ranges between any two of the listed values, for example 10-15, 10-20, 15-20, 10-26, 15-26, or 20-26 consecutive nucleotides.
  • the first primer can hybridize to a target sequence of SEQ ID NO: 10, and have at least about 90% identity to the target sequence.
  • the first primer has a length of 15-50 nucleotides and comprises at least about 10 consecutive nucleotides of SEQ ID NO: l, for example at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleotides of SEQ ID NO: 1 or its complement, including ranges between any two of the listed values, for example 10-15, 10-20, 15-20, 10-26, 15-26, or 20-26 consecutive nucleotides.
  • the first primer can hybridize to a target sequence of SEQ ID NO: 10, and have at least about 95% identity to the target sequence.
  • the first primer can have 100% identity to the target sequence.
  • the first primer of any of Alternatives 1-4 can be paired with the second primer.
  • the second primer can primer have a length of 15-50 nucleotides and comprise at least about 10 consecutive nucleotides of SEQ ID NO:2, for example at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
  • the second primer can hybridize to a target sequence of SEQ ID NO: 10, and have at least about 80% identity to the target sequence.
  • the first primer of any of Alternatives 1-4 can be paired with the second primer.
  • the second primer can have a length of 15-50 nucleotides and comprise at least about 10 consecutive nucleotides of SEQ ID NO:2, for example at least about 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
  • the second primer can hybridize to a target sequence of SEQ ID NO: 10, and have at least about 85% identity to the target sequence.
  • the first primer of any of Alternatives 1-4 can be paired with the second primer.
  • the second primer can have a length of 15-50 nucleotides and comprise at least about 10 consecutive nucleotides of SEQ ID NO:2, for example at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
  • the second primer can hybridize to a target sequence of SEQ ID NO: 10, and have at least about 90% identity to the target sequence.
  • the first primer of any of Alternatives 1-4 can be paired with the second primer.
  • the second primer have a length of 15-40 nucleotides and can comprise at least about 10 consecutive nucleotides of SEQ ID NO:2, for example at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
  • the second primer can hybridize to a target sequence of SEQ ID NO: 10, and have at least about 95% identity to the target sequence.
  • the second primer can have 100% identity to the target sequence.
  • the first primer is designed in accordance with the alignment shown in Figure 2A, so that the first primer anneals to a sequence of E. histolytica, but not to the homologous sequence of E. dispar under standard amplification conditions. In some embodiments, the first primer anneals to a sequence of E.
  • At least the 3 '-most nucleotide of the first primer is complementary to an E.
  • the second primer comprises at least about 10 consecutive nucleotides of SEQ ID NO: 2, for example about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24, consecutive nucleotides of SEQ ID NO: 2.
  • the second primer comprises a polynucleotide sequence that is at least about 41% identical of SEQ ID NO: 2, for example at least about 41, 45, 50, 54, 58, 62, 66, 70, 75, 79, 83, 87, 91, or 95% identical to SEQ ID NO: 2.
  • sequence-specific probes are provided. Probes include, but are not limited to oligonucleotides as described herein. In some embodiments, the sequence-specific probes disclosed herein specifically hybridize to a target nucleic acid sequence. In some embodiments, the sequence-specific probe can hybridize to a sequence that is found in both E. histolytica and E. dispar. In some embodiments, the sequence-specific probe can hybridize to a sequence that is found in E. histolytica, but not in E. dispar. In some embodiments, the sequence-specific probe specifically hybridizes to, and is fully or substantially complementary to a nucleotide sequence flanked by the binding sites of a pair of amplification primers disclosed herein.
  • the sequence-specific probe specifically hybridizes to, and is fully or substantially complementary a target amplification sequence of a primer set that amplifies E. histolytica, but not E. dispar, nucleic acids under standard amplification conditions.
  • the sequence-specific probe comprises the polynucleotide of SEQ ID NO: 3 (ATTGTCGTGGCATCCTAACTCA).
  • the sequence-specific probe comprises at least about 5 consecutive nucleotides of SEQ ID NO: 3, for example about 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 nucleotides of SEQ ID NO: 3.
  • the sequence-specific probe comprises a sequence that is at least about 22% identical so SEQ ID NO: 3, for example at least about 22%, 27, 31, 36, 40, 45, 54, 59, 63, 68, 72, 77, 81, 86, 90, or 95% identical so SEQ ID NO: 3.
  • the sequence-specific probe overlaps with the binding site of an amplification primer disclosed herein.
  • the probe can have a length of 15-75 nucleotides and comprise at least 10 nucleotides of SEQ ID NO: 3, SEQ ID NO: l, for example at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleotides of SEQ ID NO: 3 or its complement, including ranges between any two of the listed values, for example 10-15, 10-20, 15-20, 10-26, 15-26, or 20-26 consecutive nucleotides.
  • the probe can hybridize to a target sequence of SEQ ID NO: 10, and have at least about 80% identity to the target sequence.
  • the probe can be used in conjunction with any of the primer pairs of Alternatives 5-8.
  • the probe can also hybridize to SEQ ID NO: 1 1.
  • the probe can have a length of 15- 75 nucleotides and comprises at least 10 nucleotides of SEQ ID NO: 3, SEQ ID NO: l, for example at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleotides of SEQ ID NO: 3 or its complement, including ranges between any two of the listed values, for example 10-15, 10-20, 15-20, 10-26, 15-26, or 20-26 consecutive nucleotides.
  • the probe can hybridize to a target sequence of SEQ ID NO: 10, and have at least about 85% identity to the target sequence.
  • the probe can be used in conjunction with any of the primer pairs of Alternatives 5-8.
  • the probe can also hybridize to SEQ ID NO: 1 1.
  • the probe can have a length of 15- 75 nucleotides and comprise at least 10 nucleotides of SEQ ID NO: 3, SEQ ID NO: l, for example at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleotides of SEQ ID NO: 3 or its complement, including ranges between any two of the listed values, for example 10-15, 10-20, 15-20, 10-26, 15-26, or 20-26 consecutive nucleotides.
  • the probe can hybridize to a target sequence of SEQ ID NO: 10, and have at least about 90% identity to the target sequence.
  • the probe can be used in conjunction with any of the primer pairs of Alternatives 5-8.
  • the probe can also hybridize to SEQ ID NO: 1 1.
  • the probe can have a length of 15- 75 nucleotides and comprise at least 10 nucleotides of SEQ ID NO: 3, SEQ ID NO: l, for example at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleotides of SEQ ID NO: 3 or its complement, including ranges between any two of the listed values, for example 10-15, 10-20, 15-20, 10-26, 15-26, or 20-26 consecutive nucleotides.
  • the probe can hybridize to a target sequence of SEQ ID NO: 10, and have at least about 95% identity to the target sequence.
  • the probe can be used in conjunction with any of the primer pairs of Alternatives 5-8.
  • the probe can also hybridize to SEQ ID NO: 1 1.
  • the probe can have a length of 15- 75 nucleotides and comprise at least 10 nucleotides of SEQ ID NO: 3, SEQ ID NO: l, for example at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleotides of SEQ ID NO: 3 or its complement, including ranges between any two of the listed values, for example 10-15, 10-20, 15-20, 10-26, 15-26, or 20-26 consecutive nucleotides.
  • the probe can hybridize to a target sequence of SEQ ID NO: 10, and have 100% identity to the target sequence.
  • the probe can be used in conjunction with any of the primer pairs of Alternatives 5-8.
  • the probe can also hybridize to SEQ ID NO: 11.
  • detectable moieties have been described for the detection of amplification products.
  • One class of detectable moieties is intercalating agents, which bind non- specifically to double-stranded nucleic acid. Intercalating agents have a relatively low fluorescence when unbound, and a relatively high fluorescence upon binding to double-stranded nucleic acids. As such, intercalating agents can be used to monitor the accumulation of double strained nucleic acids during a nucleic acid amplification reaction. Examples of such non-specific dyes include intercalating agents such as SYBR Green I (Molecular Probes), PicoGreen (Molecular Probes), TOTO, YOYO, propidium iodide, ethidium bromide, and the like.
  • sequence-specific nucleic acid probes can be labeled with one or more dyes, such that upon hybridization to a template nucleic acid, a detectable change in fluorescence is generated. While non-specific dyes may be desirable for some applications, sequence-specific probes can provide more accurate measurements of amplification.
  • One configuration of sequence-specific probe can include one end of the probe tethered to a fluorophore, and the other end of the probe tethered to a quencher.
  • sequence-specific probe can include a first probe tethered to a first fluorophore of a FRET pair, and a second probe tethered to a second fluorophore of a FRET pair.
  • the first probe and second probe can be configured to hybridize to sequences of an amplicon that are within sufficient proximity to permit energy transfer by FRET when the first probe and second probe are hybridized to the same amplicon.
  • the sequence specific probe comprises an oligonucleotide as disclosed herein conjugated to a fluorophore.
  • the probe is conjugated to two or more flurophores.
  • fluorophores include: xanthene dyes, e.g., fluorescein and rhodamine dyes, such as fluorescein isothiocyanate (FITC), 2-[ethylamino)-3-(ethylimino)-2-7-dimethyl-3H-xanthen-9-yl]benzoic acid ethyl ester monohydrochloride (R6G)(emits a response radiation in the wavelength that ranges from about 500 to 560 nm), l,l,3,3,3',3'-Hexamethylindodicarbocyanine iodide (HIDC) (emits a response radiation in the wavelength that ranged from about 600 to 660 nm), 6- carboxyfluoresc
  • FITC fluorescein iso
  • Cy3, Cy5 and Cy7 dyes include coumarins, e.g., umbelliferone; benzimide dyes, e.g. Hoechst 33258; phenanthridine dyes, e.g. Texas Red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, e.g. cyanine dyes such as Cy3 (emits a response radiation in the wavelength that ranges from about 540 to 580 nm), Cy5 (emits a response radiation in the wavelength that ranges from about 640 to 680 nm), etc; BODIPY dyes and quinoline dyes.
  • Cy3 emits a response radiation in the wavelength that ranges from about 540 to 580 nm
  • Cy5 emits a response radiation in the wavelength that ranges from about 640 to 680 nm
  • fluorophores of interest include: Pyrene, Coumarin, Diethylaminocoumarin, FAM, Fluorescein Chlorotriazinyl, Fluorescein, Rl 10, Eosin, JOE, R6G, HIDC, Tetramethylrhodamine, TAMRA, Lissamine, ROX, Napthofluorescein, Texas Red, Napthofluorescein, Cy3, and Cy5, CalFluorOrange, and the like.
  • the probe is conjugated to a quencher.
  • a quencher can absorb electromagnetic radiation and dissipate it as heat, thus remaining dark.
  • Example quenchers include Dabcyl, NFQ's, such as BHQ-1 or BHQ-2 (Biosearch), IOWA BLACK FQ (IDT), and IOWA BLACK RQ (IDT).
  • the quencher is selected to pair with a fluorphore so as to absorb electromagnetic radiation emitted by the fluorophore.
  • Flourophore/quencher pairs useful in the compositions and methods disclosed herein are well-known in the art, and can be found, e.g., described in S.
  • a flourophore/quencher pair includes CalFluor Orange and BHQ-1.
  • a fluorophore is attached to a first end of the probe, and a quencher is attached to a second end of the probe. Attachment can include covalent bonding, and can optionally include at least one linker molecule positioned between the probe and the fluorophore or quencher.
  • a fluorophore is attached to a 5 ' end of a probe, and a quencher is attached to a 3 ' end of a probe.
  • a fluorphore is attached to a 3' end of a probe, and a quencher is attached to a 5' end of a probe. Examples of probes that can be used in quantitative nucleic acid amplification include molecular beacons, SCORPIONSTM probes (Sigma) and TAQMANTM probes (Life Technologies).
  • cross-react refers to yielding a detectable signal from a template of the indicated organism (e.g. a non-is. hisotofytica organism as listed below). As shown, for example in Example 6, the presence of the organisms listed in Table 4 does not result in a detectable signal for amplification using primers and probes in accordance with some embodiments herein.
  • cross-reacting can further include depression of the E.
  • the primers and probes do not cross-react with any of the following organisms: Abiotrophia defectiva, Acinetobacter baumannii, Acinetobacter Iwoffii, Aeromonas hydrophila, Alcaligenes faecalis subsp.
  • faecalis Anaerococcus tetradius, Arcobacter butzleri, Arcobacter cryaerophilus, Bacillus cereus, Bacteroides caccae, Bacteroides merdae, Bacteroides stercoris, Bifidobacterium adolescentis, Bifidobacterium longum, Camplylobacter coli, Campylobacter concisus, Campylobacter curvus, Campylobacter fetus subsp. fetus, Campylobacter fetus subsp.
  • probes, primers, and methods of detection in accordance with some embodiments herein are robust in the presence of additional pathogens.
  • primer and probe sets in accordance with embodiments herein showed provided robust results for both fixed and unfixed sample types, and provided results consistent with those of commercial ELISA kits for the detection of E. histolytica. As such, it is contemplated that primer and probe sets in accordance with embodiments herein provide robust results across a variety of sample types (e.g. fixed and unpreserved or non-fixed samples), and consistent with other methods of determining the presence of absence of is. histolytica.
  • the 95% limit of detection (LoD) for some primers and probes in accordnance with emodiments herein is about 17 is. histolytica organsims per milliliter of sample.
  • the "95% LoD,” or unless stated otherwise, “LoD” refers to the concentration that yields a positive result 95% of the time. Accordingly, in some embodiments, the primers and probes will produce a positive signal (e.g. a Ct score below the cutoff) if is.
  • histolytica is present in the amplification reaction in a quantity that is at least the 95% limit of detection (LoD), but will not produce a positive signal if only one or more of the above-listed non-is. histolytica organisms are present.
  • the LoD of E. hisolytica is about 17 is.
  • histolytica organisms (or quantity of template sequence corresponding to 17 is. histolyica organisms) per milliliter of sample.
  • the LoD is no more than about 50 organisms per militliter of reaction, for example no more than about 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 E. histolytica organisms (or genomes thereof) per mililiter. It is noted that the in some embodiments, the LoD is comparable for both fixed and non-fixed samples (see Example 8 and Tables 6-7). As such, it is understood that primer and probes in accordance with some embodiments herein yield comparable E. histolytica detection properties, for example comparable LoD values, for both fixed and non-fixed samples.
  • the amount of E. histolytica detected was not substantially altered by a high titer of Cryptosporidium parvum, Giardia lamblia, and Entamoeba dispar, nor was there substantial cross-reactivity with these organisms. Accordingly, in some embodiments, the LoD of E. histolytica organisms is not substantially altered by a high titer presence of another pathogenic organism in the sample. In some embodiments, the detection of E. histolytica organisms (measured, for example by Ct score) is not substantially altered by a high titer presence of another pathogenic organism in the sample.
  • a high titer comprises a quantity of at least lxlO 6 organisms/mL of sample, for example about lxlO 6 organisms/mL, lxlO 6 , 2xl0 6 , 3xl0 6 , 4xl0 6 , 5xl0 6 , 6xl0 6 , 7xl0 6 , 8xl0 6 , 9xl0 6 , lxlO 7 , 1.5xl0 7 , 2xl0 7 , 3xl0 7 , 4xl0 7 , 5xl0 7 , 6xl0 7 , 7xl0 7 , 8xl0 7 , 9xl0 7 , lxlO 8 , lxlO 9 , or lxlO 10 organisms/mL of sample.
  • a high titer comprises a quantity of at least about 1.5xl0 7 organisms/mL of sample.
  • kits can include at least one primer pair as described herein.
  • the primer pair can amplify an E. histolytica target sequence under standard amplification conditions, but cannot amplify an E. dispar target sequence under standard amplification conditions, as described herein.
  • the kits can include a probe as described herein.
  • the probe is specific to a nucleic acid sequence that occurs in both E. histolytica and E. dispar as described herein.
  • the primer set includes a forward primer comprising an oligonucleotide having the sequence of SEQ ID NO: 1, or a variant thereof, a reverse primer comprising an oligonucleotide having the sequence of SEQ ID NO: 2, or a variant thereof, and a probe comprising an oligonucleotide having the sequence of SEQ ID NO: 3, or a variant thereof.
  • the probe comprises a fluorophore/quencher pair as described herein.
  • the kits include samples, for example positive controls that contain E. histolytica or E. histolytica DNA as decribed herein.
  • the kits can further include negative controls, for example that contain only E. dispar, or E. dispar DNA.
  • the kits can further include packaging and/or instructions.
  • kits further include reagents for a multiplex assay for detecting at least one other parasitic organism from a human stool sample, for example at least one of Giardia lamblia, Cryptosporidium parvum, Cryptosporidium hominis, and the like.
  • a master mix can include at least two reagents for an assay that are provided in relative concentrations that are proportional to the relative concentrations of the reagents in a quantitative nucleic acid amplification assay Thus, a single a single quantity of master mix can be added to a reaction to provide appropriate relative concentrations of two or more reagents.
  • a master mix can include at least two of: polymerase, buffer, salts, for example magnesium, nucleotide triphosphates, a primer set, and water.
  • a master mix can be provided at a higher concentration than will be used in a reaction.
  • a master mix is provided in a lyophilized form, and reconstituted at a higher concentration that will be used in the reaction.
  • a master mix includes reagents at a concentration of at least about 2x of the reaction concentration, for example 2x, 2.5x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, 15x, 20x, 25x, 40x, 50x, lOOx, 200x, 250x, or 500x.
  • Samples as provided herein include substances that may or may not contain Entamoeba nucleic acids.
  • the sample includes fecal matter from a human, or a portion or derivative thereof.
  • the sample includes a biopsy, for example tissue from a human that is possibly infected with Entamoeba, such as gastrointestinal, liver, lung, or central nervous system tissue.
  • the sample includes a cell culture, for example a culture derived from human fecal matter.
  • the sample has been processed, for example to isolate nucleic acids from other substances, or to remove non-nucleic acid substances from the sample (for example to remove lipids, proteins, cellular debris, and the like).
  • the sample has been treated with protease. It has been shown that primers and probes in accordance with embodiments herein achieve comparable detection properties for fixed and unpreserved samples (see, e.g., Example 8 and Tables 6-7).
  • the sample is fixed, for example in a quantity of fixative such as formalin.
  • the sample is unpreserved (e.g. "non-fixed").
  • the sample contains E. histolytica and/or E dispar nucleic acids. In some embodiments, it is known that the sample includes at least one of E. histolytica or E. dispar, but it is unknown which one sample includes, or whether the sample includes both. In some embodiments, the sample contains both E. hisotlytica and E. dispar.
  • the sample includes a positive control, for example spiking the sample with nucleic acids of E. histolytica, E. dispar, or a combination of nucleic acids from is. histolytica, or E. dispar.
  • the sample is spiked with at least 1000 ("IK") copies of E.
  • dispar target amplification sequence for example at least about IK copies, 2K, 3K, 4K, 5K, 6K, 7K, 8K, 9K, 10K, 20K, 30K, 40K, 50K, 60K, 70K, 80K, 90K, 100K, 150K, 200K, 250K, 300K, 350K, 400K, 450K, 500K, 550K, 600K, 650K, 700K, 750K, 800K, 850K, 900K, 1000K, HOOK, 1200K, 1300K, 1400K, 1500K, 1600K, 1700K 1800K, 1900K, or 2000K copies.
  • the sample is spiked with at least 100 copies of E.
  • histolytica target amplification sequence for example at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, IK copies, 2K, 3K, 4K, 5K, 6K, 7K, 8K, 9K, 10K, 20K, 30K, 40K, 50K, 60K, 70K, 80K, 90K, 100K, 150K, 200K, 250K, 300K, 350K, 400K, 450K, 500K, 550K, 600K, 650K, 700K, 750K, 800K, 850K, 900K, 1000K, HOOK, 1200K, 1300K, 1400K, 1500K, 1600K, 1700K 1800K, 1900K, or 2000K copies.
  • the sample is spiked with E. histolytica and E. dispar target nucleic acids.
  • the sample includes nucleic acids isolated from one or more of the above. Nucleic acids can be isolated using standard techniques, well- known to one skilled in the art.
  • primer and probe sets, and methods of using the same are provided for the detection of E. histolyica.
  • the primers and probe sets and methods do not detect non-pathogenic E. dispar.
  • the primers and probe sets and methods produce robust results, that are not inhibited or interfered with in the case of a simulated mixed E. histolytica and E. dispar infection.
  • the primers and probe sets and methods detect Entamoeba histolytica from human clinical specimens identified by traditional microscopic methods (which at the time of the application represent the current standard of care).
  • the primers and probe sets and methods produce results that agree with a commercially available FDA-cleared ELISA assay for the appropriate specimen type using clinical specimens.
  • the primers and probe sets and methods do not cross-react with other organisms likely to be found in stool or a variety of other pathogens.
  • the primers and probe sets and methods do react with different Entamoeba histolytica isolates.
  • the primers and probe sets and methods are sensitive to detect down to, and below, 17 organisms per mL in the sample buffer tube (or a quantity of template sequence corresponding to 17 organisms).
  • EXAMPLE 1 Amplification in the presence of of E. histolytica and E. dispar plasmid sequences
  • a previously-described primer set and probe combination (see Verweij et al, J. .Clin. Microbiol. 42: 1220-23, 2004), which included a forward primer of SEQ ID NO: 4 (ATTGTCGTGGCATCCTAACTCA), a reverse primer of SEQ ID NO: 5 (GCGGACGGCTCATTATAACA), and a probe of SEQ ID NO: 6 (TCATTGAATGAATTGGCCATTT), which comprised a CalFluor Orange fluorphore and BHQ-1 quencher (see Figure 1) were used in a quantitative PCR reaction on a BD MAXTM system.
  • the primer set of SEQ ID NO: 4 and SEQ ID NO: 5 amplify rDNA sequences of both E. histolytica and E. dispar.
  • the probe of SEQ ID NO: 6 has 100% percent homology to the target amplification sequence (defined by the primer set of SEQ ID NOs: 4 and 5) in E. histolytica, but not E. dispar.
  • EXAMPLE 2 Detection of E. histolytica in the presence of E. histolytica and E. dispar plasmid sequences
  • a primer-probe set according to embodiments herein was used in a quantitative PCR amplification reaction performed on the BD MAXTM platform.
  • the PCR mixture was heated to 97°C for 10 minutes to activate the DNA Polymerase.
  • Two- step thermal cycling was then carried out for 45 cycles with a 15 second denaturation step at 97°C followed by an annealing/extension step for 64.5 seconds at 62°C.
  • the primer set included a forward primer of SEQ ID NO: 1, a reverse primer of SEQ ID NO: 2, and a probe of SEQ ID NO: 3, which comprised a CalFluor Orange fluorphore and BHQ-1 quencher (see Figure 1). It is noted that the primer of SEQ ID NO: 1 will anneal to E.
  • the primer of SEQ ID NO: 2 will anneal to target nucleic acids sequence on either of the E. histolytica and E. dispar small ribosomal subunit gene. Accordingly, the primer set of SEQ ID NO: 1 and SEQ ID NO: 2 will substantially amplify E. histolytica, but not E. dispar target amplification sequence.
  • the probe of SEQ ID NO: 3 has 100% complementarity to either of E. histolytica or E. dispar small ribosomal subunit gene DNA sequence.
  • Example 1 reactions were provided with plasmid that contained target rDNA gene sequence template from E. histolytica, and/or E. dispar. Unlike Example 1, cross-reactivity was not seen with E. dispar template. Moreover, the presence of E. dispar template did not depress the amplification signal (see Figure 4). In the presence of a constant amount of E. histolytica template, the presence of 0, 250,000 ("250K”), 500,000 ("500K”), 750,000 (“750K”), and 1,000,000 (“le6") copies of E. dispar template-containing plasmid did not decrease the amplification signal from E. histolytica ( Figure 4). A negative control that contained no template (“NTC”) was performed, and as expected, no signal was detected. As summarized in Figure 1, the primer/probe combination of this example did not cause any identifiable E. histolytica signal depression.
  • NTC no template
  • the primer set and probe as in Example 2 produced robust, and consistent levels of E. histolytica signal.
  • a primer set designed to amplify a sequence specific to E. histolytica, but not E. dispar can permit the detection of E. histolytica-specific signal without interference from E. dispar sequences.
  • DNA extraction from the stool specimens was performed as follows: Specimens were vortexed. A ⁇ ⁇ loop was inserted in each specimen to the depth of the loop and then expressed using a swirling motion into BD MAXTM Sample Buffer Tubes (SBT) containing Sample Buffer [50 mM Tris-HCl (pH 7.0), 1% Triton X-100, 1 mM EDTA (pH 8.0), 20 mM H 3 B0 3 , 20 mM Na 3 C 6 H 5 07. 2H 2 0]. The SBTs were closed with a septum cap and then heated on the BD Prewarm Heater to approximately 1 10°C for 20 minutes to facilitate lysis of organisms.
  • SBT Sample Buffer Tubes
  • the SBTs were cooled to room temperature by the BD Prewarm Heater, vortexed briefly, and then transferred to the BD MAXTM System.
  • a 500 ⁇ volume of sample buffer was extracted per sample for 10 minutes at 75°C using 12 units of proteinase K, 0.12% trehalose, and 104 copies of an internal control DNA in the presence of 0.5 ⁇ g/ ⁇ l PAMAM-coupled magnetic beads on the BD MAXTM System.
  • the beads, with the bound nucleic acids were washed with 500 ⁇ 1 of wash buffer [12.5 mM Tris (pH 6.8), 0.03% ProClin 300, 0.1% Tween-20].
  • Nucleic acids were then eluted by heating the beads for 3 minutes at 80°C in 12.5 ⁇ of elution buffer [20 mM NaOH]. Eluted nucleic acids were neutralized by the addition of 22.5 ⁇ of neutralization buffer [7.78 mM MgC12, 155.6 mM Tris (pH 8.0), 4.44 mM NaOH, 0.03% ProClin300, 0.016% Tween-20].
  • a PCR master mix was prepared as follows: Neutralized nucleic acids (35 ⁇ 1) were used to rehydrate dried down master mix. The final concentration of components in the PCR master mix after rehydration with is as follows: 5 mM MgCl 2 , 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.5 mM dNTPs (each), 0.6 mg/ml BSA, 0.04 U/ ⁇ Hot Gold Star DNA Polymerase. The master mix also included PCR primers and TaqMan® dual-labeled hydrolysis probes.
  • Primers and probes for Entamoeba histolytica were included at 900 nM for forward and reverse primers and 550 nM for the probe.
  • the primer set and probe set for the detection of E. histolytica included a forward primer having the nucleic acid sequence of SEQ ID NO: 1, a reverse primer having a nucleic acid sequence of SEQ ID NO: 2, and a probe having the nucleic acid sequence of SEQ ID NO: 3.
  • the probe for Entamoeba histolytica was labeled with Cal Fluor Orange 560 and Black Hole Quencher- 1.
  • Primers and probes for the internal control were included at 300 nM each. The internal control probe was labeled with Quasar 705 and Black Hole Quencher-3.
  • Primers and probes for Cryptosporidium parvum/hominis and Giardia lamblia were included at 200 nM for forward and reverse primers and 550 nM for probes.
  • the probe for Cryptosporidium parvum/hominis was labeled with CalFluor Red 610 and Black Hole Quencher-2.
  • the probe for Giardia lamblia was labeled with FAM and Black Hole Quencher- 1.
  • the BD MAXTM System dispenses approximately 12 ⁇ of PCR-ready solution into the BD MAXTM Microfluidic Cartridge. Microvalves in the BD MAXTM Microfluidic Cartridge are sealed by the system prior to initiating PCR to contain the amplification mixture thus preventing evaporation and contamination.
  • the PCR mixture was heated to 97°C for 10 minutes to activate the DNA Polymerase. Two- step thermal cycling was then carried out for 45 cycles with a 15 second denaturation step at 97°C followed by an annealing/extension step for 64.5 seconds at 62°C.
  • the BD MAXTM System monitors fluorescent signals at each cycle and interprets the data at the end of the program to report the final results.
  • Result calls were based on a Ct. Score algorithm that includes an initial static endpoint threshold for each target channel and a secondary dynamic QC threshold that changes inversely with Ct. Endpoint fluorescence must exceed both thresholds and a final Ct must be ⁇ 42 to be considered positive. Additional checks for excessively variable PCR curves were used to exclude reactions that had insufficient volume in the PCR chamber. Amplification failure of the internal control causes the system to return unresolved results for each target channel that fails to meet the Ct. Score thresholds for positivity.
  • the BD MAXTM detected E. histolytica near the limit of detection (LoD) in simulated multiple infection specimens containing high titer Cryptosporidium parvum, Giardia lamblia, and Entamoeba dispar.
  • the BD MAXTM system was used to detect the form of the Entamoeba histolytica organism shed in true human clinical specimens detected by traditional methods representing both unpreserved and 10% formalin fixed specimen types. For comparison, a commercially-available ELISA (TechLab E.histolytica II) was performed on the same samples.
  • Camplylobacter 15 1.55 NA >lxl0 8 >lxl0 6 15 ⁇ , Neg. coli CFU CFU
  • Escherichia coli 56 3.17 NA >lxl0 8 >lxl0 6 15 ⁇ Neg.
  • Escherichia coli >lxl0 8 >lxl0 6
  • Escherichia coli >lxl0 8 >lxl0 6
  • Proteus penneri 88 10.77 NA >lxl0 8 >lxl0 6 15 ⁇ , Neg.
  • Coxsackie A9 120 9.33 NA 1.6xl0 5 >lxl0 4 150 ⁇ , Neg.
  • the BD MAXTM assay was tested with multiple different isolates of Entamoeba histolytica at the assay LOD in the presence of ⁇ , of unpreserved stool matrix per test. 24 replicates per isolate were tested.
  • the BD MAXTM assay detected a variety of different Entamoeba histolytica isolates. The results are shown in Table 5.
  • the 95% LoD for each specimen type was determined by linear dilution of Entamoeba histolytica trophozoites in sample buffer with ⁇ ⁇ of the appropriate stool matrix. A minimum of 36 replicates per test level were performed. The LoD is approximately 17 organisms/ml in the sample buffer tube. The results are shown in Table 6 (unpreserved samples) and Table 7 (samples fixed in 10% formalin).

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Abstract

Provided herein are compositions, methods, and kits for detection of Entamoeba nucleic acids. Some embodiments relate to detection of E. histolytica but not E. dispar. Some embodiments relate to quantification of levels of E. histolytica.

Description

DETECTION OF ENTAMOEBA NUCLEIC ACIDS
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. Provisional App. No. 61/923086 filed January 2, 2014, which is hereby incorporated by reference in its entirety.
REFERENCE TO SEQUENCE LISTING, TABLE, OR COMPUTER PROGRAM
LISTING
[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled GENOM123WOSEQUENCE.TXT, created and last saved on December 22, 2014, which is 7,503 bytes in size. The information is incorporated herein by reference in its entirety.
Field
[0003] Embodiments herein relate generally to methods and compositions that are useful for detecting the presence of Entamoeba nucleic acids.
Background
[0004] Amebiasis is a disease that can be caused by infection with the protozoan Entamoeba histolytica. E. histolytica infection is typically in the intestinal tract, and can cause colitis, and amoebic dysentery. E. histolytica infection can also spread to other organs, including the liver, the lungs, or central nervous system. E. dispar is a non-pathogenic species, and is morphologically indistinguishable from the pathogenic E. histolytica (Verweij et al, J. Clin. Microbiol. 42: 1220-23, 2004). Moreover, E. dispar and E. histolytica genomes have a high degree of nucleic acid sequence homology. It has been estimated that E. histolytica and/or E. dispar parasitize 10% of the world's population (Verweij et al, J. Clin. Microbiol. 42: 1220-23, 2004). However, it has been estimated that only about 10% of these Entamoeba infections are pathogenic (e.g. infection by E. histolytica) so as to require treatment (Gonin et al, J. Clin. Microbiol. 41 : 237-42, 2003). Thus, distinguishing between E. dispar and E. histolytica infection is useful in guiding clinical decisions.
[0005] Quantitative nucleic acid amplification reactions can be useful for quantifying the relative and/or absolute amount of target nucleic acid sequences present in a sample. Due to the highly sensitive nature of quantitative nucleic acid amplification reactions, in order to avoid false positives, false negatives, overestimation of target or product quantity, or underestimation of target or product quantity, extreme care must be taken when selecting reagents and methods for quantitative nucleic acid amplification. Ribosomal DNA (rDNA) genes are highly conserved. The high degree of conservation of rDNA sequences can result in little variability between different organisms of the same species, a feature that can make rDNA genes useful for nucleic-acid-based detection assays directed to the detection of a desired species. However, the high degree of homology between E. histolytica and E. dispar rDNA genes can complicate quantitative nucleic acid amplification for the specific detection of the different species. For example, it has been reported that multi-template PCR amplification or rDNA genes can be subject to bias, and can produce various artifacts (Kanagawa, J. Bioscience and Bioengineering 96: 317-23, 2003; Wang et al., Microbiology 142: 1 107-14, 1996).
SUMMARY
[0005] According to some embodiments, a method of detecting the presence of an E. histolytica polynucleotide sequence in a sample. The method can comprise contacting the sample with a first primer consisting essentially of SEQ ID NO: 1 (GTACAAAATGGCCAATTCATTCAATG). The method can comprise contacting the sample with a second primer consisting essentially of SEQ ID NO: 2 (ACTACCAACTGATTGATAGATCAG). The method can comprise extending the first and second primer, thereby producing at least one amplicon if the E. histolytica polynucleotide sequence is present in the sample. The method can comprise contacting the sample with an oligonucleotide probe comprising a polynucleotide consisting essentially of SEQ ID NO: 3 (ATTGTCGTGGCATCCTAACTCA) or its complement. In some emboidmnents, the probe provides detectable signal when it is bound to a substantially complementary nucleic acid, but does not provide detectable signal when it is single-stranded. The method can comprise detecting the signal, if the amplicon is present. In some embodiments, if used under standard amplification conditions, the first primer and second primer amplify the E. histolytica polynucleotide sequence, but do not substantially amplify any E. dispar polynucleotide sequence. In some embodiments, the first primer hybridizes to the E. histolytica polynucleotide sequence if contacted with the E. histolytica polynucleotide sequence at a temperature of at least about 50°C in 5 mM MgC12, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA, but does not hybridize to any E. dispar polynucleotide sequence if contacted with any E. dispar polynucleotide sequence at a temperature of at least about 60°C in 5 mM MgCl2, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA. In some embodiments, the second primer hybridizes to the E. histolytica polynucleotide sequence if contacted with E. histolytica polynucleotide sequence at a temperature of at least about 60°C in 5 mM MgC12, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSAl .96% Trehalose, 0.6 mg/ml BSA, and hybridizes to an E. dispar polynucleotide sequence if contacted with the E. dispar polynucleotide sequence at a temperature of at least about 60°C in 5 mM MgC12, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSAl .96% Trehalose, 0.6 mg/ml BSA. In some embodiments, each of the first primer and second primer hybridizes to the E. histolytica polynucleotide sequence if contacted with the E. histolytica polynucleotide sequence at a temperature of at least about 60°C in in 5 mM MgC12, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSAl.96% Trehalose, 0.6 mg/ml BSA, but the second primer does not hybridize to any E. dispar polynucleotide sequence if contacted with any E. dispar polynucleotide sequence at a temperature of at least about 60°C in 5 mM MgC12, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA. In some embodiments, the sample comprises E. histolytica and E. dispar. In some embodiments, the sample comprises fecal material of a human. In some embodiments, the sample comprises fixed material. In some embodiments, the sample is non-fixed. In some embodiments, a 95% limit of detection for E. histolytica comprises no more than about 17 E. histolytica genomes per milliliter. In some embodiments, if used under standard amplification conditions, the primers and probes do not cross-react with any of the following organisms, if present in the sample: Abiotrophia defectiva, Acinetobacter baumannii, Acinetobacter Iwoffli, Aeromonas hydrophila, Alcaligenes faecalis subsp. faecalis, Anaerococcus tetradius, Arcobacter butzleri, Arcobacter cryaerophilus, Bacillus cereus, Bacteroides caccae, Bacteroides merdae, Bacteroides stercoris, Bifidobacterium adolescentis, Bifidobacterium longum, Camplylobacter coli, Campylobacter concisus, Campylobacter curvus, Campylobacter fetus subsp. fetus, Campylobacter fetus subsp. venerealis, Campylobacter gracilis, Campylobacter hominis, Camplylobacter jejuni, Campylobacter lari, Campylobacter rectus, Campylobacter upsaliensis, Candida albicans, Candida catenulate, Cedecea davisae, Chlamydia trachomatis, Citrobacter amalonaticus, Citrobacter fruendii, Citrobacter koseri, Citrobacter sedlakii, Clostridium difficile 17858, Clostridium difficile 43598, Clostridium difficile CCUG 8864-9689, Clostridium difficile 43255, Clostridium difficile BAA-1805, Clostridium difficile 43593, Clostridium perfringens, Collinsella aerofaciens, Corynebacterium genitalium, Desulfovibrio piger, Edwardsiella tarda, Eggerthella lenta, Enterobacter aerogenes, Enterobacter cloacae, Enterococcus casseliflavus, Enterococcus cecorum, Enterococcus dispar, Enterococus faecalis, Enterococcus gallinarum, Enterococcus hirae, Enterococcus raffinosus, Escherichia coli, Escherichia fergusonii, Escherichia hermannii, Escherichia vulneris, Fusobacterium varium, Gardnerella vaginalis, Gemella morbillorum, Hafnia alvei, Helicobacter fennelliae, Helicobacter pylori, Klebsiella oxytoca, Klebsiella pneumonia, Lactobacillus acidophilus, Lactobacillus reuteri, Lactococcus lactis, Leminorella grimontii, Listeria grayi, Listeria innocua, Listeria monocytogenes, Morganella morganii, Peptoniphilus asaccharolyticus, Peptostreptococcus anaerobius, Plesiomonas shigelloides, Porphyromonas asaccharolytica, Prevotella melaninogenica, Proteus mirabilis, Proteus penneri, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii, Pseudomonas aeruginosa, Pseudomonas fluorescens, Ruminococcus bromii, Salmonella typhimurium, Salmonella enteriditis, Serratia liquefaciens, Serratia marcescens, Shigella sonnei, Shigella flexneri, Staphylococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus intermedius, Streptococcus uberis, Trabulsiella guamensis, Veillonella parvula, Vibrio cholera, Vibrio parahaemolyticus, Yersinia bercovieri, Yersinia enterocolitica, Yersinia rohdei, Adenovirus type 2, Adenovirus type 14, Adenovirus type 40, Adenovirus type 41, Coxsackie A9, Coxsackie Bl, HHV-5, Cytomegalovirus, Enterovirus type 69, Human Papillomavirus Type 16, Human Papillomavirus Type 18, Herpes Simplex Virus I, Herpes Simplex Virus II, Norovirus I, Norovirus II, Rotavirus, Blastocystis hominis, Encephalitozoon intestinalis, Encephalitozoon helium, Encephalitozoon cuniculi, Pentatrichomonas hominis, Entamoeba barrette, Entamoeba dispar, Entamoeba gigivalis, Entamoeba invadens, Entamoeba moshkovskii, Entamobea ranarum, Citrobacter fruendii (rpt), Enterobacter cloacae (rpt), Cryptosporidium parvum, Giardia lamblia, or Cryptosporidium meleagridis. In some embodiments, if used under standard amplification conditions, the primers and probes do not cross-react with any of the following organisms, if present in the sample: Entamoeba coli, Entamoeba dispar, Entamoeba polecki, Entamoeba muris, Entamoeba nuttalli, Entamoeba hartmanni, and Entamoeba bovis. In some embodiments, if used under standard amplification conditions, the primers and probes produce fewer than 1 in 1600 false positives for samples that do not comprise E. histolytica. In some embodiments, E. dispar, if present, does not inhibit production of the amplicon if the E. histolytica polynucleotide sequence is present in the sample. In some embodiments, E. dispar, if present, does not inhibit determining the presence or absence of E. histolytica.
