WO2012009813A1 - Procédés de détection de polymorphismes génétiques à l'aide de la température de fusion de complexes sonde—amplicon - Google Patents

Procédés de détection de polymorphismes génétiques à l'aide de la température de fusion de complexes sonde—amplicon Download PDF

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WO2012009813A1
WO2012009813A1 PCT/CA2011/050443 CA2011050443W WO2012009813A1 WO 2012009813 A1 WO2012009813 A1 WO 2012009813A1 CA 2011050443 W CA2011050443 W CA 2011050443W WO 2012009813 A1 WO2012009813 A1 WO 2012009813A1
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probe
sequence
seq
primer
target
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Eric Frost
Sylvie Deslandes
Patrick Dextras Paquette
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Socpra Sciences Sante Et Humaines, S.E.C.
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    • 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
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
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    • 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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • 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
    • C12Q2600/156—Polymorphic or mutational markers
    • 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

Definitions

  • SNP single nucleotide polymorphisms
  • SNP single nucleotide polymorphisms
  • Single nucleotide polymorphisms (SNP) in bacterial or viral genes are a common cause of the acquisition of clinically significant resistance to antibiotics or antivirals. They are also at the origin of strain variation.
  • Single nucleotide polymorphisms (SNP) in bacterial genes are the most common cause of the acquisition of clinically significant resistance to the antibiotics usually employed to treat infections due to Campylobacter jejuni (C. jejuni), the most frequent bacterial cause of diarrhea.
  • SNPs can have multiple diagnostic applications so numerous strategies have been devised for their detection. Some involve temperature specific annealing of a primer or a probe to the target sequence containing the SNP. When both primer and target are derived from wild type sequences or both from the mutant sequence the annealing temperature is usually several degrees higher than when one comes from the mutant and the other from wild type. Small differences in annealing temperature between heterologous and homologous primer and target pairs can be enhanced by using locked nucleic acids at the SNP site or minor groove binders nearby.
  • Invader assaysTM or with TaqMan, Molecular Beacon, or fluorescent resonant energy transfer (FRET) probes by performing the detection step at a temperature in the window between the homologous and heterologous annealing temperatures.
  • Allele specific PCR or ligation can be used with primers with the SNP at the 3' end of the primer.
  • SNPs form part of a naturally occurring or artificially engineered restriction endonuclease site, digestion of the amplicon with such enzymes can reveal the SNP.
  • the melting temperature of FRET probes or the high resolution melting (HRM) profiles of short amplicons can also infer sequence data revealing SNP. When all else fails, amplicons can be sequenced, pyrosequenced, or hybridized to specific probes.
  • TaqManTM probes are usually designed to be located near one of the PCR primers so that when the Taq polymerase reaches the probe, it will cleave its 5' end. This will separate the fluorophore attached to the 5' end of the probe and notably from a strong quencher attached to the 3' end that would normally absorb all the light emitted from the fluorophore.
  • signal acquisition should be performed at the annealing temperature of 60°C rather than at the extension temperature of 72°C.
  • the 5' endonuclease activity is expected to reach its maximum at 72°C, while at 60°C only the fluorophores cleaved during the preceding cycle are expected to be detected. Consequently, it is noted that when the TaqManTM probe anneals to its target (thereby separating the 5' from the 3' end), even in the presence of a strong quencher, that light might escape and could be detected.
  • Molecular BeaconsTM probes have complementary sequences at the ends, often referred to as “panhandles", which when hybridized together bring a fluorophore at the 5' end in very close proximity to a quencher at the 3' end. Measurable light is emitted from the fluorophore only when the probe hybridizes to its target and the 5' and 3' ends are spatially separated.
  • Molecular BeaconsTM probes are designed so that their complementary ends reanneal near the annealing temperature of the PCR reaction. Thus, melting temperature curves could not usefully be performed below this PCR annealing temperature.
  • a recent modification called "sloppy" Molecular BeaconsTM which are characterized by relatively long probe sequences, have been used with melting temperature determinations.
  • FRET probes involve transfer of light not to a quencher but rather to a second fluorophore that is not excited by the wave length used by the analyzer which excites only the fluorophore fluorescein.
  • the second fluorophore then absorbs the light emitted by the fluorescein moiety to emit at a longer wave length. This occurs when two probes hybridize to adjoining sequences on the target such that the 3' end of one is very near the 5' end of the other.
  • the use of FRETTM probes in melting temperature determinations may fail probably due to the 5' endonuclease activity of the polymerase.
  • High resolution melting or HRM is a method based on PCR amplification of a short sequence and the use of a double-stranded specific fluorescent dye. Briefly, the sequence is amplified and the dye inserts within the double-stranded amplicon. Following PCR amplification, a very slow temperature ramping induced amplicon melting is performed and fluorescence (or loss thereof) is measured. Since each DNA amplicon possess a unique denaturation pattern and the presence of a polymorphism can modify the amplicon's denaturation pattern, high resolution melting can be used to distinguish between sequences differing in their nucleotide sequence. However, this procedure requires a technically demanding analysis protocol and controls representing each potential target. An adaptation of this method using unlabeled probes representing short specific sequences has also been described, but separate wells must be used for each probe and the presence of the full length amplicon tended to mask the fluorescent peaks obtained with the probe.
  • a probe is contacted with the unknown sequence so as to form a complex and the melting of the complex is determined and compared to the melting temperature of a complex between the known sequence and the probe.
  • the present application provides a method of detecting a nucleotide difference between a target sequence and a first standard sequence.
  • the method comprises: amplifying the target sequence to generate a target amplicon; contacting the target amplicon with a first probe to form a first target amplicon-probe complex, wherein the first probe has a first label covalently linked to the one end of the probe and a first corresponding quencher linked to the other end of the probe; measuring the signal from the first label of the first probe within a range of temperature to determine the melting temperature of the first target amplicon- probe complex; providing the melting temperature of a complex between a corresponding amplicon of the first standard sequence and the first probe; comparing the melting temperature of the first target amplicon-probe complex with the melting temperature of standard sequence amplicon-probe complex; and detecting the presence of the nucleotide difference if the melting temperature of the first target amplicon-probe complex is lower than the melting temperature of the standard sequence amplicon-probe complex, or the absence of the nucleotide difference if the melting temperature of the first target amplicon-probe complex is substantially similar to
  • the sequence of the first probe is substantially identical to a fragment of the corresponding amplicon of the first standard sequence.
  • the method further comprises contacting the target amplicon with a second probe to form a second target amplicon-probe complex, wherein the second probe has a second label covalently linked to one end of the probe and a second corresponding quencher linked to the other end of the probe and wherein the first and second label are different; measuring the signal from the second label of the second probe within a range of temperature to determine the melting temperature of the second target amplicon-probe complex; providing the melting temperature of a complex between a corresponding amplicon of the first standard sequence and the second probe; determining a first difference in the melting temperature of the first target amplicon-probe complex and the melting temperature of standard amplicon-probe complex; determining a second difference in the melting temperature of the second amplicon-probe complex with the standard amplicon-second probe complex; determining which of the first difference or the second difference is lower; and assessing
  • the sequence of the second probe is substantially identical to a fragment of a second standard sequence and wherein the second standard sequence differs by at least one nucleotide from the first standard sequence.
  • the second standard sequence differs by at least two, at least three, at least four or at least five nucleotides from the first standard sequence.
  • the first difference in melting temperature and the second difference in melting temperature is equal to or lower than about 25°C, 10°C, 5°C or between about 2°C to 4°C. ln still a further embodiment, the amplification step comprises a polymerase chain reaction.
  • a first primer is used for the polymerase chain reaction and said first primer is substantially identical to a first portion of the target sequence or complement thereof and a first corresponding portion of the first standard sequence or complement thereof and is non-linear to the first and/or second probe.
  • a second primer is used for the polymerase chain reaction and said second primer is substantially identical to a second portion of the target sequence or a complement thereof and a second corresponding portion of the first standard sequence or a complement thereof and is co-linear to the first and/or second probe.
  • the concentration of the first primer is higher than the concentration of the second primer in the polymerase chain reaction.
  • the first and/or second probe is present during the polymerase chain reaction and the concentration of the first and/or second probe is higher than the concentration of the second primer.
  • the range of temperature for determining the melting temperature is between about 30°C to about 95°C or about 45°C to about 70°C.
  • the nucleotide difference is a single nucleotide polymorphism.
  • the target sequence and the first standard sequence are derived from Campylobacter jejuni.
  • the target sequence and the first standard sequence can be derived from a 23S ribosomal RNA gene (wherein the first primer comprises the sequence of SEQ ID NO: 1 , SEQ ID NO: 2, or a complement thereof; wherein the second primer comprises the sequence of SEQ ID NO: 1 , SEQ ID NO: 2, or a complement thereof; and/or wherein the first probe and/or the second probe comprises at least one of the following sequences: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and complementary sequences thereof).
  • the target sequence and the first standard sequence are derived from a gyrA gene (wherein the first primer comprises the sequence of SEQ ID NO: 6, SEQ ID NO: 7, or a complement thereof; wherein the second primer comprises the sequence of of SEQ ID NO: 6, SEQ ID NO: 7, or a complement thereof; and/or the first probe and/or the second probe comprises is at least one of the following sequences: SEQ ID NO: 8, SEQ ID NO: 9 and complementary sequences thereof)
  • the target sequence and the first standard sequence can be derived from Influenza virus.
  • the target sequence and the first standard sequence can be from a matrix gene (wherein the first primer comprises the sequence of SEQ ID NO: 10, SEQ ID NO: 11 , or a complement thereof; wherein the second primer comprises the sequence of (i of SEQ ID NO: 10, SEQ ID NO: 11 , or a complement thereof ; and/or wherein the first probe and/or the second probe comprises at least one of the following sequences: SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and complementary sequences thereof).
  • the target sequence and the first standard sequence can be from a neuraminidase gene, (wherein the first primer comprises the sequence of SEQ ID NO: 12, SEQ ID NO: 13, or complement thereof; wherein the second primer comprises the sequence of SEQ ID NO: 12, SEQ ID NO: 13, or complement thereof; and/or wherein the first probe and/or the second probe comprises at least one of the following sequences: SEQ ID NO: 18, SEQ ID NO: 19 and complementary sequences thereof).
  • the target sequence and the first standard sequence can be derived from a 16S ribosomal RNA gene of a bacteria.
  • the bacteria can be Dialister pneumosintes (wherein the first primer comprises the sequence of SEQ ID NO: 31 , SEQ ID NO: 32, or a complement thereof; wherein the second primer comprises the sequence of SEQ ID NO: 31 , SEQ ID NO: 32, or a complement thereof; and/or wherein the first probe and/or the second probe comprises the sequence of SEQ ID NO: 33 or a complementary sequence thereof).
  • the bacteria can be Anaerococcus sp.
  • the bacteria can be Peptoniphilus sp. other than P.
  • the bacteria can be Bifidobacterium sp.
  • first primer comprises the sequence of SEQ ID NO: 40, SEQ ID NO: 41 , or a complement thereof
  • second primer comprises the sequence of SEQ ID NO: 40, SEQ ID NO: 41 , or a complement thereof
  • first probe and/or the second probe comprises the sequence of SEQ ID NO: 33 or a complementary sequence thereof
  • the present application provides a method of detecting a plurality of nucleotide differences between a target sequence and a first standard sequence.
  • the method comprises: amplifying the target sequence to generate a plurality of target amplicons; contacting the plurality of target amplicons with at least two probes to form at least two target amplicon-probe complexes, wherein the at least two probes each have a label covalently linked to the one end of the probe and a corresponding quencher linked to the other end of the probe and each of the at least two probes have a different label; measuring the signal from each label of the at least two probes within at least one temperature range to determine the melting temperatures of the at least two target amplicon-probe complexes; providing the melting temperature of complexes of at leas two corresponding amplicons of the first standard sequence and the at least two probes; comparing the melting temperature of the at least two of target amplicon-probe complexes with the melting temperatures of the corresponding standard sequence.
  • the sequence of the at least two probes are substantially identical to a fragment of a corresponding amplicon of the first standard sequence.
  • the amplification comprises a polymerase chain reaction.
  • a first primer is used for the polymerase chain reaction and said first primer is substantially identical to a first portion of the target sequence or a complement thereof and a first corresponding portion of the first standard sequence or a complement and is non-linear to the first of the at least two probes.
  • a second primer is used for the polymerase chain reaction and said second primer is substantially identical to a second portion of the target sequence or a complement thereof and a second corresponding portion of the first standard sequence or a complement thereof and is co-linear to the first of the at least two probes.
  • a third primer is used for the polymerase chain reaction and said third primer is substantially identical to a second portion of the target sequence or a complement thereof and a second corresponding portion of the first standard sequence or a complement thereof and is non-linear to the second of the at least two probes.
  • a fourth primer is used for the polymerase chain reaction and said fourth primer is substantially identical to a second portion of the target sequence or a complement thereof and a second corresponding portion of the first standard sequence or a complement thereof and is co-linear to the second of the at least two probes.
  • the concentration of the first primer is higher than the concentration of the second primer in the polymerase chain reaction.
  • the concentration of the third primer is higher than the concentration of the fourth primer in the polymerase chain reaction.
  • the at least two probes are present during the polymerase chain reaction.
  • the concentration of the first probe is higher than the concentration of the second primer.
  • the concentration of the second probe is higher than the concentration of the fourth primer.
  • the range of temperature is between about 30°C to about 95°C or between about 45°C to about 70°C.
  • the present application provides a kit for the determination of a nucleotide difference between a target sequence and a standard sequence.
  • the kit comprises a first primer and a second primer for amplifying the target sequence and generate a corresponding target amplicon; and a first probe for forming a first complex with the target amplicon, wherein the first probe has a first label covalently linked to the one end of the probe and a first quencher linked to the other end of the probe and wherein the sequence of the first probe is substantially identical to a portion of the standard sequence, wherein the concentration the concentration of the first probe is higher than the concentration of the second primer.
  • the kit further comprises a second probe for forming a second complex with the target amplicon, wherein the second probe has a second label covalently linked to the one end of the probe and a second corresponding quencher linked to the other end of the probe, wherein the first label and second label are different and wherein the sequence of the second probe is substantially identical to a portion of a second standard sequence, wherein the second standard sequence differs by at least one nucleotide from the first standard sequence.
  • the kit further comprises a thrid primer and a fourth primer for amplifying the target sequence and generate a second corresponding target amplicon; and a second probe for forming a second complex with the second target amplicon, wherein the second probe has a first label covalently linked to the one end of the probe and a first quencher linked to the other end of the probe and wherein the sequence of the second probe is substantially identical to a second portion of the standard sequence.
  • Fig. 1 illustrates High Resolution Melting curves (A) without and (B) with an unlabelled probe specific for the resistance sequence.
  • Fig. 2 illustrates asymmetric PCR with a non-fluorescent probe.
  • the 131 bp sequence amplicon from the 23S rRNA gene was amplified in HRM PCR reaction mix with asymmetric primers together with an unlabelled probe specific for the resistant sequence with DNA from erythromycin sensitive (regular line for Ery s ) or erythromycin resistant (dashed line for Ery R ) C. jejuni isolates.
  • Fig. 3 illustrates amplification profiles generated with different annealing temperatures to distinguish a sequence marker specific for erythromycin sensitivity.
  • Amplification profiles of a 131 bp sequence amplicon from the 23S rRNA gene were performed using a probe specific for the erythromycin sensitive sequence of C. jejuni at an annealing temperature of 55°C (panel A), 58°C (panel B), or 60°C (panel C).
  • the reactions contained DNA from erythromycin sensitive (regular line for Ery s ) or erythromycin resistant (bold line for Ery R ) C. jejuni isolates.
  • a negative control without C. jejuni DNA was also analysed (dashed line).
  • Fig. 4 illustrates a comparison between different polymerases used for SNP detection using TaqManTM probes with melting curve analysis.
  • the 131 bp sequence amplicon from the 23S rRNA gene and the 232 bp amplicon of the gyrA gene were simultaneously amplified in the presence of a mix of probes labelled with different fluorophores. Only the probe specific for the erythromycin sensitive sequence of C, jejuni is shown here, but the other three probes gave similar results.
  • Fig. 5 illustrates a comparison of primer ratios for asymmetric PCR.
  • the 131 bp sequence amplicon from the 23S rRNA gene and the 232 bp amplicon of the gyrA gene were simultaneously amplified with different asymmetric primer concentrations with a mix of probes labeled with different fluorophores. Only amplification profiles (A) and melting temperature profiles (B) detected with the probe specific for erythromycin sensitivitive C. jejuni are shown here.