[0006] According to some embodiments, a kit is provided. The kit can comprise a first primer. The kit can comprise a second primer. In some embodiments, if used under standard amplification conditions, the first primer and second primer amplify a E. histolytica polynucleotide sequence, thereby producing an amplicon, but do not substantially amplify any E. dispar polynucleotide sequence. The kit can comprise a probe, wherein the probe comprises a polynucleotide consisting essentially of a sequence, wherein the sequence or its complement is present in each of the amplicon, a polynucleotide sequence of E. histolytica, and a polynucleotide sequence of E. dispar. In some embodiments, the probe comprises a fluorophore; and a quencher. In some embodiments, the primers and probes amplify an E. histolytica polynucleotide sequence with a 95% limit of detection of no more than about 17 E. histolytica organisms per mililiter. In some embodiments, if used under standard amplification conditions, the primers and probes do not cross-react with any of the following organisms, if present in the sample: Abiotrophia defectiva, Acinetobacter baumannii, Acinetobacter Iwoffii, Aeromonas hydrophila, Alcaligenes faecalis subsp. faecalis, Anaerococcus tetradius, Arcobacter butzleri, Arcobacter cryaerophilus, Bacillus cereus, Bacteroides caccae, Bacteroides merdae, Bacteroides stercoris, Bifidobacterium adolescentis, Bifidobacterium longum, Camplylobacter coli, Campylobacter concisus, Campylobacter curvus, Campylobacter fetus subsp. fetus, Campylobacter fetus subsp. venerealis, Campylobacter gracilis, Campylobacter hominis, Camplylobacter jejuni, Campylobacter lari, Campylobacter rectus, Campylobacter upsaliensis, Candida albicans, Candida catenulate, Cedecea davisae, Chlamydia trachomatis, Citrobacter amalonaticus, Citrobacter fruendii, Citrobacter koseri, Citrobacter sedlakii, Clostridium difficile 17858, Clostridium difficile 43598, Clostridium difficile CCUG 8864-9689, Clostridium difficile 43255, Clostridium difficile BAA-1805, Clostridium difficile 43593, Clostridium perfringens, Collinsella aerofaciens, Corynebacterium genitalium, Desulfovibrio piger, Edwardsiella tarda, Eggerthella lenta, Enterobacter aerogenes, Enterobacter cloacae, Enterococcus casseliflavus, Enterococcus cecorum, Enterococcus dispar, Enterococus faecalis, Enterococcus gallinarum, Enterococcus hirae, Enterococcus rafflnosus, Escherichia coli, Escherichia fergusonii, Escherichia hermannii, Escherichia vulneris, Fusobacterium varium, Gardnerella vaginalis, Gemella morbillorum, Hafnia alvei, Helicobacter fennelliae, Helicobacter pylori, Klebsiella oxytoca, Klebsiella pneumonia, Lactobacillus acidophilus, Lactobacillus reuteri, Lactococcus lactis, Leminorella grimontii, Listeria grayi, Listeria innocua, Listeria monocytogenes, Morganella morganii, Peptoniphilus asaccharolyticus, Peptostreptococcus anaerobius, Plesiomonas shigelloides, Porphyromonas asaccharolytica, Prevotella melaninogenica, Proteus mirabilis, Proteus penneri, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii, Pseudomonas aeruginosa, Pseudomonas fluorescens, Ruminococcus bromii, Salmonella typhimurium, Salmonella enteriditis, Serratia liquefaciens, Serratia marcescens, Shigella sonnei, Shigella flexneri, Staphylococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus intermedius, Streptococcus uberis, Trabulsiella guamensis, Veillonella parvula, Vibrio cholera, Vibrio parahaemolyticus, Yersinia bercovieri, Yersinia enterocolitica, Yersinia rohdei, Adenovirus type 2, Adenovirus type 14, Adenovirus type 40, Adenovirus type 41, Coxsackie A9, Coxsackie Bl, HHV-5, Cytomegalovirus, Enterovirus type 69, Human Papillomavirus Type 16, Human Papillomavirus Type 18, Herpes Simplex Virus I, Herpes Simplex Virus II, Norovirus I, Norovirus II, Rotavirus, Blastocystis hominis, Encephalitozoon intestinalis, Encephalitozoon helium, Encephalitozoon cuniculi, Pentatrichomonas hominis, Entamoeba barrette, Entamoeba dispar, Entamoeba gigivalis, Entamoeba invadens, Entamoeba moshkovskii, Entamobea ranarum, Citrobacter fruendii (rpt), Enterobacter cloacae (rpt), Cryptosporidium parvum, Giardia lamblia, or Cryptosporidium meleagridis. In some embodiments, the first primer comprises a polynucleotide having at least about 90% identity to SEQ ID NO: 1 (GTACAAAATGGCCAATTCATTCAATG) or its complement. In some embodiments, the first primer consists essentially of SEQ ID NO: 1 (GTACAAAATGGCCAATTCATTCAATG) or its complement. In some embodiments, the second primer comprises a polynucleotide having at least about 90% identity to SEQ ID NO: 2 (ACTACCAACTGATTGATAGATCAG) or its complement. In some embodiments, the second primer comprises a polynucleotide having the sequence of SEQ ID NO: 2 (ACTACCAACTGATTGATAGATCAG) or its complement. In some embodiments, the probe comprises a polynucleotide having at least about 90% identity to SEQ ID NO: 3 (ATTGTCGTGGCATCCTAACTCA) or its complement. In some embodiments, the probe comprises a polynucleotide having the sequence of SEQ ID NO: 3 (ATTGTCGTGGCATCCTAACTCA) or its complement. In some embodiments, if used under standard amplification conditions, the primers and probes do not cross-react with any of the following organisms, if present in the sample: Entamoeba coli, Entamoeba dispar, Entamoeba poiecki, Entamoeba muris, Entamoeba nuttalli, Entamoeba hartmanni, and Entamoeba bovis. In some embodiments, if used under standard amplification conditions, the primers and probes produce fewer than 1 in 1600 false positives for samples that do not comprise E. histolytica. In some embodiments, E. dispar, if present, does not inhibit production of the amplicon if the E. histolytica polynucleotide sequence is present in the sample. In some embodiments, E. dispar, if present, does not inhibit determining the presence or absence of E. histolytica. [0006]
According to some embodiments, a kit is provided. The kit can comprise a first primer comprising a polynucleotide having at least about 90% identity to SEQ ID NO: 1 (GTACAAAATGGCCAATTCATTCAATG). The kit can comprise a second primer comprising polynucleotide having at least about 90% identity to SEQ ID NO: 2 (ACTACCAACTGATTGATAGATCAG). The kit can comprise a probe comprising a polynucleotide having at least about 90% identity to SEQ ID NO: 3 (ATTGTCGTGGCATCCTAACTCA) or its complement; a flurophore; and a quencher. In some embodiments, the first primer consists essentially of SEQ ID NO: 1 (GTACAAAATGGCCAATTCATTCAATG). In some embodiments, the second primer consists essentially of SEQ ID NO: 2 (ACTACCAACTGATTGATAGATCAG). In some embodiments, the probe comprises a polynucleotide consisting essentially of SEQ ID NO: 3 (ATTGTCGTGGCATCCTAACTCA) or its complement. In some embodiments, if used under standard amplification conditions, the primers and probes do not cross-react with any of the following organisms, if present in the sample: Entamoeba coli, Entamoeba dispar, Entamoeba polecki, Entamoeba muris, Entamoeba nuttalli, Entamoeba hartmanni, and Entamoeba bovis. In some embodiments, if used under standard amplification conditions, the primers and probes produce fewer than 1 in 1600 false positives for samples that do not comprise E. histolytica. In some embodiments, E. dispar, if present, does not inhibit production of the amplicon if the E. histolytica polynucleotide sequence is present in the sample. In some embodiments, E. dispar, if present, does not inhibit determining the presence or absence of E. histolytica.
[0007] In some embodiments, a method of detecting the presence of an E. histolytica polynucleotide sequence in a sample. The method can comprise contacting the sample with a first primer. The method can comprise contacting the sample with a second primer. In some embodiments, if used standard amplification conditions, the first primer and second primer amplify the E. histolytica polynucleotide sequence, but do not substantially amplify any E. dispar polynucleotide sequence. The method can comprise extending the first and second primer, thereby producing at least one amplicon if the E. histolytica polynucleotide sequence is present in the sample. The method can comprise contacting the sample with an oligonucleotide probe. In some embodiments, the probe provides detectable signal when it is bound to a substantially complementary nucleic acid, but does not provide detectable signal when it is single-stranded. In some embodiments, the probe comprises a polynucleotide consisting essentially of sequence that is a portion of the E. histolytica polynucleotide sequence, a polynucleotide sequence of E. dispar, and a sequence of the amplicon. The method can comprise detecting the signal, if the amplicon is present. In some embodiments, the first primer hybridizes to the E. histolytica polynucleotide sequence if contacted with the E. histolytica polynucleotide sequence at a temperature of at least about 50°C in 5 mM MgC12, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA, but does not hybridize to any E. dispar polynucleotide sequence if contacted with any E. dispar polynucleotide sequence at a temperature of at least about 60°C in 5 mM MgCl2, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA. In some embodiments, the second primer hybridizes to the E. histolytica polynucleotide sequence if contacted with E. histolytica polynucleotide sequence at a temperature of at least about 60°C in 5 mM MgC12, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA, and hybridizes to an E. dispar polynucleotide sequence if contacted with the E. dispar polynucleotide sequence at a temperature of at least about 60°C in 5 mM MgC12, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA. In some embodiments, each of the first primer and second primer hybridizes to the E. histolytica polynucleotide sequence if contacted with the E. histolytica polynucleotide sequence at a temperature of at least about 60°C in in 5 mM MgC12, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA, but the second primer does not hybridize to any E. dispar polynucleotide sequence if contacted with any E. dispar polynucleotide sequence at a temperature of at least about 50°C in 5 mM MgC12, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA. In some embodiments, the first primer comprises a polynucleotide having at least about 90% identity to SEQ ID NO: 1 (GTACAAAATGGCCAATTCATTCAATG) or its complement. In some embodiments, the first primer consists essentially of SEQ ID NO: 1 (GTACAAAATGGCCAATTCATTCAATG) or its complement. In some embodiments, the second primer comprises a polynucleotide having at least about 90% identity to SEQ ID NO: 2 (ACTACCAACTGATTGATAGATCAG) or its complement. In some embodiments, the second primer comprises a polynucleotide having the sequence of SEQ ID NO: 2 (ACTACCAACTGATTGATAGATCAG) or its complement. In some embodiments, the probe comprises a polynucleotide having at least about 90% identity to SEQ ID NO: 3 (ATTGTCGTGGCATCCTAACTCA) or its complement. In some embodiments, the probe comprises a polynucleotide having the sequence of SEQ ID NO: 3 (ATTGTCGTGGCATCCTAACTCA) or its complement. In some embodiments, the amplicon comprises a polynucleotide having at least about 95% identity to SEQ ID NO: 7 (GTACAAAATGGCCAATTCATTCAATGAATTGAGAAATGACATTCTAAGTGAG TTAGGATGCCACGACAATTGTAGAACACACAGTGTTTAACAAGTAACCAATG AGAATTTCTGATCTATCAATCAGTTGGTAGT). In some embodiments, the amplicon comprises a polynucleotide having the sequence of SEQ ID NO: 7 (GTACAAAATGGCCAATTCATTCAATGAATTGAGAAATGACATTCTAAGTGAG TTAGGATGCCACGACAATTGTAGAACACACAGTGTTTAACAAGTAACCAATG AGAATTTCTGATCTATCAATCAGTTGGTAGT). In some embodiments, the sample comprises E. histolytica and E. dispar. In some embodiments, the sample comprises fecal material of a human. In some embodiments, the sample comprises fixed material. In some embodiments, the sample is non-fixed. In some embodiments, a 95% limit of detection for E. histolytica comprises no more than about 17 E. histolytica genomes per milliliter. In some embodiments, if used under standard amplification conditions, the primers and probes do not cross-react with any of the following organisms, if present in the sample: Abiotrophia defectiva, Acinetobacter baumannii, Acinetobacter Iwoffli, Aeromonas hydrophila, Alcaligenes faecalis subsp. faecalis, Anaerococcus tetradius, Arcobacter butzleri, Arcobacter cryaerophilus, Bacillus cereus, Bacteroides caccae, Bacteroides merdae, Bacteroides stercoris, Bifidobacterium adolescentis, Bifidobacterium longum, Camplylobacter coli, Campylobacter concisus, Campylobacter curvus, Campylobacter fetus subsp. fetus, Campylobacter fetus subsp. venerealis, Campylobacter gracilis, Campylobacter hominis, Camplylobacter jejuni, Campylobacter lari, Campylobacter rectus, Campylobacter upsaliensis, Candida albicans, Candida catenulate, Cedecea davisae, Chlamydia trachomatis, Citrobacter amalonaticus, Citrobacter fruendii, Citrobacter koseri, Citrobacter sedlakii, Clostridium difficile 17858, Clostridium difficile 43598, Clostridium difficile CCUG 8864-9689, Clostridium difficile 43255, Clostridium difficile BAA-1805, Clostridium difficile 43593, Clostridium perfringens, Collinsella aerofaciens, Corynebacterium genitalium, Desulfovibrio piger, Edwardsiella tarda, Eggerthella lenta, Enterobacter aerogenes, Enterobacter cloacae, Enterococcus casselifiavus, Enterococcus cecorum, Enterococcus dispar, Enterococus faecalis, Enterococcus gallinarum, Enterococcus hirae, Enterococcus raffinosus, Escherichia coli, Escherichia fergusonii, Escherichia hermannii, Escherichia vulneris, Fusobacterium varium, Gardnerella vaginalis, Gemella morbillorum, Hafnia alvei, Helicobacter fennelliae, Helicobacter pylori, Klebsiella oxytoca, Klebsiella pneumonia, Lactobacillus acidophilus, Lactobacillus reuteri, Lactococcus lactis, Leminorella grimontii, Listeria grayi, Listeria innocua, Listeria monocytogenes, Morganella morganii, Peptoniphilus asaccharolyticus, Peptostreptococcus anaerobius, Plesiomonas shigelloides, Porphyromonas asaccharolytica, Prevotella melaninogenica, Proteus mirabilis, Proteus penneri, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii, Pseudomonas aeruginosa, Pseudomonas fluorescens, Ruminococcus bromii, Salmonella typhimurium, Salmonella enteriditis, Serratia liquefaciens, Serratia marcescens, Shigella sonnei, Shigella flexneri, Staphylococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus intermedius, Streptococcus uteris, Trabuisieiia guamensis, Veillonella parvula, Vibrio cholera, Vibrio parahaemolyticus, Yersinia bercovieri, Yersinia enterocolitica, Yersinia rohdei, Adenovirus type 2, Adenovirus type 14, Adenovirus type 40, Adenovirus type 41, Coxsackie A9, Coxsackie Bl, HHV-5, Cytomegalovirus, Enterovirus type 69, Human Papillomavirus Type 16, Human Papillomavirus Type 18, Herpes Simplex Virus I, Herpes Simplex Virus II, Norovirus I, Norovirus II, Rotavirus, Blastocystis hominis, Encephalitozoon intestinalis, Encephalitozoon helium, Encephalitozoon cuniculi, Pentatrichomonas hominis, Entamoeba barrette, Entamoeba dispar, Entamoeba gigivalis, Entamoeba invadens, Entamoeba moshkovskii, Entamobea ranarum, Citrobacter fruendii (rpt), Enterobacter cloacae (rpt), Cryptosporidium parvum, Giardia lamblia, or Cryptosporidium meleagridis. In some embodiments, if used under standard amplification conditions, the primers and probes do not cross-react with any of the following organisms, if present in the sample: Entamoeba coli, Entamoeba dispar, Entamoeba polecki, Entamoeba muris, Entamoeba nuttalli, Entamoeba hartmanni, and Entamoeba bovis. In some embodiments, if used under standard amplification conditions, the primers and probes produce fewer than 1 in 1600 false positives for samples that do not comprise E. histolytica. In some embodiments, E. dispar, if present, does not inhibit production of the amplicon if the E. histolytica polynucleotide sequence is present in the sample. In some embodiments, E. dispar, if present, does not inhibit determining the presence or absence of E. histolytica.
[0008] In some embodiments, a method of determining the presence or absence of an E. histolytica nucleic acid sequence in a sample. The method can comprise performing a nucleic acid amplification reaction on the sample, the nucleic acid amplification comprising a first oligonucleotide primer and a second oligonucleotide primer, in which the first oligonucleotide primer has a length of 15-75 nucleotides and hybridizes under standard conditions to SEQ ID NO: 10 or its complement, if present,, but does not hybridize under standard conditions to SEQ ID NO: 1 1 or its complement, if present, and in which the second oligonucleotide primer has a length of 15-75 nucleotides and hybridizes under standard conditions to a SEQ ID NO: 10 or its complement, if present, and wherein the second oligonucleotide primer hybridizes under standard conditions to SEQ ID NO: 11 or its complement, if present. The method can comprise detecting a signal, if present, from a detectably labeled probe that hybridizes to an amplicon of the the first and second oligonucleotide primers under standard hybridization conditions if the amplicon is present, in which the signal indicates the presence or absence of the amplicon, and in which the amplicon has a length of 75-350 nucleotides. Optionally, the first oligonucleotide primer comprises at least 10 consecutive nucleotides of SEQ ID NO: 1, and wherein the first oligonucleotide primer has at least 80% identity to a target sequence of SEQ ID NO: 10 or its complement. Optionally, the second oligonucleotide primer comprises at least 10 consecutive nucleotides of SEQ ID NO: 2, and wherein the second oligonucleotide primer has at least 80% identity to a target sequence of SEQ ID NO: 10 or its complement. Optionally, the first oligonucleotide primer comprises at least 12 consecutive nucleotides of SEQ ID NO: 1. Optionally, the first oligonucleotide primer comprises at least 15 consecutive nucleotides of SEQ ID NO: 1. Optionally, the first oligonucleotide primer comprises at least 20 consecutive nucleotides of SEQ ID NO: 1. Optionally, the first oligonucleotide primer has at least 85% identity to a target sequence of SEQ ID NO: 10 or its complement. Optionally, the first oligonucleotide primer has at least 90% identity to a target sequence of SEQ ID NO: 10 or its complement. Optionally, the first oligonucleotide primer has at least 95% identity to a target sequence of SEQ ID NO: 10 or its complement. Optionally, the first oligonucleotide primer has 100% identity to a target sequence of SEQ ID NO: 10 or its complement. Optionally, the second oligonucleotide primer comprises at least 12 consecutive nucleotides of SEQ ID NO: 2. Optionally, the second oligonucleotide primer comprises at least 15 consecutive nucleotides of SEQ ID NO: 2. Optionally, the second oligonucleotide primer comprises at least 20 consecutive nucleotides of SEQ ID NO: 2. Optionally, the second oligonucleotide primer has at least 85% identity to a target sequence of SEQ ID NO: 10 or its complement. Optionally, the second oligonucleotide primer has at least 90% identity to a target sequence of SEQ ID NO: 10 or its complement. Optionally, the second oligonucleotide primer has at least 95% identity to a target sequence of SEQ ID NO: 10 or its complement. Optionally, the second oligonucleotide primer has 100% identity to a target sequence of SEQ ID NO: 10 or its complement. Optionally, the probe comprises at least 10 consecutive nucleotides of SEQ ID NO: 3, and wherien the probe has at least 80% identity to a target sequence of SEQ ID NO: 10 or its complement. Optionally, the probe comprises at least 12 consecutive nucleotides of SEQ ID NO: 3. Optionally, the probe comprises at least 15 consecutive nucleotides of SEQ ID NO: 3. Optionally, the probe comprises at least 20 consecutive nucleotides of SEQ ID NO: 3. Optionally, the probe has at least 85% identity to a target sequence of SEQ ID NO: 10 or its complement. Optionally, the probe has at least 90% identity to a target sequence of SEQ ID NO: 10 or its complement. Optionally, the probe has at least 95% identity to a target sequence of SEQ ID NO: 10 or its complement. Optionally, the probe has 100% identity to a target sequence of SEQ ID NO: 10 or its complement. Optionally, the first oligonucleotide primer is about 20-50 nucleotides long. Optionally, the first oligonucleotide primer is about 23-45 nucleotides long. Optionally, the second oligonucleotide primer is about 20-50 nucleotides long. Optionally, the second oligonucleotide primer is about 23-45 nucleotides long. Optionally, the detectably labeled probe is about 15-75 nucleotides long. Optionally, the detectably labeled probe is about 20-45 nucleotides long. Optionally, the probe is capable of hybridizing to SEQ ID NO: 10 and toSEQ ID NO: 11 under standard hybridization conditions. Optionally, the probe is capable of hybridizing to SEQ ID NO: 10 but not toSEQ ID NO: 11 under standard hybridization conditions. Optionally, the probe comprises a fluorophore or a quencher. Optionally, the amplicon has a length of 100-150 nucleotides. Optionally, the amplicon comprises SEQ ID NO: 7. In some embodiments, a kit comprising any of the first oligonucleotide primer, the second oligonucleotide primer, and the detectably labeled probe as described above is provided. In some embodiments, E. dispar, if present, does not inhibit production of the amplicon if the E. histolytica polynucleotide sequence is present in the sample. In some embodiments, E. dispar, if present, does not inhibit determining the presence or absence of E. histolytica.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] Figure 1 is a diagram showing primers and probes as used in some of the embodiments disclosed herein.
[0010] Figure 2A is an alignment showing E. dispar and E. histolytica nucleic acids sequences.
[0011] Figure 2B is an annotated diagram of an E. histolytica gene encoding small subunit ribosomal RNA (GenBank: AB608092.1) (SEQ ID NO: 10)
[0012] Figure 3 is a graph showing quantitative PCR signal detection using previously-known primers and probes, for which the presence of E. dispar depresses amplification signal and can cause false negatives. [0013] Figure 4 is a graph showing quantitative PCR signal detection using primers and probes in embodiments as described herein, for which the presence of E. dispar does not interfere with amplification signal.
DETAILED DESCRIPTION
[0014] Detection of E. histolytica, and quantification of relative levels of E. histolytica can be useful in guiding clinical decisions. Quantitative nucleic acid amplification, for example quantitative assays involving nucleic acid amplification, such as polymerase chain reaction (qPCR) can be highly sensitive, and useful for quantification of nucleic acid levels, and thus can be used to infer relative quantities of E. histolytica based on quantification of nucleic acid. However, it has been appreciated herein that the presence of E. dispar can interfere with the specificity and efficiency of some qPCR reagents for detecting E. histolytica, and can cause cross-reactivity, signal suppression, or even false negatives. Accordingly, some embodiments herein provide methods and reagents for detecting and quantifying E. hisotolytica nucleic acids, without substantial interference from the presence of E. dispar. Some embodiments herein provide methods of detecting E. hisotolytica nucleic acids by qPCR. Some embodiments herein provide reagents and/or kits for detecting E. hisotolytica without substantial interference from the presence of E. dispar.
[0015] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to limit the scope of the current teachings. In this application, the use of the singular includes the plural unless specifically stated otherwise. Also, the use of "comprise", "contain", and "include", or modifications of those root words, for example but not limited to, "comprises", "contained", and "including", are not intended to be limiting. Use of "or" means "and/or" unless stated otherwise. The term "and/or" means that the terms before and after can be taken together or separately. For illustration purposes, but not as a limitation, "X and/or Y" can mean "X" or "Y" or "X and Y".
[0016] Whenever a range of values is provided herein, the range is meant to include the starting value and the ending value and any value or value range there between unless otherwise specifically stated. For example, "from 0.2 to 0.5" means 0.2, 0.3, 0.4, 0.5; ranges there between such as 0.2-0.3, 0.3-0.4, 0.2-0.4; increments there between such as 0.25, 0.35, 0.225, 0.335, 0.49; increment ranges there between such as 0.26-0.39; and the like.
[0017] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way. All literature and similar materials cited in this application including, but not limited to, patents, patent applications, articles, books, treatises, and internet web pages, regardless of the format of such literature and similar materials, are expressly incorporated by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines or uses a term in such a way that it contradicts that term's definition in this application, this application controls. While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.
[0018] Various embodiments of this disclosure describe compositions, and kits, and methods of using the same, for use in detecting and/or distinguishing or identifying Entamoeba nucleic acids. Accordingly, some embodiments provide nucleic acid sequences for use in nucleic acid detection assays, e.g., in amplification assays. A person skilled in the art will appreciate that for any nucleic acid sequence, the reverse compliment can be readily obtained, and that a disclosure of a nucleic acid sequence also provides a disclosure of the reverse compliment of that sequence. A person skilled in the art will appreciate that for any DNA sequence disclosed herein, a corresponding RNA sequence can be readily obtained, and that for any RNA sequence, a corresponding DNA can readily be obtained, for example by reverse transcription. A person skilled in the art will appreciate that subsequences of the nucleic sequences disclosed herein can be readily obtained. As used herein, "upstream" refers one or more locations 5' of a position on a nucleic acid sequence, and "downstream" refers to one or more locations 3' of a position on a nucleic acid sequence.
[0019] The nucleic acids provided herein can be in various forms. For example, in some embodiments, the nucleic acids are dissolved (either alone or in combination with various other nucleic acids) in solution, for example buffer. In some embodiments, nucleic acids are provided, either alone or in combination with other isolated nucleic acids, as a salt. In some embodiments, nucleic acids are provided in a lyophilized form that can be reconstituted. For example, in some embodiments, the isolated nucleic acids disclosed herein can be provided in a lyophilized pellet alone, or in a lyophilized pellet with other isolated nucleic acids. In some embodiments, nucleic acids are provided affixed to a solid substance, such as a bead, a membrane, or the like. In some embodiments, nucleic acids are provided in a host cell, for example a cell line carrying a plasmid, or a cell line carrying a stably integrated sequence. In some embodiments, nucleic acids are isolated from a host cell, for example one or more Entamoeba cells. In some embodiments, nucleic acids are synthesized, for example chemically or in a cell-free system.
Nucleic Acid Amplification
[0020] In some embodiments, nucleic acid amplification can include qualitative nucleic acid amplification, e.g. to determine whether a nucleic acid sequence is present or absent in a sample, for example, an E. histolytica-specific or E. dispar- specific nucleic acid sequence. In some embodiments, nucleic acid amplification can include quantitative nucleic acids amplification, e.g. to measure the relative or absolute amount of nucleic acid present in a sample. In some embodiments, nucleic acid amplification can include quantitative and qualitative nucleic acid amplification, e.g. to determine whether a nucleic acid sequence is present in a sample, and if present, to measure the relative or absolute amount of nucleic acid sequence present in the sample. In some embodiments, the method of amplification includes a multiplex assay for identifying the presence of two or more parasitic organisms from a sample, such as a human stool sample, for example at least two or more of E. histolytica, E. dispar, Giardia lamblia, Cryptosporidium parvum, Cryptosporidium hominis, and the like.
[0021] Methods of nucleic acid amplification can include, but are not limited to: polymerase chain reaction (PCR), strand displacement amplification (SDA), for example multiple displacement amplification (MDA), loop-mediated isothermal amplification (LAMP), ligase chain reaction (LCR), immuno-amplification, and a variety of transcription-based amplification procedures, including transcription-mediated amplification (TMA), nucleic acid sequence based amplification (NASBA), self- sustained sequence replication (3SR), and rolling circle amplification. See, e.g., Mullis, "Process for Amplifying, Detecting, and/or Cloning Nucleic Acid Sequences," U.S. Pat. No. 4,683, 195; Walker, "Strand Displacement Amplification," U.S. Pat. No. 5,455, 166; Dean et al, "Multiple displacement amplification," U.S. Pat. No. 6,977, 148; Notomi et al, "Process for Synthesizing Nucleic Acid," U.S. Pat. No. 6,410,278; Landegren et al. U.S. Pat. No. 4,988,617 "Method of detecting a nucleotide change in nucleic acids"; Birkenmeyer, "Amplification of Target Nucleic Acids Using Gap Filling Ligase Chain Reaction," U.S. Pat. No. 5,427,930; Cashman, "Blocked-Polymerase Polynucleotide Immunoassay Method and Kit," U.S. Pat. No. 5,849,478; Kacian et al, "Nucleic Acid Sequence Amplification Methods," U.S. Pat. No. 5,399,491 ; Malek et al, "Enhanced Nucleic Acid Amplification Process," U.S. Pat. No. 5, 130,238; Lizardi et al, BioTechnology, 6: 1197 (1988); Lizardi et al, U.S. Pat. No. 5,854,033 "Rolling circle replication reporter systems." In some embodiments, two or more of the listed nucleic acid amplification methods are performed, for example sequentially. In some embodiments, a target RNA sequence is amplified. In some embodiments, the target RNA sequence is reverse-transcribed, and the reverse transcript includes a DNA that is amplified using a nucleic acid amplification method described herein.
[0022] In some embodiments, the nucleic acid amplification is quantitative. Quantitative nucleic acid amplification can include detection of the amount of amplicon produced. The detection can be performed continuously or periodically. For example, detection can be performed at a certain point, e.g., at the end of every Nth cycle or fraction thereof, where N is one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 21, 23, 24, 25, 26, 27, 28, 29, 30, 21, 32, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 75, 80, 85, 95, 100 or the like. In some embodiments, detection can include measuring fluorescence, for example the intensity of electromagnetic radiation at the emission wavelength of a fluorophore tethered to a probe as described herein, or a wavelength range including the emission wavelength of the fluorophore tethered to the probe. As noted herein, exemplary probes include molecular beacons, SCORPIONS™ probes (Sigma), TAQMAN™ probes (Life Technologies), and the like. In some embodiments, detection can include detecting FRET. In some embodiments, detection can include detecting intensity of a non-specific detectable marker that binds to dsDNA, but does not bind to ssDNA. Examples of such non-specific dyes include intercalating agents such as SYBR Green I (Molecular Probes), PicoGreen (Molecular Probes), and the like. [0023] As used herein, "substantial" amplification, and modifications of these root words (e.g. "substantially amplify," "amplify substantially," and the like), refers to amplification that produces exponential yields of an amplicon or amplicons under standard amplification conditions. For example, PCR-derived forms of amplification and LAMP can produce discrete, double stranded amplicons, for which each strand can serve as a template in successive rounds of amplification, thus permitting exponential amplification. It is contemplated herein that a template can be substantially amplified and detected by polynucleotide that have less than 100% complementarity to the template, for example primers and/or probes having degenerate nucleotides, inosines, or the like at one or more positions. On the other hand, if a forward primer anneals to a target non- specifically, or anneals to a region that is not flanked by a reverse primer binding site on the opposite strand, there can be low-level amplification of by extension of the forward primer in the 3 ' direction to produce a new single strand, but the inability of this new single strand to serve as a template for successive amplification can result in non- exponential (for example linear), insubstantial amplification.
[0024] The skilled artisan will appreciate that the compositions disclosed herein can be used in various types of nucleic acid amplification reactions, as disclosed herein. In some embodiments, the compositions disclosed herein can be used in polymerase chain reaction (PCR). For a review of PCR technology, including amplification conditions, applied to clinical microbiology, see DNA Methods in Clinical Microbiology, Singleton P., published by Dordrecht ; Boston: Kluwer Academic, (2000) Molecular Cloning to Genetic Engineering White, B.A. Ed. in Methods in Molecular Biology 67: Humana Press, Totowa (1997) and "PCR Methods and Applications", from 1991 to 1995 (Cold Spring Harbor Laboratory Press), each of which is hereby incorporated by reference in its entirity. As used herein "standard amplification conditions" refer to 5 mM MgCl2, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA with a denaturation temperature of 97°C, and an annealing temperature of 62°C. While "standard amplification conditions" are described herein for reference purposes, it is contemplated that oligonucleotides in conjunction with some embodiments herein can readily be used under other "amplification conditions," including but not limited to, modifications and variations of such "standard amplification conditions." Non-limiting examples of "amplification conditions" include the conditions disclosed in the references cited herein, such as, for example, 5 mM MgCl2, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA with an annealing temperature of 72°C; 5 mM MgCl2; 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA with an annealing temperature of 62°C; 5 mM MgCi2, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA with an annealing temperature of 60°C; 5 mM MgCl2; 5 mM MgCl2, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA with an annealing temperature of 55°C; 50 mM KC1, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgC12, with an annealing temperature of 72°C; or 4mM MgC12, lOOmM Tris, pH 8.3, lOmM KC1, 5mM (NH4)2S04, 0.15mg BSA, 4% Trehalose, with an annealing temperature of 62°C; 4mM MgC12, lOOmM Tris, pH 8.3, lOmM KC1, 5mM (NH4)2S04, 0.15mg BSA, 4% Trehalose, with an annealing temperature of 60°C; or 50 mM KC1, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgC12, with an annealing temperature of 55°C, or the like. In some embodiments, an annealing temperatures as described herein is modified, for example to at least about 50°C, for example 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, or 75°C.
[0025] In some embodiments, at least one polymerase is provided. The polymerase can be used for quantitative PCR. Different nucleic acid polymerases are available for use, including but not limited to the FASTSTART™ Taq DNA polymerase (Roche), the KlenTaq 1 (AB peptides Inc.), the HOTGOLDSTAR™ DNA polymerase (Eurogentec), the KAPATAQ™ HotStart DNA polymerase or the KAPA2G™ Fast HotStart DNA polymerase (Kapa Biosystemss), and the PHUSION™ Hot Start (Finnzymes).
Thermal Cycling
[0026] Thermal cycling conditions can vary in time as well as in temperature for each of the different steps, depending on the thermal cycler used as well as other variables that could modify the amplification's performance. In some embodiments, a 2- step protocol is performed, in which the protocol combines the annealing and elongation steps at a common temperature, optimal for both the annealing of the primers and probes as well as for the extension step. In some embodiments, a 3 -step protocol is performed, in which a denaturation step, an annealing step, and an elongation step are performed.
[0027] In some embodiments, the compositions disclosed herein can be used in connection with devices for real-time amplification reactions, e.g., the BD MAX® (Becton Dickinson and Co., Franklin Lakes, NJ), the VIPER® (Becton Dickinson and Co., Franklin Lakes, NJ), the VIPER LT® (Becton Dickinson and Co., Franklin Lakes, NJ), the SMARTCYLCER® (Cepheid, Sunnyvale, CA), ABI PRISM 7700® (Applied Biosystems, Foster City, CA), ROTOR-GENE™ (Corbett Research, Sydney, Australia), LIGHTCYCLER® (Roche Diagnostics Corp, Indianapolis, IN), ICYCLER® (BioRad Laboratories, Hercules, CA), IMX4000® (Stratagene, La Jolla, CA), CFX96™ Real- Time PCR System (Bio-Rad Laboratories Inc), and the like.
Isothermal amplification
[0028] In some embodiments, the compositions disclosed herein can be used in methods comprising isothermal amplification of nucleic acids. Isothermal amplification conditions can vary in time as well as temperature, depending on variables such as the method, enzyme, template, and primer or primers used. Examples of amplification methods that can be performed under isothermal conditions include, but are not limited to, some versions of LAMP, SDA, and the like.