  • Ratios used for Forward primer/Reverse Primer were: 1 :7 (0.1 ⁇ and 0.7 ⁇ ), 1 :10 (0.07 ⁇ and 0.7 ⁇ ), 1 :14 (0.05 ⁇ and 0.7 ⁇ ), 1 :23 (0.03 ⁇ and 0.7 ⁇ ) and 1 :70 (0.01 ⁇ and 0.7 ⁇ ).
  • Profiles for DNA from erythromycin sensitive (regular line for Ery s ), erythromycin resistant (dashed line for Ery R ), C. jejuni isolates are shown.
  • Fig. 6 illustrates SNP detection using labelled probes with melting curve analysis.
  • the 131 bp sequence amplicon from the 23S rRNA gene and the 232 bp amplicon of the gyrA gene were simultaneously amplified with asymmetric probe concentrations together with a mix of probes labeled with different fluorophores.
  • Amplification profiles and melting temperature profiles detected with the probe specific for erythromycin sensitivity are shown in panels A and E, those for the erythromycin resistance specific probe in panels B and F, those for the ciprofloxacin sensitive sequence in panels C and G and those for the ciprofloxacin resistance sequences in panels D and H.
  • Fig. 7 illustrates a distinction of heterozygotes in the 23S rRNA gene by amplification profiles and by melting curves with labeled probes.
  • Artificial mixes of DNA from bacteria sensitive to erythromycin with that from resistant bacteria were made to simulate bacteria with two 23S resistant copies and one 23S sensitive copy; with two 23S sensitive copies and one 23S resistant copy, or with all of three 23S gene copies that are resistant; or sensitive.
  • Amplification curves (panel A) from sensitive bacteria (regular line), for resistant bacteria (dashed line), and mixtures of sensitive and resistant bacteria (bold line) are shown.
  • Fig. 8 illustrates a sequence alignment between the matrix coding sequence of various representative influenza strains downloaded from GenBank including seasonal H1 N1 isolates A/Maryland/1/2006(H1 N1) (referred to as 2006(H1 N1)), and A/New Jersey/06/2008(H1 N1 ) (referred to as 2008(H1 N1 )), a reference pandemic strain A California/04/2009(H1 N1 ) (referred to as Pandemic 2009(H1 N1 )), a seasonal H3N2 isolate A/California/UR07-0053/2008(H3N2) (referred to as 2008(H3N2)), and an H5N1 isolate A/lndonesia/CDC1046/2007(H5N1) (referred to as 2007(H5N1)).
  • Consensus sequence is shown above the other sequences. Dots indicate the same sequence as the consensus. Primers and the universal probe FluAun209F Tx615 are underlined. The probes used for subtyping purposes are identified as "subtype specific probes" and are underlined.
  • Fig. 9 illustrates the distinction of Influenza A virus sub-types by amplification and melting curves using labeled probes.
  • Amplification curves panels A, C, and E
  • melting temperatures panels B, D, and F
  • a and B H3N2 probe
  • C and D PanH1 N1 probe
  • E and F Sea H1 N1 probe
  • universal probe not shown three probes.
  • Only samples containing the corresponding virus type yielded amplification and melting curves, except for the pandemic H1 1 samples which gave melting temperature peaks at 57°C with the H3N2 probe (panel B), but not amplification curves (panel A).
  • Fig. 10 illustrates a sequence alignment of a portion of the neuraminidase gene surrounding the H275Y mutation responsible for oseltamivir resistance between various influenza strains (the pandemic probe used in experiments, a seasonal H1 N1 sensitive strain A/New York/UR06- 0253/2007(H1 N1 ) (referred to as Seasonal H1 N1 S), a seasonal H1 N1 resistant strain A/Pennsylvania/20172008(H1 N1 ) (referred to as Seasonal H1 N1 R), a pandemic sensitive strain A/California/04/2009(H1 N1 ) (referred to as Pandemic H1 N1 S), two H5N1 sensitive strains A/lndonesia/CDC1032T/2007(H5N1 ) and A/Cambodia/R0405050/2007(H5N1) (both referred to as H5N1 S) and an inosine probe. Resistance and sensitivity are in relationship with o
  • Fig. 11 illustrates the detection of oseltamivir sensitivity in Influenza A viruses carrying neuraminidase type 1.
  • the amplification curves (panel B) and melting temperatures (panel A) are shown for the neuraminidase probe (universal probe not shown).
  • Fig. 12 illustrates a sequence alignment of a portion of the 16S ribosomal gene for Anaerococcus, Dialister, Bifidobacter, Peptinophilus sp., and Peptinophilus lacrimalis as well as the position and sequence identity of various primers and probes used in the subsequent PCR reactions. The numerous differences in the sequences within the primers and probes should assure specificity for each reaction to the desired microorganism.
  • Fig. 13 illustrates a sequence alignment of a portion of the 16S ribosomal gene for Peptinophilus lacrimalis, Peptinophilus indolicus, Peptinophilus stomatis, Anaerobranca californiensis, Anaerobranca horikoshii, Anaerobranca gottschalkii, Dialister pneumosintes, Dialister invisus and Dialister micraerophilus as well as the position and sequence identity of various primers and probes. There are 4 single nucleotide polymorphisms between the probe sequence for Dialister pneumosintes and the 2 other Dialister species tested.
  • Fig 14 illustrates the classification and identification of bacteria by amplification of 16S ribosomal RNA genes and performing melting curves using TaqManTM probes.
  • DNA from patient vaginal samples was analysed by TaqTm probing using primers and probes specific for different bacteria.
  • Left panels (A, C and E) show melting curves whereas right panels (B, D and F) show amplification curves.
  • Results for Peptinophilus species other than P. lacrimalis are shown in the top panels (A and B), for Dialister pseumosintes in the middle panels (C and D), for Anaerococcus sp. in the bottom panels (E and F).
  • Positively identified bacteria are presented as regular lines, negative samples as clashed lines, and variant bacteria as bold lines.
  • a method for the detection of differences between at least two corresponding sequences is based on the amplification of a corresponding portion of the sequence to be analyzed (herein referred to as the target or unknown sequence).
  • the resulting amplicon is then contacted with a probe so as to form a complex with the amplicon.
  • the probe is covalently linked at one of its ends with a detectable label and at its other end with a corresponding quencher.
  • the melting temperature of the resulting complex (between the probe and the amplicon) is then determined, based on the acquisition of the signal from the probe, and compared with a standard melting temperature to characterize the presence or absence of the nucleotide difference.
  • the method can be conveniently modified to accommodate the use of more than one probe, the detection of more than one nucleotide difference as well as to determine the copy number of an allele, on a semiquantitative basis.
  • the method can be particularly convenient to detect mutations associated with antibiotic or antiviral resistance, bacterial and viral subtypes, or human haplotypes.
  • both strands produced by the PCR reaction are present in large enough amounts that they will reanneal in less than 10 seconds and thereby prevent smaller fluorescent probes from reannealing with their complementary strand.
  • each primer that initiates synthesis of a strand will degrade the probe hybridized to the strand being copied so 0.7 ⁇ of primer might consume 0.7 ⁇ of probe.
  • a fluorescent probe could not be used to determine the melting temperature of the amplicons.
  • the use of a PCR enzyme that does not have a 5' endonuclease activity would not degrade the probe and so could be employed to preserve intact probe. It would not generate a typical PCR curve, however, because it would not degrade the TaqManTM probe, so signal acquisition must be determined rapidly at the annealing temperature before the polymerase displaced the probe in the course of synthesis of a new DNA strand and even this action did not generate amplification curves with crossing points as low and fluorescent intensities as high as polymerases with a 5' endonuclease activity.
  • the methods described use the determination of the melting temperature of a complex between a labeled probe and an amplicon to indicate the presence or absence of base or nucleotide mismatch(es) between the probe and the amplicon.
  • base refers to any nitrogen-containing heterocyclic moiety capable of forming Watson-Crick type hydrogen bonds in pairing with a complementary base or base analog.
  • bases include purines and pyrimidines, and modified forms thereof.
  • the naturally occurring bases include, but are not limited to, adenine (A), guanine (G), cytosine (C), uracil (U) and thymine (T).
  • nucleoside refers to a compound consisting of a base linked to the C-1' carbon of a sugar, for example, ribose or deoxyribose.
  • nucleotide refers to a phosphate ester of a nucleoside, as a monomer unit or within a polynucleotide.
  • Nucleotide 5'-triphosphate refers to a nucleotide with a triphosphate ester group attached to the sugar 5 -carbon position, and are sometimes denoted as “NTP", or “dNTP” and “ddNTP”.
  • a modified nucleotide is any nucleotide (e.g., ATP, TTP, GTP or CTP) that has been chemically modified, typically by modification of the base moiety.
  • Modified nucleotides include, for example but not limited to, methylcytosine, 6-mercaptopurine, 5- fluorouracil, 5-iodo-2'-deoxyuridine and 6-thioguanine.
  • nucleotide analog refers to any nucleotide that is non-naturally occurring.
  • polynucleotide refers to a polymeric arrangement of monomers that corresponded to a sequence of nucleotide bases, e.g., a DNA, RNA, peptide nucleic acid, or the like.
  • a polynucleotide can be single- or double-stranded, and can be complementary to the sense or antisense strand of a gene sequence, for example.
  • a polynucleotide can hybridize with a complementary portion of a target polynucleotide to form a duplex, which can be a homoduplex or a heteroduplex.
  • a homoduplex being a duplex between two perfectly complementary DNA strands such as those from a single organism whereas a heteroduplex is a duplex that is not perfectly complementary where one strand of DNA (for example the sense strand) comes from one organism and the other strand (for example the antisense strand) and has a different sequence of bases and thus forms a duplex that is not perfectly complementary.
  • the length of a polynucleotide is not limited in any respect. Linkages between nucleotides can be internucleotide-type phosphodiester linkages, or any other type of linkage.
  • a "polynucleotide sequence" refers to the sequence of nucleotide monomers along the polymer.
  • a "polynucleotide” is not limited to any particular length or range of nucleotide sequence, as the term “polynucleotide” encompasses polymeric forms of nucleotides of any length.
  • a polynucleotide can be produced by biological means (e.g., enzymatically), or synthesized using an enzyme-free system.
  • a polynucleotide can be enzymatically extendable or enzymatically non-extendable.
  • Polynucleotides that are formed by 3'-5' phosphodiester linkages are said to have 5 -ends and 3 -ends because the nucleotide monomers that are reacted to make the polynucleotide are joined in such a manner that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen (hydroxyl) of its neighbor in one direction via the phosphodiester linkage.
  • the 5'-end of a polynucleotide molecule has a free phosphate group, a hydroxyl, or other group at the 5' position of the pentose ring of the nucleotide, while the 3' end of the polynucleotide molecule has a free phosphate, hydroxyl, or other group at the 3' position of the pentose ring.
  • a position or sequence that is oriented 5' relative to another position or sequence is said to be located "upstream”, while a position that is 3' to another position is said to be "downstream".
  • polynucleotides As used herein, it is not intended that the term "polynucleotides” be limited to naturally occurring polynucleotides sequences or polynucleotide structures, naturally occurring backbones or naturally occurring internucleotide linkages.
  • polynucleotide analogues unnatural nucleotides, non-natural phosphodiester bond linkages and internucleotide analogs that find use with the invention.
  • unnatural structures include non-ribose sugar backbones, 3'-5' and 2'- 5' phosphodiester linkages, internucleotide inverted linkages (e.g., 3 -3' and 5 -5'), and branched structures.
  • unnatural structures also include unnatural internucleotide analogs, e.g., peptide nucleic acids (PNAs), locked nucleic acids (LNAs), C -C alkylphosphonate linkages such as methylphosphonate, phosphoramidate, Ci-C 6 alkyl-phosphotriester, phosphorothioate and phosphorodithioate internucleotide linkages.
  • PNAs peptide nucleic acids
  • LNAs locked nucleic acids
  • C -C alkylphosphonate linkages such as methylphosphonate, phosphoramidate, Ci-C 6 alkyl-phosphotriester, phosphorothioate and phosphorodithioate internucleot
  • a polynucleotide can be composed entirely of a single type of monomeric subunit and one type of linkage, or can be composed of mixtures or combinations of different types of subunits and different types of linkages (a polynucleotide can be a chimeric molecule).
  • a polynucleotide analog retains the essential nature of natural polynucleotides in that they hybridize to a single-stranded nucleic acid target in a manner similar to naturally occurring polynucleotides.
  • sequence of a polynucleotide refers to the order of nucleotides in the polynucleotide.
  • a “sequence” refers more specifically to the order and identity of the bases that are each attached to the nucleotides.
  • a sequence is typically read (written or provided) in the 5' to 3' direction.
  • a particular polynucleotide sequence of the invention optionally encompasses complementary sequences, in addition to the sequence explicitly indicated.
  • the terms "subsequence”, “fragment” or “portion” and the like refer to any portion of a larger sequence (e.g., a polynucleotide or polypeptide sequence), up to and including the complete sequence.
  • the minimum length of a subsequence is generally not limited, except that a minimum length may be useful in view of its intended function.
  • a polynucleotide portion can be amplified to produce an amplicon, which in turn can be used in a hybridization reaction that includes a polynucleotide probe.
  • the amplified portion should be long enough to specifically hybridize to a polynucleotide probe.
  • Portions of polynucleotides can be any length, for example, at least 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 150 or 200 nucleotides or more in length.
  • amplification means the production of multiple copies of a polynucleotide molecule, or a portion of a polynucleotide molecule, typically starting from a small amount (undetectable without amplification) of a polynucleotide, until, typically, the amplified material becomes detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other detection.
  • Amplification of polynucleotides encompasses a variety of chemical and enzymatic processes.
  • PCR polymerase chain reaction
  • SDA strand displacement amplification
  • TMA transcription mediated amplification
  • NASBA nucleic acid sequence-based amplification
  • LCR ligase chain reaction
  • PCR polymerase chain reaction
  • the term "polymerase chain reaction” refers to a method for amplification well known in the art for increasing the concentration of a segment of a target polynucleotide in a sample, where the sample can be a single polynucleotide species, or multiple polynucleotides.
  • the PCR process consists of introducing a molar excess of two or more extendable oligonucleotide primers to a reaction mixture comprising the desired target sequence(s), where the primers are complementary to opposite strands of the double stranded target sequence.
  • the use of the primers enable the production of amplicons that represent a target or standard sequence.
  • RT-PCR Reverse transcriptase PCR
  • RT-PCR is a PCR reaction that uses an RNA template and a reverse transcriptase, or an enzyme having reverse transcriptase activity, to first generate a single stranded DNA molecule prior to the multiple cycles of DNA- dependent DNA polymerase primer elongation.
  • Multiplex PCR refers to PCR reactions that produce multiple copies of more than one product or amplicon in a single reaction, typically by the inclusion of more than two different primers in a single reaction.
  • the cycles can consist of an initial denaturation step at 94 or 95°C where double stranded DNA is denatured to provide single strands that can be copied; an annealing step usually at 55°C or 60°C allows primers and probes to anneal to the single strands.
  • Primers can initiate synthesis of new DNA copies and this process can be completed in the following step of synthesis at 72°C which is the usual optimal temperature for Taq DNA polymerase activity.
  • Probe fluorescence can be measured for some probes at the annealing temperature while the probes are annealed to the single strand separating the fluorophore from its quencher or bringing together the probes and before the Taq polymerase degrades or displaces the probes to complete synthesis of the new strand.
  • fluorescence may be measured during the annealing cycle or at 72°C. Since the melting temperature of most probes is below 72°C, they will have dissociated from the complex and no longer be fluorescent.
  • asymmetric PCR refers to the preferential PCR amplification of one strand of a DNA target by adjusting the molar concentration of the primers in a primer pair so that they are unequal.
  • An asymmetric PCR reaction produces a predominantly single- stranded product and a smaller quantity of a double-stranded product as a result of the unequal primer concentrations.
  • the lower concentration primer is quantitatively incorporated into a double-stranded DNA amplicon, but the higher concentration primer continues to prime DNA synthesis, resulting in continued accumulation of a single stranded product.
  • Asymmetric PCR also includes the use of a single primer for amplification.
  • real-time PCR or “kinetic PCR” refer to real-time detection and/or quantitation of amplicon generated in a PCR, without the need for a detection or quantitation step following the completion of the amplification.
  • the TaqManTM PCR reaction uses a thermostable DNA-dependent DNA polymerase that possesses a 5'-3' nuclease activity.