[0029] Isothermal amplification can include an optional denaturation step, followed by an isothermal incubation in which nucleic acid is amplified. In some embodiments, an isothermal incubation is performed without an initial denaturing step. In some embodiments, the isothermal incubation is performed at least about 25°C, for example about 25°C, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 °C, including ranges between any of the listed values. In some embodiments, the isothermal incubation is performed at about 37°C. In some embodiments, the isothermal incubation is performed at about 64°C. In some embodiments, the isothermal incubation is performed for 180 minutes or less, for example about 180, 165, 150, 135, 120, 105, 90, 75, 60, 45, 30, or 15 minutes, including ranges between any two of the listed values. Oligonucleotides
[0030] In some embodiments, oligonucleotides are provided, for example primers and/or probes. As used herein, the terms "primer" and "probe" include, but are not limited to oligonucleotides. Preferably, the oligonucleotide primers and/or probes disclosed herein can be between 8 and 45 nucleotides in length. For example, the primers and or probes can be at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or more nucleotides in length. Primers and/or probes can be provided in any suitable form, included bound to a solid support, liquid, and lyophilized, for example. The primer and probe sequences disclosed herein can be modified to contain additional nucleotides at the 5' or the 3' terminus, or both. The skilled artisan will appreciate, however, that additional bases to the 3' terminus of amplification primers (not necessarily probes) are generally complementary to the template sequence. The primer and probe sequences disclosed herein can also be modified to remove nucleotides at the 5' or the 3' terminus.
[0031] Oligonucleotide primers and probes can bind to their targets at an annealing temperature, which is a temperature less than the melting temperature (Tm). As used herein, "Tm" and "melting temperature" are interchangeable terms which refer to the temperature at which 50% of a population of double-stranded polynucleotide molecules becomes dissociated into single strands. Formulae for calculating the Tm of polynucleotides are well known in the art. For example, the Tm may be calculated by the following equation: Tm =69.3+0.41 x.(G+C)%-6- 50/L, wherein L is the length of the probe in nucleotides. The Tm of a hybrid polynucleotide may also be estimated using a formula adopted from hybridization assays in 1 M salt, and commonly used for calculating Tm for PCR primers: [(number of A+T) x 2°C +(number of G+C) x 4°C]. See, e.g., C. R. Newton et al. PCR, 2nd Ed., Springer-Verlag (New York: 1997), p. 24. Other more sophisticated computations exist in the art, which take structural as well as sequence characteristics into account for the calculation of Tm. The melting temperature of an oligonucleotide can depend on complementarity between the oligonucleotide primer or probe and the binding sequence, and on salt conditions. In some embodiments, an oligonucleotide primer or probe provided herein has a Tm of less than about 90°C in 50mM KC1, 10 mM Tris-HCl buffer, for example about 89°C, 88, 87, 86, 85, 84, 83, 82, 81, 80 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39°C, or less, including ranges between any two of the listed values. In some embodiments, an oligonucleotide primer or probe provided herein has a Tm of less than about 90°C in 5 mM MgCi2, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA, for example about 89°C, 88, 87, 86, 85, 84, 83, 82, 81, 80 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39°C, or less, including ranges between any two of the listed values. As discussed in further detail below, in some embodiments, the primers disclosed herein are provided as an amplification primer set, e.g., comprising a forward primer and a reverse primer. Preferably, the forward and reverse primers have Tm's that do not differ by more than 10°C, e.g., that differ by less than 10°C, less than 9°C, less than 8°C, less than 7°C, less than 6°C, less than 5°C, less than 4°C, less than 3°C, less than 2°C, or less than 1°C.
[0032] The primer and probe sequences may be modified by having nucleotide substitutions (relative to the target nucleic acid sequence) within the oligonucleotide sequence, provided that the oligonucleotide contains enough complementarity to hybridize specifically to the target nucleic acid sequence. In this manner, at least 1, 2, 3, 4, or up to about 5 nucleotides can be substituted. As used herein, the term "complementary" refers to sequence complementarity between regions of two polynucleotide strands or between two regions of the same polynucleotide strand. A first region of a polynucleotide is complementary to a second region of the same or a different polynucleotide if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide of the first region is capable of base pairing with a base of the second region. Therefore, it is not required for two complementary polynucleotides to base pair at every nucleotide position. "Fully complementary" refers to a first polynucleotide that is 100% or "fully" complementary to a second polynucleotide and thus forms a base pair at every nucleotide position. "Partially complementary" also refers to a first polynucleotide that is not 100% complementary (e.g., 90%, or 80% or 70% complementary) and contains mismatched nucleotides at one or more nucleotide positions. In some embodiments, an oligonucleotide includes a universal base.
[0033] As used herein, the term "hybridization" is used in reference to the pairing of complementary (including partially complementary) polynucleotide strands. Hybridization and the strength of hybridization (i.e., the strength of the association between polynucleotide strands) is impacted by many factors well known in the art including the degree of complementarity between the polynucleotides, stringency of the conditions involved affected by such conditions as the concentration of salts, the melting temperature of the formed hybrid, the presence of other components (e.g., the presence or absence of polyethylene glycol), the molarity of the hybridizing strands and the G:C content of the polynucleotide strands. In some embodiments, the primers are designed such that the Tm of one primer in the set is within 2°C of the Tm of the other primer in the set. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al, eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley- Interscience, New York). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York). As discussed further herein, the term "specific hybridization" or "specifically hybridizes" refers to the hybridization of a polynucleotide, e.g., an oligonucleotide primer or probe or the like to a target sequence, such as a sequence to be quantified in a sample, a positive control target nucleic acid sequence, or the like, and not to unrelated sequences, under conditions typically used for nucleic acid amplification.
[0034] In some embodiments, the primers and/or probes include oligonucleotides that hybridize to a target nucleic acid sequence over the entire length of the oligonucleotide sequence. Such sequences can be referred to as "fully complementary" with respect to each other. Where an oligonucleotide is referred to as "substantially complementary" with respect to a nucleic acid sequence herein, the two sequences can be fully complementary, or they may form mismatches upon hybridization, but retain the ability to hybridize under stringent conditions or standard PCR conditions as discussed below. As used herein, the term "substantially complementary" refers to the complementarity between two nucleic acids, e.g., the complementary region of the oligonucleotide and the target sequence. The complementarity need not be perfect; there may be any number of base pair mismatches that between the two nucleic acids. However, if the number of mismatches is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence is not a substantially complementary sequence. When two sequences are referred to as "substantially complementary" herein, it is meant that the sequences are sufficiently complementary to the each other to hybridize under the selected reaction conditions. The relationship of nucleic acid complementarity and stringency of hybridization sufficient to achieve specificity is well known in the art and described further below in reference to sequence identity, melting temperature and hybridization conditions. Therefore, substantially complementary sequences can be used in any of the detection methods disclosed herein. Such probes can be, for example, perfectly complementary or can contain from 1 to many mismatches so long as the hybridization conditions are sufficient to allow, for example discrimination between a target sequence and a non-target sequence. Accordingly, substantially complementary sequences can refer to sequences ranging in percent identity from 100%, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 75, 70% or less, or any number in between, compared to the reference sequence. For example, the oligonucleotides disclosed herein can contain 1, 2, 3, 4, 5, or more mismatches and/or degenerate bases (e.g. "variant oligonucleotides"), as compared to the target sequence to which the oligonucleotide hybridizes, with the proviso that the oligonucleotides are capable of specifically hybridizing to the target sequence under, for example, standard nucleic acid amplification conditions.
[0035] The primers described herein can be prepared using techniques known in the art, including, but not limited to, cloning and digestion of the appropriate sequences and direct chemical synthesis. Chemical synthesis methods that can be used to make the primers of the described herein, include, but are not limited to, the phosphotriester method described by Narang et al. (1979) Methods in Enzymology 68:90, the phosphodiester method disclosed by Brown et al. (1979) Methods in Enzymology 68: 109, the diethylphosphoramidate method disclosed by Beaucage et al. (1981) Tetrahedron Letters 22: 1859, and the solid support method described in U.S. Patent No. 4,458,066. The use of an automated oligonucleotide synthesizer to prepare synthetic oligonucleotide primers described herein is also contemplated herein. Additionally, if desired, the primers can be labeled using techniques known in the art and described below.
Primer Sets
[0036] In some embodiments, a set of amplification primers is provided. The set of amplification primers can include one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, or more primer pairs. As used herein, the term "primer pair" can refer to two amplification primers that individually hybridize to opposite strands of a target nucleic acid sequence (e.g., a sequence of E. histolytica, a sequence of E. dispar, or a sequence found in both E. histolytic and E. dispar), in which each primer can be extended at its 3' end to form a target amplification product, for example in PCR. The target amplification product can include an amplicon. A primer pair can include a forward primer and a reverse primer. The skilled artisan will appreciate that the terms "forward primer" and "reverse primer" are frequently used for convenience in identifying each primer in a primer pair, for example with reference to a which strand is identified as the "+" strand or "top" strand of a target nucleic acid sequence, but that no further limitation should be inferred from "forward" or "reverse," unless stated otherwise.
[0037] In some embodiments, the primer set includes amplification primers that will anneal to, and amplify, a sequence of E. histolytica under standard amplification conditions, but will not anneal to a sequence of E. dispar, or will anneal to a sequence of E. dispar, but not substantially amplify this sequence of E. dispar under the same or similar amplification conditions. Accordingly, in some embodiments, the primer set is used to detect the presence of E. histolytica, but not E. dispar. Due to the high degree of homology between E. histolytica and E. dispar, an alternative approach for quantitative amplification of E. histolytica sequences would be to select a primer set that amplifies a polynucleotide sequence found in both E. histolytica and E. dispar (e.g., a homologous sequence), and then use a probe that hybridizes only to the polynucleotide sequence of E. histolytica to detect amplification of E. histolytica product (see Verweij, et al, Clin. Microbiol. 42: 1220-23, 2004). Unexpectedly, it has been discovered herein that undertaking such an approach can result in reduction of the expected amplification signal from E. histolytica, especially as the dose of E. dispar target nucleic acid sequence increases. As E. histolytica and E. dispar may both infect the same individual, it is contemplated that previous approaches (e.g. of Verweij, et al, Clin. Microbiol. 42: 1220- 23, 2004) could result in false negatives. As shown in Example 1 and Figure 3, when a primer set that amplifies both E. histolytica and E. dispar nucleic acid sequences was used, a known copy number of E. histolytica target nucleic acid sequence became nearly undetectable in the presence of a high copy number of E. dispar target nucleic acid sequences. Without being bound by any one theory, it is contemplated that homo-and hetero duplex formation between amplification products of E. histolytica and E. dispar can block available E. histolytica probe binding sequences as the proportion of E. dispar amplicons increases in the reaction. Performing quantitative nucleic acid amplification of E. histolytica according to some embodiments herein can minimize or eliminiate interfering effects of E. dispar nucleic acids. Thus, in some embodiments, depression of E. histolytica signal by E. dispar can be minimized. In some embodiments, depression of E. histolytica signal by E. dispar can be effectively eliminated. In some embodiments, primers are designed to substantially amplify a E. histolytica target nucleic acid sequence under standard amplification conditions, without substantially amplifying any E. dispar nucleic acid sequences.
[0038] In some embodiments, the primers of the primer set will individually hybridize to opposite strands of a target nucleic acid of E. histolytica under standard amplification conditions, so as to define a target amplification product. In some embodiments, when extended at their respective 3 ' ends, the primers will produce a target amplification product. Accordingly, in some embodiments, when extended, the primers will substantially amplify an E. histolytica target nucleic acid sequence. In some embodiments, neither primer of the primer pair will hybridize to a strand of E. dispar nucleic acid under standard amplification conditions, and thus will not substantially amplify any sequence of E. dispar. In some embodiments, only one primer of the primer pair will hybridize to a strand of E. dispar nucleic acid under standard amplification conditions, while the other primer will not hybridize to any E. dispar nucleic acid under these conditions, so that the primer set will fail to substantially amplify any E. dispar sequence. In some embodiments, each primer of the primer pair will hybridize to E. dispar nucleic acid under standard amplification conditions, but these primers will not hybridize in an orientation that will form an amplification product when each primer is extended at its 3 ' end (e.g. the primers may hybridize to the same strand, or hybridize too far apart to form an amplification product when extended, or hybridize in an orientation so that when extended at its 3' end, at least one primer extends "away" from the other primer). Accordingly, in some embodiments, the primers of the primer pair will not substantially amplify any nucleic acid sequence of E. dispar.
[0039] In some embodiments, in designing primer sets that reliably amplify sequences of E. histolytica but not E. dispar, it can be useful to select primers that amplify a conserved region of E. histolytica, so as to minimize false negatives due to strain-to-strain variation among E. histolytica, but do not amplify a conserved region of E. dispar, so as to minimize false positives that could otherwise be caused by the presence of E. dispar. For example, a highly conserved sequence with ancestral differences between E. dispar and E. histolytica can be a useful region from which to select a target nucleic acid (e.g. a "template") for a target amplification sequence. In some embodiments, the target amplification sequence includes an rDNA gene or portion thereof. In some embodiments, a gene product (for example, an rRNA or portion thereof) is reverse-transcribed, and used as a target nucleic acid sequence for qualitative and/or quantitative nucleic acid amplification. While small ribosomal subunit genes are highly conserved, there are some apparently ancestral differences between the sequences of the small ribosomal subunit gene of E. histolytica and E. dispar, as shown in Figure 2A. The skilled artisan will appreciate that these differences can be used to design primer sets that can produce a target amplification sequence of E. histolytica, but not E. dispar. In some embodiments, the primer pair amplifies a polynucleotide sequence that includes at least a portion of the gene encoding the E. histolytica small subunit ribosomal RNA (GenBank Accession No: AB608092.1)(SEQ ID NO: 10). An annotated diagram of the E. histolytica small subunit ribosomal RNA gene is illustrated in Figure 2B. In some embodiments, the target sequence includes a polynucleotide having SEQ ID NO: 7 (e.g. positions 191-325 of SEQ ID NO: 10)
(GTACAAAATGGCCAATTCATTCAATGAATTGAGAAATGACATTCTAAGTGAG TTAGGATGCCACGACAATTGTAGAACACACAGTGTTTAACAAGTAACCAATG AGAATTTCTGATCTATCAATCAGTTGGTAGT). In some embodiments the target amplification product includes at least about 30 continuous nucleotides of SEQ ID NO: 7, for example at least about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135 continuous nucleotides of SEQ ID NO: 7, including ranges between any two of the listed values. In some embodiments, the target amplification sequence includes at least about 30-135 continuous nucleotides of SEQ ID NO: 7, for example about 30-100, 30-110, SOUS, 30-120, 30-125, 30-130, 30-135, 40-100, 40-110, 40-115, 40-120, 40-125, 40-130, 40-135, 50-100, 50-110, 50-115, 50-120, 50-125, 50-130, 50-135, 60-100, 60-110, 60- 115, 60-120, 60-125, 60-130, 60-135, 70-100, 70-110, 70-115, 70-120, 70-125, 70-130, 70-135, 80-100, 80-110, 80-115, 80-120, 80-125, 80-130, 80-135, 90-100, 90-110, 90- 115, 90-120, 90-125, 90-130, 90-135, 100-110, 100-115, 100-120, 100-125, 100-130, 100-135, 110-115, 110-120, 110-125, 110-130, 110-135, 115-120, 115-125, 115-130, 115-135, 120-125, 120-130, 120-135, 125-130, 125-135, or 130-135 continuous nucleotides of SEQ ID NO: 7. In some embodiments, the target amplification produce has at least 70% nt-nt identity to SEQ ID NO: 7, for example at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.2% nt- nt identity, including ranges between any two of the listed values. In some embodiments, the target amplification sequence includes a polynucleotide having the sequence of SEQ ID NO: 7, and at least one additional polynucleotide upstream and/or downstream of a 5' end or 3 ' end of SEQ ID NO: 7 (e.g. positions 191-325 of SEQ ID NO: 10). In some embodiment, the target amplification sequence includes at least about 1 nucleotide upstream of the 5' end of SEQ ID NO: 7 as shown in SEQ ID NO: 10 (see Figure 2B), for example at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides upstream. In some embodiment, the target amplification includes at least about 1 nucleotide downstream of the 3' end of SEQ ID NO: 7 as shown in SEQ ID NO: 10 (see Figure 2B), for example at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides upstream. In some embodiments, the target amplification sequence includes nucleotides both upstream and downstream of the ends of SEQ ID NO: 7 (as shown in SEQ ID NO: 10), as described herein. In some embodiments, the pimer pair does not amplify, under standard amplification conditions, the E. dispar small subunit rRNA. By way of example, a sequence of the E. dispar small subunit rRNA can be found Genbank accession umber AB282661, which is provided herein as SEQ ID NO: 11
(TGGATATAAATACAAAAGAGAAGTAAGAATAAAGAATCCTTCCTTTTAAAAA GGAAGAAGAATAAAATATCTGGTTGATCCTGCCAGTATTATATGCTGATGTTA AAGATTAAGCCATGCATGTGTAAGTATAAAGACCAAGTAGGATGAAACTGCG GACGGCTCATTATAACAGTAATAGTTTCTTTGGTTAGTAAAGTACAAGGATAG CTTTGTGAATGATAAAGATAATACTTGAGACGATCCAATTTGTATTAGTACAA AGTGGCCAATTTATGTAAGTAAATTGAGAAATGACATTCTAAGTGAGTTAGG ATGCCACGACAATTGTAGAACACACAGTGTTTAACAAGTAACCAATGAGAAT TTCTGATCTATCAATCAGTTGGTAGTATCGAGGACTACCAAGATTATAACGGA TAACGAGGAATTGGGGTTCGACATCGGAGAGGGAGCTTTACAGATGGCTACC ACTTCTAAGGAAGGCAGCAGGCGCGTAAATTACCCACTTTCGAATTGAAGAG
GTAGTGACGACACATAACTCTAGAGTTGAGTAAAATCAATTCTTGAAGGAAT
GAGTAGGAGGTAAATTCTCCTACGAAATCAATTGGAGGGCAAGTCTGGTGCC
AGCAGCCGCGGTAATTCCAGCTCCAATAGTGTATATTAAAGTTGCTGTGATTA
AAACGCTCGTAGTTGAATTAAAATGTGATTTTATACATTTTGAAGACTTTACA
TTAAGTGAAGTTTCTAGAAATGTTAAATTAAAATCAAAGAAGGAGACAATTC
AAGTAATTGAGTTGTCATTACTTTGAATAAAATAAGGTGTTTAAAGCAAAACA
TTATGTTAATGAATATTCAAGCATGGGACAATGCTGAGGAGATGTCAATTAG
ACATTTCGAGAGAAGGATTAAAAGGAACAATTGGGGTGATTCAGAAAATAAC
GGGAGAGGTGAAAATCCATGATCGCTATAAGATGCACGAGAGCGAAAGCATT
TCACTCAACTGGGTCCATTAATCAAGAACGAAAGTTAGGGGATCGAAGACGA
TCAGATACCGTCGTAGTCCTAACTATAAACGATGTCAACCAAGGATTGGATG
AAATTCAGATGTACAAAGATGAAGAAACATTGTTTCTAAATCCAAGTATATC
AATACTACCTTGTTCAGAACTTAAAGAGAAATCTTGAGTTTATGGACTTCAGG
GGGAGTATGGTCACAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACACC
AGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGAAAACTTACCAAGA
CCGAACAGTAGAAGGAATGACAGATTAAGAGTTCTTTCATGATTTATTGGGTA
GTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCAGGTTAATTCCGGT
AACGAACGAGACTGAAACCTATTAATTAGTTTTCTGCCTATAAGACAGAAAT
GTTCGCAAGAACAGGTGCGTAAGTACCACTTCTTAAAGGGACACATTTCAATT
GTCCTATTTTAATTGTTAGTTATCTAATTTCGATTAGAACTCTTTTAACGTGGG
AAAAAGAAAAAGGAAGCATTCAGCAATAACAGGTCTGTGATGCCCTTAGACA
TCTTGGGCCGCACGCGCGCTACAATGGAGTTACTAGAGAGCATTTTATCATTT
ACACCTTATTTATTAGGCTATGTCTAATAGGTAGGGATAGTAAGTGGTGTACC
GAGATTGAAATAGTTAAGGAAAACTCAAAAGAACGTACATGACAGGGATAA
ATGATTGGAATTATTTGTTTTGAACGAGGAATTCCTTGTAATATCGAGTCATT
AACTCGAGATGAATACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTCCTAC
CGATTGAATAAAGAGGTGAAATTCTAGGATTCTGTCTTATAGATAGAAAAAT
GGATTTAAATCTCCTTATTTAGAGGAAGGAGAAGTCGTAACAAGGTTTCCGTA
GGTGAACCTGCGGAAGGATCATTAAAAGAAAAGAAATAATCTTTTAAAATAA
AACAAGAAATTTATAGAATAAGATAATCTACAAAGAAAATAATAAAAGTAAG
AATAAAAGGAATTAGAATATAAGAAGAAAGAAAAAGTATAATAAAATATTA
CTTTGGATAGTTTAGTTTCCTGTGCGATGAAGAACGCAATGAATTGCGATAAG TGATAGGAACAATAAAATGTGAATATCCAAACTTTGAATGCTTGAAAGTATA
CTTATGAACTTCAAGGTATATATGATATTCAATATCCAAAATAAAAGAGTATA
TTAAAAGCAAATATTAGTAGAAGTGAGAAGTAGCTAGTGGGTAAAAGAGAG
AAGAAGTAAAGAGCTTTAACCAGATATCTATAAGTGAGTTAATAAATAAAGA
TTTGAGTATCGTAAGAG).
[0040] In some embodiments, the primer set includes a first primer that comprises SEQ ID NO: 1 (GTACAAAATGGCCAATTCATTCAATG). In some embodiments the primer set includes a second primer that comprises SEQ ID NO: 2 (ACTACCAACTGATTGATAGATCAG). As shown in Figure 2A, a primer that comprises SEQ ID NO: 1 can have high (up to 100%) identity to a sequence encoding the E. histolytica small subunit rRNA, while having lower identity to a homologous region of the E. dispar genome. Accordingly, in some embodiments, a primer comprising SEQ ID NO: 1 hybridizes to E. histolytica genomic DNA, but does not hybridize to E. dispar genomic DNA under standard amplification conditions. A primer that comprises SEQ ID NO: 2 can hybridize to genomic DNA in the region of the small subunit rRNA gene of both E. histolytica and E. dispar. Accordingly, in some embodiments, a primer set that includes a first primer comprising SEQ ID NO: 1, and a second primer comprising SEQ ID NO: 2, can amplify a target sequence of E. histolytica nucleic acid, but not E. dispar nucleic acids. In some embodiments, the nucleic acid includes genomic DNA. In some embodiments, the nucleic acid includes nucleic acid reverse-transcribed from a gene product, for example an mRNA or rRNA.
[0041] In some embodiments, the first primer comprises at least about 10 consecutive nucleotides of SEQ ID NO: l, for example at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleotides of SEQ ID NO: 1 or its complement. In some embodiments, the first primer comprises a polynucleotide sequence that is at least about 38% identical of SEQ ID NO: 1, for example at least about 38%, 42, 46, 50, 53, 57, 61, 65, 69, 73, 76, 80, 84, 88, 92, or 96% identical to SEQ ID NO: 1. In some embodiments, the first primer comprises SEQ ID NO: 1, and at least 1 additional nucleotide 5' of the 5' terminus of SEQ ID NO: 1, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides 5' of the 5' terminus of SEQ ID NO: 1. In some embodiments, one or more of the nucleotides 5' of SEQ ID NO: 1 are complementary to the template strand of SEQ ID NO: 10 as shown in Figure 2B. [0042] A number of Alternatives are contemplated for primers in accordance with some embodiments herein:
[0043] In accordance with Alternative 1, the first primer has a length of 15-50 nucleotides and comprises at least about 10 consecutive nucleotides of SEQ ID NO: l, for example at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleotides of SEQ ID NO: 1 or its complement, including ranges between any two of the listed values, for example 10-15, 10-20, 15-20, 10-26, 15-26, or 20-26 consecutive nucleotides. The first primer can hybridize to a target sequence of SEQ ID NO: 10, and have at least about 80% identity to the target sequence.
[0044] In accordance with Alternative 2, the first primer has a length of 15-50 nucleotides and comprises at least about 10 consecutive nucleotides of SEQ ID NO: l, for example at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleotides of SEQ ID NO: 1 or its complement, including ranges between any two of the listed values, for example 10-15, 10-20, 15-20, 10-26, 15-26, or 20-26 consecutive nucleotides. The first primer can hybridize to a target sequence of SEQ ID NO: 10, and have at least about 85% identity to the target sequence.
[0045] In accordance with Alternative 3, the first primer has a length of 15-50 nucleotides and comprises at least about 10 consecutive nucleotides of SEQ ID NO: l, for example at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleotides of SEQ ID NO: 1 or its complement, including ranges between any two of the listed values, for example 10-15, 10-20, 15-20, 10-26, 15-26, or 20-26 consecutive nucleotides. The first primer can hybridize to a target sequence of SEQ ID NO: 10, and have at least about 90% identity to the target sequence.
[0046] In accordance with Alternative 4, the first primer has a length of 15-50 nucleotides and comprises at least about 10 consecutive nucleotides of SEQ ID NO: l, for example at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleotides of SEQ ID NO: 1 or its complement, including ranges between any two of the listed values, for example 10-15, 10-20, 15-20, 10-26, 15-26, or 20-26 consecutive nucleotides. The first primer can hybridize to a target sequence of SEQ ID NO: 10, and have at least about 95% identity to the target sequence. Optionally, the first primer can have 100% identity to the target sequence.
[0047] In accordance with Alternative 5, the first primer of any of Alternatives 1-4 can be paired with the second primer. The second primer can primer have a length of 15-50 nucleotides and comprise at least about 10 consecutive nucleotides of SEQ ID NO:2, for example at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, or 26 consecutive nucleotides of SEQ ID NO: 2 or its complement, including ranges between any two of the listed values, for example 10-15, 10-20, 15-20, 10-26, 15-26, or 20-26 consecutive nucleotides. The second primer can hybridize to a target sequence of SEQ ID NO: 10, and have at least about 80% identity to the target sequence.
[0048] In accordance with Alternative 6, the first primer of any of Alternatives 1-4 can be paired with the second primer. The second primer can have a length of 15-50 nucleotides and comprise at least about 10 consecutive nucleotides of SEQ ID NO:2, for example at least about 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, or 26 consecutive nucleotides of SEQ ID NO: 2 or its complement, including ranges between any two of the listed values, for example 10-15, 10-20, 15-20, 10-26, 15- 26, or 20-26 consecutive nucleotides. The second primer can hybridize to a target sequence of SEQ ID NO: 10, and have at least about 85% identity to the target sequence.
[0049] In accordance with Alternative 7, the first primer of any of Alternatives 1-4 can be paired with the second primer. The second primer can have a length of 15-50 nucleotides and comprise at least about 10 consecutive nucleotides of SEQ ID NO:2, for example at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, or 26 consecutive nucleotides of SEQ ID NO: 2 or its complement, including ranges between any two of the listed values, for example 10-15, 10-20, 15-20, 10-26, 15- 26, or 20-26 consecutive nucleotides. The second primer can hybridize to a target sequence of SEQ ID NO: 10, and have at least about 90% identity to the target sequence.
[0050] In accordance with Alternative 8, the first primer of any of Alternatives 1-4 can be paired with the second primer. The second primer have a length of 15-40 nucleotides and can comprise at least about 10 consecutive nucleotides of SEQ ID NO:2, for example at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, or 26 consecutive nucleotides of SEQ ID NO: 2 or its complement, including ranges between any two of the listed values, for example 10-15, 10-20, 15-20, 10-26, 15- 26, or 20-26 consecutive nucleotides. The second primer can hybridize to a target sequence of SEQ ID NO: 10, and have at least about 95% identity to the target sequence. Optionally, the second primer can have 100% identity to the target sequence. [0051] In some embodiments, the first primer is designed in accordance with the alignment shown in Figure 2A, so that the first primer anneals to a sequence of E. histolytica, but not to the homologous sequence of E. dispar under standard amplification conditions. In some embodiments, the first primer anneals to a sequence of E. histolytica, but not to the homologous sequence of E. dispar under typical PCR conditions, e.g., at 50°C in 50mM KC1, lOmM Tris-HCl buffer (pH 8.0), or, e.g., 62°C in 5 mM MgCl2, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA. In some embodiments, at least the 3 '-most nucleotide of the first primer is complementary to an E. histolytica nucleotide at a conserved position that differs in E.dispar, so that the first primer, when hybridized, can typically extend from its 3' end in E. histolytica, but typically not in E. dispar. In some embodiments, the second primer comprises at least about 10 consecutive nucleotides of SEQ ID NO: 2, for example about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24, consecutive nucleotides of SEQ ID NO: 2. In some embodiments, the second primer comprises a polynucleotide sequence that is at least about 41% identical of SEQ ID NO: 2, for example at least about 41, 45, 50, 54, 58, 62, 66, 70, 75, 79, 83, 87, 91, or 95% identical to SEQ ID NO: 2.
Probes
[0052] In some embodiments, sequence-specific probes are provided. Probes include, but are not limited to oligonucleotides as described herein. In some embodiments, the sequence-specific probes disclosed herein specifically hybridize to a target nucleic acid sequence. In some embodiments, the sequence-specific probe can hybridize to a sequence that is found in both E. histolytica and E. dispar. In some embodiments, the sequence-specific probe can hybridize to a sequence that is found in E. histolytica, but not in E. dispar. In some embodiments, the sequence-specific probe specifically hybridizes to, and is fully or substantially complementary to a nucleotide sequence flanked by the binding sites of a pair of amplification primers disclosed herein. In some embodiments, the sequence-specific probe specifically hybridizes to, and is fully or substantially complementary a target amplification sequence of a primer set that amplifies E. histolytica, but not E. dispar, nucleic acids under standard amplification conditions. In some embodiments, the sequence-specific probe comprises the polynucleotide of SEQ ID NO: 3 (ATTGTCGTGGCATCCTAACTCA). In some embodiments, the sequence-specific probe comprises at least about 5 consecutive nucleotides of SEQ ID NO: 3, for example about 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 nucleotides of SEQ ID NO: 3. In some embodiments, the sequence-specific probe comprises a sequence that is at least about 22% identical so SEQ ID NO: 3, for example at least about 22%, 27, 31, 36, 40, 45, 54, 59, 63, 68, 72, 77, 81, 86, 90, or 95% identical so SEQ ID NO: 3. In some embodiments, the sequence-specific probe overlaps with the binding site of an amplification primer disclosed herein.
[0053] A number Alternatives are contemplated for probes in accordance with some embodiments herein.
[0054] In accordance with Alternative 9, the probe can have a length of 15-75 nucleotides and comprise at least 10 nucleotides of SEQ ID NO: 3, SEQ ID NO: l, for example at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleotides of SEQ ID NO: 3 or its complement, including ranges between any two of the listed values, for example 10-15, 10-20, 15-20, 10-26, 15-26, or 20-26 consecutive nucleotides. The probe can hybridize to a target sequence of SEQ ID NO: 10, and have at least about 80% identity to the target sequence. The probe can be used in conjunction with any of the primer pairs of Alternatives 5-8. Optionally, the probe can also hybridize to SEQ ID NO: 1 1.
[0055] In accordance with Alternative 10, the probe can have a length of 15- 75 nucleotides and comprises at least 10 nucleotides of SEQ ID NO: 3, SEQ ID NO: l, for example at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleotides of SEQ ID NO: 3 or its complement, including ranges between any two of the listed values, for example 10-15, 10-20, 15-20, 10-26, 15-26, or 20-26 consecutive nucleotides. The probe can hybridize to a target sequence of SEQ ID NO: 10, and have at least about 85% identity to the target sequence. The probe can be used in conjunction with any of the primer pairs of Alternatives 5-8. Optionally, the probe can also hybridize to SEQ ID NO: 1 1.
[0056] In accordance with Alternative 1 1, the probe can have a length of 15- 75 nucleotides and comprise at least 10 nucleotides of SEQ ID NO: 3, SEQ ID NO: l, for example at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleotides of SEQ ID NO: 3 or its complement, including ranges between any two of the listed values, for example 10-15, 10-20, 15-20, 10-26, 15-26, or 20-26 consecutive nucleotides. The probe can hybridize to a target sequence of SEQ ID NO: 10, and have at least about 90% identity to the target sequence. The probe can be used in conjunction with any of the primer pairs of Alternatives 5-8. Optionally, the probe can also hybridize to SEQ ID NO: 1 1.
[0057] In accordance with Alternative 12, the probe can have a length of 15- 75 nucleotides and comprise at least 10 nucleotides of SEQ ID NO: 3, SEQ ID NO: l, for example at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleotides of SEQ ID NO: 3 or its complement, including ranges between any two of the listed values, for example 10-15, 10-20, 15-20, 10-26, 15-26, or 20-26 consecutive nucleotides. The probe can hybridize to a target sequence of SEQ ID NO: 10, and have at least about 95% identity to the target sequence. The probe can be used in conjunction with any of the primer pairs of Alternatives 5-8. Optionally, the probe can also hybridize to SEQ ID NO: 1 1.
[0058] In accordance with Alternative 13, the probe can have a length of 15- 75 nucleotides and comprise at least 10 nucleotides of SEQ ID NO: 3, SEQ ID NO: l, for example at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleotides of SEQ ID NO: 3 or its complement, including ranges between any two of the listed values, for example 10-15, 10-20, 15-20, 10-26, 15-26, or 20-26 consecutive nucleotides. The probe can hybridize to a target sequence of SEQ ID NO: 10, and have 100% identity to the target sequence. The probe can be used in conjunction with any of the primer pairs of Alternatives 5-8. Optionally, the probe can also hybridize to SEQ ID NO: 11.
[0059] Different types of detectable moieties have been described for the detection of amplification products. One class of detectable moieties is intercalating agents, which bind non- specifically to double-stranded nucleic acid. Intercalating agents have a relatively low fluorescence when unbound, and a relatively high fluorescence upon binding to double-stranded nucleic acids. As such, intercalating agents can be used to monitor the accumulation of double strained nucleic acids during a nucleic acid amplification reaction. Examples of such non-specific dyes include intercalating agents such as SYBR Green I (Molecular Probes), PicoGreen (Molecular Probes), TOTO, YOYO, propidium iodide, ethidium bromide, and the like. Other types of detectable moities employ derivatives of sequence-specific nucleic acid probes. For example, oligonucleotide probes can be labeled with one or more dyes, such that upon hybridization to a template nucleic acid, a detectable change in fluorescence is generated. While non-specific dyes may be desirable for some applications, sequence-specific probes can provide more accurate measurements of amplification. One configuration of sequence-specific probe can include one end of the probe tethered to a fluorophore, and the other end of the probe tethered to a quencher. When the probe is unhybridized, it can maintain a stem-loop configuration, in which the fluorophore is quenched by the quencher, thus preventing the fluorophore from fluorescing. When the probe is hybridized to a template nucleic sequence, it is linearized, distancing the fluorophore from the quencher, and thus permitting the fluorophore to fluoresce. Another configuration of sequence-specific probe can include a first probe tethered to a first fluorophore of a FRET pair, and a second probe tethered to a second fluorophore of a FRET pair. The first probe and second probe can be configured to hybridize to sequences of an amplicon that are within sufficient proximity to permit energy transfer by FRET when the first probe and second probe are hybridized to the same amplicon.
[0060] In some embodiments, the sequence specific probe comprises an oligonucleotide as disclosed herein conjugated to a fluorophore. In some embodiments, the probe is conjugated to two or more flurophores. Examples of fluorophores include: xanthene dyes, e.g., fluorescein and rhodamine dyes, such as fluorescein isothiocyanate (FITC), 2-[ethylamino)-3-(ethylimino)-2-7-dimethyl-3H-xanthen-9-yl]benzoic acid ethyl ester monohydrochloride (R6G)(emits a response radiation in the wavelength that ranges from about 500 to 560 nm), l,l,3,3,3',3'-Hexamethylindodicarbocyanine iodide (HIDC) (emits a response radiation in the wavelength that ranged from about 600 to 660 nm), 6- carboxyfluorescein (commonly known by the abbreviations FAM and F), 6-carboxy- 2',4',7',4,7-hexachlorofluorescein (HEX), 6-carboxy-4',5'-dichloro-2',7'- dimethoxyfluorescein (JOE or J), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA or T), 6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G5 or G5), 6- carboxyrhodamine-6G (R6G6 or G6), and rhodamine 1 10; cyanine dyes, e.g. Cy3, Cy5 and Cy7 dyes; coumarins, e.g., umbelliferone; benzimide dyes, e.g. Hoechst 33258; phenanthridine dyes, e.g. Texas Red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, e.g. cyanine dyes such as Cy3 (emits a response radiation in the wavelength that ranges from about 540 to 580 nm), Cy5 (emits a response radiation in the wavelength that ranges from about 640 to 680 nm), etc; BODIPY dyes and quinoline dyes. Specific fluorophores of interest include: Pyrene, Coumarin, Diethylaminocoumarin, FAM, Fluorescein Chlorotriazinyl, Fluorescein, Rl 10, Eosin, JOE, R6G, HIDC, Tetramethylrhodamine, TAMRA, Lissamine, ROX, Napthofluorescein, Texas Red, Napthofluorescein, Cy3, and Cy5, CalFluorOrange, and the like.
[0061] In some embodiments, the probe is conjugated to a quencher. A quencher can absorb electromagnetic radiation and dissipate it as heat, thus remaining dark. Example quenchers include Dabcyl, NFQ's, such as BHQ-1 or BHQ-2 (Biosearch), IOWA BLACK FQ (IDT), and IOWA BLACK RQ (IDT). In some embodiments, the quencher is selected to pair with a fluorphore so as to absorb electromagnetic radiation emitted by the fluorophore. Flourophore/quencher pairs useful in the compositions and methods disclosed herein are well-known in the art, and can be found, e.g., described in S. Marras, "Selection of Fluorophore and Quencher Pairs for Fluorescent Nucleic Acid Hybridization Probes" available at the world wide web site molecular- beacons. org/download/marras,mmb06%28335%293.pdf. In some embodiments, a flourophore/quencher pair includes CalFluor Orange and BHQ-1.
[0062] In some embodiments, a fluorophore is attached to a first end of the probe, and a quencher is attached to a second end of the probe. Attachment can include covalent bonding, and can optionally include at least one linker molecule positioned between the probe and the fluorophore or quencher. In some embodiments, a fluorophore is attached to a 5 ' end of a probe, and a quencher is attached to a 3 ' end of a probe. In some embodiments, a fluorphore is attached to a 3' end of a probe, and a quencher is attached to a 5' end of a probe. Examples of probes that can be used in quantitative nucleic acid amplification include molecular beacons, SCORPIONS™ probes (Sigma) and TAQMAN™ probes (Life Technologies).
[0063] It has been shown that primers and probes in accordance with embodiments herein detect E. histolytica if present, but do not cross-react when any of a number of other pathogens are present in the sample (see, e.g., Examples 4 and 6). As used herein "cross-react" refers to yielding a detectable signal from a template of the indicated organism (e.g. a non-is. hisotofytica organism as listed below). As shown, for example in Example 6, the presence of the organisms listed in Table 4 does not result in a detectable signal for amplification using primers and probes in accordance with some embodiments herein. In some embodiments, cross-reacting can further include depression of the E. histolytica signal when a template from the indicated organism is present. As shown, for example, in Examples 4 and 6, the presence of Cryptosporidium parvum, Giardia lamblia, or Entamoeba dispar (even at high-titer) neither yield a detectable signal, nor substantially suppresses the detectable signal from E. histolytica for primers and probes in accordance with some embodiments herein. In some embodiments, the primers and probes do not cross-react with any of the following organisms: Abiotrophia defectiva, Acinetobacter baumannii, Acinetobacter Iwoffii, Aeromonas hydrophila, Alcaligenes faecalis subsp. faecalis, Anaerococcus tetradius, Arcobacter butzleri, Arcobacter cryaerophilus, Bacillus cereus, Bacteroides caccae, Bacteroides merdae, Bacteroides stercoris, Bifidobacterium adolescentis, Bifidobacterium longum, Camplylobacter coli, Campylobacter concisus, Campylobacter curvus, Campylobacter fetus subsp. fetus, Campylobacter fetus subsp. venerealis, Campylobacter gracilis, Campylobacter hominis, Camplylobacter jejuni, Campylobacter lari, Campylobacter rectus, Campylobacter upsaliensis, Candida albicans, Candida catenulate, Cedecea davisae, Chlamydia trachomatis, Citrobacter amalonaticus, Citrobacter fruendii, Citrobacter koseri, Citrobacter sedlakii, Clostridium difficile 17858, Clostridium difficile 43598, Clostridium difficile CCUG 8864-9689, Clostridium difficile 43255, Clostridium difficile BAA-1805, Clostridium difficile 43593, Clostridium perfringens, Collinsella aerofaciens, Corynebacterium genitalium, Desulfovibrio piger, Edwardsiella tarda, Eggerthella lenta, Enterobacter aerogenes, Enterobacter cloacae, Enterococcus casseliflavus, Enterococcus cecorum, Enterococcus dispar, Enterococus faecalis, Enterococcus gallinarum, Enterococcus hirae, Enterococcus raffinosus, Escherichia coli, Escherichia fergusonii, Escherichia hermannii, Escherichia vulneris, Fusobacterium varium, Gardnerella vaginalis, Gemella morbillorum, Hafnia alvei, Helicobacter fennelliae, Helicobacter pylori, Klebsiella oxytoca, Klebsiella pneumonia, Lactobacillus acidophilus, Lactobacillus reuteri, Lactococcus lactis, Leminorella grimontii, Listeria grayi, Listeria innocua, Listeria monocytogenes, Morganella morganii, Peptoniphilus asaccharolyticus, Peptostreptococcus anaerobius, Plesiomonas shigelloides, Porphyromonas asaccharolytica, Prevotella melaninogenica, Proteus mirabilis, Proteus penneri, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii, Pseudomonas aeruginosa, Pseudomonas fluorescens, Ruminococcus bromii, Salmonella typhimurium, Salmonella enteriditis, Serratia liquefaciens, Serratia marcescens, Shigella sonnei, Shigella flexneri, Staphylococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus intermedius, Streptococcus uberis, Trabulsiella guamensis, Veillonella parvula, Vibrio cholera, Vibrio parahaemolyticus, Yersinia bercovieri, Yersinia enterocolitica, Yersinia rohdei, Adenovirus type 2, Adenovirus type 14, Adenovirus type 40, Adenovirus type 41, Coxsackie A9, Coxsackie Bl, HHV-5, Cytomegalovirus, Enterovirus type 69, Human Papillomavirus Type 16, Human Papillomavirus Type 18, Herpes Simplex Virus I, Herpes Simplex Virus II, Norovirus I, Norovirus II, Rotavirus, Blastocystis hominis, Encephalitozoon intestinalis, Encephalitozoon helium, Encephalitozoon cuniculi, Pentatrichomonas hominis, Entamoeba barrette, Entamoeba gigivalis, Entamoeba invadens, Entamoeba moshkovskii, Entamobea ranarum, Citrobacter fruendii (rpt), Enterobacter cloacae (rpt), Cryptosporidium parvum, Giardia lamblia, or Cryptosporidium meleagridis.
[0064] It is noted that while a number of the above-listed organisms are typically found in human stool, several listed organisms are not. As such, it is contemplated herein that probes, primers, and methods of detection in accordance with some embodiments herein are robust in the presence of additional pathogens.
[0065] Furthermore, as shown in Examples 5 and 8, primer and probe sets in accordance with embodiments herein showed provided robust results for both fixed and unfixed sample types, and provided results consistent with those of commercial ELISA kits for the detection of E. histolytica. As such, it is contemplated that primer and probe sets in accordance with embodiments herein provide robust results across a variety of sample types (e.g. fixed and unpreserved or non-fixed samples), and consistent with other methods of determining the presence of absence of is. histolytica.
[0066] As shown in Example 7, the 95% limit of detection (LoD) for some primers and probes in accordnance with emodiments herein is about 17 is. histolytica organsims per milliliter of sample. As used herein, the "95% LoD," or unless stated otherwise, "LoD," refers to the concentration that yields a positive result 95% of the time. Accordingly, in some embodiments, the primers and probes will produce a positive signal (e.g. a Ct score below the cutoff) if is. histolytica is present in the amplification reaction in a quantity that is at least the 95% limit of detection (LoD), but will not produce a positive signal if only one or more of the above-listed non-is. histolytica organisms are present. In some embodiments, the LoD of E. hisolytica is about 17 is. histolytica organisms (or quantity of template sequence corresponding to 17 is. histolyica organisms) per milliliter of sample. In some embodiments, the LoD is no more than about 50 organisms per militliter of reaction, for example no more than about 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 E. histolytica organisms (or genomes thereof) per mililiter. It is noted that the in some embodiments, the LoD is comparable for both fixed and non-fixed samples (see Example 8 and Tables 6-7). As such, it is understood that primer and probes in accordance with some embodiments herein yield comparable E. histolytica detection properties, for example comparable LoD values, for both fixed and non-fixed samples.
[0067] As shown in Example 4, the amount of E. histolytica detected was not substantially altered by a high titer of Cryptosporidium parvum, Giardia lamblia, and Entamoeba dispar, nor was there substantial cross-reactivity with these organisms. Accordingly, in some embodiments, the LoD of E. histolytica organisms is not substantially altered by a high titer presence of another pathogenic organism in the sample. In some embodiments, the detection of E. histolytica organisms (measured, for example by Ct score) is not substantially altered by a high titer presence of another pathogenic organism in the sample. In some embodiments a high titer comprises a quantity of at least lxlO6 organisms/mL of sample, for example about lxlO6 organisms/mL, lxlO6, 2xl06, 3xl06, 4xl06, 5xl06, 6xl06, 7xl06, 8xl06, 9xl06, lxlO7, 1.5xl07, 2xl07, 3xl07, 4xl07, 5xl07, 6xl07, 7xl07, 8xl07, 9xl07, lxlO8, lxlO9, or lxlO10 organisms/mL of sample. In some embodiments a high titer comprises a quantity of at least about 1.5xl07 organisms/mL of sample.
Kits
[0068] Some embodiments include kits. The kits can include at least one primer pair as described herein. In some embodiments, the primer pair can amplify an E. histolytica target sequence under standard amplification conditions, but cannot amplify an E. dispar target sequence under standard amplification conditions, as described herein. The kits can include a probe as described herein. In some embodiments, the probe is specific to a nucleic acid sequence that occurs in both E. histolytica and E. dispar as described herein. In some embodiments, the primer set includes a forward primer comprising an oligonucleotide having the sequence of SEQ ID NO: 1, or a variant thereof, a reverse primer comprising an oligonucleotide having the sequence of SEQ ID NO: 2, or a variant thereof, and a probe comprising an oligonucleotide having the sequence of SEQ ID NO: 3, or a variant thereof. In some embodiments, the probe comprises a fluorophore/quencher pair as described herein. In some embodiments, the kits include samples, for example positive controls that contain E. histolytica or E. histolytica DNA as decribed herein. The kits can further include negative controls, for example that contain only E. dispar, or E. dispar DNA. The kits can further include packaging and/or instructions.
[0069] In some embodiments, the kits further include reagents for a multiplex assay for detecting at least one other parasitic organism from a human stool sample, for example at least one of Giardia lamblia, Cryptosporidium parvum, Cryptosporidium hominis, and the like.
Master Mix
[0070] In some embodiments, a master mix is provided. A master mix can include at least two reagents for an assay that are provided in relative concentrations that are proportional to the relative concentrations of the reagents in a quantitative nucleic acid amplification assay Thus, a single a single quantity of master mix can be added to a reaction to provide appropriate relative concentrations of two or more reagents. In some embodiments, a master mix can include at least two of: polymerase, buffer, salts, for example magnesium, nucleotide triphosphates, a primer set, and water. In some embodiments, a master mix can be provided at a higher concentration than will be used in a reaction. In some embodiments, a master mix is provided in a lyophilized form, and reconstituted at a higher concentration that will be used in the reaction. In some embodiments a master mix includes reagents at a concentration of at least about 2x of the reaction concentration, for example 2x, 2.5x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, 15x, 20x, 25x, 40x, 50x, lOOx, 200x, 250x, or 500x.
Samples
[0071] Samples as provided herein include substances that may or may not contain Entamoeba nucleic acids. In some embodiments, the sample includes fecal matter from a human, or a portion or derivative thereof. In some embodiments, the sample includes a biopsy, for example tissue from a human that is possibly infected with Entamoeba, such as gastrointestinal, liver, lung, or central nervous system tissue. In some embodiments, the sample includes a cell culture, for example a culture derived from human fecal matter. In some embodiments, the sample has been processed, for example to isolate nucleic acids from other substances, or to remove non-nucleic acid substances from the sample (for example to remove lipids, proteins, cellular debris, and the like). In some embodiments, the sample has been treated with protease. It has been shown that primers and probes in accordance with embodiments herein achieve comparable detection properties for fixed and unpreserved samples (see, e.g., Example 8 and Tables 6-7). In some embodiments, the sample is fixed, for example in a quantity of fixative such as formalin. In some embodiments the sample is unpreserved (e.g. "non-fixed").
[0072] In some embodiments, it is unknown whether the sample contains E. histolytica and/or E dispar nucleic acids. In some embodiments, it is known that the sample includes at least one of E. histolytica or E. dispar, but it is unknown which one sample includes, or whether the sample includes both. In some embodiments, the sample contains both E. hisotlytica and E. dispar.
[0073] In some embodiments, the sample includes a positive control, for example spiking the sample with nucleic acids of E. histolytica, E. dispar, or a combination of nucleic acids from is. histolytica, or E. dispar. In some embodiments, the sample is spiked with at least 1000 ("IK") copies of E. dispar target amplification sequence, for example at least about IK copies, 2K, 3K, 4K, 5K, 6K, 7K, 8K, 9K, 10K, 20K, 30K, 40K, 50K, 60K, 70K, 80K, 90K, 100K, 150K, 200K, 250K, 300K, 350K, 400K, 450K, 500K, 550K, 600K, 650K, 700K, 750K, 800K, 850K, 900K, 1000K, HOOK, 1200K, 1300K, 1400K, 1500K, 1600K, 1700K 1800K, 1900K, or 2000K copies. In some embodiments, the sample is spiked with at least 100 copies of E. histolytica target amplification sequence, for example at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, IK copies, 2K, 3K, 4K, 5K, 6K, 7K, 8K, 9K, 10K, 20K, 30K, 40K, 50K, 60K, 70K, 80K, 90K, 100K, 150K, 200K, 250K, 300K, 350K, 400K, 450K, 500K, 550K, 600K, 650K, 700K, 750K, 800K, 850K, 900K, 1000K, HOOK, 1200K, 1300K, 1400K, 1500K, 1600K, 1700K 1800K, 1900K, or 2000K copies. In some embodiments, the sample is spiked with E. histolytica and E. dispar target nucleic acids.
[0074] In some embodiments, the sample includes nucleic acids isolated from one or more of the above. Nucleic acids can be isolated using standard techniques, well- known to one skilled in the art.
Additional Embodiments
[0075] In accordance with some embodiments, primer and probe sets, and methods of using the same are provided for the detection of E. histolyica. In some embodiments, the primers and probe sets and methods do not detect non-pathogenic E. dispar. In some embodiments, the primers and probe sets and methods produce robust results, that are not inhibited or interfered with in the case of a simulated mixed E. histolytica and E. dispar infection. In some embodiments, the primers and probe sets and methods detect Entamoeba histolytica from human clinical specimens identified by traditional microscopic methods (which at the time of the application represent the current standard of care). In some embodiments, the primers and probe sets and methods produce results that agree with a commercially available FDA-cleared ELISA assay for the appropriate specimen type using clinical specimens. In some embodiments, the primers and probe sets and methods do not cross-react with other organisms likely to be found in stool or a variety of other pathogens. In some embodiments, the primers and probe sets and methods do react with different Entamoeba histolytica isolates. In some embodiments, the primers and probe sets and methods are sensitive to detect down to, and below, 17 organisms per mL in the sample buffer tube (or a quantity of template sequence corresponding to 17 organisms).
EXAMPLE 1 : Amplification in the presence of of E. histolytica and E. dispar plasmid sequences
[0076] A previously-described primer set and probe combination (see Verweij et al, J. .Clin. Microbiol. 42: 1220-23, 2004), which included a forward primer of SEQ ID NO: 4 (ATTGTCGTGGCATCCTAACTCA), a reverse primer of SEQ ID NO: 5 (GCGGACGGCTCATTATAACA), and a probe of SEQ ID NO: 6 (TCATTGAATGAATTGGCCATTT), which comprised a CalFluor Orange fluorphore and BHQ-1 quencher (see Figure 1) were used in a quantitative PCR reaction on a BD MAX™ system. It is noted that the primer set of SEQ ID NO: 4 and SEQ ID NO: 5 amplify rDNA sequences of both E. histolytica and E. dispar. The probe of SEQ ID NO: 6 has 100% percent homology to the target amplification sequence (defined by the primer set of SEQ ID NOs: 4 and 5) in E. histolytica, but not E. dispar.
[0077] Reactions were provided with template plasmid that contained target rDNA gene sequence from E. histolytica, and/or E. dispar. Low-level cross-reactivity was observed between E. histolytica and E. dispar target DNA sequence. Furthermore, when plasmid containing E. dispar target nucleic acid was spiked into the PCR reaction at a higher proportion than plasmid containing E. histolytica nucleic acid, the specific signal from E. histolytica was drastically reduced (see Figure 3). While quantitative PCR reactions with E. histolytica template produced detectable signal, the presence of 5,000 ("5K"), 25,000 ("25K"), 50,000("50K"), 75,000 (75K), and 100,000 ("100K") copies of E. dispar template plasmid, in addition to a constant level of E. histolytica template, decreased the amount of detectable signal in a dose-dependent manner (Figure 3). As summarized in Figure 1, the primer/probe combination of this example resulted in E. histolytica signal depression.
[0078] Without being limited by any one theory, it is contemplated that the use of primers that amplify both E. histolytica and E. dispar DNA, and reliance on an E. /zzsto/ ft'ca-sequence-specific probe resulted in both cross-reactivity, and signal suppression in the presence of E. dispar, possibly due to homo- and hetero- duplex formation between amplification products of E. histolytica and E. dispar that blocks the availability of E. histolytica probe binding sites.
EXAMPLE 2: Detection of E. histolytica in the presence of E. histolytica and E. dispar plasmid sequences
[0079] A primer-probe set according to embodiments herein was used in a quantitative PCR amplification reaction performed on the BD MAX™ platform. The PCR mixture was heated to 97°C for 10 minutes to activate the DNA Polymerase. Two- step thermal cycling was then carried out for 45 cycles with a 15 second denaturation step at 97°C followed by an annealing/extension step for 64.5 seconds at 62°C. The primer set included a forward primer of SEQ ID NO: 1, a reverse primer of SEQ ID NO: 2, and a probe of SEQ ID NO: 3, which comprised a CalFluor Orange fluorphore and BHQ-1 quencher (see Figure 1). It is noted that the primer of SEQ ID NO: 1 will anneal to E. histolytica, but not E. dispar target nucleic acids sequence of the small ribosomal subunit gene under standard amplification conditions (see Figure 2), while the primer of SEQ ID NO: 2 will anneal to target nucleic acids sequence on either of the E. histolytica and E. dispar small ribosomal subunit gene. Accordingly, the primer set of SEQ ID NO: 1 and SEQ ID NO: 2 will substantially amplify E. histolytica, but not E. dispar target amplification sequence. The probe of SEQ ID NO: 3 has 100% complementarity to either of E. histolytica or E. dispar small ribosomal subunit gene DNA sequence.
[0080] As in Example 1, reactions were provided with plasmid that contained target rDNA gene sequence template from E. histolytica, and/or E. dispar. Unlike Example 1, cross-reactivity was not seen with E. dispar template. Moreover, the presence of E. dispar template did not depress the amplification signal (see Figure 4). In the presence of a constant amount of E. histolytica template, the presence of 0, 250,000 ("250K"), 500,000 ("500K"), 750,000 ("750K"), and 1,000,000 ("le6") copies of E. dispar template-containing plasmid did not decrease the amplification signal from E. histolytica (Figure 4). A negative control that contained no template ("NTC") was performed, and as expected, no signal was detected. As summarized in Figure 1, the primer/probe combination of this example did not cause any identifiable E. histolytica signal depression.
[0081] Thus, even in the presence of a high copy number of E. dispar template, the primer set and probe as in Example 2 produced robust, and consistent levels of E. histolytica signal. Without being bound to any one theory, it is contemplated that a primer set designed to amplify a sequence specific to E. histolytica, but not E. dispar can permit the detection of E. histolytica-specific signal without interference from E. dispar sequences.
EXAMPLES 3-13 Detection of E. histolytica sequences
[0082] The following methods were used in Examples 3-13.
[0083] Stool specimens were collected from patients and transported to the laboratory unpreserved in a clean container (unpreserved) or fixed (10% formalin).
[0084] DNA extraction from the stool specimens was performed as follows: Specimens were vortexed. A ΙΟμΙ^ loop was inserted in each specimen to the depth of the loop and then expressed using a swirling motion into BD MAX™ Sample Buffer Tubes (SBT) containing Sample Buffer [50 mM Tris-HCl (pH 7.0), 1% Triton X-100, 1 mM EDTA (pH 8.0), 20 mM H3B03, 20 mM Na3C6H507. 2H20]. The SBTs were closed with a septum cap and then heated on the BD Prewarm Heater to approximately 1 10°C for 20 minutes to facilitate lysis of organisms. The SBTs were cooled to room temperature by the BD Prewarm Heater, vortexed briefly, and then transferred to the BD MAX™ System. A 500 μΐ volume of sample buffer was extracted per sample for 10 minutes at 75°C using 12 units of proteinase K, 0.12% trehalose, and 104 copies of an internal control DNA in the presence of 0.5 μg/μl PAMAM-coupled magnetic beads on the BD MAX™ System. The beads, with the bound nucleic acids, were washed with 500μ1 of wash buffer [12.5 mM Tris (pH 6.8), 0.03% ProClin 300, 0.1% Tween-20]. Nucleic acids were then eluted by heating the beads for 3 minutes at 80°C in 12.5 μΐ of elution buffer [20 mM NaOH]. Eluted nucleic acids were neutralized by the addition of 22.5 μΐ of neutralization buffer [7.78 mM MgC12, 155.6 mM Tris (pH 8.0), 4.44 mM NaOH, 0.03% ProClin300, 0.016% Tween-20].
[0085] A PCR master mix was prepared as follows: Neutralized nucleic acids (35μ1) were used to rehydrate dried down master mix. The final concentration of components in the PCR master mix after rehydration with is as follows: 5 mM MgCl2, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.5 mM dNTPs (each), 0.6 mg/ml BSA, 0.04 U/μΙ Hot Gold Star DNA Polymerase. The master mix also included PCR primers and TaqMan® dual-labeled hydrolysis probes. Primers and probes for Entamoeba histolytica were included at 900 nM for forward and reverse primers and 550 nM for the probe. The primer set and probe set for the detection of E. histolytica included a forward primer having the nucleic acid sequence of SEQ ID NO: 1, a reverse primer having a nucleic acid sequence of SEQ ID NO: 2, and a probe having the nucleic acid sequence of SEQ ID NO: 3. The probe for Entamoeba histolytica was labeled with Cal Fluor Orange 560 and Black Hole Quencher- 1. Primers and probes for the internal control were included at 300 nM each. The internal control probe was labeled with Quasar 705 and Black Hole Quencher-3. Primers and probes for Cryptosporidium parvum/hominis and Giardia lamblia were included at 200 nM for forward and reverse primers and 550 nM for probes. The probe for Cryptosporidium parvum/hominis was labeled with CalFluor Red 610 and Black Hole Quencher-2. The probe for Giardia lamblia was labeled with FAM and Black Hole Quencher- 1.
[0086] After rehydration, the BD MAX™ System dispenses approximately 12 μΐ of PCR-ready solution into the BD MAX™ Microfluidic Cartridge. Microvalves in the BD MAX™ Microfluidic Cartridge are sealed by the system prior to initiating PCR to contain the amplification mixture thus preventing evaporation and contamination. The PCR mixture was heated to 97°C for 10 minutes to activate the DNA Polymerase. Two- step thermal cycling was then carried out for 45 cycles with a 15 second denaturation step at 97°C followed by an annealing/extension step for 64.5 seconds at 62°C. The BD MAX™ System monitors fluorescent signals at each cycle and interprets the data at the end of the program to report the final results. Result calls were based on a Ct. Score algorithm that includes an initial static endpoint threshold for each target channel and a secondary dynamic QC threshold that changes inversely with Ct. Endpoint fluorescence must exceed both thresholds and a final Ct must be <42 to be considered positive. Additional checks for excessively variable PCR curves were used to exclude reactions that had insufficient volume in the PCR chamber. Amplification failure of the internal control causes the system to return unresolved results for each target channel that fails to meet the Ct. Score thresholds for positivity.
Example 3
[0087] Two lots of Entamoeba histolytica trophozoites were detected by the BD MAX™ assay as described. The BD MAX™ assay does not detect low (2,550 trophozoites per ml in specimen) or high (1.5e6 trophozoites per ml in specimen) titer Entamoeba dispar. The results are shown in Table 1.
Table 1
Figure imgf000049_0001
E. histolytica lot 1 2550 7168.81 23.75 Positive
E. dispar Low 2550 0.72 NA Negative
E. histolytica lot 2 2550 6774.47 26.05 Positive
E. dispar High 1.50E+06 1 .16 NA Negative
E. histolytica lot 1 2550 8523.08 24.45 Positive
E. dispar Low 2550 0.81 NA Negative
Example 4
[0088] The BD MAX™ detected E. histolytica near the limit of detection (LoD) in simulated multiple infection specimens containing high titer Cryptosporidium parvum, Giardia lamblia, and Entamoeba dispar.
Table 2
Figure imgf000050_0001
E. histolytica + 450 (E.
C.parvum, G.lamblia, histolytica)
E.dispar 1.5e7 (Cp, Gl, Ed) 4681.89 23.49 Positive
E. histolytica + 450 (E.
C.parvum, G.lamblia, histolytica)
E.dispar 1.5e7 (Cp, Gl, Ed) 4351.27 24.95 Positive
E. histolytica + 450 (E.
C.parvum, G.lamblia, histolytica)
E.dispar 1.5e7 (Cp, Gl, Ed) 8041.45 24.41 Positive
E. histolytica + 450 (E.
C.parvum, G.lamblia, histolytica)
E.dispar 1.5e7 (Cp, Gl, Ed) 6941.23 25.1 1 Positive
Example 5
[0089] The BD MAX™ system was used to detect the form of the Entamoeba histolytica organism shed in true human clinical specimens detected by traditional methods representing both unpreserved and 10% formalin fixed specimen types. For comparison, a commercially-available ELISA (TechLab E.histolytica II) was performed on the same samples.
[0090] The results of the BD MAX™ assay closely agree with a commercially available ELISA result (TechLab E.histolytica II) in unpreserved specimens for which the ELISA is cleared. It is noted that TechLab ELISA assay is not cleared for fixed specimens and therefore, the negative result for the fixed specimens is in-line with the properties of the TechLab ELISA assay.
Table 3
Figure imgf000051_0001
EH21 Unpreserved 245.52 NA UNR Positive
EH22 Unpreserved 3053.63 33.71 Positive Positive
EH23 Unpreserved 3835.47 27.65 Positive Positive
EH24 Unpreserved 4079.21 31 .79 Positive Positive
EH25 Unpreserved 3965.72 30.79 Positive Positive
+ CTRL
(spike) N/A 4279.29 26.1 1 Positive Positive
NEG CTRL N/A 1 .25 NA Negative Negative
EH01 10% Formalin 4850 27.19 Positive Negative
EH02 10% Formalin 5462.54 23.44 Positive Negative
EH03 10% Formalin 4773.22 23.69 Positive Negative
EH04 10% Formalin 4998.17 25.81 Positive Negative
EH05 10% Formalin 5338.4 23.96 Positive Negative
EH06 10% Formalin 5233.45 25.48 Positive Negative
EH07 10% Formalin 5199.61 28.69 Positive Negative
EH08 10% Formalin 5595.57 24.32 Positive Negative
EH09 10% Formalin 5000.83 28.98 Positive Negative
EH10 10% Formalin 2839.61 29.24 Positive Negative
EH11 10% Formalin 5951 .38 22.89 Positive Negative
EH12 10% Formalin 4592.81 28.92 Positive Negative
EH13 10% Formalin 5071 .64 29.26 Positive Negative
EH14 10% Formalin 5038.97 24.5 Positive Negative
EH15 10% Formalin 4545.1 1 29.06 Positive Negative
Example 6
[0091] To determine whether the BD MAX™ assay cross-reacts with other organisms E. histolytica sequences were detected in the presence of template from other organsms, including organisms likely to be found in stool, and well as exemplary organisms that were not likely to be found in stool. Challenge organisms were spiked into an SBT without stool matrix. Each organism was tested in triplicate.
[0092] The results are shown in Table 4. The BD MAX™ assay does not cross-react with other organisms likely (or unlikely) to be found in stool. Table 4
Figure imgf000053_0001
Figure imgf000054_0001
Bacteroides caccae 10 1.71 NA >lxl08 >lxl06 15μΙ, Neg.
Figure imgf000055_0001
Camplylobacter 15 1.55 NA >lxl08 >lxl06 15μΙ, Neg. coli CFU CFU
Campylobacter >lxl08 >lxl06
16 0.57 NA Neg. concisus CFU CFU 15μί
Campylobacter >lxl08 >lxl06
16 0.26 NA Neg. concisus CFU CFU 15μί
Campylobacter >lxl08 >lxl06
16 0.83 NA Neg. concisus CFU CFU 15μί
Campylobacter >lxl08 >lxl06
17 0.33 NA Neg. curvus CFU CFU 15μί
Campylobacter >lxl08 >lxl06
17 2.34 NA Neg. curvus CFU CFU 15μί
Campylobacter >lxl08 >lxl06
17 1.46 NA Neg. curvus CFU CFU 15μί
Campylobacter >lxl08 >lxl06
18 0.65 NA Neg. fetus subsp. fetus CFU CFU 15μί
Campylobacter >lxl08 >lxl06
18 0.57 NA Neg. fetus subsp. fetus CFU CFU 15μί
Campylobacter >lxl08 >lxl06
18 3.49 NA Neg. fetus subsp. fetus CFU CFU 15μί
Campylobacter
>lxl08 >lxl06
fetus subsp. 19 0.66 NA Neg.
CFU CFU
venerealis 15μί
Campylobacter
>lxl08 >lxl06
fetus subsp. 19 0.09 NA Neg.
CFU CFU
venerealis 15μί
Campylobacter
>lxl08 >lxl06
fetus subsp. 19 0.51 NA Neg.
CFU CFU
venerealis 15μί
Campylobacter >lxl08 >lxl06
20 0.85 NA Neg. gracilis CFU CFU 15μί
Campylobacter >lxl08 >lxl06
20 0.31 NA Neg. gracilis CFU CFU 15μί
Figure imgf000057_0001
ups aliens is CFU CFU 15μΙ,
Figure imgf000058_0001
fruendii CFU CFU 15μΙ,
Figure imgf000059_0001
Clostridium
>lxl08 >lxl06
difficile CCUG 36 0.66 NA Neg.
CFU CFU
8864-9689 15μί
Clostridium
>lxl08 >lxl06
difficile CCUG 36 4.21 NA Neg.
CFU CFU
8864-9689 15μί
Clostridium >lxl08 >lxl06
37 1.99 NA Neg. difficile 43255 CFU CFU 15μί
Clostridium >lxl08 >lxl06
37 1.3 NA Neg. difficile 43255 CFU CFU 15μί
Clostridium >lxl08 >lxl06
37 0.64 NA Neg. difficile 43255 CFU CFU 15μί
Clostridium
>lxl08 >lxl06
difficile BAA- 38 4.9 NA Neg.
CFU CFU
1805 15μί
Clostridium
>lxl08 >lxl06
difficile BAA- 38 0.99 NA Neg.
CFU CFU
1805 15μί
Clostridium
>lxl08 >lxl06
difficile BAA- 38 4.86 NA Neg.
CFU CFU
1805 15μί
Clostridium >lxl08 >lxl06
39 2.78 NA Neg. difficile 43593 CFU CFU 15μί
Clostridium >lxl08 >lxl06
39 0.69 NA Neg. difficile 43593 CFU CFU 15μί
Clostridium >lxl08 >lxl06
39 16.21 NA Neg. difficile 43593 CFU CFU 15μί
Clostridium >lxl08 >lxl06
40 0.47 NA Neg. perfringens CFU CFU 15μί
Clostridium >lxl08 >lxl06
40 1.22 NA Neg. perfringens CFU CFU 15μί
Clostridium 40 3.69 NA >lxl08 >lxl06 15μί Neg.
Figure imgf000061_0001
Enterobacter 46 7.62 NA >lxl08 >lxl06 15μί Neg. aerogenes CFU CFU
Enterobacter >lxl08 >lxl06
46 7.57 NA Neg. aerogenes CFU CFU 15μί
Enterobacter >lxl08 >lxl06
46 8.36 NA Neg. aerogenes CFU CFU 15μί
Enterobacter >lxl08 >lxl06
47 7.2 NA Neg. cloacae CFU CFU 15μί
Enterobacter >lxl08 >lxl06
47 9.51 NA Neg. cloacae CFU CFU 15μί
Enterobacter >lxl08 >lxl06
47 9.35 NA Neg. cloacae CFU CFU 15μί
Enterococcus >lxl08 >lxl06
48 9.7 NA Neg. casseliflavus CFU CFU 15μί
Enterococcus >lxl08 >lxl06
48 7.86 NA Neg. casseliflavus CFU CFU 15μί
Enterococcus >lxl08 >lxl06
48 8.66 NA Neg. casseliflavus CFU CFU 15μί
Enterococcus >lxl08 >lxl06
49 11.59 NA Neg. cecorum CFU CFU 15μί
Enterococcus >lxl08 >lxl06
49 10.93 NA Neg. cecorum CFU CFU 15μί
Enterococcus >lxl08 >lxl06
49 9.84 NA Neg. cecorum CFU CFU 15μί
Enterococcus >lxl08 >lxl06
50 6.68 NA Neg. dispar CFU CFU 15μί
Enterococcus >lxl08 >lxl06
50 7.06 NA Neg. dispar CFU CFU 15μί
Enterococcus >lxl08 >lxl06
50 6.2 NA Neg. dispar CFU CFU 15μί
Enterococus >lxl08 >lxl06
51 8.56 NA Neg. faecalis CFU CFU 15μί
Enterococus 51 8.93 NA >lxl08 >lxl06 15μί Neg.
Figure imgf000063_0001
Escherichia coli 56 3.17 NA >lxl08 >lxl06 15μί Neg.
Figure imgf000064_0001
Escherichia coli 62 5.42 NA >lxl08 >lxl06 15μί Neg. 33605 CFU CFU
Escherichia coli >lxl08 >lxl06
62 19.56 NA Neg. 33605 CFU CFU 15μί
Escherichia coli >lxl08 >lxl06
62 2.2 NA Neg. 33605 CFU CFU 15μί
Escherichia >lxl08 >lxl06
63 1.84 NA Neg. fergusonii CFU CFU 15μί
Escherichia >lxl08 >lxl06
63 5.88 NA Neg. fergusonii CFU CFU 15μί
Escherichia >lxl08 >lxl06
63 11.36 NA Neg. fergusonii CFU CFU 15μί
Escherichia >lxl08 >lxl06
64 17.27 NA Neg. hermannii CFU CFU 15μί
Escherichia >lxl08 >lxl06
64 4.04 NA Neg. hermannii CFU CFU 15μί
Escherichia >lxl08 >lxl06
64 2.54 NA Neg. hermannii CFU CFU 15μί
Escherichia >lxl08 >lxl06
65 6.71 NA Neg. vulneris CFU CFU 15μί
Escherichia >lxl08 >lxl06
65 11.1 NA Neg. vulneris CFU CFU 15μί
Escherichia >lxl08 >lxl06
65 6.4 NA Neg. vulneris CFU CFU 15μί
Fusobacterium >lxl08 >lxl06
66 5.59 NA Neg. varium CFU CFU 15μί
Fusobacterium >lxl08 >lxl06
66 2.6 NA Neg. varium CFU CFU 15μί
Fusobacterium >lxl08 >lxl06
66 4.26 NA Neg. varium CFU CFU 15μί
Gardnerella >lxl08 >lxl06
67 3.45 NA Neg. vaginalis CFU CFU 15μί
Gardnerella 67 5.99 NA >lxl08 >lxl06 15μί Neg.
Figure imgf000066_0001
Klebsiella oxytoca 72 4.13 NA >lxl08 >lxl06 15μΙ, Neg.
Figure imgf000067_0001
Listeria grayi 78 1.27 NA >lxl08 >lxl06 15μί Neg.
Figure imgf000068_0001
Peptostreptococcu 83 0.27 NA >lxl08 >lxl06 15μΙ, Neg.
Figure imgf000069_0001
Proteus penneri 88 10.77 NA >lxl08 >lxl06 15μΙ, Neg.
Figure imgf000070_0001
Pseudomonas 94 2.03 NA >lxl08 >lxl06 15μΙ, Neg. fluorescens CFU CFU
Pseudomonas >lxl08 >lxl06
94 2.32 NA Neg. fluorescens CFU CFU 15μΙ,
Pseudomonas >lxl08 >lxl06
94 5.55 NA Neg. fluorescens CFU CFU 15μΙ,
Ruminococcus >lxl08 >lxl06
95 1.25 NA Neg. bromii CFU CFU 15μΙ,
Ruminococcus >lxl08 >lxl06
95 0.73 NA Neg. bromii CFU CFU 15μΙ,
Ruminococcus >lxl08 >lxl06
95 0.73 NA Neg. bromii CFU CFU 15μΙ,
Salmonella >lxl08 >lxl06
96 0.77 NA Neg. typhimurium CFU CFU 15μΙ,
Salmonella >lxl08 >lxl06
96 3.81 NA Neg. typhimurium CFU CFU 15μΙ,
Salmonella >lxl08 >lxl06
96 2.84 NA Neg. typhimurium CFU CFU 15μΙ,
Salmonella >lxl08 >lxl06
97 1.28 NA Neg. enteriditis CFU CFU 15μΙ,
Salmonella >lxl08 >lxl06
97 1.36 NA Neg. enteriditis CFU CFU 15μΙ,
Salmonella >lxl08 >lxl06
97 1.13 NA Neg. enteriditis CFU CFU 15μΙ,
Serratia >lxl08 >lxl06
98 2.19 NA Neg. liquefaciens CFU CFU 15μΙ,
Serratia >lxl08 >lxl06
98 1.69 NA Neg. liquefaciens CFU CFU 15μΙ,
Serratia >lxl08 >lxl06
98 0.91 NA Neg. liquefaciens CFU CFU 15μΙ,
Serratia >lxl08 >lxl06
99 1.79 NA Neg. marcescens CFU CFU 15μΙ,
Serratia 99 4.78 NA >lxl08 >lxl06 15μΙ, Neg.