  • the 5 -3' nuclease activity of the DNA polymerase cleaves the labeled probe that is hybridized to the amplicon in a template-dependent manner.
  • the resultant probe fragments dissociate from the primer/template complex, and the reporter fluorophore typically, but not exclusively attached to the 5' end of the probe is then free from the quenching effect of the quencher moiety typically, but not exclusively attached to the 3' end of the probe.
  • reporter fluorophore Approximately one molecule of reporter fluorophore is liberated for each new amplicon molecule synthesized, and detection of the unquenched reporter fluorophore provides the basis for quantitative interpretation of the data, such that the amount of released fluorescent reporter is directly proportional to the amount of amplicon template.
  • C T One measure of the TaqManTM assay data is typically expressed as the threshold cycle (C T ). Fluorescence levels are recorded during each PCR cycle and are proportional to the amount of product amplified to that point in the amplification reaction. The PCR cycle when the fluorescence signal is first recorded as statistically significant, or where the fluorescence signal is above some other arbitrary level (e.g., the arbitrary fluorescence level, or AFL), is the threshold cycle (C T ).
  • Variations in methodologies for real-time amplicon detection are also known, and in particular, where the 5'-nuclease probe is replaced by double-stranded DNA intercalating dye resulting in fluorescence that is dependent on the amount of double-stranded amplicon that is present in the amplification reaction.
  • DNA-dependent DNA polymerase refers to a DNA polymerase enzyme that uses deoxyribonucleic acid (DNA) as a template for the synthesis of a complementary and antiparallel DNA strand.
  • Thermostable DNA-dependent DNA polymerases find use in PCR amplification reactions. Suitable reaction conditions (and reaction buffers) for DNA-dependent DNA polymerase enzymes, and indeed any polymerase enzyme, are widely known in the art.
  • Reaction buffers for DNA-dependent DNA polymerase enzymes can comprise, for example, free deoxyribonucleotide triphosphates, salts and buffering agents.
  • RNA-dependent DNA polymerase refers to a DNA polymerase enzyme that uses ribonucleic acid (RNA) as a template for the synthesis of a complementary and antiparallel DNA strand.
  • RNA ribonucleic acid
  • RT reverse transcription
  • the enzyme that accomplishes that is a “reverse transcriptase” Some naturally-occurring and mutated DNA polymerases also possess reverse transcription activity.
  • thermalostable refers to an enzyme that retains its biological activity at elevated temperatures (e.g., at 55°C or higher), or retains its biological activity following repeated cycles of heating and cooling.
  • Thermostable polynucleotide polymerases find particular use in PCR amplification reactions, and, in an embodiment, they can retain their biological activity even at 94°C or 95°C.
  • primer refers to an enzymatically extendable oligonucleotide, generally with a defined sequence that is designed to hybridize in an antiparallel manner with a complementary, primer-specific portion of a sequence (e.g. target sequence). Further, a primer can initiate the polymerization of nucleotides in a template-dependent manner to yield a polynucleotide that is complementary to the target polynucleotide.
  • the extension of a primer annealed to a target uses a suitable DNA or RNA polymerase in suitable reaction conditions.
  • polymerization reaction conditions and reagents are well established in the art, and are described in a variety of sources.
  • a primer nucleic acid does not need to be 100% complementary with its template subsequence (such as the target sequence or the standard sequence) for primer elongation or probe binding to occur; primers and probes with less than 100% complementarity can be sufficient for hybridization and polymerase elongation to occur depending on the annealing temperature used. If the melting temperature of the primer, even with less than 100% complementarity, is still above the annealing temperature then elongation will occur. Sequences with less than 100% complementarity can be excluded by choosing an annealing temperature below the standard, but above the target annealing temperature.
  • a primer nucleic acid can be labeled, if desired.
  • the label used on a primer can be any suitable label, and can be detected by, for example, by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other detection means.
  • a pair of primers (also referred to as a first primer and a second primer or a forward primer and a reverse primer) can be used in the amplification reaction.
  • Such pair of primers are designed to hybridize to complementary strands of the standard sequence and allow the specific extension of a portion of the standard sequence.
  • the pair of primers are also designed to hybridize to corresponding complementary strands of the target sequence and allow the specific extension of a corresponding portion of the target sequence.
  • the first primer is non-linear to the probe, i.e. it specifically produces the strand that will hybridize to the probe and the second primer is co-linear to the probe, i.e. it specifically hybridizes to the same strand as the probe and produces the strand that contains the sequence of the probe.
  • condition wherein base-pairing occurs refers to any hybridization conditions that permit complementary polynucleotides or partially complementary polynucleotides to form a stable hybridization complex.
  • stringent As used herein, the terms “stringent”, “stringent conditions”, “high stringency” and the like denote hybridization conditions of generally low ionic strength and high temperature, as is well known in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3 rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); Current Protocols in Molecular Biology (Ausubel et al., ed., J. Wiley & Sons Inc., New York, 1997), which are incorporated herein by reference.
  • stringent conditions for polynucleotides longer than 1000 bases are selected to be about 5-30°C lower than the thermal melting point (T m ) for the hybridization complex comprising the specified sequence at a defined ionic strength and pH.
  • stringent conditions for shorter polynucleotides are selected to be about 5-10°C lower than the T m for the specified sequence at a defined ionic strength and pH.
  • the T m is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the hybridization complexes comprising complementary (or partially complementary) polynucleotides become dissociated.
  • the expression "low stringency” denotes hybridization conditions of generally high ionic strength and lower temperature. Under low stringency hybridization conditions, polynucleotides with imperfect complementarity can more readily form hybridization complexes.
  • complementary or “complementarity” are used in reference to antiparallel strands of polynucleotides related by the Watson-Crick and Hoogsteen-type base- pairing rules.
  • sequence 5'-AGTTC-3' is complementary to the sequence 5'- GAACT-3'.
  • completely complementary or “100% complementary” and the like refer to complementary sequences that have perfect Watson-Crick pairing of bases between the antiparallel strands (no mismatches in the polynucleotide duplex).
  • complementarity need not be perfect; stable duplexes, for example, may contain mismatched base pairs or unmatched bases.
  • partial complementarity refers to any alignment of bases between antiparallel polynucleotide strands that is less than 100% perfect (e.g., there exists at least one mismatch or unmatched base in the polynucleotide duplex).
  • the alignment of bases between the antiparallel polynucleotide strands can be at least 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50%, or any value between.
  • a "complement" of a target polynucleotide refers to a polynucleotide that can combine (e.g., hybridize) in an antiparallel association with at least a portion of the target polynucleotide.
  • the antiparallel association can be intramolecular, e.g., in the form of a hairpin loop within a nucleic acid molecule, or intermolecular, such as when two or more single- stranded nucleic acid molecules hybridize with one another.
  • the hybridization complex formed as a result of the annealing of a polynucleotide with a probe is termed a "polynucleotide-probe complex".
  • the hybridization complex can form in solution (and is therefore soluble), or one or more component of the hybridization complex can be affixed to a solid phase (e.g., a dot blot, affixed to a bead system to facilitate removal or isolation of the hybridization complexes, or in a microarray).
  • the structure of the probe is not limited, and can be composed of DNA, RNA, analogs thereof, or combinations thereof, and can be single-stranded or double-stranded.
  • a probe can be derived from any source.
  • the term "probe” refers typically to a polynucleotide that is capable of hybridizing to a standard sequence (as well as fragment or amplicon thereof) and a target sequence (as well as fragment or amplicon thereof).
  • the probe is associated (preferably in a covalent manner) with a suitable label(s) or reporter moiety(ies) so that the probe (and the complex it can form with the standard or the target sequence) can be detected, visualized, measured and/or quantitated.
  • the probe may also be linked at its other end to a quencher. The presence of a quencher is advantageous to suppress the fluorescent signal when the probe is not hybridized to its target or has not been degraded.
  • the probe is preferentially substantially identical or even identical over its entire length to a portion of a standard sequence.
  • the probe is free (or devoid) of complementary, self-annealing 3' and 5' ends that are that are not present in the standard sequence.
  • Detection systems for labeled probes include, but are not limited to, the detection of fluorescence, fluorescence quenching, fluorescence resonant energy transfer (FRET), enzymatic activity, absorbance, molecular mass, radioactivity, luminescence or binding properties that permit specific binding of the reporter (e.g., where the reporter is an antibody, antigen, or small molecule with high affinity binding properties such as biotin).