Figure imgf000072_0001
Stenotrophomonas 104 2.62 NA >lxl08 >lxl06 15μΙ, Neg. maltophilia CFU CFU
Streptococcus >lxl08 >lxl06
105 5.5 NA Neg. agalactiae CFU CFU 15μΙ,
Streptococcus >lxl08 >lxl06
105 2.98 NA Neg. agalactiae CFU CFU 15μΙ,
Streptococcus >lxl08 >lxl06
105 8.92 NA Neg. agalactiae CFU CFU 15μΙ,
Streptococcus >lxl08 >lxl06
106 6.85 NA Neg. dysgalactiae CFU CFU 15μΙ,
Streptococcus >lxl08 >lxl06
106 1.45 NA Neg. dysgalactiae CFU CFU 15μΙ,
Streptococcus >lxl08 >lxl06
106 4.53 NA Neg. dysgalactiae CFU CFU 15μΙ,
Streptococcus >lxl08 >lxl06
107 5.55 NA Neg. intermedius CFU CFU 15μΙ,
Streptococcus >lxl08 >lxl06
107 1.27 NA Neg. intermedius CFU CFU 15μΙ,
Streptococcus >lxl08 >lxl06
107 1.56 NA Neg. intermedius CFU CFU 15μΙ,
Streptococcus >lxl08 >lxl06
108 5.03 NA Neg. uberis CFU CFU 15μΙ,
Streptococcus >lxl08 >lxl06
108 6.12 NA Neg. uberis CFU CFU 15μΙ,
Streptococcus >lxl08 >lxl06
108 6.26 NA Neg. uberis CFU CFU 15μΙ,
Trabulsiella >lxl08 >lxl06
109 8.68 NA Neg. guamensis CFU CFU 15μΙ,
Trabulsiella >lxl08 >lxl06
109 9.48 NA Neg. guamensis CFU CFU 15μΙ,
Trabulsiella >lxl08 >lxl06
109 8.58 NA Neg. guamensis CFU CFU 15μΙ,
Veillonella parvula 110 9.54 NA >lxl08 >lxl06 15μΙ, Neg.
Figure imgf000074_0001
Yersinia rohdei 115 1.79 NA >lxl08 >lxl06 15μΙ, Neg.
Coxsackie A9 120 9.33 NA 1.6xl05 >lxl04 150μΙ, Neg.
Figure imgf000076_0001
Human 125 3.11 NA HPV Highest 150μί Neg.
Figure imgf000077_0001
Norovirus II 129 3.59 NA 8.5xl07 >lxl04 150μΙ, Neg.
Figure imgf000078_0001
cuniculi cells/mL cells/mL 15μί Encephalitozoon 3.40xl07 >lxl05
134 2.25 NA Neg. cuniculi cells/mL cells/mL 15μί
Encephalitozoon 3.40xl07 >lxl05
134 1.23 NA Neg. cuniculi cells/mL cells/mL 15μί
Pentatrichomonas 8.50xl06 >lxl05
135 3.61 NA Neg. hominis cells/mL cells/mL 150μί
Pentatrichomonas 8.50xl06 >lxl05
135 27.13 NA Neg. hominis cells/mL cells/mL 150μί
Pentatrichomonas 8.50xl06 >lxl05
135 15.04 NA Neg. hominis cells/mL cells/mL 150μί
Unknown Highest
Entamoeba barretti 136 1.63 NA Neg.
Titer Spike 150μί
Unknown Highest
Entamoeba barretti 136 5.63 NA Neg.
Titer Spike 150μί
Unknown Highest
Entamoeba barretti 136 1.02 NA Neg.
Titer Spike 150μί
~1.50xl05 Highest
Entamoeba dispar 137 1.82 NA Neg.
cells/mL Spike 150μί
~1.50xl05 Highest
Entamoeba dispar 137 2.43 NA Neg.
cells/mL Spike 150μί
~1.50xl05 Highest
Entamoeba dispar 137 32.69 NA Neg.
cells/mL Spike 150μί
Entamoeba -l.OOxlO5 Highest
138 5.88 NA Neg. gigivalis cells/mL Spike 150μί
Entamoeba -l.OOxlO5 Highest
138 8.41 NA Neg. gigivalis cells/mL Spike 150μί
Entamoeba -l.OOxlO5 Highest
138 4.77 NA Neg. gigivalis cells/mL Spike 150μί
Entamoeba 2.90xl06 >lxl05
139 3.15 NA Neg. invadens cells/mL cells/mL 150μί
Entamoeba 2.90xl06 >lxl05
139 4.52 NA Neg. invadens cells/mL cells/mL 150μί Entamoeba 2.90xl06 >lxl05
139 1.15 NA Neg. invadens cells/mL cells/mL 150μί
Entamoeba 2.20xl05 >lxl04
140 1.51 NA Neg. moshkovskii cells/mL cells/mL 150μί
Entamoeba 2.20xl05 >lxl04
140 8.7 NA Neg. moshkovskii cells/mL cells/mL 150μί
Entamoeba 2.20xl05 >lxl04
140 4 NA Neg. moshkovskii cells/mL cells/mL 150μί
Entamobea 7.33xl05 >lxl04
141 13.14 NA Neg. ranarum cells/mL cells/mL 150μί
Entamobea 7.33xl05 >lxl04
141 12.42 NA Neg. ranarum cells/mL cells/mL 150μί
Entamobea 7.33xl05 >lxl04
141 9.68 NA Neg. ranarum cells/mL cells/mL 150μί
Citrobacter >lxl08 >lxl06
31 2.11 NA Neg. fruendii (rpt) CFU CFU 15μί
Enterobacter >lxl08 >lxl06
47 0.85 NA Neg. cloacae (rpt) CFU CFU 15μί
Cryptosporidium 6.25xl06 >lxl05
143 3.85 NA Neg. parvum cells/mL cells/mL 150μί
Cryptosporidium 6.25xl06 >lxl05
143 2.83 NA Neg. parvum cells/mL cells/mL 150μί
Cryptosporidium 6.25xl06 >lxl05
143 6.22 NA Neg. parvum cells/mL cells/mL 150μί
Entamoeba l. lOxlO6 >lxl05
144 4691.2 15.5 Pos. histolytica cells/mL cells/mL 150μί
Entamoeba l. lOxlO6 >lxl05
144 4740.92 14.61 Pos. histolytica cells/mL cells/mL 150μί
Entamoeba l. lOxlO6 >lxl05
144 4941.93 14.48 Pos. histolytica cells/mL cells/mL 150μί
6.25xl06 >lxl05
Giardia lamblia 145 9.81 NA Neg.
cells/mL cells/mL 150μί
Figure imgf000081_0001
meleagridis cells/mL cells/mL 15μΙ, Cryptosporidium 3.0xl07 >lxl05
142 1.18 NA Neg. meleagridis cells/mL cells/mL 15μΙ,
Cryptosporidium 3.0xl07 >lxl05
142 2.82 NA Neg. meleagridis cells/mL cells/mL 15μΙ,
Cryptosporidium 3.0xl07 >lxl05
142 1.83 NA Neg. meleagridis cells/mL cells/mL 15μΙ,
Cryptosporidium 3.0xl07 >lxl05
142 0.34 NA Neg. meleagridis cells/mL cells/mL 15μΙ,
Cryptosporidium 3.0xl07 >lxl05
142 3.25 NA Neg. meleagridis cells/mL cells/mL 15μΙ,
Cryptosporidium 3.0xl07 >lxl05
142 0.42 NA Neg. meleagridis cells/mL cells/mL 15μΙ,
Cryptosporidium 3.0xl07 >lxl05
142 0.14 NA Neg. meleagridis cells/mL cells/mL 15μΙ,
Cryptosporidium 3.0xl07 >lxl05
142 1.49 NA Neg. meleagridis cells/mL cells/mL 15μΙ,
Cryptosporidium 3.0xl07 >lxl05
142 0.58 NA Neg. meleagridis cells/mL cells/mL 15μΙ,
Cryptosporidium 3.0xl07 >lxl05
142 1.55 NA Neg. meleagridis cells/mL cells/mL 15μΙ,
Example 7:
[0093] The BD MAX™ assay was tested with multiple different isolates of Entamoeba histolytica at the assay LOD in the presence of ΙΟμΙ, of unpreserved stool matrix per test. 24 replicates per isolate were tested. The BD MAX™ assay detected a variety of different Entamoeba histolytica isolates. The results are shown in Table 5.
Table 5
Figure imgf000082_0001
4445.23 29.16 Positive
3623.57 31.24 Positive
4248.16 30.84 Positive
4993.12 27.91 Positive
4143.38 30.82 Positive
4718.04 29.98 Positive
4322.62 31.11 Positive
4827.17 30.9 Positive
4938.83 29.19 Positive
2513.48 29.57 Positive
2953.5 32.62 Positive
3036.1 1 30.18 Positive
3657.4 31.59 Positive
2761.39 29.94 Positive
2886.84 30.98 Positive
3633.27 29.05 Positive
3160.31 31.27 Positive
332.57 35.89 Positive
3020.09 32.98 Positive
3448.17 28.43 Positive
2856.23 31.77 Positive
4208.86 31.92 Positive
3852.52 29.38 Positive
4600.73 28.83 Positive
4093.43 28.42 Positive
3440.67 28.84 Positive
H- Cultured
30889 13 org/mL 3705.75 29.69 Positive
458:CDC Isolate
3676.13 28.74 Positive
3725.05 30.58 Positive
4989.86 28.27 Positive
4775.94 29.92 Positive
4591.33 29.98 Positive 4110.95 32.51 Positive
4566.19 31.58 Positive
4467.29 29.24 Positive
4157.47 31.47 Positive
4042.66 30.3 Positive
4053.97 32.32 Positive
5247.62 28.93 Positive
5267.41 27.97 Positive
4754.97 28.65 Positive
4415.17 29.59 Positive
4797.29 28.48 Positive
4915.8 31.86 Positive
3839.02 28.51 Positive
890.79 37.16 Positive
1312.98 36.43 Positive
2225.24 35.43 Positive
2.99 NA Negative
26.41 NA Negative
2201.85 33.34 Positive
1 179.67 36.23 Positive
1510.96 36.22 Positive
3368.77 32.84 Positive
Cultured
DKB 50007 13 org/mL 3177.34 35.02 Positive
Isolate
743.44 40.11 Positive
1865.96 35.09 Positive
2163.25 35.18 Positive
1050.86 36.33 Positive
1655 35.52 Positive
1791.46 35.21 Positive
1941.9 35.09 Positive
7.77 NA Negative
1070.97 37.1 Positive 17.03 NA Negative
1.91 NA Negative
0.32 NA Negative
2789.16 36.18 Positive
2537.6 36.42 Positive
3766.93 27.82 Positive
4708.97 27.44 Positive
5143.31 27.74 Positive
4078.25 27.7 Positive
4641.32 26.09 Positive
4880.21 27.54 Positive
4477.49 27.97 Positive
4558.3 27.66 Positive
5068.43 26.51 Positive
5213.9 27.68 Positive
5122.26 27.46 Positive
Frozen 5252.53 27.4 Positive
200:NIH 30458 13 org/mL
Isolate 3436.73 27.8 Positive
3654.58 27.66 Positive
4203.64 28.36 Positive
3587.22 27.78 Positive
3374.32 27.81 Positive
3649.92 27.34 Positive
3285.35 27.83 Positive
3358.72 27.56 Positive
3556.5 27.81 Positive
3893.07 27.91 Positive
3909.42 27.84 Positive
3860.65 27.68 Positive
3741.63 21.85 Positive
HM- Frozen
30459 13 org/mL 4025.64 21.8 Positive
1 :IMSS Isolate
4224.04 21.73 Positive 4408.48 21.9 Positive
4033.02 21.86 Positive
4035.99 21.79 Positive
4606.63 21.8 Positive
3689.37 21.76 Positive
4306.67 21.78 Positive
4485.92 21.63 Positive
4236.68 21.72 Positive
4025.16 21.66 Positive
4731.57 21.61 Positive
3861.55 21.67 Positive
4472.74 21.9 Positive
4661.65 21.77 Positive
5151.08 21.82 Positive
3788.4 21.65 Positive
5016.46 21.81 Positive
4821.04 23.1 Positive
5269.22 21.62 Positive
4122.29 22.18 Positive
4838.41 21.77 Positive
4535.04 21.85 Positive
4558.59 27.03 Positive
4282.77 26.7 Positive
4403.06 26.71 Positive
4519.79 26.6 Positive
4464.05 27.17 Positive
PRA- Frozen
IP: 1 182:2 13 org/mL 4514.94 26.94 Positive
357 Isolate
4740.94 26.89 Positive
4092.67 26.65 Positive
5027.85 26.96 Positive
5064.84 26.46 Positive
5109.14 26.79 Positive 4958.97 26.86 Positive
4407 27.6 Positive
4535.17 27.23 Positive
4571.14 27.15 Positive
4196.92 26.6 Positive
4401.92 26.75 Positive
4685.94 26.89 Positive
1735.56 35.5 Positive
391 1.61 26.87 Positive
4558.06 27.33 Positive
4681.03 26.91 Positive
4736.92 27.56 Positive
5206.35 26.91 Positive
4547.02 32.7 Positive
846.46 38.73 Positive
4040.67 35.61 Positive
3912.84 35.34 Positive
3868.84 34.78 Positive
2494.87 36.65 Positive
3765.99 33.92 Positive
3399.28 35.09 Positive
4459.57 32.73 Positive
HM- Cultured
30890 13 org/mL 3083.3 35.02 Positive
3 :IMSS Isolate
3188.32 33.77 Positive
4784.16 31.72 Positive
2379.38 35.63 Positive
1.7 NA Negative
3686.26 33.6 Positive
2440.99 35.4 Positive
4049.49 29.89 Positive
525.31 41.28 Positive
3221.64 35.16 Positive 1822.64 36.33 Positive
2621.45 35.09 Positive
2244.92 34.96 Positive
1.4 NA Negative
3558.48 34.14 Positive
Example 8:
[0094] The 95% LoD for each specimen type was determined by linear dilution of Entamoeba histolytica trophozoites in sample buffer with ΙΟμΙ^ of the appropriate stool matrix. A minimum of 36 replicates per test level were performed. The LoD is approximately 17 organisms/ml in the sample buffer tube. The results are shown in Table 6 (unpreserved samples) and Table 7 (samples fixed in 10% formalin).
Table 6 (unpreserved samples)
Figure imgf000088_0001
0 7.19 NA Negative
0 54.92 NA Negative
0 2.83 NA Negative
0 10.48 NA UNR
0 8.33 NA Negative
0 9.47 NA Negative
0 2.64 NA Negative
0 6.22 NA Negative
0 2.65 NA Negative
0 1.73 NA Negative
0 7.28 NA Negative
0 13.06 NA Negative
0 0.52 NA Negative
0 24.9 NA Negative
0 3.8 NA Negative
0 10.57 NA Negative
0 0.81 NA Negative
0 18.32 NA Negative
0 5.25 NA Negative
1.5 3491.96 28.83 Positive
1.5 2.31 NA Negative
1.5 10.87 NA Negative
1.5 3.16 NA Negative
1.5 21.73 NA Negative
1.5 0.16 NA Negative
1.5 45.07 NA Negative
1.5 6.79 NA Negative
1.5 1.67 NA Negative
1.5 4715.47 29.13 Positive
1.5 7.24 NA Negative
1.5 5.99 NA Negative
1.5 3095.48 33.29 Positive 1.5 3482.41 34.47 Positive
1.5 4300.99 34.14 Positive
1.5 19.73 NA Negative
1.5 24.09 NA Negative
1.5 4456.34 34.2 Positive
1.5 54.38 NA Negative
1.5 3.01 NA Negative
1.5 8.39 NA Negative
1.5 7.88 NA Negative
1.5 1.75 NA Negative
1.5 7.07 NA Negative
1.5 33.9 NA Negative
1.5 2.56 NA Negative
1.5 8.51 NA Negative
1.5 2.04 NA Negative
1.5 17.23 NA Negative
1.5 1.06 NA Negative
1.5 67.26 NA Negative
1.5 5.14 NA Negative
1.5 8.24 NA Negative
1.5 0.4 NA Negative
1.5 10.67 NA Negative
1.5 3.11 NA Negative
3 3567.46 31.39 Positive
3 0.64 NA Negative
3 3462.65 32.5 Positive
3 3.23 NA Negative
3 4948.93 28.59 Positive
3 2.4 NA Negative
3 3143.06 32.1 Positive
3 2.41 NA Negative
3 12.22 NA Negative 3 0.57 NA Negative
3 12.03 NA Negative
3 3948.89 31.95 Positive
3 13.89 NA Negative
3 3678.22 33.52 Positive
3 23.9 NA Negative
3 34.43 NA Negative
3 50.52 NA Negative
3 7.36 NA Negative
3 50.44 NA Negative
3 36.33 NA Negative
3 24.59 NA Negative
3 4797.13 31.82 Positive
3 7.94 NA Negative
3 5156.56 27.71 Positive
3 13.05 NA Negative
3 4059.67 30.97 Positive
3 4.88 NA Negative
3 4.07 NA Negative
3 5148.21 32.48 Positive
3 5848.21 27.65 Positive
3 23.72 NA Negative
3 3971.89 26.78 Positive
3 13.25 NA Negative
3 4462.91 29.75 Positive
3 5246.72 27.84 Positive
3 5685.43 30.5 Positive
6 3904.47 29.53 Positive
6 4222.69 26.97 Positive
6 16.53 NA Negative
6 31.52 NA Negative
6 6.81 NA Negative 6 0.59 NA Negative
6 8.42 NA Negative
6 3240.6 31.37 Positive
6 4490.59 28.01 Positive
6 4083.43 33.06 Positive
6 7.7 NA Negative
6 2.8 NA Negative
6 3524.76 33.5 Positive
6 4533.07 27.87 Positive
6 20.68 NA Negative
6 8.97 NA Negative
6 2756.57 37.21 Positive
6 4436.58 32.45 Positive
6 2664.51 34.96 Positive
6 33.21 NA Negative
6 6077.66 28.47 Positive
6 4.8 NA Negative
6 24.36 NA Negative
6 4964.91 28.27 Positive
6 4318 25.96 Positive
6 2354.93 34.63 Positive
6 3905.86 30.62 Positive
6 4.91 NA Negative
6 4549.72 28.82 Positive
6 1.85 NA Negative
6 3215.39 32.45 Positive
6 49.2 NA Negative
6 3309.9 33.41 Positive
6 2.33 NA Negative
6 15.14 NA Negative
6 6286.84 31.04 Positive
12 1.82 NA Negative 12 3898.36 28.51 Positive
12 2577.56 32.74 Positive
12 3947.57 31.65 Positive
12 4232.4 29.27 Positive
12 4056.49 30.95 Positive
12 3445.95 28.9 Positive
12 3205.26 29.07 Positive
12 4172.74 31.9 Positive
12 4480.14 26.57 Positive
12 3929.88 30.82 Positive
12 3.12 A Negative
12 4313.96 26.69 Positive
12 1185.52 36.43 Positive
12 5597.67 29.16 Positive
12 4376.16 27.52 Positive
12 5636.53 27.57 Positive
12 3860.33 34.42 Positive
12 2750.81 34.32 Positive
12 3349.49 28.92 Positive
12 5062.72 27.54 Positive
12 4569.68 31.42 Positive
12 3937.91 33.31 Positive
12 4276.11 30.54 Positive
12 3854.83 25.66 Positive
12 4152.17 28.01 Positive
12 4215.86 27.49 Positive
12 3870.53 32.41 Positive
12 2043.52 36.35 Positive
12 5068.25 25.19 Positive
12 3218 31.61 Positive
12 1152.03 32.64 Positive
12 3687.64 29.77 Positive 12 3955.63 30.17 Positive
12 5469.84 25.48 Positive
12 4896.75 31.41 Positive
24 3386.13 28.93 Positive
24 4359.59 30.68 Positive
24 4561.76 26.86 Positive
24 5393.38 25.78 Positive
24 4074.4 26.36 Positive
24 5100.02 25.65 Positive
24 3279.79 27.87 Positive
24 3909.66 26.05 Positive
24 4640.2 24.72 Positive
24 4026.76 31.43 Positive
24 4491.63 27.01 Positive
24 4446.85 27.16 Positive
24 3212.29 32.23 Positive
24 3943.76 30.58 Positive
24 5016.95 30.6 Positive
24 4219.82 31.99 Positive
24 52.57 A Negative
24 4963.69 29.01 Positive
24 2642.13 33.08 Positive
24 3991.36 28.02 Positive
24 4739.55 26.39 Positive
24 5324.49 26.34 Positive
24 4533.39 33.16 Positive
24 5013.46 28.74 Positive
24 3740.59 26.8 Positive
24 41 14.65 24.72 Positive
24 5059.97 28.01 Positive
24 4404.69 29.08 Positive
24 4268.5 26.44 Positive 24 4405.85 29.75 Positive
24 3104.97 27 Positive
24 3671.19 25.34 Positive
24 3847.73 26.56 Positive
24 4490.29 25.81 Positive
24 5898.68 26.59 Positive
24 5421.98 28.57 Positive
Table 7 (Samples fixed in 10% formalin)
Figure imgf000095_0001
0 13.37 NA Negative
0 22.34 NA Negative
0 5.02 NA Negative
0 22.35 NA Negative
0 5.09 NA Negative
0 6.65 NA Negative
0 4.91 NA Negative
0 14.62 NA Negative
0 7.49 NA Negative
0 4.98 NA Negative
0 7.55 NA Negative
0 9.14 NA Negative
0 3.71 NA Negative
0 95.74 NA Negative
0 5.05 NA Negative
1.5 2593.13 33.67 Positive
1.5 5.04 NA Negative
1.5 40.45 NA Negative
1.5 14.97 NA Negative
1.5 3763.41 29.38 Positive
1.5 9.24 NA Negative
1.5 2788.37 30.25 Positive
1.5 11.37 NA Negative
1.5 4.45 NA Negative
1.5 10.18 NA Negative
1.5 32.84 NA Negative
1.5 4376.92 31.41 Positive
1.5 2837.85 30.61 Positive
1.5 7.01 NA Negative
1.5 28.18 NA Negative
1.5 19.62 NA Negative
1.5 3.05 NA Negative 1.5 4116.38 34.29 Positive
1.5 26.08 NA Negative
1.5 2954.88 31.9 Positive
1.5 14.31 NA Negative
1.5 7.44 NA Negative
1.5 16 NA Negative
1.5 2307.69 32.03 Positive
1.5 16.65 NA Negative
1.5 5.7 NA Negative
1.5 32.94 NA Negative
1.5 26.7 NA Negative
1.5 12.55 NA Negative
1.5 3.14 NA Negative
1.5 55.11 NA Negative
1.5 2985.7 33.85 Positive
1.5 3257.04 35.16 Positive
1.5 4.76 NA Negative
1.5 18.07 NA Negative
1.5 21.08 NA Negative
3 3153.69 32.16 Positive
3 3694.7 33.31 Positive
3 2278.04 34.18 Positive
3 3876.12 29.43 Positive
3 27.92 NA Negative
3 5065.1 29.76 Positive
3 23.23 NA Negative
3 11.5 NA Negative
3 10.1 NA Negative
3 12.91 NA Negative
3 44.64 NA Negative
3 3501.07 33.35 Positive
3 29.05 NA Negative 3 8.96 NA Negative
3 2.24 NA Negative
3 2390.42 32.18 Positive
3 4.19 NA Negative
3 4616.76 31.14 Positive
3 31.35 NA Negative
3 9.45 NA Negative
3 2.9 NA Negative
3 4153.36 32.83 Positive
3 33.42 NA Negative
3 21.34 NA Negative
3 3384.98 32.33 Positive
3 1.43 NA Negative
3 6.79 NA Negative
3 3324.58 32.66 Positive
3 3294.33 31.54 Positive
3 13.62 NA Negative
3 3.85 NA Negative
3 3590.8 33.27 Positive
3 2.18 NA Negative
3 4876.76 30.19 Positive
3 68.35 NA Negative
3 24.04 NA Negative
6 10.62 NA Negative
6 3579.07 34.67 Positive
6 20.58 NA Negative
6 4019.37 28.65 Positive
6 4506.32 27.55 Positive
6 4583.14 30.85 Positive
6 3318.77 31.31 Positive
6 3132.25 28.12 Positive
6 4535.77 26.08 Positive 6 5.31 NA Negative
6 3849.97 30.09 Positive
6 36.99 NA Negative
6 3080.65 31.44 Positive
6 6.35 NA Negative
6 2186.43 30.14 Positive
6 4122.31 29.24 Positive
6 4615.67 28.06 Positive
6 4671.13 32.32 Positive
6 3.47 NA Negative
6 3341.52 29.83 Positive
6 16.04 NA Negative
6 4549.83 32.4 Positive
6 5203.16 28.67 Positive
6 3739.9 30.76 Positive
6 4.01 NA Negative
6 4856.83 33.09 Positive
6 3898.6 31.38 Positive
6 3887.04 29.64 Positive
6 5.94 NA Negative
6 4947.47 27.78 Positive
6 3198.17 30.84 Positive
6 2987.71 31.81 Positive
6 7.67 NA Negative
6 3910.92 33.44 Positive
6 8.25 NA Negative
6 24.5 NA Negative
12 3621.08 29.5 Positive
12 3719.92 27.32 Positive
12 2277.49 32.02 Positive
12 3896.73 29.38 Positive
12 3731.83 30.55 Positive 12 3593.57 32.18 Positive
12 3000.19 31.1 Positive
12 3227.4 30.55 Positive
12 3637.22 32.33 Positive
12 4641.37 29.24 Positive
12 3335.53 32.74 Positive
12 3266.99 31.75 Positive
12 3393.99 29 Positive
12 26.89 NA Negative
12 3387.11 31.86 Positive
12 3685.94 29.31 Positive
12 4538.31 31.05 Positive
12 3873.71 31.47 Positive
12 1216.2 35.98 Positive
12 14.66 NA Negative
12 3660.51 29.7 Positive
12 3925.71 29. NA Positive
12 3891.51 30.67 Positive
12 33.19 NA Negative
12 3308.03 32.32 Positive
12 3753.85 29.96 Positive
12 3798.92 29.85 Positive
12 3863.38 32.1 1 Positive
12 4.91 NA Negative
12 5013.19 27.47 Positive
12 3495.4 27.87 Positive
12 3673.51 29.83 Positive
12 3532.78 31.31 Positive
12 4185.77 28.92 Positive
12 5206.82 28.89 Positive
12 4941.56 29.81 Positive
24 3474.34 27.41 Positive 24 3701.02 28.45 Positive
24 3292.8 27.48 Positive
24 2971.68 32.76 Positive
24 4027.63 27.64 Positive
24 4598.32 26.39 Positive
24 2909.48 30.82 Positive
24 3795.26 27.74 Positive
24 4331.92 27.15 Positive
24 4939.44 26.47 Positive
24 3931.84 30.56 Positive
24 4517.49 29.47 Positive
24 3195.57 27.8 Positive
24 3213.45 31.64 Positive
24 3810.56 30.76 Positive
24 3794.57 28.82 Positive
24 3823.73 27.67 Positive
24 3995.24 29.56 Positive
24 2261.02 33.99 Positive
24 2942.54 29.82 Positive
24 3945.75 26.62 Positive
24 4596.59 28.05 Positive
24 3531.07 31.65 Positive
24 3676.28 30.64 Positive
24 3102.46 30.05 Positive
24 4364.4 27.12 Positive
24 4463.09 29.61 Positive
24 4207.13 28.5 Positive
24 4370.07 28.92 Positive
24 4883.31 26.95 Positive
24 3342.28 28.06 Positive
24 3213.18 28.92 Positive
24 3638.74 29.5 Positive 24 4580.64 28.87 Positive
24 4936.54 30.09 Positive
24 5545.29 28.4 Positive
Example 9: Detection of E. histolytica in mixed infections
[0095] E. histolytica detection was validated in samples comprising mixtures of two or more organisms, which can simulate multiple infection specimens. A low level of one target (Low Target) was spiked into unpreserved stool with high levels (High Level) of other organisms.
[0096] As shown in Table 8, the BD MAX™ assay detected E. histolytica near the LoD in simulated multiple infection specimens containing high titer Cryptosporidium parvum, Giardia lamblia, and Entamoeba dispar. E. dispar was included in High Level mixes to confirm that presence of E. dispar does not block amplification of E. histolytica.
Table 8
Figure imgf000102_0001
Figure imgf000103_0001
Giardia (14.8 Crypto, E.hist 8090 17.11 Positive
Figure imgf000104_0001
E.dispar
Figure imgf000105_0001
E.dispar
[0097] As shown in Table 8, the BD MAX™ assay detected the presence of E. histolytica at low levels, and at high levels. Moreover, the presence of high levels of E. dispar did not interfere with detection of E. histolytica. Accordingly, it is contemplated that methods of detecting E. histolytica in accordance with some embodiments herein are sensitive to very low levels of E. histolytica, and are not compromised by the presence of high levels of E. dispar.
Example 10: Validation of BD MAX™ results by sequencing
[0098] The BD MAX™ assay was compared to a validated alternate PCR and bi-directional sequencing approach. A clinical simulation study was performed using retrospective archived stool specimens representing both unpreserved and 10% formalin fixed stool types. The BD MAX™ assay was performed on the specimens. A validated alternate PCR and bi-directional sequencing assay was also performed on the specimens. Specimens were considered positive for the alternate PCR and bi-directional sequencing assay if their top BLAST hit was E. histolytica. Only specimens for which the alternate PCR/bi-directional sequencing results agreed with the original site reference method were included in performance calculations. The results are summarized in Tables 9.1, 9.2 and
9.3.
Table 9.1
Figure imgf000106_0001
Table 9.2
Figure imgf000106_0002
Table 9.3
Figure imgf000106_0003
[0099] Both unpreserved specimens and 10% formalin-fixed specimens exhibited 100% concordance between the BDMAX™ assay and the alternate PCR and sequencing method. Furthermore, a number of specimens were found to contain nonpathogenic Entamoeba species which the BD MAX™ E. histolytica assay correctly called as "negative". Accordingly, it is contemplated that methods of detecting E. histolytica nucleic acids in accordance with some embodiments herein provide highly accurate results, characterized by minimizing cross -reactivity with other organisms, and minimizing both false negatives and false positives.
[0100] The raw data for Tables 9.1-9.3 are shown in Tables 10.1-10.2.
Table 10.1
Figure imgf000107_0001
E20 Fixed 46 3.51 Negative Negative NA NA
12S0000765 Unpres 46 4.47 Negative NA NA NA erved
Leiden 12 Unpres 46 2.57 Crypto NA NA Positive erved
12S0000693 Unpres 46 60.46 Negative Negative NA NA erved
Leiden 78 Unpres 46 1.02 Giardia Negative Positive NA erved
EH17 Unpres 34.89 2249.05 Entamoeba NA NA NA erved Histolytica
CCF06 Fixed 46 1.82 Giardia NA NA Negativ e
EH19 Unpres 33.95 2976.38 Entamoeba NA NA NA erved Histolytica
EH20 Unpres 35.48 2753.42 Entamoeba NA NA NA erved Histolytica
1438 Fixed 46 8.86 Crypto NA NA NA
13S0000005 Unpres 46 2.81 Negative NA NA Negativ erved e
13S0000159 Fixed 46 9.3 Negative NA NA NA
12S0000722 Unpres 46 7.03 Negative NA NA NA erved
DLS13- Fixed 46 0.68 Giardia Negative Positive NA 05812-01-01
12S0000706 Unpres 46 7.28 Negative NA NA NA erved
Leiden 64 Unpres 46 30.24 Giardia NA Positive NA erved
13S0000559 Fixed 46 15.83 Negative NA NA NA
12S0000673 Unpres 46 21 Negative NA NA NA erved
DLS13- Fixed 46 5.1 Giardia NA NA NA 05780-01-01
12S0000788 Unpres 46 8.01 Negative Negative NA NA erved
13S0000059 Unpres 46 7.52 Negative NA NA NA erved
DLS13- Unpres 46 6.56 Crypto Negative NA Positive 05960 erved
13S0000139 Fixed 46 11.4 Negative Negative NA Negativ e
13S0000037 Unpres 46 0.36 Negative NA NA NA erved
CIN01005 Fixed 46 1.94 Giardia NA NA NA
13S0000185 Fixed 46 15.96 Negative NA NA NA
12S0000699 Unpres 46 20.76 Negative Negative NA NA erved
55 Fixed 46 6.23 Giardia Negative Positive NA
CCF08 Fixed 46 38.38 Giardia NA NA NA
13S0000564 Fixed 46 4.82 Negative NA NA NA 13S0000131 Fixed 46 21.12 Negative Negative NA NA
12S0000561 Fixed 46 1.43 Negative NA NA NA
EH23 Unpres 28.12 4057.88 Entamoeba Positive NA NA erved Histolytica
564 Fixed 46 4.66 Crypto NA NA Positive
Leiden 26 Unpres 46 1.44 Crypto NA NA Positive erved
12S0000771 Unpres 46 4.71 Negative NA NA NA erved
12S0000729 Unpres 46 1.32 Negative Negative NA NA erved
Leiden 57 Unpres 46 2.32 Giardia NA Positive NA erved
12997 Fixed 46 4.22 Crypto NA NA Positive
13S0000070 Unpres 46 14.93 Negative NA NA NA erved
13S0000171 Fixed 46 22.03 Negative NA NA NA
DLS13- Unpres 46 0.94 Negative Negative NA NA 06001 erved
Leiden 39 Unpres 46 7.58 Crypto Negative NA Positive erved
DLS13- Unpres 46 10.82 Crypto Negative NA Positive 05967 erved
EH18 Unpres 32.94 3130.69 Entamoeba NA NA NA erved Histolytica
13S0000122 Fixed 46 0.8 Negative NA NA NA
13S0000057 Unpres 46 24.3 Negative NA NA NA erved
12S0000575 Fixed 46 16.63 Negative NA NA NA
12S0000737 Unpres 46 20.8 Negative NA NA NA erved
13S0000188 Fixed 46 3.77 Negative NA NA NA
12S0000569 Fixed 46 21.08 Negative NA NA NA
12S0000692 Unpres 46 49.89 Negative NA NA NA erved
13S0000036 Unpres 46 32.29 Negative Negative NA NA erved
13S0000566 Fixed 46 17.56 Negative NA NA NA
13S0000109 Unpres 46 23.01 Negative NA NA NA erved
13S0000090 Unpres 46 7.58 Negative NA NA NA erved
36 Fixed 46 4.42 Giardia NA Positive NA
12S0000697 Unpres 46 34.47 Negative NA NA NA erved
12S0000572 Fixed 46 15.66 Negative NA NA NA
13S0000106 Unpres 46 2.5 Negative NA NA NA erved
F35 Fixed 46 2.28 Giardia NA Positive NA
12S0000735 Unpres 46 2.02 Negative NA NA NA erved 13S0000177 Fixed 46 2.16 Negative NA NA NA
12S0000707 Unpres 46 2.37 Negative NA NA NA erved
12S0000708 Unpres 46 13.91 Negative Negative NA NA erved
DLS13- Unpres 46 2.83 Negative NA NA NA 05974 erved
13S0000008 Unpres 46 5.71 Negative NA NA NA erved
13S0000011 Unpres 46 3.13 Negative NA NA NA erved
DLS13- Fixed 46 1.36 Giardia, NA NA Positive 05782-01-01 Crypto
12S0000670 Unpres 46 2.58 Negative NA NA NA erved
CIN01020 Fixed 46 2.86 Giardia NA NA NA
13S0000156 Fixed 46 3.5 Negative NA NA NA
13S0000039 Unpres 46 11.25 Negative NA NA NA erved
13S0000035 Unpres 46 25.79 Negative Negative NA NA erved
12S0000570 Fixed 46 18.56 Negative NA NA NA
11334 Fixed 46 23.01 Crypto NA NA Positive
13S0000569 Fixed 46 4.48 Negative NA NA NA
13S0000065 Unpres 46 2.09 Negative NA NA NA erved
EH12 Fixed 30.93 3244.67 Entamoeba NA NA NA
Histolytica
12S0000777 Unpres 46 4.09 Negative NA NA NA erved
13S0000116 Unpres 46 4.06 Negative NA NA NA erved
DLS13- Unpres 46 1.53 Crypto NA NA Positive 05994 erved
13S0000138 Fixed 46 1.32 Negative NA NA NA
Leiden 68 Unpres 46 1.09 Giardia NA Positive NA erved
Leiden 63 Unpres 46 1.64 Giardia NA Positive NA erved
13S0000149 Fixed 46 9.42 Negative NA NA NA
12S0000732 Unpres 46 8.91 Negative NA NA NA erved
DLS13- Unpres 46 32.29 Giardia, Negative Positive Positive 05976 erved Crypto
12S0000688 Unpres 46 20.19 Negative NA NA NA erved
EH08 Fixed 25.49 4426.18 Entamoeba Positive NA NA
Histolytica
CIN01004 Fixed 46 1.77 Giardia NA NA NA
DLS13- Unpres 46 18.49 Crypto NA NA Positive 05956 erved Leiden 10 Unpres 46 22.73 Crypto NA NA Positive erved
Leiden 37 Unpres 46 3.65 Crypto Negative NA Positive erved
DLS13- Fixed 46 5.62 Crypto NA NA Positive 05790-01-01
DLS13- Unpres 46 1.52 Negative NA NA NA 05996 erved
F33 Fixed 46 3.66 Giardia NA Positive NA
Leiden 96 Unpres 46 6.72 Giardia NA Positive NA erved
Leiden 99 Unpres 46 2.14 Giardia NA Positive NA erved
13S0000183 Fixed 46 3.56 Negative NA NA NA
12S0000503 Fixed 46 25.82 Negative NA NA NA
13S0000092 Unpres 46 27.75 Negative NA NA NA erved
Leiden 84 Unpres 46 2.92 Giardia Negative Positive NA erved
12S0000786 Unpres 46 1.75 Negative NA NA NA erved
DLS13- Fixed 46 4.16 Giardia, Negative Positive Positive 05817-01-01 Crypto
Leiden 79 Unpres 46 10.49 Giardia NA NA NA erved
13S0000111 Unpres 46 5.26 Negative NA NA NA erved
CCF11 Fixed 46 3.61 Giardia NA NA NA
124 Fixed 46 3.64 Giardia NA Positive NA
13S0000563 Fixed 46 2.05 Negative NA NA NA
12S0000678 Unpres 46 8.8 Negative NA NA NA erved
12S0000698 Unpres 46 2.76 Negative NA NA NA erved
77 Fixed 46 2.32 Giardia Negative Positive NA
13S0000084 Unpres 46 0.51 Negative NA NA NA erved
12S0000746 Unpres 46 14.43 Negative NA NA NA erved
CIN01040 Fixed 46 2.92 Giardia NA Positive NA
13S0000117 Unpres 46 1.03 Negative NA NA NA erved
Leiden 32 Unpres 46 6.42 Crypto NA NA Positive erved
13S0000112 Unpres 46 2.19 Negative NA NA NA erved
12S0000711 Unpres 46 2.05 Negative NA NA NA erved
DLS13- Unpres 46 6.28 Negative Negative NA NA 05982 erved
13S0000032 Unpres 46 5.8 Negative NA NA NA erved 13S0000565 Fixed 46 0.54 Negative NA NA NA
DLS13- Fixed 46 13.03 Crypto NA NA Positive 05787-01-01
12S0000760 Unpres 46 1.75 Negative NA NA NA erved
Leiden 98 Unpres 46 0.99 Giardia NA Positive NA erved
13S0000087 Unpres 46 0.98 Negative NA NA NA erved
F41 Fixed 46 2.97 Giardia NA Positive NA
6289 Fixed 46 3.55 Crypto NA NA Positive
DLS13- Unpres 46 15.37 Crypto Negative NA Positive 05947 erved
E18 Fixed 46 2.05 Negative Negative NA NA
13S0000568 Fixed 46 2.96 Negative NA NA NA
F37 Fixed 46 1.77 Giardia NA Positive NA
Leiden 36 Unpres 46 7.32 Crypto Negative NA Positive erved
13S0000158 Fixed 46 1.21 Negative NA NA NA
DLS13- Unpres 46 23.69 Negative NA NA NA 05973 erved
13S0000053 Unpres 46 1.93 Negative NA NA NA erved
Leiden 91 Unpres 46 0.13 Giardia NA Positive NA erved
DLS13- Unpres 46 13.22 Crypto Negative NA Positive 05966 erved
12S0000778 Unpres 46 10.25 Negative NA NA NA erved
13S0000151 Fixed 46 2.11 Negative NA NA NA
13S0000147 Fixed 46 6.24 Negative NA NA NA
13S0000179 Fixed 46 1.09 Negative NA NA NA
13S0000006 Unpres 46 11.64 Negative NA NA NA erved
13S0000189 Fixed 46 8.69 Negative NA NA NA
12S0000573 Fixed 46 11.49 Negative NA NA NA
DLS13- Unpres 46 16.94 Crypto Negative NA Positive 05980 erved
12S0000791 Unpres 46 20.37 Negative NA NA NA erved
Leiden 25 Unpres 46 16.23 Crypto Negative NA Positive erved
DLS13- Unpres 46 17.92 Crypto Negative NA Positive 05952 erved
13S0000019 Unpres 46 10.66 Negative NA NA NA erved
91 Fixed 46 2.56 Giardia NA Positive NA
DLS13- Unpres 46 6.19 Negative Negative NA NA 05972 erved
13S0000043 Unpres 46 7 Negative NA NA NA erved 12S0000714 Unpres 46 1.87 Negative NA NA NA erved
12S0000675 Unpres 46 1.75 Negative NA NA NA erved
DLS13- Fixed 46 1.94 Crypto NA NA Positive 05801-01-01
12S0000682 Unpres 46 23.33 Negative NA NA NA erved
DLS13- Fixed 46 5.22 Crypto NA NA Positive 05802-01-01
113 Fixed 46 21.51 Giardia Negative Positive NA
Leiden 85 Unpres 46 63.6 Giardia NA Positive NA erved
EH14 Fixed 26.09 3348.86 Entamoeba Positive NA NA
Histolytica
13S0000096 Unpres 46 48.77 Negative NA NA NA erved
12S0000733 Unpres 46 37 Negative Negative NA NA erved
12S0000689 Unpres 46 0.68 Negative NA NA NA erved
CCF03 Fixed 46 5.54 Giardia NA NA NA
13S0000049 Unpres 46 5.73 Negative NA NA NA erved
Leiden 8 Unpres 46 53.94 Crypto NA NA Positive erved
Leiden 29 Unpres 46 8.26 Crypto Negative NA Positive erved
12S0000576 Fixed 46 35.02 Negative NA NA NA
Leiden 6 Unpres 46 17.46 Crypto Negative NA Positive erved
13S0000007 Unpres 46 26.52 Negative NA NA NA erved
12S0000717 Unpres 46 9.55 Negative NA NA NA erved
13S0000001 Unpres 46 12.88 Negative NA NA NA erved
13S0000134 Fixed 46 36.35 Negative NA Negative NA
13S0000104 Unpres 46 3.96 Negative NA NA NA erved
13S0000089 Unpres 46 12.21 Negative NA NA NA erved
12S0000762 Unpres 46 7.7 Negative NA NA NA erved
13S0000562 Fixed 46 4.05 Negative NA NA NA
13S0000010 Unpres 46 17.88 Negative NA NA NA erved
125 Fixed 46 6.4 Giardia NA Positive NA
13S0000135 Fixed 46 5.82 Negative NA NA NA
13S0000118 Unpres 46 9.18 Negative NA NA NA erved
12S0000713 Unpres 46 38.22 Negative NA NA NA erved
13S0000162 Fixed 46 26.34 Negative NA NA NA
DLS13- Unpres 46 14.32 Crypto Negative NA Positive 05970 erved
12S0000727 Unpres 46 3.79 Negative Negative NA NA erved
39546 Fixed 46 5.5 Giardia Negative Positive NA
13S0000083 Unpres 46 2.22 Negative NA NA NA erved
Leiden 49 Unpres 46 11.51 Crypto Negative NA Positive erved
EH09 Fixed 31.04 4213.82 Entamoeba NA NA NA
Histolytica
F31 Fixed 46 7.62 Giardia Negative Positive NA
12S0000751 Unpres 46 7.09 Negative Negative NA NA erved
13S0000153 Fixed 46 9.34 Negative Negative NA NA
13S0000166 Fixed 46 0.22 Negative Negative NA NA
12S0000685 Unpres 46 17.56 Negative NA NA NA erved
CCF10 Fixed 46 4.23 Giardia NA NA NA
EH10 Fixed 32.34 2999.22 Entamoeba NA NA NA
Histolytica
13S0000098 Unpres 46 15.43 Negative NA NA NA erved
81 Fixed 46 7.28 Giardia Negative Positive NA
13S0000095 Unpres 46 22.84 Negative NA NA NA erved
Leiden 1 Unpres 46 4.94 Crypto Negative NA Positive erved
13S0000173 Fixed 46 18.66 Negative Negative NA NA
DLS13- Fixed 46 19.18 Giardia NA Positive NA 05784-01-01
Leiden 51 Unpres 46 32.95 Giardia Negative Positive NA erved
12S0000725 Unpres 46 52.6 Negative NA NA NA erved
162 Fixed 46 38.96 Giardia Negative Positive NA
12S0000781 Unpres 46 22.39 Negative NA NA NA erved
13S0000143 Fixed 46 45.21 Negative NA NA NA
13S0000003 Unpres 46 18.9 Negative NA NA NA erved
12S0000710 Unpres 46 88.74 Crypto NA NA NA erved
Leiden 86 Unpres 46 6.46 Giardia Negative Positive NA erved
13S0000027 Unpres 46 47.45 Negative NA NA NA erved
DLS13- Unpres 46 5.75 Negative NA NA NA 05949 erved 12S0000702 Unpres 46 7.58 Negative Negative NA NA erved
Leiden 82 Unpres 46 4.01 Giardia Negative Positive NA erved
1247 Fixed 46 6.93 Negative NA NA Positive
13S0000140 Fixed 46 9.5 Negative Negative NA NA
Leiden 59 Unpres 46 10.33 Giardia Negative Positive NA erved
13S0000558 Fixed 46 2.85 Negative Negative NA NA
13S0000157 Fixed 46 12.44 Negative Negative NA NA
8174 Fixed 46 15.07 Crypto NA NA NA
F29 Fixed 46 30.36 Giardia Negative Negative NA
CIN01026 Fixed 46 3.99 Giardia Negative Positive NA
Leiden 58 Unpres 46 12.14 Giardia NA Positive NA erved
13S0000165 Fixed 46 10.87 Negative Negative NA NA
13S0000164 Fixed 46 3.14 Negative Negative NA NA
DLS13- Fixed 46 8.14 Crypto Negative NA Positive 05808-01-01
12S0000716 Unpres 46 10.74 Negative Negative NA NA erved
12S0000784 Unpres 46 3.05 Negative Negative NA NA erved
EH24 Unpres 31.84 2717.46 Entamoeba Negative NA NA erved Histolytica
12S0000681 Unpres 46 13.88 Negative Negative NA NA erved
DLS13- Unpres 46 24.35 Crypto Negative NA Positive 05969 erved
Leiden 62 Unpres 46 9.24 Giardia Negative Positive NA erved
13S0000002 Unpres 46 1.57 Negative Negative NA NA erved
12S0000770 Unpres 46 6.25 Negative Negative NA NA erved
DLS13- Fixed 46 1.48 Giardia NA NA Positive 05789-01-01
13S0000560 Fixed 46 3.8 Negative Negative NA NA
13S0000145 Fixed 46 5.11 Negative Negative NA NA
DLS13- Unpres 46 1.49 Crypto Negative NA Positive 05958 erved
12S0000563 Fixed 46 2.25 Negative Negative NA NA
12S0000683 Unpres 46 2.48 Negative Negative NA NA erved
13S0000168 Fixed 46 1.16 Negative NA NA NA
Leiden 46 Unpres 46 2.6 Negative NA NA Positive erved
E24 Fixed 46 2.28 Negative Negative NA NA
13S0000101 Unpres 46 2.28 Negative NA NA NA erved 13S0000155 Fixed 46 6.29 Negative NA NA NA
Leiden 71 Unpres 46 1.65 Giardia NA Positive NA erved
EH06 Fixed 27.49 3596.16 Entamoeba NA NA NA
Histolytica
DLS13- Fixed 46 2.68 Giardia, NA NA Positive 05816-01-01 Crypto
F27 Fixed 46 1.22 Giardia NA Positive NA
DLS13- Unpres 46 2.71 Negative Negative NA NA 05997 erved
EH04 Fixed 30.16 3073.89 Entamoeba NA NA NA
Histolytica
12S0000726 Unpres 46 2.46 Negative NA NA NA erved
DLS13- Unpres 46 2.55 Negative Negative NA NA 05963 erved
DLS13- Fixed 46 0.47 Crypto NA NA Positive 05793-01-01
13S0000126 Fixed 46 2.6 Negative NA NA NA
EH25 Unpres 33.04 2731.07 Entamoeba NA NA NA erved Histolytica
40015 Fixed 46 0.24 Negative NA NA Positive
13S0000181 Fixed 46 4.58 Negative Negative NA NA
58 Fixed 46 6.16 Giardia Negative Positive NA
CCF02 Fixed 46 3.45 Giardia NA Positive NA
12S0000568 Fixed 46 5.8 Negative NA NA NA
96 Fixed 46 2.94 Giardia NA Positive NA
Leiden 43 Unpres 46 11.23 Crypto NA NA Positive erved
13S0000123 Fixed 46 18.72 Negative NA NA NA
12S0000705 Unpres 46 6.71 Negative NA NA NA erved
DLS13- Fixed 46 1.34 Crypto NA NA Positive 05794-01-01
13S0000136 Fixed 46 7.28 Negative NA NA NA
Leiden 97 Unpres 46 1.83 Giardia Negative Positive NA erved
DLS13- Fixed 46 20.49 Crypto NA NA Positive 05798-01-01
12S0000719 Unpres 46 0.23 Negative Negative NA NA erved
12S0000668 Unpres 46 1.12 Negative NA NA NA erved
Leiden 80 Unpres 46 1.93 Giardia NA NA NA erved
12S0000756 Unpres 46 0.55 Negative NA NA NA erved
13S0000107 Unpres 46 0.63 Negative NA NA NA erved
13S0000068 Unpres 46 1.3 Negative Negative NA NA erved 12S0000749 Unpres 46 1.61 Negative NA NA NA erved
Leiden 16 Unpres 46 16.82 Crypto Negative NA Positive erved
Leiden 72 Unpres 46 1.56 Giardia NA Positive NA erved
12S0000763 Unpres 46 1.78 Negative Negative NA NA erved
12S0000709 Unpres 46 0.52 Negative Negative NA NA erved
13S0000072 Unpres 46 0.92 Negative NA NA NA erved
13S0000013 Unpres 46 4.63 Negative NA NA NA erved
CCF01 Fixed 46 4.11 Giardia NA Positive NA
DLS13- Unpres 46 12.38 Crypto Negative NA Positive 05953 erved
12S0000761 Unpres 46 0.27 Negative NA NA NA erved
Leiden 9 Unpres 46 22.34 Negative NA NA Positive erved
13S0000054 Unpres 46 1.61 Negative NA NA NA erved
EH16 Unpres 30.2 3188.71 Entamoeba NA NA NA erved Histolytica
13S0000081 Unpres 46 24.01 Negative NA NA NA erved
DLS13- Unpres 46 11.67 Crypto Negative NA Positive 05951 erved
DLS13- Fixed 46 9.97 Giardia Negative Positive NA 05820-01-01
Leiden 5 Unpres 46 9.52 Crypto Negative NA Positive erved
EH07 Fixed 30.45 2634.49 Entamoeba NA NA NA
Histolytica
13S0000146 Fixed 46 0.59 Negative NA NA NA
CIN01012 Fixed 46 2.03 Giardia NA Positive NA
DLS13- Fixed 46 0.67 Giardia Negative Positive NA 05818-01-01
13S0000169 Fixed 46 0.73 Negative Negative NA NA
13S0000186 Fixed 46 1.8 Negative NA NA NA
13S0000152 Fixed 46 5.8 Negative Negative NA NA
12S0000669 Unpres 46 2.63 Negative NA NA NA erved
12S0000566 Fixed 46 1.25 Negative NA NA NA
14790 Fixed 46 20.38 Crypto NA NA Positive
Leiden 18 Unpres 46 28.25 Crypto Negative NA Positive erved
13S0000094 Unpres 46 26.47 Negative Negative NA NA erved
13S0000154 Fixed 46 1.94 Negative NA NA NA 12S0000724 Unpres 46 4.83 Negative NA NA NA erved
13S0000161 Fixed 46 1.42 Negative Negative NA NA
12S0000562 Fixed 46 0.23 Negative Negative NA NA
13S0000040 Unpres 46 0.86 Negative NA NA NA erved
13S0000024 Unpres 46 2.28 Negative NA NA NA erved
13S0000148 Fixed 46 14.85 Negative NA NA NA
DLS13- Unpres 46 9.67 Crypto Negative NA Positive 05983 erved
159 Fixed 46 51.22 Giardia Negative Positive NA
Leiden 74 Unpres 46 1.51 Giardia NA Positive NA erved
13S0000031 Unpres 46 18.76 Negative NA NA NA erved
EH05 Fixed 24.52 4316.84 Entamoeba Positive NA NA
Histolytica
3645 Fixed 46 1.56 Crypto NA NA Positive
13S0000119 Unpres 46 0.63 Negative NA NA NA erved
13S0000021 Unpres 46 3.81 Negative Negative NA NA erved
13S0000132 Fixed 46 3.39 Negative NA NA NA
12S0000764 Unpres 46 2.81 Negative Negative NA NA erved
13S0000064 Unpres 46 14.37 Negative NA NA NA erved
183 Fixed 46 35.62 Giardia NA Positive NA
DLS13- Unpres 46 28.06 Crypto Negative NA Positive 05950 erved
13S0000557 Fixed 46 24.46 Negative NA NA NA
CIN01038 Fixed 46 0.33 Giardia NA Positive NA
13S0000120 Unpres 46 20.14 Negative NA NA NA erved
EH01 Fixed 27.55 3452.35 Entamoeba Positive NA NA
Histolytica
12S0000679 Unpres 46 11.58 Negative NA NA NA erved
Leiden 73 Unpres 46 26.9 Giardia NA Positive NA erved
DLS13- Fixed 46 1.5 Giardia, NA Positive NA 05781-01-01 Crypto
13S0000150 Fixed 46 0.65 Negative NA NA NA
13S0000115 Unpres 46 6.8 Negative NA NA NA erved
13S0000142 Fixed 46 30.68 Negative NA NA NA
Leiden 70 Unpres 46 9.98 Giardia NA Positive NA erved
Leiden 69 Unpres 46 24.98 Giardia NA Positive NA erved 12S0000704 Unpres 46 43.32 Negative NA NA NA erved
129 Fixed 46 35.34 Giardia NA Positive NA
Leiden 61 Unpres 46 30.72 Giardia Negative Positive NA erved
EH03 Fixed 27.63 4138.66 Entamoeba NA NA NA
Histolytica
13S0000121 Fixed 46 8.94 Negative Negative NA NA
7458 Fixed 46 21.52 Negative NA NA NA
13S0000129 Fixed 46 13.08 Negative NA NA NA
12S0000703 Unpres 46 15.65 Negative NA NA NA erved
13S0000056 Unpres 46 9.93 Negative Negative NA NA erved
12S0000766 Unpres 46 4.49 Negative NA NA NA erved
F23 Fixed 46 3.28 Giardia NA Positive NA
13S0000103 Unpres 46 2.52 Negative NA NA NA erved
12S0000723 Unpres 46 38.51 Negative NA NA NA erved
130 Fixed 46 14.19 Giardia NA Positive NA
Leiden 52 Unpres 46 18.59 Giardia NA Positive NA erved
Leiden 60 Unpres 46 12.77 Giardia NA Positive NA erved
13S0000038 Unpres 46 29.99 Negative NA NA NA erved
13S0000100 Unpres 46 11.06 Negative NA NA NA erved
13S0000102 Unpres 46 7.13 Negative NA NA NA erved
13S0000576 Fixed 46 14.16 Negative NA NA NA
12S0000567 Fixed 46 11.31 Negative NA NA NA
12S0000672 Unpres 46 9.87 Negative NA NA NA erved
12S0000757 Unpres 46 1.05 Negative NA NA NA erved
12S0000742 Unpres 46 1.37 Negative NA NA NA erved
Leiden 17 Unpres 46 2.26 Crypto NA NA Positive erved
Leiden 15 Unpres 46 73.62 Crypto NA NA Positive erved
13S0000009 Unpres 46 0.24 Negative NA NA NA erved
CIN01028 Fixed 46 3.36 Giardia NA Positive NA
13S0000127 Fixed 46 0.85 Negative Negative NA NA
KH12-5156 Unpres 46 4.73 Giardia Negative Positive NA erved
12S0000674 Unpres 46 1.41 Negative NA NA NA erved
13S0000571 Fixed 46 2.72 Negative NA NA NA
13S0000128 Fixed 46 1.01 Negative NA NA NA
140 Fixed 46 1.12 Giardia Negative Positive NA
12S0000738 Unpres 46 2.45 Negative NA NA NA erved
CIN01039 Fixed 46 1.62 Giardia NA Positive NA
KH 12-4357 Unpres 46 2.14 Giardia NA Positive NA erved
Leiden 30 Unpres 46 1.76 Crypto Negative NA Positive erved
13S0000114 Unpres 46 0.3 Negative NA NA NA erved
Leiden 55 Unpres 46 4.4 Giardia Negative Positive NA erved
12S0000680 Unpres 46 2.94 Negative NA NA NA erved
E22 Fixed 46 3.31 Negative Negative NA NA
39543 Fixed 46 0.36 Giardia NA Positive NA
12S0000776 Unpres 46 12.99 Negative NA NA NA erved
KH 12-6359 Unpres 46 0.2 Giardia NA Positive NA erved
13S0000160 Fixed 46 56.76 Negative NA NA NA
DLS13- Unpres 46 6.49 Giardia, NA Positive Positive 05945 erved Crypto
11796 Fixed 46 11.69 Crypto NA NA Positive
DLS13- Unpres 46 23.82 Giardia NA Positive NA 05978 erved
13S0000187 Fixed 46 28.62 Negative NA NA NA
13S0000063 Unpres 46 1.54 Negative Negative NA NA erved
12S0000779 Unpres 46 6.07 Negative NA NA NA erved
DLS13- Fixed 46 24.79 Crypto Negative NA Positive 05800-01-01
13S0000110 Unpres 46 4.48 Negative NA NA NA erved
13S0000113 Unpres 46 7.12 Negative NA NA NA erved
CIN01027 Fixed 46 5.72 Giardia NA NA NA
13S0000180 Fixed 46 4.41 Negative NA NA NA
12S0000564 Fixed 46 2.75 Negative NA NA NA
CCF05 Fixed 46 4.06 Giardia NA NA NA
DLS13- Unpres 46 3.96 Negative Negative NA NA 05991 erved
12S0000667 Unpres 46 39.87 Negative NA NA NA erved
13S0000182 Fixed 46 48.02 Negative NA NA NA
12S0000790 Unpres 46 3.34 Negative NA NA NA erved
CIN01003 Fixed 46 12.21 Giardia NA NA NA
Leiden 19 Unpres 46 20.7 Crypto Negative NA Positive erved
DLS13- Fixed 46 4.47 Crypto NA NA Positive 05792-01-01
13S0000099 Unpres 46 1.5 Negative NA NA NA erved
CIN01024 Fixed 46 3.55 Giardia NA NA NA
13S0000062 Unpres 46 1.41 Negative Negative NA NA erved
EH13 Fixed 32.23 2934.41 Entamoeba NA NA NA
Histolytica
12S0000715 Unpres 46 8.69 Negative NA NA NA erved
DLS13- Unpres 46 6.92 Giardia, Negative Positive Positive 05961 erved Crypto
DLS13- Unpres 46 24.25 Giardia, Negative Positive Positive 05981 erved Crypto
CCF04 Fixed 46 2.56 Giardia NA NA NA
Leiden 22 Unpres 46 22.76 Crypto NA NA Positive erved
EH02 Fixed 24.05 4776.64 Entamoeba Positive NA NA
Histolytica
13S0000184 Fixed 46 5.29 Negative NA NA NA
Leiden 35 Unpres 46 5.77 Negative Negative NA Positive erved
DLS13- Fixed 46 2.82 Giardia NA NA NA 05807-01-01
13S0000175 Fixed 46 6.57 Negative NA NA NA
13S0000091 Unpres 46 4.62 Negative NA NA NA erved
13S0000075 Unpres 46 12.08 Negative NA NA NA erved
DLS13- Fixed 46 8.41 Crypto NA NA Positive 05795-01-01
12998 Fixed 46 15.49 Crypto Negative NA Positive
13S0000174 Fixed 46 7.46 Negative NA NA NA
13S0000055 Unpres 46 21.59 Negative Negative NA NA erved
12S0000782 Unpres 46 8.4 Negative NA NA NA erved
13S0000163 Fixed 46 12.96 Negative NA NA NA
13S0000574 Fixed 46 15.3 Negative NA NA NA
13S0000071 Unpres 46 7.56 Negative NA NA NA erved
Leiden 2 Unpres 46 19.52 Crypto Negative Positive Positive erved
13S0000167 Fixed 46 16.57 Negative NA NA NA
13S0000133 Fixed 46 3.83 Negative NA NA NA
13S0000046 Unpres 46 29.37 Negative NA NA NA erved
12S0000759 Unpres 46 4.19 Negative NA NA NA erved
Leiden 7 Unpres 46 17.57 Crypto Negative NA Positive erved
13S0000041 Unpres 46 8.81 Negative NA NA NA erved
12S0000671 Unpres 46 9.1 Negative Negative NA NA erved
13S0000093 Unpres 46 2.36 Negative Negative NA NA erved
CIN01006 Fixed 46 1.33 Giardia NA NA NA
Leiden 33 Unpres 46 26.84 Crypto NA NA Positive erved
DLS13- Unpres 46 17.62 Negative Negative NA NA 05882 erved
13S0000144 Fixed 46 6.03 Negative Negative NA NA
DLS13- Unpres 46 0.96 Negative Negative NA NA 05893 erved
13S0000088 Unpres 46 7.76 Negative Negative NA NA erved
DLS13- Unpres 46 9.72 Crypto Negative NA Positive 05954 erved
20 Fixed 46 0.47 Giardia Negative Positive NA
13S0000060 Unpres 46 2.91 Negative Negative NA NA erved
64 Fixed 46 2.57 Giardia NA Positive NA
Leiden 67 Unpres 46 29.44 Giardia NA Positive NA erved
13S0000066 Unpres 46 13.65 Negative NA NA NA erved
13S0000178 Fixed 46 11.61 Negative Negative NA NA
13S0000570 Fixed 46 23.22 Negative NA NA NA
12S0000712 Unpres 46 26 Negative Negative NA NA erved
DLS13- Unpres 46 8.49 Crypto NA NA Positive 05962 erved
73 Fixed 46 10.18 Giardia NA Positive NA
EH15 Fixed 32.49 3811.37 Entamoeba NA NA NA
Histolytica
Leiden 81 Unpres 46 0.37 Giardia NA Positive NA erved
Leiden 13 Unpres 46 4.25 Crypto NA NA Positive erved
12S0000731 Unpres 46 7.83 Negative Negative NA NA erved
12S0000686 Unpres 46 12.69 Negative NA NA NA erved
12S0000676 Unpres 46 1.85 Negative NA NA NA erved
13S0000067 Unpres 46 6.33 Negative NA NA NA erved CINOlOl l Fixed 46 1.44 Giardia NA Positive NA
Leiden 20 Unpres 46 12.19 Negative Negative NA NA erved
EH11 Fixed 24.65 4407.66 Entamoeba Positive NA NA
Histolytica
Leiden 23 Unpres 46 1.37 Crypto NA NA Positive erved
161 Fixed 46 0.43 Giardia NA NA NA
13S0000058 Unpres 46 1.45 Negative NA NA NA erved
13S0000077 Unpres 46 12.28 Negative NA NA NA erved
DLS13- Fixed 46 3.4 Crypto NA NA Positive 05809-01-01
DLS13- Fixed 46 32.89 Crypto Negative NA Positive 05788-01-01
DLS13- Unpres 46 36.67 Crypto Negative NA Positive 05987 erved
Leiden 95 Unpres 46 20.81 Giardia Negative Positive NA erved
68 Fixed 46 68.16 Giardia NA Positive NA
Leiden 53 Unpres 46 17.42 Negative NA Positive NA erved
12S0000677 Unpres 46 37.95 Crypto NA NA NA erved
12S0000721 Unpres 46 12.76 Negative NA NA NA erved
DLS13- Fixed 46 26.94 Giardia NA Positive NA 05814-01-01
Leiden 11 Unpres 46 20.87 Crypto NA NA Positive erved
Leiden 3 Unpres 46 14.15 Crypto NA NA Positive erved
12S0000577 Fixed 46 0.9 Negative NA NA NA
12S0000701 Unpres 46 9.16 Negative NA NA NA erved
37 Fixed 46 0.75 Giardia NA Positive NA
13S0000076 Unpres 46 7.89 Negative NA NA NA erved
DLS13- Unpres 46 8.78 Crypto NA NA Positive 05968 erved
DLS13- Fixed 46 9.6 Crypto NA NA Positive 05805-01-01
13S0000078 Unpres 46 6.16 Negative NA NA NA erved
Leiden 65 Unpres 46 4.28 Giardia Negative Positive NA erved
13S0000573 Fixed 46 7.2 Negative NA NA NA
13S0000097 Unpres 46 4.94 Negative NA NA NA erved
DLS13- Fixed 46 10.14 Crypto NA NA Positive 05791-01-01 12S0000691 Unpres 46 7.04 Negative NA NA NA erved
13S0000105 Unpres 46 3.83 Negative NA NA NA erved
13S0000108 Unpres 46 1.46 Negative NA NA NA erved
12S0000560 Fixed 46 3.5 Negative NA NA NA
13S0000042 Unpres 46 6.1 Negative NA NA NA erved
12S0000690 Unpres 46 18.57 Negative NA NA NA erved
Leiden 92 Unpres 46 53.63 Giardia NA Positive NA erved
DLS13- Fixed 46 16.64 Crypto NA NA Positive 05804-01-01
11556 Fixed 46 42.2 Crypto NA NA Positive
13S0000014 Unpres 46 48.81 Negative NA NA NA erved
EH21 Unpres 27.75 3533.77 Entamoeba NA NA NA erved Histolytica
CCF07 Fixed 46 0.64 Giardia NA NA NA
DLS13- Fixed 46 4.03 Giardia, NA Positive Positive 05799-01-01 Crypto
CINOIOIO Fixed 46 3.1 Giardia NA Positive NA
13S0000137 Fixed 46 1.84 Negative Negative NA NA
13S0000034 Unpres 46 15.23 Negative NA NA NA erved
4369 Fixed 46 3.26 Crypto NA NA Positive
12S0000787 Unpres 46 7.78 Negative Negative NA NA erved
DLS13- Unpres 46 21.38 Crypto Negative NA Positive 05977 erved
13S0000125 Fixed 46 5.03 Negative Negative NA NA
Leiden 28 Unpres 46 46.83 Negative NA NA Positive erved
138 Fixed 46 0.67 Giardia NA Positive NA
CIN01013 Fixed 46 1.94 Giardia NA Negative NA
13S0000124 Fixed 46 19.7 Negative NA NA NA
13S0000170 Fixed 46 0.88 Negative NA NA NA
13S0000176 Fixed 46 0.7 Negative NA NA NA
13S0000052 Unpres 46 0.65 Negative Negative NA NA erved
13S0000073 Unpres 46 13.93 Negative NA NA NA erved
13S0000051 Unpres 46 0.77 Negative NA NA NA erved
13S0000567 Fixed 46 4.26 Negative NA NA NA
DLS13- Unpres 46 2.24 Negative NA NA Positive 05986 erved
Leiden 66 Unpres 46 1.32 Giardia NA Positive NA erved DLS13- Unpres 46 3.76 Crypto NA NA Positive 05959 erved
Leiden 4 Unpres 46 12.08 Giardia NA Positive NA erved
KH 12-6358 Unpres 46 4.31 Giardia NA Positive NA erved
CCF12 Fixed 46 2.65 Giardia NA NA NA
F21 Fixed 46 3 Giardia NA Positive NA
DLS13- Fixed 46 2.64 Crypto NA NA Positive 05796-01-01
Leiden 21 Unpres 46 8.02 Crypto NA NA Positive erved
13S0000572 Fixed 46 1.6 Negative NA NA NA
Leiden 34 Unpres 46 7.64 Crypto NA NA Positive erved
Table 10.2
Figure imgf000125_0001
13S0000069 Negative Amp Negative Negative
E20 Entamoeba Amp Negative Negative Entamoeba
Histolytica dispar genes for 18S rRNA, ITS1. 5.8S rRNA, ITS2, complete sequence
12S0000765 Negative Negative Negative Negative
Leiden 12 Crypto Negative Negative Amp
12S0000693 Negative Amp Negative Negative
Leiden 78 Giardia Amp Amp Negative
EH17 Entamoeba Negative Negative Negative
Histolytica
CCF06 Giardia Negative Negative Amp
EH19 Entamoeba Negative Negative Negative
Histolytica
EH20 Entamoeba Negative Negative Negative
Histolytica
1438 Crypto Negative Negative Negative
13S0000005 Negative Negative Negative Amp
13S0000159 Negative Negative Negative Negative
12S0000722 Negative Negative Negative Negative
DLS13- Giardia Amp Amp Negative
05812-01-01
12S0000706 Negative Negative Negative Negative
Leiden 64 Giardia Negative Amp Negative
13S0000559 Negative Negative Negative Negative
12S0000673 Negative Negative Negative Negative
DLS13- Giardia Negative Negative Negative
05780-01-01
12S0000788 Negative Amp Negative Negative
13S0000059 Negative Negative Negative Negative
DLS13- Crypto Amp Negative Amp
05960
13S0000139 Negative Amp Negative Amp
13S0000037 Negative Negative Negative Negative
CIN01005 Giardia Negative Negative Negative
13S0000185 Negative Negative Negative Negative
12S0000699 Negative Amp Negative Negative
55 Giardia Amp Amp Negative
CCF08 Giardia Negative Negative Negative
13S0000564 Negative Negative Negative Negative
13S0000131 Negative Amp Negative Negative
12S0000561 Negative Negative Negative Negative
EH23 Entamoeba Amp Negative Negative Entamoeba
Histolytica histolytica gene for small subunit ribosomal RNA, strain: BF-841 ell
564 Crypto Negative Negative Amp
Leiden 26 Crypto Negative Negative Amp
12S0000771 Negative Negative Negative Negative
12S0000729 Negative Amp Negative Negative
Leiden 57 Giardia Negative Amp Negative
12997 Crypto Negative Negative Amp
13S0000070 Negative Negative Negative Negative
13S0000171 Negative Negative Negative Negative
DLS13- Crypto Amp Negative Negative
06001
Leiden 39 Crypto Amp Negative Amp
DLS13- Crypto Amp Negative Amp
05967
EH18 Entamoeba Negative Negative Negative
Histolytica
13S0000122 Negative Negative Negative Negative
13S0000057 Negative Negative Negative Negative
12S0000575 Negative Negative Negative Negative
12S0000737 Negative Negative Negative Negative
13S0000188 Negative Negative Negative Negative
12S0000569 Negative Negative Negative Negative
12S0000692 Negative Negative Negative Negative
13S0000036 Negative Amp Negative Negative
13S0000566 Negative Negative Negative Negative
13S0000109 Negative Negative Negative Negative
13S0000090 Negative Negative Negative Negative
36 Giardia Negative Amp Negative
12S0000697 Negative Negative Negative Negative
12S0000572 Negative Negative Negative Negative
13S0000106 Negative Negative Negative Negative
F35 Giardia Negative Amp Negative
12S0000735 Negative Negative Negative Negative
13S0000177 Negative Negative Negative Negative
12S0000707 Negative Negative Negative Negative
12S0000708 Negative Amp Negative Negative
DLS13- Giardia Negative Negative Negative
05974
13S0000008 Negative Negative Negative Negative
13S0000011 Negative Negative Negative Negative
DLS13- Crypto Negative Negative Amp
05782-01-01
12S0000670 Negative Negative Negative Negative
CIN01020 Giardia Negative Negative Negative 13S0000156 Negative Negative Negative Negative
13S0000039 Negative Negative Negative Negative
13S0000035 Negative Amp Negative Negative
12S0000570 Negative Negative Negative Negative
11334 Crypto Negative Negative Amp
13S0000569 Negative Negative Negative Negative
13S0000065 Negative Negative Negative Negative
EH12 Entamoeba Negative Negative Negative
Histolytica
12S0000777 Negative Negative Negative Negative
13S0000116 Negative Negative Negative Negative
DLS13- Crypto Negative Negative Amp
05994
13S0000138 Negative Negative Negative Negative
Leiden 68 Giardia Negative Amp Negative
Leiden 63 Giardia Negative Amp Negative
13S0000149 Negative Negative Negative Negative
12S0000732 Negative Negative Negative Negative
DLS13- Giardia Amp Amp Amp
05976
12S0000688 Negative Negative Negative Negative
EH08 Entamoeba Amp Negative Negative Entamoeba
Histolytica histolytica gene for small subunit ribosomal RNA, strain: BF-841 ell
CIN01004 Giardia Negative Negative Negative
DLS13- Crypto Negative Negative Amp
05956
Leiden 10 Crypto Negative Negative Amp
Leiden 37 Crypto Amp Negative Amp
DLS13- Giardia Negative Negative Amp
05790-01-01
DLS13- Entamoeba Negative Negative Negative
05996 Histolytica
F33 Giardia Negative Amp Negative
Leiden 96 Giardia Negative Amp Negative
Leiden 99 Giardia Negative Amp Negative
13S0000183 Negative Negative Negative Negative
12S0000503 Negative Negative Negative Negative
13S0000092 Negative Negative Negative Negative
Leiden 84 Giardia Amp Amp Negative
12S0000786 Negative Negative Negative Negative
DLS13- Crypto Amp Amp Amp
05817-01-01
Leiden 79 Giardia Negative Negative Negative 13S0000111 Negative Negative Negative Negative
CCF11 Giardia Negative Negative Negative
124 Giardia Negative Amp Negative
13S0000563 Negative Negative Negative Negative
12S0000678 Negative Negative Negative Negative
12S0000698 Negative Negative Negative Negative
77 Giardia Amp Amp Negative Entamoeba coli partial 18S rRNA gene, isolate EM049
13S0000084 Negative Negative Negative Negative
12S0000746 Negative Negative Negative Negative
CIN01040 Giardia Negative Amp Negative
13S0000117 Negative Negative Negative Negative
Leiden 32 Crypto Negative Negative Amp
13S0000112 Negative Negative Negative Negative
12S0000711 Negative Negative Negative Negative
DLS13- Entamoeba Amp Negative Negative
05982 Histolytica
13S0000032 Negative Negative Negative Negative
13S0000565 Negative Negative Negative Negative
DLS13- Crypto Negative Negative Amp
05787-01-01
12S0000760 Negative Negative Negative Negative
Leiden 98 Giardia Negative Amp Negative
13S0000087 Negative Negative Negative Negative
F41 Giardia Negative Amp Negative
6289 Crypto Negative Negative Amp
DLS13- Crypto Amp Negative Amp
05947
E18 Entamoeba Amp Negative Negative Entamoeba
Histolytica dispar genes for 18S rRNA, ITS1, 5.8S rRNA, ITS2, complete sequence
13S0000568 Negative Negative Negative Negative
F37 Giardia Negative Amp Negative
Leiden 36 Crypto Amp Negative Amp