  • a probe can be an antibody or DNA binding protein, rather than a polynucleotide, that has binding specificity for a nucleic acid nucleotide sequence of interest. It is not intended that the present invention be limited to any particular probe label or probe detection system.
  • the source of the polynucleotide used in the probe is not limited, and can be produced synthetically in a non-enzymatic system, or can be a polynucleotide (or a portion of a polynucleotide) that is produced using a biological (e.g., enzymatic) system (e.g., in a bacterial cell).
  • a biological e.g., enzymatic
  • a probe is sufficiently complementary (e.g. identical) to the standard sequence (or fragment or amplicon thereof) to form a stable hybridization complex with the standard sequence under a selected hybridization condition, such as, but not limited to, a stringent hybridization condition.
  • the probe is sufficiently complementary to the target sequence (or fragment or amplicon thereof) to form a stable hybridization complex with the target sequence under a selected hybridization condition, such as, but not limited to, a stringent hybridization condition.
  • the length of the probe smaller than the length of the amplicon.
  • label or “reporter”, in their broadest sense, refer to any moiety or property that is detectable, or allows the detection of, that which is associated with it.
  • a polynucleotide that comprises a label is detectable (and in some aspects is referred to as a probe).
  • a labeled polynucleotide permits the detection of a hybridization complex that comprises the polynucleotide.
  • a label is attached (preferably covalently) to a probe.
  • a label can, alternatively or in combination: (i) provide a detectable signal; (ii) interact with a second label to modify the detectable signal provided by the second label, e.g., FRET; (iii) stabilize hybridization, e.g., duplex formation; (iv) confer a capture function, e.g., hydrophobic affinity, antibody/antigen, ionic complexation, or (v) change a physical property, such as electrophoretic mobility, hydrophobicity, hydrophilicity, solubility, or chromatographic behavior. Labels vary widely in their structures and their mechanisms of action.
  • labels include, but are not limited to, fluorescent labels (including, e.g., quenchers or absorbers), non-fluorescent labels, colorimetric labels, chemiluminescent labels, bioluminescent labels, radioactive labels, mass-modifying groups, antibodies, antigens, biotin, haptens, enzymes (including, e.g., peroxidase, phosphatase, etc.), and the like.
  • fluorescent labels may include dyes that are negatively charged, such as dyes of the fluorescein family, or dyes that are neutral in charge, such as dyes of the rhodamine family, or dyes that are positively charged, such as dyes of the cyanine family.
  • Dyes of the fluorescein family include, e.g., FAM, HEX, TET, JOE, NAN and ZOE.
  • Dyes of the rhodamine family include, e.g., ROX, R110, R6G, Carboxytetramethylrhodamine (TAMRA), tetramethylrhodamine (TMR) and its isothiocyanate derivative (TRITC), sulforhodamine 101 (and its sulfonyl chloride form Texas Red) and Rhodamine Red.
  • Alternatives to rhodamine dyes include, but are not limited to, Alexa 546TM, Alexa 555TM, Alexa 633TM, DyLight 549TM and DyLight 633TM.
  • Dyes of the cyanine family include, e.g., Cy2, Cy3, Cy5, Cy 5.5 and Cy7.
  • Alternatives to cyanine dyes include, but are not limited to, Freedom Dyes TEXTM, and TYETM, Alexa FluorTM dyes, DylightTM, IRISTM Dyes, SetaTM dyes, SeTauTM dyes, SRfluorTM dyes and SquareTM dyes.
  • a quencher can be matched to limit signal emission from the fluorescent label when the probe is not hybridized with a corresponding sequence.
  • quenchers that can be linked to the probe include, but are not limited to DDQ-I A, Dabcyl, Eclipse B, Iowa Black FQ C, BHQ-1 D, QSY-7 E, BHQ-2 D, DDQ-II A, Iowa Black RQ C, QSY- 21 E and BHQ-3 D.
  • the first step of the method described herein comprises the amplification of a target sequence.
  • a target sequence is an unknown sequence suspected of different from the standard sequence (e.g. being difference in at least one nucleotide), similar or identical to the probe and/or the standard (known) sequence or sequences.
  • the nucleotide identity of a "standard sequence” is known and is similar (preferably identical) to the probe used.
  • the method must be realized so that a fraction of the initial probe population remains intact (e.g. label remains attached to one of its ends) and is available to form a complex with the amplification product or amplicon.
  • the amplification can be performed in the presence of the probe, in conditions allowing that a fraction of the initial population of probes remains intact after the amplification reaction.
  • the amplification step can be performed in the absence of the probe and the probe can be added after the amplification step.
  • the amplification step can also be preceded by a reverse- transcription step when the staring material is RNA.
  • the nucleic acid identity and length of the amplification primer pair must be adjusted in order that, during the amplification step, a single fragment of the target sequence is amplified.
  • the primers When more than one amplification primer pairs are used, for example in a multiplex application, the primers must be designed to limit the interference between the primer pairs and allow the specific amplification of the various fragments of the target sequence.
  • the amplification primers used in the amplification step can, in an embodiment, have a length between 10 and 30 nucleotides. Modifications known to those skilled in the art, for example the used of locked nucleic acid bases or inosine, can help reducing the length of the primer without altering the specificity.
  • the amplification reaction results in the formation of amplicons of the target sequence (also referred to as target amplicon).
  • amplicon refers to a polynucleotide molecule (or collectively the plurality of molecules) produced following the amplification of a particular nucleic acid.
  • the amplification method used to generate the amplicon can be any suitable method, most typically, for example, by using a PCR methodology.
  • An amplicon is typically, but not exclusively, a DNA amplicon.
  • An amplicon can be single-stranded or double-stranded, or in a mixture thereof in any concentration ratio.
  • the amplicon is generated using real-time qPCR.
  • the amplicon has a length between about 50 bp to 500 bp or even between 100 bp and 300 bp.
  • an amplicon is generated, it is contacted with a probe to form a complex.
  • the probe can be present in the initial amplification mixture or can be added once amplification is terminated.
  • the contact between the amplicon and the probe should be made under conditions favoring the hybridization or annealing of the amplicon to the probe.
  • hybridization and “annealing” and the like are used interchangeably and refer to the base- pairing interaction of one polynucleotide with another polynucleotide (typically an antiparallel polynucleotide) that results in formation of a duplex or other higher-ordered structure, typically termed a hybridization complex or complex.
  • the primary interaction between the antiparallel polynucleotide molecules is typically base specific, e.g., A/T and G/C, by Watson/Crick and/or Hoogsteen-type hydrogen bonding. It is not a requirement that two polynucleotides have 100% complementarity over their full length to achieve hybridization.
  • the present method used the ability of the mismatch probe and amplicon to anneal together to determine the presence of possible nucleotide mismatch.
  • the probe is intended to specifically hybridize (e.g. being substantially identical) with the standard sequence or a portion of the standard sequence.
  • the probe is identical to the standard sequence or a complement thereof.
  • the probe possesses an increased specificity towards the standard sequence in comparison with its specificity to the target sequence, when the standard sequence and the target sequence differ in their nucleotide sequence.
  • the phrases "specifically hybridize”, “specific hybridization” and the like refer to hybridization resulting in a complex where the annealing pair show complementarity, and preferentially bind to each other to the exclusion of other potential binding partners in the hybridization reaction.
  • the term "specifically hybridize” does not require that a resulting hybridization complex have 100% complementarity; hybridization complexes that have mismatches can also specifically hybridize and form a hybridization complex.
  • the degree of specificity of the hybridization can be measured using a distinguishing hybridization property, e.g., the melting temperature of the hybridization complex (T m ).
  • T m melting temperature of the hybridization complex
  • the nucleic acid identity and length of the probe must be adjusted in order that, during the annealing step, the probe will specifically associate with its corresponding amplicon. When more than one probes are used, they must be designed to limit the interference with other amplicons.
  • the probes can, in an embodiment, have a length between 15 and 40 nucleotides, preferably between 20 and 40 nucleotides. Modifications known to those skilled in the art, for example the used of locked nucleic acid bases, minor groove binders, or inosine, can help reducing the length of the probe without altering its specific
  • the number of nucleotides mismatch between the two probes is, in an embodiment, five, less than five nucleotides, less than four nucleotides, less than three nucleotides, less than two nucleotides or one nucleotides. In a further embodiment, the number of nucleotide mismatches between the two probes is between one and five, preferably between two and four.