13S0000158 Negative Negative Negative Negative
DLS13- Entamoeba Negative Negative Negative
05973 Histolytica
13S0000053 Negative Negative Negative Negative
Leiden 91 Giardia Negative Amp Negative
DLS13- Giardia Amp Negative Amp
05966 12S0000778 Negative Negative Negative Negative
13S0000151 Negative Negative Negative Negative
13S0000147 Negative Negative Negative Negative
13S0000179 Negative Negative Negative Negative
13S0000006 Negative Negative Negative Negative
13S0000189 Negative Negative Negative Negative
12S0000573 Negative Negative Negative Negative
DLS13- Crypto Amp Negative Amp
05980
12S0000791 Negative Negative Negative Negative
Leiden 25 Crypto Amp Negative Amp
DLS13- Crypto Amp Negative Amp
05952
13S0000019 Negative Negative Negative Negative
91 Giardia Negative Amp Negative
DLS13- Crypto Amp Negative Negative
05972
13S0000043 Negative Negative Negative Negative
12S0000714 Negative Negative Negative Negative
12S0000675 Negative Negative Negative Negative
DLS13- Crypto Negative Negative Amp
05801-01-01
12S0000682 Negative Negative Negative Negative
DLS13- Crypto Negative Negative Amp
05802-01-01
113 Giardia Amp Amp Negative
Leiden 85 Giardia Negative Amp Negative
EH14 Entamoeba Amp Negative Negative Entamoeba
Histolytica histolytica gene for small subunit ribosomal RNA, strain: BF-841 ell
13S0000096 Negative Negative Negative Negative
12S0000733 Negative Amp Negative Negative
12S0000689 Negative Negative Negative Negative
CCF03 Giardia Negative Negative Negative
13S0000049 Negative Negative Negative Negative
Leiden 8 Crypto Negative Negative Amp
Leiden 29 Crypto Amp Negative Amp
12S0000576 Negative Negative Negative Negative
Leiden 6 Crypto Amp Negative Amp
13S0000007 Negative Negative Negative Negative
12S0000717 Negative Negative Negative Negative
13S0000001 Negative Negative Negative Negative
13S0000134 Negative Negative Amp Negative 13S0000104 Negative Negative Negative Negative
13S0000089 Negative Negative Negative Negative
12S0000762 Negative Negative Negative Negative
13S0000562 Negative Negative Negative Negative
13S0000010 Negative Negative Negative Negative
125 Giardia Negative Amp Negative
13S0000135 Negative Negative Negative Negative
13S0000118 Negative Negative Negative Negative
12S0000713 Negative Negative Negative Negative
13S0000162 Negative Negative Negative Negative
DLS13- Crypto Amp Negative Amp
05970
12S0000727 Negative Amp Negative Negative
39546 Giardia Amp Amp Negative
13S0000083 Negative Negative Negative Negative
Leiden 49 Crypto Amp Negative Amp
EH09 Entamoeba Negative Negative Negative
Histolytica
F31 Giardia Amp Amp Negative
12S0000751 Negative Amp Negative Negative
13S0000153 Negative Amp Negative Negative
13S0000166 Negative Amp Negative Negative
12S0000685 Negative Negative Negative Negative
CCF10 Giardia Negative Negative Negative
EH10 Entamoeba Negative Negative Negative
Histolytica
13S0000098 Negative Negative Negative Negative
81 Giardia Amp Amp Negative
13S0000095 Negative Negative Negative Negative
Leiden 1 Crypto Amp Negative Amp
13S0000173 Negative Amp Negative Negative
DLS13- Giardia Negative Amp Negative
05784-01-01
Leiden 51 Giardia Amp Amp Negative
12S0000725 Negative Negative Negative Negative
162 Giardia Amp Amp Negative Entamoeba coli partial 18S rRNA gene, isolate J65
12S0000781 Negative Negative Negative Negative
13S0000143 Negative Negative Negative Negative
13S0000003 Negative Negative Negative Negative
12S0000710 Negative Negative Negative Negative
Leiden 86 Giardia Amp Amp Negative
13S0000027 Negative Negative Negative Negative
DLS13- Crypto Negative Negative Negative 05949
12S0000702 Negative Amp Negative Negative
Leiden 82 Giardia Amp Amp Negative
1247 Crypto Negative Negative Amp
13S0000140 Negative Amp Negative Negative
Leiden 59 Giardia Amp Amp Negative Entamoeba dispar genes for 18S rRNA, ITS1. 5.8S rRNA, ITS2, complete sequence
13S0000558 Negative Amp Negative Negative
13S0000157 Negative Amp Negative Negative
8174 Crypto Negative Negative Negative
F29 Giardia Amp Amp Negative
CIN01026 Giardia Amp Amp Negative
Leiden 58 Giardia Negative Amp Negative
13S0000165 Negative Amp Negative Negative
13S0000164 Negative Amp Negative Negative
DLS13- Crypto Amp Negative Amp
05808-01-01
12S0000716 Negative Amp Negative Negative
12S0000784 Negative Amp Negative Negative
EH24 Entamoeba Amp Negative Negative
Histolytica
12S0000681 Negative Amp Negative Negative
DLS13- Crypto Amp Negative Amp
05969
Leiden 62 Giardia Amp Amp Negative
13S0000002 Negative Amp Negative Negative
12S0000770 Negative Amp Negative Negative
DLS13- Giardia Negative Negative Amp
05789-01-01
13S0000560 Negative Amp Negative Negative
13S0000145 Negative Amp Negative Negative
DLS13- Crypto Amp Negative Amp
05958
12S0000563 Negative Amp Negative Negative
12S0000683 Negative Amp Negative Negative
13S0000168 Negative Negative Negative Negative
Leiden 46 Crypto Negative Negative Amp
E24 Entamoeba Amp Negative Negative Entamoeba
Histolytica dispar genes for 18S rRNA, ITS1, 5.8S rRNA, ITS2, complete sequence 13S0000101 Negative Negative Negative Negative
13S0000155 Negative Negative Negative Negative
Leiden 71 Giardia Negative Amp Negative
EH06 Entamoeba Negative Negative Negative
Histolytica
DLS13- Crypto Negative Negative Amp
05816-01-01
F27 Giardia Negative Amp Negative
DLS13- Crypto Amp Negative Negative
05997
EH04 Entamoeba Negative Negative Negative
Histolytica
12S0000726 Negative Negative Negative Negative
DLS13- Entamoeba Amp Negative Negative
05963 Histolytica
DLS13- Crypto Negative Negative Amp
05793-01-01
13S0000126 Negative Negative Negative Negative
EH25 Entamoeba Negative Negative Negative
Histolytica
40015 Crypto Negative Negative Amp
13S0000181 Negative Amp Negative Negative
58 Giardia Amp Amp Negative Entamoeba hartmanni partial 18S rRNA gene, isolate EM061a
CCF02 Giardia Negative Amp Negative
12S0000568 Negative Negative Negative Negative
96 Giardia Negative Amp Negative
Leiden 43 Crypto Negative Negative Amp
13S0000123 Negative Negative Negative Negative
12S0000705 Negative Negative Negative Negative
DLS13- Crypto Negative Negative Amp
05794-01-01
13S0000136 Negative Negative Negative Negative
Leiden 97 Giardia Amp Amp Negative
DLS13- Crypto Negative Negative Amp
05798-01-01
12S0000719 Negative Amp Negative Negative
12S0000668 Negative Negative Negative Negative
Leiden 80 Giardia Negative Negative Negative
12S0000756 Negative Negative Negative Negative
13S0000107 Negative Negative Negative Negative
13S0000068 Negative Amp Negative Negative
12S0000749 Negative Negative Negative Negative
Leiden 16 Crypto Amp Negative Amp
Leiden 72 Giardia Negative Amp Negative 12S0000763 Negative Amp Negative Negative Entamoeba coli partial 18S rRNA gene, isolate J65
12S0000709 Negative Amp Negative Negative
13S0000072 Negative Negative Negative Negative
13S0000013 Negative Negative Negative Negative
CCF01 Giardia Negative Amp Negative
DLS13- Crypto Amp Negative Amp
05953
12S0000761 Negative Negative Negative Negative
Leiden 9 Crypto Negative Negative Amp
13S0000054 Negative Negative Negative Negative
EH16 Entamoeba Negative Negative Negative
Histolytica
13S0000081 Negative Negative Negative Negative
DLS13- Crypto Amp Negative Amp
05951
DLS13- Giardia Amp Amp Negative
05820-01-01
Leiden 5 Crypto Amp Negative Amp
EH07 Entamoeba Negative Negative Negative
Histolytica
13S0000146 Negative Negative Negative Negative
CIN01012 Giardia Negative Amp Negative
DLS13- Giardia Amp Amp Negative
05818-01-01
13S0000169 Negative Amp Negative Negative
13S0000186 Negative Negative Negative Negative
13S0000152 Negative Amp Negative Negative
12S0000669 Negative Negative Negative Negative
12S0000566 Negative Negative Negative Negative
14790 Crypto Negative Negative Amp
Leiden 18 Crypto Amp Negative Amp
13S0000094 Negative Amp Negative Negative
13S0000154 Negative Negative Negative Negative
12S0000724 Negative Negative Negative Negative
13S0000161 Negative Amp Negative Negative
12S0000562 Negative Amp Negative Negative
13S0000040 Negative Negative Negative Negative
13S0000024 Negative Negative Negative Negative
13S0000148 Negative Negative Negative Negative
DLS13- Crypto Amp Negative Amp
05983
159 Giardia Amp Amp Negative
Leiden 74 Giardia Negative Amp Negative
13S0000031 Negative Negative Negative Negative EH05 Entamoeba Amp Negative Negative Entamoeba Histolytica histolytica gene for small subunit ribosomal RNA, strain: BF-841 ell
3645 Crypto Negative Negative Amp
13S0000119 Negative Negative Negative Negative
13S0000021 Negative Amp Negative Negative
13S0000132 Negative Negative Negative Negative
12S0000764 Negative Amp Negative Negative
13S0000064 Negative Negative Negative Negative
183 Giardia Negative Amp Negative
DLS13- Crypto Amp Negative Amp
05950
13S0000557 Negative Negative Negative Negative
CIN01038 Giardia Negative Amp Negative
13S0000120 Negative Negative Negative Negative
EH01 Entamoeba Amp Negative Negative Entamoeba
Histolytica histolytica gene for small subunit ribosomal RNA, strain: BF-841 ell
12S0000679 Negative Negative Negative Negative
Leiden 73 Giardia Negative Amp Negative
DLS13- Crypto Negative Amp Negative
05781-01-01
13S0000150 Negative Negative Negative Negative
13S0000115 Negative Negative Negative Negative
13S0000142 Negative Negative Negative Negative
Leiden 70 Giardia Negative Amp Negative
Leiden 69 Giardia Negative Amp Negative
12S0000704 Negative Negative Negative Negative
129 Giardia Negative Amp Negative
Leiden 61 Giardia Amp Amp Negative
EH03 Entamoeba Negative Negative Negative
Histolytica
13S0000121 Negative Amp Negative Negative
7458 Crypto Negative Negative Negative
13S0000129 Negative Negative Negative Negative
12S0000703 Negative Negative Negative Negative
13S0000056 Negative Amp Negative Negative
12S0000766 Negative Negative Negative Negative
F23 Giardia Negative Amp Negative
13S0000103 Negative Negative Negative Negative 12S0000723 Negative Negative Negative Negative
130 Giardia Negative Amp Negative
Leiden 52 Giardia Negative Amp Negative
Leiden 60 Giardia Negative Amp Negative
13S0000038 Negative Negative Negative Negative
13S0000100 Negative Negative Negative Negative
13S0000102 Negative Negative Negative Negative
13S0000576 Negative Negative Negative Negative
12S0000567 Negative Negative Negative Negative
12S0000672 Negative Negative Negative Negative
12S0000757 Negative Negative Negative Negative
12S0000742 Negative Negative Negative Negative
Leiden 17 Crypto Negative Negative Amp
Leiden 15 Crypto Negative Negative Amp
13S0000009 Negative Negative Negative Negative
CIN01028 Giardia Negative Amp Negative
13S0000127 Negative Amp Negative Negative Entamoeba hartmanni partial 18S rRNA gene, isolate EM042
KH12-5156 Giardia Amp Amp Negative
12S0000674 Negative Negative Negative Negative
13S0000571 Negative Negative Negative Negative
13S0000128 Negative Negative Negative Negative
140 Giardia Amp Amp Negative Entamoeba coli strain IH:96/135 16S-like small subunit ribosomal RNA gene, complete sequence
12S0000738 Negative Negative Negative Negative
CIN01039 Giardia Negative Amp Negative
KH12-4357 Giardia Negative Amp Negative
Leiden 30 Crypto Amp Negative Amp
13S0000114 Negative Negative Negative Negative
Leiden 55 Giardia Amp Amp Negative
12S0000680 Negative Negative Negative Negative
E22 Entamoeba Amp Negative Negative Entamoeba
Histolytica dispar genes for 18S rRNA, ITS1, 5.8S rRNA, ITS2, complete sequence 39543 Giardia Negative Amp Negative
12S0000776 Negative Negative Negative Negative
KH12-6359 Giardia Negative Amp Negative
13S0000160 Negative Negative Negative Negative
DLS13- Giardia Negative Amp Amp
05945
11796 Crypto Negative Negative Amp
DLS13- Crypto Negative Amp Negative
05978
13S0000187 Negative Negative Negative Negative
13S0000063 Negative Amp Negative Negative
12S0000779 Negative Negative Negative Negative
DLS13- Crypto Amp Negative Amp
05800-01-01
13S0000110 Negative Negative Negative Negative
13S0000113 Negative Negative Negative Negative
CIN01027 Giardia Negative Negative Negative
13S0000180 Negative Negative Negative Negative
12S0000564 Negative Negative Negative Negative
CCF05 Giardia Negative Negative Negative
DLS13- Crypto Amp Negative Negative
05991
12S0000667 Negative Negative Negative Negative
13S0000182 Negative Negative Negative Negative
12S0000790 Negative Negative Negative Negative
CIN01003 Giardia Negative Negative Negative
Leiden 19 Crypto Amp Negative Amp
DLS13- Crypto Negative Negative Amp
05792-01-01
13S0000099 Negative Negative Negative Negative
CIN01024 Giardia Negative Negative Negative
13S0000062 Negative Amp Negative Negative
EH13 Entamoeba Negative Negative Negative
Histolytica
12S0000715 Negative Negative Negative Negative
DLS13- Crypto Amp Amp Amp
05961
DLS13- Crypto Amp Amp Amp
05981
CCF04 Giardia Negative Negative Negative
Leiden 22 Crypto Negative Negative Amp
EH02 Entamoeba Amp Negative Negative Entamoeba
Histolytica histolytica gene for small subunit ribosomal RNA, strain: BF-841 ell 13S0000184 Negative Negative Negative Negative
Leiden 35 Crypto Amp Negative Amp
DLS13- Giardia Negative Negative Negative 05807-01-01
13S0000175 Negative Negative Negative Negative
13S0000091 Negative Negative Negative Negative
13S0000075 Negative Negative Negative Negative
DLS13- Crypto Negative Negative Amp 05795-01-01
12998 Crypto Amp Negative Amp
13S0000174 Negative Negative Negative Negative
13S0000055 Negative Amp Negative Negative
12S0000782 Negative Negative Negative Negative
13S0000163 Negative Negative Negative Negative
13S0000574 Negative Negative Negative Negative
13S0000071 Negative Negative Negative Negative
Leiden 2 Crypto Amp Amp Amp
13S0000167 Negative Negative Negative Negative
13S0000133 Negative Negative Negative Negative
13S0000046 Negative Negative Negative Negative
12S0000759 Negative Negative Negative Negative
Leiden 7 Crypto Amp Negative Amp
13S0000041 Negative Negative Negative Negative
12S0000671 Negative Amp Negative Negative
13S0000093 Negative Amp Negative Negative
CIN01006 Giardia Negative Negative Negative
Leiden 33 Crypto Negative Negative Amp
DLS13- Crypto Amp Negative Negative 05882
13S0000144 Negative Amp Negative Negative
DLS13- Crypto Amp Negative Negative 05893
13S0000088 Negative Amp Negative Negative
DLS13- Crypto Amp Negative Amp 05954
20 Giardia Amp Amp Negative
13S0000060 Negative Amp Negative Negative
64 Giardia Negative Amp Negative
Leiden 67 Giardia Negative Amp Negative
13S0000066 Negative Negative Negative Negative
13S0000178 Negative Amp Negative Negative
13S0000570 Negative Negative Negative Negative
12S0000712 Negative Amp Negative Negative
DLS13- Crypto Negative Negative Amp 05962
73 Giardia Negative Amp Negative
EH15 Entamoeba Negative Negative Negative Histolytica
Leiden 81 Giardia Negative Amp Negative
Leiden 13 Crypto Negative Negative Amp
12S0000731 Negative Amp Negative Negative
12S0000686 Negative Negative Negative Negative
12S0000676 Negative Negative Negative Negative
13S0000067 Negative Negative Negative Negative
CINOlOl l Giardia Negative Amp Negative
Leiden 20 Crypto Amp Negative Negative
EH11 Entamoeba Amp Negative Negative Entamoeba
Histolytica histolytica gene for small subunit ribosomal RNA, strain: BF-841 ell
Leiden 23 Crypto Negative Negative Amp
161 Giardia Negative Negative Negative
13S0000058 Negative Negative Negative Negative
13S0000077 Negative Negative Negative Negative
DLS13- Crypto Negative Negative Amp
05809-01-01
DLS13- Crypto Amp Negative Amp
05788-01-01
DLS13- Crypto Amp Negative Amp
05987
Leiden 95 Giardia Amp Amp Negative
68 Giardia Negative Amp Negative
Leiden 53 Giardia Negative Amp Negative
12S0000677 Negative Negative Negative Negative
12S0000721 Negative Negative Negative Negative
DLS13- Crypto/Giardi Negative Amp Negative
05814-01-01 a
Leiden 11 Crypto Negative Negative Amp
Leiden 3 Crypto Negative Negative Amp
12S0000577 Negative Negative Negative Negative
12S0000701 Negative Negative Negative Negative
37 Giardia Negative Amp Negative
13S0000076 Negative Negative Negative Negative
DLS13- Crypto Negative Negative Amp
05968
DLS13- Crypto Negative Negative Amp
05805-01-01
13S0000078 Negative Negative Negative Negative
Leiden 65 Giardia Amp Amp Negative
13S0000573 Negative Negative Negative Negative
13S0000097 Negative Negative Negative Negative
DLS13- Crypto Negative Negative Amp 05791-01-01
12S0000691 Negative Negative Negative Negative
13S0000105 Negative Negative Negative Negative
13S0000108 Negative Negative Negative Negative
12S0000560 Negative Negative Negative Negative
13S0000042 Negative Negative Negative Negative
12S0000690 Negative Negative Negative Negative
Leiden 92 Giardia Negative Amp Negative
DLS13- Crypto Negative Negative Amp 05804-01-01
11556 Crypto Negative Negative Amp
13S0000014 Negative Negative Negative Negative
EH21 Entamoeba Negative Negative Negative
Histolytica
CCF07 Giardia Negative Negative Negative
DLS13- Crypto Negative Amp Amp 05799-01-01
CINOIOIO Giardia Negative Amp Negative
13S0000137 Negative Amp Negative Negative
13S0000034 Negative Negative Negative Negative
4369 Crypto Negative Negative Amp
12S0000787 Negative Amp Negative Negative
DLS13- Crypto Amp Negative Amp 05977
13S0000125 Negative Amp Negative Negative
Leiden 28 Crypto Negative Negative Amp
138 Giardia Negative Amp Negative
CIN01013 Giardia Negative Amp Negative
13S0000124 Negative Negative Negative Negative
13S0000170 Negative Negative Negative Negative
13S0000176 Negative Negative Negative Negative
13S0000052 Negative Amp Negative Negative
13S0000073 Negative Negative Negative Negative
13S0000051 Negative Negative Negative Negative
13S0000567 Negative Negative Negative Negative
DLS13- Crypto Negative Negative Amp 05986
Leiden 66 Giardia Negative Amp Negative
DLS13- Crypto Negative Negative Amp 05959
Leiden 4 Crypto Negative Amp Negative
KH12-6358 Giardia Negative Amp Negative
CCF12 Giardia Negative Negative Negative
F21 Giardia Negative Amp Negative
DLS13- Crypto Negative Negative Amp 05796-01-01
Leiden 21 Crypto Negative Negative Amp 13S0000572 Negative Negative Negative Negative
Leiden 34 Crypto Negative Negative Amp
Example 11 : Clinical simulation
[0101] To further confirm the ability of the BD MAX™ assay to detect E. histolytica in samples positive for E. histolytica, a contrived clinical simulation was performed. Individual unpreserved and 10% formalin- fixed stool specimens screened as negative for Entamoeba histolytica were spiked with E. histolytica trophozoites near the assay LOD. Contrived specimens were tested by blinded operators with the BD MAX™ Enteric Parasite Panel. The results of this clinical simulation are shown in Table 11.
Table 11
Figure imgf000141_0001
Unpreserved Negative 23.37 46 Negative
Unpreserved 2X LoD 2881.86 33.79 Positive
Unpreserved Negative 0.77 46 Negative
Unpreserved Negative 25.47 46 Negative
Unpreserved 2X LoD 3627.53 27.41 Positive
Unpreserved 2X LoD 3549.91 28.78 Positive
Unpreserved 2X LoD 2557.79 33.6 Positive
Unpreserved Negative 3.43 46 Negative
Unpreserved Negative 3.34 46 Negative
Unpreserved 2X LoD 2914.65 28.07 Positive
Unpreserved Negative 1.52 46 Negative
Unpreserved Negative 6.69 46 Negative
Unpreserved 2X LoD 3216.07 25.18 Positive
Unpreserved Negative 0.59 46 Negative
Unpreserved Negative 2.75 46 Negative
Unpreserved Negative 4.93 46 Negative
Unpreserved 2X LoD 4301.96 27.85 Positive
Unpreserved 2X LoD 4383.23 28.45 Positive
Unpreserved 2X LoD 2292.14 31.58 Positive
Unpreserved 2X LoD 3490.01 30.58 Positive
10% Formalin 2X LoD 3905.11 27.33 Positive Fixed
10% Formalin 2X LoD 3587.14 24.96 Positive Fixed
10% Formalin 2X LoD 4042.64 28.14 Positive Fixed
10% Formalin 2X LoD 4088.09 25.63 Positive Fixed
10% Formalin 2X LoD 3217.87 26.04 Positive Fixed
10% Formalin 2X LoD 3611.21 23.54 Positive Fixed
10% Formalin 2X LoD 3407.33 26.3 Positive Fixed
10% Formalin 2X LoD 3522.62 24.42 Positive Fixed
10% Formalin 2X LoD 4156.1 25.21 Positive Fixed
10% Formalin 2X LoD 4722.12 26.83 Positive Fixed
10% Formalin Negative 0.55 46 Negative Fixed
10% Formalin Negative 0.55 46 Negative Fixed
10% Formalin Negative 12.92 46 Negative Fixed
10% Formalin Negative 3.54 46 Negative Fixed
10% Formalin Negative 5.95 46 Negative Fixed
10% Formalin 2X LoD 3460.87 29.1 Positive Fixed
10% Formalin 2X LoD 3207.24 24.99 Positive Fixed
10% Formalin Negative 1.21 46 Negative Fixed
10% Formalin Negative 2.57 46 Negative Fixed
10% Formalin 2X LoD 4342.59 25.23 Positive Fixed
10% Formalin 2X LoD 2809.65 27.47 Positive Fixed
10% Formalin Negative 5.87 46 Negative Fixed
10% Formalin 2X LoD 3467.59 24.63 Positive Fixed
10% Formalin 2X LoD 3940.15 24.39 Positive Fixed
10% Formalin Negative 1.73 46 Negative Fixed
10% Formalin Negative 5.04 46 Negative Fixed
10% Formalin 2X LoD 3898.36 26 Positive Fixed
10% Formalin Negative 1.6 46 Negative Fixed
10% Formalin Negative 2.28 46 Negative Fixed
10% Formalin Negative 0.89 46 Negative Fixed
10% Formalin 2X LoD 4136.67 27.6 Positive Fixed
10% Formalin Negative 8.34 46 Negative Fixed
10% Formalin 2X LoD 3609.34 27.12 Positive Fixed
10% Formalin 2X LoD 4351.88 26.7 Positive Fixed
10% Formalin Negative 1.37 46 Negative Fixed
10% Formalin Negative 0.55 46 Negative Fixed
10% Formalin Negative 0.86 46 Negative Fixed
10% Formalin Negative 6.67 46 Negative Fixed
10% Formalin 2X LoD 3951.5 29.41 Positive
Fixed
10% Formalin Negative 1.27 46 Negative
Fixed
10% Formalin Negative 13.13 46 Negative
Fixed
10% Formalin 2X LoD 2784.26 30.06 Positive
Fixed
10% Formalin 2X LoD 4015.27 26.76 Positive
Fixed
10% Formalin Negative 6.14 46 Negative
Fixed
10% Formalin Negative 2.61 46 Negative
Fixed
10% Formalin 2X LoD 2553.31 27.45 Positive
Fixed
10% Formalin Negative 0.74 46 Negative
Fixed
10% Formalin Negative 8.64 46 Negative
Fixed
[0102] 100% of spiked specimens were positive and 100% of non-spiked specimens were negative. As such, it is contemplated that methods of detecting E. histolytica nucleic acids in accordance with some embodiments herein accurately detect E. histolytica, with minimal false negatives.
Example 12: Comparison of BP MAX™ assay to reference methods
[0103] The results of the BD MAX™ assay were compared to various reference methods. A "final reference method (RM) result" (also referred to herein as a "composite RM") was based on a combined input from a Trichrome Entamoeba spp. assay and an alternative PCR and sequencing approach. For E. histolytica, the composite refernece method (RM) included 1) a microscopic examination of a trichrome staining of PVA fixed stool, in parallel with 2) an analytically validated alternate PCR and bidirectional sequencing. The study involved a total of five (5) US investigational Clinical Centers where specimens were collected as part of the routine patient care, enrolled in the trial and tested with the BD MAX™ Enteric Parasite Panel. Three specimen collection centers and additional specimen brokers sent specimens to investigational clinical centers for testing.
[0104] For prospective samples, the inclusion criteria were as follows: Specimens were obtained from pediatric or adult patients suspected of acute gastroenteritis or colitis for which target parasitic diagnostic tests have been ordered by a healthcare provider. A stool specimen was collected either unpreserved or 10% formalin- fixed. Only one specimen of each specimen type (fixed or unpreserved), collected from a single patient was allowed. The study required a sufficient volume of stool to be available for adequate reference method testing (depending on each clinical center standard procedure) and a minimum of 0.5 mL or 0.5 gram of stool to be available for BD MAX™ EPP testing.
[0105] For retrospective samples, the inclusion criteria were as follows: Unpreserved and fixed specimen for which the original results of the routine test method were available, for at least one (1) of the three (3) EPP targets. Each specimen had a known collection date. Each specimen was stored at -20°C or colder if unpreserved or 2- 8°C if preserved in formalin throughout the entire storage period.
[0106] As summarized in Table 12.1, a "final RM result" was scored as positive if both Trichrome Entamoeba spp. assay and alterantive PCR and sequencing were positive, and was scored as a negative if either or both of these methods was negative. As summarized in Table 12.2, a result was scored as a "true positive" if the BD MAX™ assay and final RM result were both positive, and a "true negative" if the BD MAX™ assay and final RM result were both negative. A result was scored as a "false positive" if the BD MAX™ assay was positive and final RM result was negative, and a "false negative" if the BD MAX™ assay was negative and the final RM result was positive (see Table 12.2).
[0107] Abbreviations used include: P = Positive; N = Negative; LB = Lower Bound; UB = Upper Bound; PPA = Positive Percent Agreement (Sensitivity); NPA = Negative Percent Agreement (Specificity).
Table 12.1
Figure imgf000145_0001
Table 12.2
Figure imgf000145_0002
EPP RM
Positive Positive Concordant True Positive
Positive Negative Discrepant False Positive
Negative Positive Discrepant False Negative
Negative Negative Concordant True Negative
[0108] The overall performance results are summarized in Table 12.3. It is noted that for 1660 samples screened, there were 11 "true positives", 1649 "true negatives", 0 "false positives", and 0 "false negatives".
Table 12.3
Figure imgf000146_0001
[0109] As such, the results summarized in Table 12.3 showed a high degree concordance between the BD MAX™ assay and reference methods. There were no false positives or false negatives among 1660 samples. Accordingly, it is contemplated that methods of detecting E. histolytica nucleic acids in accordance with some embodiments herein accurately detect E. histolytica, with minimal false negatives and minimal false positives, for example fewer than one false negative in 1660, and fewer than one false negative in 1660.
[0110] The overall performance results were further analyzed for prospective and retrospective specome origins, as described herein. These results of this analysis are summarized in Table 12.4.
Table 12.4
Figure imgf000146_0002
Negative 0 245 245
Total 11 245 256
PPA (95% CI (LB, UB)): 100% (74.1%, 100%)
NPA (95% CI (LB, UB)): 100% (98.5%, 100%)
[0111] As shown in Table 12.4, the BD Max™ assay yielded accurate results for both prospective and retrospective specimens. Accordingly, it is contemplated that methods of detecting E. histolytica nucleic acids in accordance with some embodiments herein accurately detect E. histolytica, with minimal false negatives.
[0112] The overall performance results were further analyzed for unpreserved specimens, and specimens fixed in 10% formalin. The results of this analysis are summarized in Table 12.5.
Table 12.5
Figure imgf000147_0001
N 0 768 768
Total 1 1 768 779
PPA (95% CI (LB, UB)): 100% (74.1%, 100%)
NPA (95% CI (LB, UB)): 100% (99.5%, 100%)
[0113] As shown in Table 12.5, the BD Max™ assay yielded accurate results for both formalin fixed and unpreserved specimens. Accordingly, it is contemplated that methods of detecting E. histolytica nucleic acids in accordance with some embodiments herein are suitable for a variety of sample formats, including, but not limited to unpreserved samples and, fixed samples. As such, methods in accordance with some embodimetns herein can be suitable for screening samples at clinical sites, at off-site testing cenetrs that may require fixing samples and/or a substantial lag time between sample collection and testing.
[0114] Particular sequence features (e.g. organisms, genes and/or portions therof) identified by the analytically validated alternate PCR and bi-directional sequencing were consistent with the BD MAX™ assay result. As summarized in Table 12.6 below, samples that yielded non-is. histolytica sequencing results were identified as negative by the BD MAX™ assay, and samples that yielded sequence characteristic of E. histolytica sequencing results were identified as positive by the BD MAX™ assay.
Table 12.6
Figure imgf000148_0001
Entamoeba coli partial 18S rRNA gene,
NEG isolate J65 NEG NA NEG TN
Entamoeba coli partial 18S rRNA gene,
NEG isolate J65 NEG NA NEG TN
Entamoeba coli partial 18S rRNA gene,
NEG isolate J65 NEG NA NEG TN
Entamoeba dispar genes for 18S rRNA,
POS ITS1, 5.8S rRNA, ITS2, complete sequence NEG NA NEG TN
Entamoeba sp. RL2 partial 18S rRNA gene,
NEG isolate Cow350 NEG NA NEG TN
NEG Entamoeba gingivalis SrRNA gene NEG NA NEG TN
Entamoeba hartmanni partial 18S rRNA
NEG gene, isolate 08/1040 NEG NA NEG TN
Entamoeba coli partial 18S rRNA gene,
NEG isolate EM047 NEG NA NEG TN
Entamoeba coli partial 18S rRNA gene,
NEG isolate J65 NEG NA NEG TN
Entamoeba dispar genes for 18S rRNA,
NEG ITS1, 5.8S rRNA, ITS2, complete sequence NEG NA NEG TN
NEG Entamoeba gingivalis SrRNA gene NEG NA NEG TN
Entamoeba coli partial 18S rRNA gene,
NEG isolate EM047 NEG NA NEG TN
Entamoeba histolytica gene for small
POS subunit ribosomal RNA, strain : BF-841 cll POS 22.7 POS TP
Entamoeba histolytica gene for small
POS subunit ribosomal RNA, strain : BF-841 cll POS 19.8 POS TP
Entamoeba histolytica gene for small
POS subunit ribosomal RNA, strain : BF-841 cll POS 31.4 POS TP
Entamoeba histolytica gene for small
POS subunit ribosomal RNA, strain : BF-841 cll POS 24.8 POS TP
Entamoeba histolytica gene for small
POS subunit ribosomal RNA, strain : BF-841 cll POS 23.7 POS TP
Entamoeba histolytica gene for small
POS subunit ribosomal RNA, strain : BF-841 cll POS 33.5 POS TP
Entamoeba histolytica gene for small
POS subunit ribosomal RNA, strain : BF-841 cll POS 24.9 POS TP
Entamoeba histolytica gene for small
POS subunit ribosomal RNA, strain : BF-841 cll POS 27.6 POS TP
Entamoeba histolytica gene for small
POS subunit ribosomal RNA, strain : BF-841 cll POS 27.1 POS TP
Entamoeba histolytica gene for small
POS subunit ribosomal RNA, strain : BF-841 cll POS 25.2 POS TP
Entamoeba hartmanni partial 18S rRNA
NEG gene, isolate EM042 NEG NA NEG TN
Entamoeba dispar genes for 18S rRNA,
NEG ITS1, 5.8S rRNA, ITS2, complete sequence NEG NA NEG TN
Entamoeba coli partial 18S rRNA gene,
NEG isolate J65 NEG NA NEG TN
Entamoeba coli partial 18S rRNA gene,
NEG isolate J65 NEG NA NEG TN Entamoeba coli partial 18S r NA gene,
NEG isolate EM049 NEG NA NEG TN
Entamoeba hartmanni partial 18S rRNA
NEG gene, isolate J92 NEG NA NEG TN
Entamoeba histolytica gene for small
POS subunit ribosomal RNA, strain: BF-841 ell POS 30.9 POS TP
Entamoeba hartmanni partial 18S rRNA
NEG gene, isolate EM061a NEG NA NEG TN
[0115] As such, it is contemplated that methods of detecting E. histolytica nucleic acids in accordance with some embodiments herein are highly accurate, and yield results in line with particular sequence features of the samples examined.
[0116] Samples that did not mee the criteria for the study were excluded. By way of example, trichrome, sequencing, and BD MAX™ assay results for the samples excluded from the study are provided in Table 12.7.
Table 12.7
Figure imgf000150_0001
Unpreserved NEG Entamoeba dispar genes for NA NA NEG
18S r NA, ITS1, 5.8S rRNA,
ITS2, complete sequence
Formalin 10% NEG Entamoeba dispar genes for NA NA N G
18S rRNA, ITS1, 5.8S rRNA,
ITS2, complete sequence
Formalin 10% NEG Entamoeba coli partial 18S NA NA N G rRNA gene, isolate EM049
Formalin 10% NEG Entamoeba dispar genes for NA NA NEG
18S rRNA, ITS1, 5.8S rRNA,
ITS2, complete sequence
Formalin 10% Non-Compliant Entamoeba coli partial 18S NA NA NEG rRNA gene, isolate EM047
Formalin 10% Non-Compliant Entamoeba coli partial 18S NA NA UEG rRNA gene, isolate EM077
Formalin 10% Non-Compliant Entamoeba coli partial 18S NA NA NEG rRNA gene, isolate J65
Formalin 10% Non-Compliant Entamoeba hartmanni NA NA NEG partial 18S rRNA gene,
isolate EM061a
Unpreserved Non-Compliant Entamoeba hartmanni NA NA NEG partial 18S rRNA gene,
isolate EM061a
Formalin 10% Non-Compliant Entamoeba hartmanni NA NA NEG partial 18S rRNA gene,
isolate 09/1140
Unpreserved Non-Compliant Entamoeba bovis 18S rRNA NA NA NEG gene, isolate Sheep310
[0117] It is noted that the sequencing results of the non-compliant samples shown in Table 12.7 indicated that the BD MAX™ assay is not cross-reactive with other Etnamoeba sequences (such as Entamoeba coli, Entamoeba dispar, Entamoeba polecki, Entamoeba muris, Entamoeba nuttalli, Entamoeba hartmanni, and Entamoeba bovis). As such, it is contemplated that methods of detecting E. histolytica nucleic acids in accordance with some embodiments herein do not cross react with Entamoeba coli, Entamoeba dispar, Entamoeba polecki, Entamoeba muris, Entamoeba nuttalli, Entamoeba hartmanni, and/or Entamoeba bovis.
Example 13 : Contrived Clinical Supplemental Study
[0118] It is noted that E. histolytica infection can be relatively rare, and consistent with this relative rarity, a number of the clinical studies produced many more negative results than positive results. So as to characterize additional positive results for the BD MAX™ assay, a contrived clinical supplemental study was designed and performed, in which a number of specimens were spiked with E. histolytica trophozites. [0119] In particular, individual unpreserved and 10% formalin-fixed stool specimens screened as negative for Entamoeba histolytica were spiked with E. histolytica trophozoites at levels spanning the assay range. Contrived specimens were tested by blinded operators with the BD MAX™ Enteric Parasite Panel (EPP). The results of the contrived clinical study are shown in Table 13.