  • the difference in melting temperature between the standard amplicon-first probe complex and the standard amplicon-second probe complex is 25°C, less than 25°C, less than 20°C, less than 15°C, less than 10°C, less than 5°C, less than 4°C, less than 3°C or less than 2°C. In still a further embodiment, the difference in melting temperature between the standard amplicon-first probe complex and the standard amplicon-second probe complex is between about 2°C to about 4°C.
  • the probe When the probe is not associated to the target sequence (fragment or amplicon thereof), it adopts a random coil structure and the quencher at one end captures the signal emitted by the fluorescent dye at the other end. However, once the probe associates with the target sequence (fragment or amplicon thereof) and a complex between the probe and the target is formed, the quencher is too far from the fluorescent dye for it to efficiently capture the fluorescence emitted. This fluorescent emission can then be measured, for examplem by a fluorometer incorporated in the real-time PCR cycler or by an independent fluorometer generating a signal.
  • the presence of the probe in the complex is associated with a signal from the label of the probe and the dissociation of the probe from the complex is associate with a loss of signal from the label of the probe.
  • This very specific property enables the determination of the melting temperature of the amplicon-probe complex.
  • the melting temperature (T m ) is a temperature-dependent distinguishing hybridization property of the complex.
  • the melting temperature of an amplicon-probe complex is dependant on the degree of identity between the probe. The higher the degree of identity between the amplicon and the probe, the higher the melting temperature.
  • the melting temperature is the temperature at which one half of a population of double-stranded polynucleotides or nucleobase oligomers (e.g., hybridization complexes), in homoduplexes or heteroduplexes, become dissociated into single strands.
  • the prediction of a T m of a duplex polynucleotide takes into account the base sequence as well as other factors including structural and sequence characteristics and nature of the oligomeric linkages. Methods for predicting and experimentally determining T m are known in the art.
  • a T m is traditionally determined using a melting curve, wherein a duplex nucleic acid molecule is heated in a controlled temperature program, and the state of association/dissociation of the two single strands in the duplex is monitored (in the present method with by assessing the signal from the probe) and plotted until reaching a temperature where the two strands are completely dissociated.
  • the T m is read from this melting curve.
  • a signal from the probe is measured within a specified temperature range. At temperatures below the melting temperature, the complexes are in majority annealed and the signal measured from the probe is high. As the temperature increases, the complex begins its dissociation and the probe is progressively released from the complex, thereby decreasing the measured value of the signal. At temperatures above the melting temperature, the majority of the complexes are dissociated. As the temperatures continue to increase, the probe has been completely released from the complex and the value of the signal is at a minimum. Fluctuation in signal may still occur for the probes as the random coil assumed by the free probe may fold differently depending on temperature, thus creating minor signal variation. Melting temperature curves are usually presented as second derivatives of fluorescence and represent the change in fluorescent signal so they appear as peaks with a maximum at the melting temperature.
  • a T m can be determined by an annealing curve, wherein a duplex nucleic acid molecule is heated to a temperature where the two strands are completely dissociated. The temperature is then lowered in a controlled temperature program, and the state of association/dissociation of the two single strands in the duplex is monitored and plotted until reaching a temperature where the two strands are completely annealed. The T m is read from this annealing curve.
  • a melting curve when used to determine the melting temperature, a signal from the probe is measured within a specified temperature range. At temperatures below the melting temperature, the complexes are in majority associated and the signal measured from the probe is high. As the temperature increases, the complex begins to melt and the probe is progressively separated from the complex, thereby decreasing the measured value of the signal. At temperatures above the melting temperature, the majority of the probes is dissociated from the amplicon and the signal is low.
  • the melting curve is a first derivative of the temperature change where the midpoint of the transition is considered the melting temperature. This should reflect the temperature where exactly half of the DNA strands have separated.
  • an annealing curve (or its first or second derivative) can be used to determine the melting temperature.
  • the melting temperature is not the sole temperature-dependent hybridization property that can be used in the method to provide useful information on the presence (or absence) of a mismatch between the probe and the target sequence.
  • the temperature at which 25% of a population of double-stranded polynucleotides or nucleobase oligomers (e.g., hybridization complexes), in homoduplexes or heteroduplexes, become dissociated into single strands (T 25 ) is also a defining characteristic of the hybridization complex.
  • the temperature at which 75% of a population of double-stranded polynucleotides or nucleobase oligomers (e.g., hybridization complexes), in homoduplexes or heteroduplexes, become dissociated into single strands is also a defining property of the hybridization complex.
  • the percentage of dissociation of a hybridization complex at any series of defined temperatures is a quantitative temperature dependent hybridization property and this usually forms the basis for High Resolution Melting (HRM).
  • the melting temperature of the complex is compared to the melting temperature of a complex between a corresponding amplicon of a standard sequence and the probe.
  • the amplicon of the standard sequence "corresponds" to the amplicon of the target sequence, in the sense that both amplicons are derived from the same corresponding region and were generated using similar methods (e.g. same primer oligonucleotide pair). Both amplicons are approximately the same length and also possess a certain degree of identity. This similarity in length and sequence is necessary to generate melting temperature that can be compared to detect nucleotide differences between the target and the standard sequences.
  • the melting temperature of the complex between the amplicon of the target sequence and the probe will be different from (in an embodiment, it will be lower than) the melting temperature of the complex between the amplicon of the standard sequence and the probe.
  • This difference in melting temperature is attributed to the presence of a mismatch or difference between the target sequence and the probe and/or between the standard sequence and the probe.
  • the melting temperature of the complex between the amplicon of the target sequence and the probe will be substantially similar (i.e. indistinguishable).
  • This lack of difference in melting temperature is attributed to the absence of a mismatch or difference between the target sequence and the probe.
  • a single base mismatch between the probe and the target sequence will typically lower the melting temperature of the probe-amplicon complex by at least 2°C (with respect to the standard amplicon-probe complex).
  • a difference of less than 1°C is usually not considered to be a substantially dissimilar temperature.
  • the method described herein can be used to detect a specific known nucleotide difference between the target sequence and standard sequences. However, in some instances, some additional unknown nucleotide differences can be present between the target and the standard sequences. In those instances, the unknown additional nucleotide differences can modify the melting temperature of the probe-amplicon complex and the detection of the "known" nucleotide difference can be difficult to perform. As such, it may be advantageous to use a further probe (e.g. second probe) in the method. This further probe is linked to a detectable label (different from the label of the first probe so as to provide a clear distinct signal from the first probe) and, optionally, a corresponding quencher.
  • the method is not limited to the addition of a single further probe, multiple probes can be used, as long as they can be labeled with different labels that will generate distinct signals.
  • the first probe is substantially identical to a first standard sequence (or amplicon or fragment thereof) and the second probe is substantially identical to a second standard sequence (or amplicon or fragment thereof).
  • the first and second standard sequence differ in nucleotide identity and this difference is known.
  • the first and second standard sequence may be overlapping and, in an embodiment, may only differ at a single nucleotide.
  • the first standard sequence can correspond to a wild-type sequence, whereas the second standard sequence can correspond to a known mutant sequence.
  • the melting temperature of the target amplicon-probe complexes with both of the probes is then determined and compared to the melting temperature derived from the standard amplicons-probes complexes with both of the probes.
  • Dissimilarity in melting temperature between the target amplicon-first probe complex and the first standard amplicon-first probe complex indicates the presence of a nucleotide difference between the target sequence and the standard sequence (or first probe). This result can be optionally confirmed (and the identity of this difference be reasonably inferred) by the similarity in melting temperature between the target amplicon- second probe complex and the second standard amplicon-second probe complex. If a dissimilarity still exists between the target amplicon-second probe complex and the second standard amplicon-second probe complex, it can be assumed that the difference in the nucleotide sequence of the target is different than the one(s) known between the first and second standard sequence.
  • the nucleotide sequence varies at a nucleotide other than the one known between the first and second standard sequences, it is still possible to distinguish the presence of the wild type or known mutant sequence as either the first or second probe will have a higher melting temperature then the other probe, because this probe will have only one nucleotide mismatch whereas the other probe will have this mismatch in addition to the mismatch at the wild type/mutant site.
  • both a standard and the target sequence do not contain a nucleotide difference at the level of the probe, even though they contain differences elsewhere, then the melting temperature of all their amplicon-probe complexes derived from this same probe, would be similar.