Table 13
Figure imgf000152_0001
CF035 Fixed lOOX LoD 22.14 POS
CF036 Fixed 4X LoD 24.84 POS
CF037 Fixed Negative 46 NEG
CF038 Fixed Negative 46 NEG
CF039 Fixed 4X LoD 24.47 POS
CF040 Fixed Negative 46 NEG
CF041 Fixed 2X LoD 26.28 POS
CF042 Fixed Negative 46 NEG
CF043 Fixed 2X LoD 25.76 POS
CF044 Fixed 4X LoD 24.24 POS
CF045 Fixed Negative 46 NEG
CF046 Fixed Negative 46 NEG
CF047 Fixed Negative 46 NEG
CF048 Fixed 4X LoD 24.77 POS
CF049 Fixed 2X LoD 27.53 POS
CF050 Fixed Negative 46 NEG
CF051 Fixed Negative 46 NEG
CF052 Fixed 10X LoD 23.33 POS
CF053 Fixed Negative 46 NEG
CF054 Fixed Negative 46 NEG
CF055 Fixed 10X LoD 24.69 POS
CF056 Fixed Negative 46 NEG
CF057 Fixed Negative 46 NEG
CF058 Fixed 2X LoD 27.31 POS
CF059 Fixed Negative 46 NEG
CF060 Fixed Negative 46 NEG
CF061 Fixed Negative 46 NEG
CF062 Fixed 2X LoD 26.19 POS
CF063 Fixed 50X LoD 21.11 POS
CF064 Fixed 2X LoD 27.83 POS
CF065 Fixed Negative 46 NEG
CF066 Fixed Negative 46 NEG
CF067 Fixed Negative 46 NEG
CF068 Fixed 2X LoD 24.63 POS
CF069 Fixed Negative 46 NEG
CF070 Fixed Negative 46 NEG
CF071 Fixed Negative 46 NEG
CF072 Fixed 2X LoD 25.43 POS
CF073 Fixed 100X LoD 20.38 POS
CF074 Fixed 10X LoD 23.5 POS
CF075 Fixed Negative 46 NEG
CF076 Fixed Negative 46 NEG
CF077 Fixed 2X LoD 26.37 POS
CF078 Fixed 10X LoD 23.89 POS
CF079 Fixed 100X LoD 21.87 POS CF080 Fixed 2X LoD 26.67 POS
CF081 Fixed Negative 46 NEG
CF082 Fixed Negative 46 NEG
CF083 Fixed 2X LoD 27.09 POS
CF084 Fixed 100X LoD 21.05 POS
CF085 Fixed Negative 46 NEG
CF086 Fixed 2X LoD 26.25 POS
CF087 Fixed 50X LoD 21.96 POS
CF088 Fixed 2X LoD 25.46 POS
CF089 Fixed Negative 46 NEG
CF090 Fixed Negative 46 NEG
CF091 Fixed Negative 46 NEG
CF092 Fixed Negative 46 NEG
CF093 Fixed Negative 46 NEG
CF094 Fixed 2X LoD 25.92 POS
CF095 Fixed 2X LoD 24.12 POS
CF096 Fixed Negative 46 NEG
CF097 Fixed Negative 46 NEG
CF098 Fixed Negative 46 NEG
CF099 Fixed Negative 46 NEG
CF100 Fixed Negative 46 NEG
CUOOl Unpreserved 2X LoD 28.7 POS
CU002 Unpreserved 50X LoD 22.18 POS
CU003 Unpreserved 2X LoD 24.27 POS
CU004 Unpreserved Negative 46 NEG
CU005 Unpreserved Negative 46 NEG
CU006 Unpreserved Negative 46 NEG
CU007 Unpreserved Negative 46 NEG
CU008 Unpreserved 2X LoD 24.73 POS
CU009 Unpreserved 100X LoD 24.43 POS
CUOIO Unpreserved 2X LoD 28 POS
CU011 Unpreserved Negative 46 NEG
CU012 Unpreserved Negative 46 NEG
CU013 Unpreserved 2X LoD 27.6 POS
CU014 Unpreserved Negative 46 NEG
CU015 Unpreserved Negative 46 NEG
CU016 Unpreserved Negative 46 NEG
CU017 Unpreserved Negative 46 NEG
CU018 Unpreserved 4X LoD 25.95 POS
CU019 Unpreserved Negative 46 NEG
CU020 Unpreserved Negative 46 NEG
CU021 Unpreserved 2X LoD 27 POS
CU022 Unpreserved 100X LoD 19.77 POS
CU023 Unpreserved 2X LoD 22.86 POS
CU024 Unpreserved Negative 46 NEG CU025 Unpreserved Negative 46 NEG
CU026 Unpreserved 4X LoD 22.39 POS
CU027 Unpreserved Negative 46 NEG
CU028 Unpreserved 10X LoD 22.54 POS
CU029 Unpreserved Negative 46 NEG
CU030 Unpreserved Negative 46 NEG
CU031 Unpreserved Negative 46 NEG
CU032 Unpreserved 10X LoD 21.78 POS
CU033 Unpreserved Negative 46 NEG
CU034 Unpreserved Negative 46 NEG
CU035 Unpreserved 2X LoD 25.11 POS
CU036 Unpreserved 4X LoD 23.61 POS
CU037 Unpreserved 100X LoD 19.23 POS
CU038 Unpreserved 2X LoD 27.79 POS
CU039 Unpreserved Negative 46 NEG
CU040 Unpreserved Negative 46 NEG
CU041 Unpreserved Negative 46 NEG
CU042 Unpreserved Negative 46 NEG
CU043 Unpreserved Negative 46 NEG
CU044 Unpreserved 2X LoD 24.24 POS
CU045 Unpreserved 100X LoD 20.28 POS
CU046 Unpreserved 4X LoD 23.88 POS
CU047 Unpreserved 2X LoD 25.09 POS
CU048 Unpreserved Negative 46 NEG
CU049 Unpreserved Negative 46 NEG
CU050 Unpreserved Negative 46 NEG
CU051 Unpreserved 4X LoD 23.86 POS
CU052 Unpreserved 50X LoD 20.96 POS
CU053 Unpreserved 4X LoD 24.41 POS
CU054 Unpreserved 10X LoD 22.37 POS
CU055 Unpreserved Negative 46 NEG
CU056 Unpreserved 2X LoD 25.03 POS
CU057 Unpreserved 50X LoD 22 POS
CU058 Unpreserved 2X LoD 25.57 POS
CU059 Unpreserved Negative 46 NEG
CU060 Unpreserved Negative 46 NEG
CU061 Unpreserved 2X LoD 23.58 POS
CU062 Unpreserved 100X LoD 21.18 POS
CU063 Unpreserved 2X LoD 25.34 POS
CU064 Unpreserved Negative 46 NEG
CU065 Unpreserved 2X LoD 25.46 POS
CU066 Unpreserved Negative 46 NEG
CU067 Unpreserved Negative 46 NEG
CU068 Unpreserved 2X LoD 23.55 POS
CU069 Unpreserved 50X LoD 20.95 POS CU070 Unpreserved 2X LoD 23.14 POS
CU071 Unpreserved Negative 46 NEG
CU072 Unpreserved 10X LoD 21.97 POS
CU073 Unpreserved Negative 46 NEG
CU074 Unpreserved Negative 46 NEG
CU075 Unpreserved 2X LoD 24.1 POS
CU076 Unpreserved Negative 46 NEG
CU077 Unpreserved 2X LoD 23.25 POS
CU078 Unpreserved 50X LoD 19.7 POS
CU079 Unpreserved 2X LoD 22.96 POS
CU080 Unpreserved Negative 46 NEG
CU081 Unpreserved 2X LoD 25.73 POS
CU082 Unpreserved Negative 46 NEG
CU083 Unpreserved Negative 46 NEG
CU084 Unpreserved Negative 46 NEG
CU085 Unpreserved 10X LoD 21.41 POS
CU086 Unpreserved Negative 46 NEG
CU087 Unpreserved Negative 46 NEG
CU088 Unpreserved 4X LoD 23.59 POS
CU089 Unpreserved 100X LoD 18.47 POS
CU090 Unpreserved 2X LoD 23.44 POS
CU091 Unpreserved Negative 46 NEG
CU092 Unpreserved Negative 46 NEG
CU093 Unpreserved Negative 46 NEG
CU094 Unpreserved 2X LoD 24.55 POS
CU095 Unpreserved 50X LoD 18.7 POS
CU096 Unpreserved 2X LoD 24.53 POS
CU097 Unpreserved Negative 46 NEG
CU098 Unpreserved Negative 46 NEG
CU099 Unpreserved 10X LoD 21.36 POS
CUIOO Unpreserved Negative 46 NEG
[0120] As shown in Table 13, 100% of the spiked specimens were positive and 100% of non-spiked specimens were negative. As such, it is contemplated that methods of detecting E. histolytica nucleic acid in accordance with some embodiments herein are robust and accurate among samples that contain E. histolytica, as well as samples that do not contain E. histolytica.
[0121] With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity. [0122] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles "a" or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an" (e.g., "a" and/or "an" should be interpreted to mean "at least one" or "one or more"); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of "two recitations," without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., " a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to "at least one of A, B, or C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., " a system having at least one of A, B, or C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."
[0123] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.
[0124] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as "up to," "at least," "greater than," "less than," and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.
[0125] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.
[0126] While the present invention has been described in some detail for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope of the invention.
[0127] The term "comprising" as used herein is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. [0128] All numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth herein are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of any claims in any application claiming priority to the present application, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.
[0129] The above description discloses several methods and materials of the present invention. This invention is susceptible to modifications in the methods and materials, as well as alterations in the fabrication methods and equipment. Such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the invention disclosed herein. Consequently, it is not intended that this invention be limited to the specific embodiments disclosed herein, but that it cover all modifications and alternatives coming within the true scope and spirit of the invention.
[0130] The foregoing description and Examples detail certain embodiments. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the invention may be practiced in many ways and the invention should be construed in accordance with the appended claims and any equivalents thereof.

Claims

WHAT IS CLAIMED IS:
1. A method of detecting the presence of an E. histolytica polynucleotide sequence in a sample, the method comprising:
contacting the sample with a first primer consisting essentially of SEQ ID NO: 1 (GTACAAAATGGCCAATTCATTCAATG);
contacting the sample with a second primer consisting essentially of SEQ ID NO: 2 (ACTACCAACTGATTGATAGATCAG);
extending the first and second primer, thereby producing at least one amplicon if the E. histolytica polynucleotide sequence is present in the sample; and
contacting the sample with an oligonucleotide probe comprising a polynucleotide consisting essentially of SEQ ID NO: 3 (ATTGTCGTGGCATCCTAACTCA) or its complement,
wherein the probe provides detectable signal when it is bound to a substantially complementary nucleic acid, but does not provide detectable signal when it is single-stranded, and
detecting the signal, if the amplicon is present.
2. The method of claim 1, wherein, if used under standard amplification conditions, the first primer and second primer amplify the E. histolytica polynucleotide sequence, but do not substantially amplify any E. dispar polynucleotide sequence;
3. The method of any one of claims 1-2, wherein the first primer hybridizes to the E. histolytica polynucleotide sequence if contacted with the E. histolytica polynucleotide sequence at a temperature of at least about 50°C in 5 mM MgC12, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA, but does not hybridize to any E. dispar polynucleotide sequence if contacted with any E. dispar polynucleotide sequence at a temperature of at least about 60°C in 5 mM MgCl2, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA.
4. The method of any one of claims 1-3, wherein the second primer hybridizes to the E. histolytica polynucleotide sequence if contacted with E. histolytica polynucleotide sequence at a temperature of at least about 60°C in 5 mM MgC12, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA, and hybridizes to an E. dispar polynucleotide sequence if contacted with the E. dispar polynucleotide sequence at a temperature of at least about 60°C in 5 mM MgC12, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA.
5. The method of any of claims 1-3, wherein each of the first primer and second primer hybridizes to the E. histolytica polynucleotide sequence if contacted with the E. histolytica polynucleotide sequence at a temperature of at least about 60°C in in 5 mM MgC12, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA, but the second primer does not hybridize to any E. dispar polynucleotide sequence if contacted with any E. dispar polynucleotide sequence at a temperature of at least about 60°C in 5 mM MgC12, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA.
6. The method of any one of claims 1-5, wherein the sample comprises E. histolytica and E. dispar.
7. The method of any one of claims 1-6, wherein the sample comprises fecal material of a human.
8. The method of any one of claims 1-7, wherein the sample comprises fixed material.
9. The method of any one of claims 1-7, wherein the sample is non-fixed.
10. The method of any one of claims 1-9, wherein a 95% limit of detection for E. histolytica comprises no more than about 17 E. histolytica genomes per milliliter.
1 1. The method of any of claims 1-10, wherein if used under standard amplification conditions, the primers and probes do not cross-react with any of the following organisms, if present in the sample: Abiotrophia defectiva, Acinetobacter baumannii, Acinetobacter Iwoffli, Aeromonas hydrophila, Alcaligenes faecalis subsp. faecalis, Anaerococcus tetradius, Arcobacter butzleri, Arcobacter cryaerophilus, Bacillus cereus, Bacteroides caccae, Bacteroides merdae, Bacteroides stercoris, Bifidobacterium adolescentis, Bifidobacterium longum, Camplylobacter coli, Campylobacter concisus, Campylobacter curvus, Campylobacter fetus subsp. fetus, Campylobacter fetus subsp. venerealis, Campylobacter gracilis, Campylobacter hominis, Camplylobacter jejuni, Campylobacter lari, Campylobacter rectus, Campylobacter upsaliensis, Candida albicans, Candida catenulate, Cedecea davisae, Chlamydia trachomatis, Citrobacter amalonaticus, Citrobacter fruendii, Citrobacter koseri, Citrobacter sedlakii, Clostridium difficile 17858, Clostridium difficile 43598, Clostridium difficile CCUG 8864-9689, Clostridium difficile 43255, Clostridium difficile BAA-1805, Clostridium difficile 43593, Clostridium perfringens, Collinsella aerofaciens, Corynebacterium genitalium, Desulfovibrio piger, Edwardsiella tarda, Eggerthella lenta, Enterobacter aerogenes, Enterobacter cloacae, Enterococcus casseliflavus, Enterococcus cecorum, Enterococcus dispar, Enterococus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus hirae, Enterococcus raffinosus, Escherichia coli, Escherichia fergusonii, Escherichia hermannii, Escherichia vulneris, Fusobacterium varium, Gardnerella vaginalis, Gemella morbillorum, Hafnia alvei, Helicobacter fennelliae, Helicobacter pylori, Klebsiella oxytoca, Klebsiella pneumonia, Lactobacillus acidophilus, Lactobacillus reuteri, Lactococcus lactis, Leminorella grimontii, Listeria grayi, Listeria innocua, Listeria monocytogenes, Morganella morganii, Peptoniphilus asaccharolyticus, Peptostreptococcus anaerobius, Plesiomonas shigelloides, Porphyromonas asaccharolytica, Prevotella melaninogenica, Proteus mirabilis, Proteus penneri, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii, Pseudomonas aeruginosa, Pseudomonas fluorescens, Ruminococcus bromii, Salmonella typhimurium, Salmonella enteriditis, Serratia liquefaciens, Serratia marcescens, Shigella sonnei, Shigella flexneri, Staphylococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus intermedius, Streptococcus uberis, Trabulsiella guamensis, Veillonella parvula, Vibrio cholera, Vibrio parahaemolyticus, Yersinia bercovieri, Yersinia enterocolitica, Yersinia rohdei, Adenovirus type 2, Adenovirus type 14, Adenovirus type 40, Adenovirus type 41, Coxsackie A9, Coxsackie Bl, HHV-5, Cytomegalovirus, Enterovirus type 69, Human Papillomavirus Type 16, Human Papillomavirus Type 18, Herpes Simplex Virus I, Herpes Simplex Virus II, Norovirus I, Norovirus II, Rotavirus, Blastocystis hominis, Encephalitozoon intestinalis, Encephalitozoon helium, Encephalitozoon cuniculi, Pentatrichomonas hominis, Entamoeba barrette, Entamoeba dispar, Entamoeba gigivalis, Entamoeba invadens, Entamoeba moshkovskii, Entamobea ranarum, Citrobacter fruendii (rpt), Enterobacter cloacae (rpt), Cryptosporidium parvum, Giardia lamblia, or Cryptosporidium meleagridis.
12. A kit comprising:
a first primer;
a second primer,
wherein, if used under standard amplification conditions, the first primer and second primer amplify a E. histolytica polynucleotide sequence, thereby producing an amplicon, but do not substantially amplify any E. dispar polynucleotide sequence; and
a probe, wherein the probe comprises a polynucleotide consisting essentially of a sequence, wherein the sequence or its complement is present in each of the amplicon, a polynucleotide sequence of E. histolytica, and a polynucleotide sequence of E. dispar.
13. The kit of claim 12, wherein the probe comprises:
a fluorophore; and
a quencher.
14. The kit of any one of claims 12-13, wherein the primers and probes amplify an E. histolytica polynucleotide sequence with a 95% limit of detection of no more than about 17 E. histolytica organisms per mililiter.
15. The kit of any one of claims 12-14, wherein if used under standard amplification conditions, the primers and probes do not cross-react with any of the following organisms, if present in the sample: Abiotrophia defectiva, Acinetobacter baumannii, Acinetobacter Iwqffii, Aeromonas hydrophila, Alcaligenes faecalis subsp. faecalis, Anaerococcus tetradius, Arcobacter butzleri, Arcobacter cryaerophilus, Bacillus cereus, Bacteroides caccae, Bacteroides merdae, Bacteroides stercoris, Bifidobacterium adolescentis, Bifidobacterium longum, Camplylobacter coli, Campylobacter concisus, Campylobacter curvus, Campylobacter fetus subsp. fetus, Campylobacter fetus subsp. venerealis, Campylobacter gracilis, Campylobacter hominis, Campylobacter jejuni, Campylobacter lari, Campylobacter rectus, Campylobacter upsaliensis, Candida albicans, Candida catenulate, Cedecea davisae, Chlamydia trachomatis, Citrobacter amalonaticus, Citrobacter fruendii, Citrobacter koseri, Citrobacter sedlakii, Clostridium difficile 17858, Clostridium difficile 43598, Clostridium difficile CCUG 8864-9689, Clostridium difficile 43255, Clostridium difficile BAA-1805, Clostridium difficile 43593, Clostridium perfringens, Collinsella aerofaciens, Corynebacterium genitalium, Desulfovibrio piger, Edwardsiella tarda, Eggerthella lenta, Enterobacter aerogenes, Enterobacter cloacae, Enterococcus casseliflavus, Enterococcus cecorum, Enterococcus dispar, Enterococus faecalis, Enterococcus gallinarum, Enterococcus hirae, Enterococcus raffinosus, Escherichia coli, Escherichia fergusonii, Escherichia hermannii, Escherichia vulneris, Fusobacterium varium, Gardnerella vaginalis, Gemella morbillorum, Hafnia alvei, Helicobacter fennelliae, Helicobacter pylori, Klebsiella oxytoca, Klebsiella pneumonia, Lactobacillus acidophilus, Lactobacillus reuteri, Lactococcus lactis, Leminorella grimontii, Listeria grayi, Listeria innocua, Listeria monocytogenes, Morganella morganii, Peptoniphilus asaccharolyticus, Peptostreptococcus anaerobius, Plesiomonas shigelloides, Porphyromonas asaccharolytica, Prevotella melaninogenica, Proteus mirabilis, Proteus penneri, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii, Pseudomonas aeruginosa, Pseudomonas fluorescens, Ruminococcus bromii, Salmonella typhimurium, Salmonella enteriditis, Serratia liquefaciens, Serratia marcescens, Shigella sonnei, Shigella flexneri, Staphylococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus intermedius, Streptococcus uberis, Trabulsiella guamensis, Veillonella parvula, Vibrio cholera, Vibrio parahaemolyticus, Yersinia bercovieri, Yersinia enterocolitica, Yersinia rohdei, Adenovirus type 2, Adenovirus type 14, Adenovirus type 40, Adenovirus type 41, Coxsackie A9, Coxsackie Bl, HHV-5, Cytomegalovirus, Enterovirus type 69, Human Papillomavirus Type 16, Human Papillomavirus Type 18, Herpes Simplex Virus I, Herpes Simplex Virus II, Norovirus I, Norovirus II, Rotavirus, Blastocystis hominis, Encephalitozoon intestinalis, Encephalitozoon helium, Encephalitozoon cuniculi, Pentatrichomonas hominis, Entamoeba barrette, Entamoeba dispar, Entamoeba gigivalis, Entamoeba invadens, Entamoeba moshkovskii, Entamobea ranarum, Citrobacter fruendii (rpt), Enterobacter cloacae (rpt), Cryptosporidium parvum, Giardia lamblia, or Cryptosporidium meleagridis.
16. A kit comprising:
a first primer comprising a polynucleotide having at least about 90% identity to SEQ ID NO: 1 (GTACAAAATGGCCAATTCATTCAATG);
a second primer comprising polynucleotide having at least about 90% identity to SEQ ID NO: 2 (ACTACCAACTGATTGATAGATCAG); and
a probe comprising:
a polynucleotide having at least about 90% identity to SEQ ID NO: 3 (ATTGTCGTGGCATCCTAACTCA) or its complement;
a flurophore; and
a quencher.
17. The kit of claim 16, wherein
the first primer consists essentially of SEQ ID NO: 1 (GTACAAAATGGCCAATTCATTCAATG),
the second primer consists essentially of SEQ ID NO: 2 (ACTACCAACTGATTGATAGATCAG); and
the probe comprises a polynucleotide consisting essentially of SEQ ID NO: 3 (ATTGTCGTGGCATCCTAACTCA) or its complement.
18. A method of detecting the presence of an E. histolytica polynucleotide sequence in a sample, the method comprising:
contacting the sample with a first primer;
contacting the sample with a second primer,
wherein, under if used standard amplification conditions, the first primer and second primer amplify the E. histolytica polynucleotide sequence, but do not substantially amplify any E. dispar polynucleotide sequence;
extending the first and second primer, thereby producing at least one amplicon if the E. histolytica polynucleotide sequence is present in the sample; and
contacting the sample with an oligonucleotide probe, wherein the probe provides detectable signal when it is bound to a substantially complementary nucleic acid, but does not provide detectable signal when it is single-stranded, and
wherein the probe comprises a polynucleotide consisting essentially of sequence that is a portion of the E. histolytica polynucleotide sequence, a polynucleotide sequence of E. dispar, and a sequence of the amplicon; and detecting the signal, if the amplicon is present.
19. The method of claim 18, wherein the first primer hybridizes to the E. histolytica polynucleotide sequence if contacted with the E. histolytica polynucleotide sequence at a temperature of at least about 50°C in 5 mM MgC12, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA, but does not hybridize to any E. dispar polynucleotide sequence if contacted with any E. dispar polynucleotide sequence at a temperature of at least about 60°C in 5 mM MgCl2, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA.
20. The method of any one of claims 18-19, wherein the second primer hybridizes to the E. histolytica polynucleotide sequence if contacted with E. histolytica polynucleotide sequence at a temperature of at least about 60°C in 5 mM MgC12, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA, and hybridizes to an E. dispar polynucleotide sequence if contacted with the E. dispar polynucleotide sequence at a temperature of at least about 60°C in 5 mM MgC12, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA.
21. The method of any one of claims 18-20, wherein each of the first primer and second primer hybridizes to the E. histolytica polynucleotide sequence if contacted with the E. histolytica polynucleotide sequence at a temperature of at least about 60°C in in 5 mM MgC12, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA, but the second primer does not hybridize to any E. dispar polynucleotide sequence if contacted with any E. dispar polynucleotide sequence at a temperature of at least about 50°C in 5 mM MgC12, 100 mM Tris, 10 mM NaOH, 0.019% ProClin300, 0.010% Tween-20, 1.96% Trehalose, 0.6 mg/ml BSA.
22. The method of any one of claims 18-21 or 40-75, wherein the first primer comprises a polynucleotide having at least about 90% identity to SEQ ID NO: 1 (GTACAAAATGGCCAATTCATTCAATG) or its complement.
23. The method of any one of claims 18-22, wherein the first primer consists essentially of SEQ ID NO: 1 (GTACAAAATGGCCAATTCATTCAATG) or its complement.
24. The method of any one of claims 18-23, or 40-75, wherein the second primer comprises a polynucleotide having at least about 90% identity to SEQ ID NO: 2 (ACTACCAACTGATTGATAGATCAG) or its complement.
25. The method of any of claims 18-24, or 40-75, wherein the second primer comprises a polynucleotide having the sequence of SEQ ID NO: 2 (ACTACCAACTGATTGATAGATCAG) or its complement.
26. The method of any of claims 18-25, or 40-75, wherein the probe comprises a polynucleotide having at least about 90% identity to SEQ ID NO: 3 (ATTGTCGTGGCATCCTAACTCA) or its complement.
27. The method of any of claims 18-26, or 40-75, wherein the probe comprises a polynucleotide having the sequence of SEQ ID NO: 3 (ATTGTCGTGGCATCCTAACTCA) or its complement.
28. The method of any of claims 18-27, or 40-75, wherein the amplicon comprises a polynucleotide having at least about 95% identity to SEQ ID NO: 7 (GTACAAAATGGCCAATTCATTCAATGAATTGAGAAATGACATTCTAAGTGAG TTAGGATGCCACGACAATTGTAGAACACACAGTGTTTAACAAGTAACCAATG AGAATTTCTGATCTATCAATCAGTTGGTAGT).
29. The method of any of claims 18-28, or 40-75, wherein the amplicon comprises a polynucleotide having the sequence of SEQ ID NO: 7 (GTACAAAATGGCCAATTCATTCAATGAATTGAGAAATGACATTCTAAGTGAG TTAGGATGCCACGACAATTGTAGAACACACAGTGTTTAACAAGTAACCAATG AGAATTTCTGATCTATCAATCAGTTGGTAGT).
30. The method of any of claims 18-29, or 40-75, wherein the sample comprises E. histolytica and E. dispar.
31. The method of any of claims 18-30, or 40-75, wherein the sample comprises fecal material of a human.
32. The method of any of claims 18-31, or 40-75, wherein the sample comprises fixed material.
33. The method of any of claims 18-32, or 40-75, wherein the sample is non- fixed.
34. The method of any of claims 18-33, or 40-75, wherein a 95% limit of detection for E. histolytica comprises no more than about 17 E. histolytica genomes per milliliter.
35. The method of any of claims 18-34, or 40-75, wherein if used under standard amplification conditions, the primers and probes do not cross-react with any of the following organisms, if present in the sample: Abiotrophia defectiva, Acinetobacter baumannii, Acinetobacter Iwoffli, Aeromonas hydrophila, Alcaligenes faecalis subsp. faecalis, Anaerococcus tetradius, Arcobacter butzleri, Arcobacter cryaerophilus, Bacillus cereus, Bacteroides caccae, Bacteroides merdae, Bacteroides stercoris, Bifidobacterium adolescentis, Bifidobacterium longum, Camplylobacter coli, Campylobacter concisus, Campylobacter curvus, Campylobacter fetus subsp. fetus, Campylobacter fetus subsp. venerealis, Campylobacter gracilis, Campylobacter hominis, Camplylobacter jejuni, Campylobacter lari, Campylobacter rectus, Campylobacter upsaliensis, Candida albicans, Candida catenulate, Cedecea davisae, Chlamydia trachomatis, Citrobacter amalonaticus, Citrobacter fruendii, Citrobacter koseri, Citrobacter sedlakii, Clostridium difficile 17858, Clostridium difficile 43598, Clostridium difficile CCUG 8864-9689, Clostridium difficile 43255, Clostridium difficile BAA-1805, Clostridium difficile 43593, Clostridium perfringens, Collinsella aerofaciens, Corynebacterium genitalium, Desulfovibrio piger, Edwardsiella tarda, Eggerthella lenta, Enterobacter aerogenes, Enterobacter cloacae, Enterococcus casseliflavus, Enterococcus cecorum, Enterococcus dispar, Enterococus faecalis, Enterococcus gallinarum, Enterococcus hirae, Enterococcus raffinosus, Escherichia coli, Escherichia fergusonii, Escherichia hermannii, Escherichia vulneris, Fusobacterium varium, Gardnerella vaginalis, Gemella morbillorum, Hafnia alvei, Helicobacter fennelliae, Helicobacter pylori, Klebsiella oxytoca, Klebsiella pneumonia, Lactobacillus acidophilus, Lactobacillus reuteri, Lactococcus lactis, Leminorella grimontii, Listeria grayi, Listeria innocua, Listeria monocytogenes, Morganella morganii, Peptoniphilus asaccharolyticus, Peptostreptococcus anaerobius, Plesiomonas shigelloides, Porphyromonas asaccharolytica, Prevotella melaninogenica, Proteus mirabilis, Proteus penneri, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii, Pseudomonas aeruginosa, Pseudomonas fluorescens, Ruminococcus bromii, Salmonella typhimurium, Salmonella enteriditis, Serratia liquefaciens, Serratia marcescens, Shigella sonnei, Shigella flexneri, Staphylococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus intermedius, Streptococcus uberis, Trabulsiella guamensis, Veillonella parvula, Vibrio cholera, Vibrio parahaemolyticus, Yersinia bercovieri, Yersinia enterocolitica, Yersinia rohdei, Adenovirus type 2, Adenovirus type 14, Adenovirus type 40, Adenovirus type 41, Coxsackie A9, Coxsackie Bl, HHV-5, Cytomegalovirus, Enterovirus type 69, Human Papillomavirus Type 16, Human Papillomavirus Type 18, Herpes Simplex Virus I, Herpes Simplex Virus II, Norovirus I, Norovirus II, Rotavirus, Blastocystis hominis, Encephalitozoon intestinalis, Encephalitozoon helium, Encephalitozoon cuniculi, Pentatrichomonas hominis, Entamoeba barrette, Entamoeba dispar, Entamoeba gigivalis, Entamoeba invadens, Entamoeba moshkovskii, Entamobea ranarum, Citrobacter fruendii (rpt), Enterobacter cloacae (rpt), Cryptosporidium parvum, Giardia lamblia, or Cryptosporidium meleagridis.
36. The method of any one of claims 1-11 or 18-35 or 40-75, wherein if used under standard amplification conditions, the primers and probes do not cross-react with any of the following organisms, if present in the sample: Entamoeba coli, Entamoeba dispar, Entamoeba polecki, Entamoeba muris, Entamoeba nuttalli, Entamoeba hartmanni, and Entamoeba bovis.
37. The method of any one of claims 1-11 or 18-36 or 40-75, wherein if used under standard amplification conditions, the primers and probes produce fewer than 1 in 1600 false positives for samples that do not comprise E. histolytica.
38. The kit of any one of claims 12-17, wherein if used under standard amplification conditions, the primers and probes do not cross-react with any of the following organisms, if present in the sample: Entamoeba coli, Entamoeba dispar, Entamoeba polecki, Entamoeba muris, Entamoeba nuttalli, Entamoeba hartmanni, and Entamoeba bovis.
39. The kit of any one of claims 12-17 or 38, wherein if used under standard amplification conditions, the primers and probes produce fewer than 1 in 1600 false positives for samples that do not comprise E. histolytica.
40. A method of determining the presence or absence of an E. histolytica nucleic acid sequence in a sample, the method comprising:
performing a nucleic acid amplification reaction on the sample, the nucleic acid amplification comprising a first oligonucleotide primer and a second oligonucleotide primer,
wherein the first oligonucleotide primer has a length of 15-75 nucleotides and hybridizes under standard conditions to SEQ ID NO: 10 or its complement, if present,, but does not hybridize under standard conditions to SEQ ID NO: 1 1 or its complement, if present, and
wherein the second oligonucleotide primer has a length of 15-75 nucleotides and hybridizes under standard conditions to a SEQ ID NO: 10 or its complement, if present, and wherein the second oligonucleotide primer hybridizes under standard conditions to SEQ ID NO: 11 or its complement, if present; detecting a signal, if present, from a detectably labeled probe that hybridizes to an amplicon of the the first and second oligonucleotide primers under standard hybridization conditions if the amplicon is present, wherein the signal indicates the presence or absence of the amplicon,
wherein the amplicon has a length of 75-350 nucleotides.
41. The method of claim 40, wherein the first oligonucleotide primer comprises at least 10 consecutive nucleotides of SEQ ID NO: 1, and wherein the first oligonucleotide primer has at least 80% identity to a target sequence of SEQ ID NO: 10 or its complement.
42. The method of claim 40 or claim 41, wherein the second oligonucleotide primer comprises at least 10 consecutive nucleotides of SEQ ID NO: 2, and wherein the second oligonucleotide primer has at least 80% identity to a target sequence of SEQ ID NO: 10 or its complement.
43. The method of claim 41 or claim 42, wherein the first oligonucleotide primer comprises at least 12 consecutive nucleotides of SEQ ID NO: 1.
44. The method of claim 41 or claim 42, wherein the first oligonucleotide primer comprises at least 15 consecutive nucleotides of SEQ ID NO: 1.
45. The method of claim 41 or claim 42, wherein the first oligonucleotide primer comprises at least 20 consecutive nucleotides of SEQ ID NO: 1.
46. The method of any one of claims 41-45, wherein the first oligonucleotide primer has at least 85% identity to a target sequence of SEQ ID NO: 10 or its complement.
47. The method of any one of claims 41-45, wherein the first oligonucleotide primer has at least 90% identity to a target sequence of SEQ ID NO: 10 or its complement.
48. The method of any one of claims 41-45, wherein the first oligonucleotide primer has at least 95% identity to a target sequence of SEQ ID NO: 10 or its complement.
49. The method of any one of claims 41-45, wherein the first oligonucleotide primer has 100% identity to a target sequence of SEQ ID NO: 10 or its complement.
50. The method of any one of claims 42-49, wherein the second oligonucleotide primer comprises at least 12 consecutive nucleotides of SEQ ID NO: 2.
51. The method of any one of claims 42-49, wherein the second oligonucleotide primer comprises at least 15 consecutive nucleotides of SEQ ID NO: 2.
52. The method of any one of claims 42-49, wherein the second oligonucleotide primer comprises at least 20 consecutive nucleotides of SEQ ID NO: 2.
53. The method of any one of claims 42-52, wherein the second oligonucleotide primer has at least 85% identity to a target sequence of SEQ ID NO: 10 or its complement.
54. The method of any one of claims 42-52, wherein the second oligonucleotide primer has at least 90% identity to a target sequence of SEQ ID NO: 10 or its complement.
55. The method of any one of claims 42-52, wherein the second oligonucleotide primer has at least 95% identity to a target sequence of SEQ ID NO: 10 or its complement.
56. The method of any one of claims 42-52, wherein the second oligonucleotide primer has 100% identity to a target sequence of SEQ ID NO: 10 or its complement.
57 The method of any one of claims 40-56, wherien the probe comprises at least 10 consecutive nucleotides of SEQ ID NO: 3, and wherien the probe has at least 80% identity to a target sequence of SEQ ID NO: 10 or its complement.
58. The method of claim 57, wherein the probe comprises at least 12 consecutive nucleotides of SEQ ID NO: 3.
59. The method of claim 57, wherein the probe comprises at least 15 consecutive nucleotides of SEQ ID NO: 3.
60. The method of claim 57 wherein the probe comprises at least 20 consecutive nucleotides of SEQ ID NO: 3.
61. The method of any one of claims 57-60, wherein the probe has at least 85% identity to a target sequence of SEQ ID NO: 10 or its complement.
62. The method of any one of claims 57-60, wherein the probe has at least 90% identity to a target sequence of SEQ ID NO: 10 or its complement.
63. The method of any one of claims 57-60, wherein the probe has at least 95% identity to a target sequence of SEQ ID NO: 10 or its complement.
64. The method of any one of claims 57-60, wherein the probe has 100% identity to a target sequence of SEQ ID NO: 10 or its complement.
65. The method of any one of claims 40-64, wherein the first oligonucleotide primer is about 20-50 nucleotides long.
66. The method of any one of claims 40-64, wherein the first oligonucleotide primer is about 23-45 nucleotides long.
67. The method of any one of claims 40-66, wherein the second oligonucleotide primer is about 20-50 nucleotides long.
68. The method of any one of claims 40-66, wherein the second oligonucleotide primer is about 23-45 nucleotides long.
69. The method of any one of claims 40-68, wherein the detectably labeled probe is about 15-75 nucleotides long.
70. The method of any one of claims 40-68, wherein the detectably labeled probe is about 20-45 nucleotides long.
71. The method of any one of claims 40-70 wherein the detectably labeled probe probe is capable of hybridizing to SEQ ID NO: 10 and toSEQ ID NO: 11 under standard hybridization conditions.
72. The method of any one of claims 40-70, wherein the detectably labeled probe probe is capable of hybridizing to SEQ ID NO: 10 but not toSEQ ID NO: 1 1 under standard hybridization conditions.
73. The method of any one of claims 40-72, wherien the detectably labeled probe probe comprises a fluorophore or a quencher.
74. The method of any one of claims 40-73, wherein the amplicon has a length of 100- 150 nucleotides.
75. The method of any one of claims 40-74, wherein the amplicon comprises SEQ ID NO: 7.
76. The method of any of claims 18-26, or 40-75, wherein E. dispar, if present, does not inhibit determining the presence or absence of E. histolytica.
77. The method of any of claims 18-26, wherein E. dispar, if present, does not inhibit production of the amplicon if the E. histolytica polynucleotide sequence is present in the sample.
78. The method of any of claims 40-75, wherein E. dispar, if present, does not inhibit production of the amplicon of the the first and second oligonucleotide primers.
79. A kit comprising the first oligonucleotide primer, the second oligonucleotide primer, and the detectably labeled probe of any one of claims 40-77.
80. The kit of any of claims 12-17, 38, or 79, wherein E. dispar, if present, does not inhibit determining the presence or absence of E. histolytica.
81. The kit of any of claims 12-17, 38, or 79, wherein E. dispar, if present, does not inhibit production of the amplicon if the E. histolytica polynucleotide sequence is present in the sample.
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