  • the method presented herewith can also be adapted for multiplex PCR.
  • Amplicons from more than one portion of the standard and/or target sequence can be generated. These different amplicons can be contacted with a single or a combination of probes, under conditions favoring the formation of a complex between the plurality of amplicons and the probe(s). The melting temperatures of these complexes are determined and compared to the melting temperatures of complexes between the plurality of amplicons of the standard sequences and the probe(s). If one of the amplicons of the target sequence is dissimilar to the probe (and consequently to the standard sequence), then its melting temperature will be different from the corresponding amplicon from the standard sequence.
  • the first standard sequence can correspond to a sequence in one gene
  • the second standard sequence can correspond to a sequence in another gene (and even in another organism).
  • the melting temperature of the target amplicon-probe complexes with both of the probes is then determined and compared to the melting temperature derived from the standard amplicons-probes complexes with both of the probes. Absence of a melting temperature between an amplicon and a probe indicates the absence of the corresponding standard sequence (or gene) in the target sequence. Alternatively, the presence of a melting temperature between an amplicon and a probe indicates the presence of the corresponding standard sequence (or gene) in the target sequence.
  • Similarity/dissimilarity in melting temperature between the target amplicon-probe complexes indicates the absence/presence of a nucleotide difference between the target sequence and the standard sequence. This result can be optionally confirmed (and the identity of this difference be reasonably inferred) by the similarity/dissimilarity in melting temperature between the target amplicon and a further probe complex, as discussed above.
  • the method presented herewith can be used in a sample for detecting various target sequences (overlapping or not) as well as the presence of a nucleotide difference between the target and the standard sequence.
  • sample is used in its broadest sense, and refers to any material subject to analysis.
  • sample refers typically to any type of material of biological origin, for example, any type of material obtained from animals or plants.
  • a sample can be, for example, any fluid or tissue such as blood or serum, and furthermore, can be human blood or human serum.
  • a sample can be cultured cells or tissues, cultures of microorganisms (prokaryotic or eukaryotic), viruses or any fraction or products produced from or derived from biological materials (living or once living).
  • a sample can be purified, partially purified, unpurified, enriched or amplified.
  • the sample can comprise principally one component, e.g., nucleic acid. More specifically, for example, a purified or amplified sample can comprise total cellular RNA, total cellular mRNA, DNA, cDNA, cRNA, or an amplified product derived there from.
  • sample used in the methods of the invention can be from any source, and is not limited.
  • sample can be an amount of tissue or fluid isolated from an individual or individuals, including, but not limited to, for example, skin, plasma, serum, whole blood, blood products, spinal fluid, saliva, peritoneal fluid, lymphatic fluid, aqueous or vitreous humor, synovial fluid, urine, feces, tears, blood cells, blood products, semen, seminal fluid, nasal secretions, vaginal or cervical secretions, pulmonary effusion, serosal fluid, organs, bronchio-alveolar lavage, tumors, paraffin embedded tissues, lesion scrapings, etc.
  • Samples also can include constituents and components of in vitro cell cultures, including, but not limited to, conditioned medium resulting from the growth of cells in the cell culture medium, recombinant cells, cell components, etc.
  • the target and standard sequences can be derived from various organisms.
  • the expression "derived from” refers to a component that is isolated from or made using a specified sample, molecule, organism or information from the specified molecule or organism.
  • a nucleic acid molecule that is derived from an influenza virus can be a molecule of the influenza genome, or alternatively, a transcript from the influenza genome.
  • Kits approved for clinical use are often based on fluorescent resonant energy transfer (FRET) where melting temperatures between the target and a probe distinguish mutant from wild type sequences, which is a more robust methodology (simpler to interpret) for clinical laboratories.
  • FRET fluorescent resonant energy transfer
  • the present application does provide a kit for determining the presence or absence as well as the identity of a nucleotide difference between a target sequence and a standard sequence.
  • the kit for use in the methods described herewith, comprises at least two components: a pair of primers for amplifying a fragment of the target sequence and a probe that is able to bind specifically to the amplified target sequence.
  • the kit may also comprise a second probe that is able to specifically bind to the amplified target sequence.
  • the second is similar to the first probe and its nucleic acid identity is known.
  • the second is identical to the first probe except in at least one nucleotide.
  • kit If the kit is to be used in a multiplex PCR, it also comprise a second pair of primers for amplifying a fragment in the target sequence as well as an additional probe that is able to bind specifically to the amplified target sequence.
  • the comparison, assessment and detection steps may be assisted with a computer for ease of use.
  • Providing melting temperature Since the sequence of the probe is known and the sequence of the corresponding amplicon of the standard sequence is known, it is possible to store the properties of the melting temperature and/or melting curve of the standard amplicon-probe in a memory card and retrieve such information when the comparison is performed.
  • the comparison can be made in a comparison module.
  • Such comparison module may comprise a processor and a memory card to perform an application.
  • the processor may access the memory to retrieve data (such as the stored standard amplicon-probe melting temperature).
  • the processor may be any device that can perform operations on data. Examples are a central processing unit (CPU), a front-end processor, a microprocessor, a graphics processing unit (PPU/VPU), a physics processing unit (PPU), a digital signal processor and a network processor.
  • the application is coupled to the processor and configured to determine if a difference exists between the target amplicon-probe melting temperature and the standard amplicon-probe melting temperature. An output of this comparison may be transmitted to a display device.
  • the memory accessible by the processor, receives and stores data, such as measured melting temperatures or any other information generated or used.
  • the memory may be a main memory (such as a high speed Random Access Memory or RAM) or an auxiliary storage unit (such as a hard disk, a floppy disk or a magnetic tape drive).
  • the memory may be any other type of memory (such as a Read-Only Memory or ROM) or optical storage media (such as a videodisc or a compact disc).
  • Detection of a presence of a nucleotide difference and/or nucleotide identity is possible because, as shown herein, the presence of a nucleotide difference on the target sequence (with respect to the standard sequence) modifies (e.g. lowers) the resulting melting temperature of the target amplicon-probe with respect to the standard amplicon-probe. As such, the presence or absence of the nucleotide difference is based on that premise.
  • a nucleotide difference is detected, its identity can be determined by comparing the melting temperature of additional amplicon-probe complexes as indicated above.
  • the determination can be made with a processor and a memory card to perform an application.
  • the processor may access the memory to retrieve data.
  • the processor may be any device that can perform operations on data. Examples are a central processing unit (CPU), a front-end processor, a microprocessor, a graphics processing unit (PPU/VPU), a physics processing unit (PPU), a digital signal processor and a network processor.
  • the application is coupled to the processor and configured to determine the presence (or absence) of a nucleotide difference in the target sequence.
  • the memory accessible by the processor, receives and stores data, such as measured parameters or any other information generated or used.
  • the memory may be a main memory (such as a high speed Random Access Memory or RAM) or an auxiliary storage unit (such as a hard disk, a floppy disk or a magnetic tape drive).
  • the memory may be any other type of memory (such as a Read-Only Memory or ROM) or optical storage media (such as a videodisc or a compact disc).
  • the present application also provides a screening system for determining the presence or absence of a nucleotide difference between the target sequence and the standard sequence.
  • This screening system comprises a reaction vessel for combining the target amplicon and the probe, a processor in a computer system, a memory accessible by the processor and an application coupled to the processor.
  • the application or group of applications is(are) configured for receiving a value of a melting temperature of the target amplicon-probe; comparing the value of the melting temperature of the target amplicon-probe to the melting temperature of the standard amplicon-probe and/or determining the presence of a nucleotide difference in the target sequence with the compared melting temperatures are different. Further assessment can be made with respect to the identity of the nucleotide difference when more than one probe is used.
  • the present application also provides a software product embodied on a computer readable medium.
  • This software product comprises instructions for determining the presence of a nucleotide sequence in a target sequence.
  • the software product comprises a receiving module for receiving a value of a melting temperature of a target amplicon-probe complex; a comparison module receiving input from the receiving module for determining if the melting temperature of the target amplicon-probe complex different from the melting temperature of the standard amplicon-probe complex; a determination module receiving input from the comparison module for determining the presence of a nucleotide sequence in a target sequence.
  • the nucleotide difference is considered present when a difference in the melting temperatures is observed. On the other hand, the nucleotide difference is considered absent when no (or a statistically insignificant) difference in the melting temperatures is observed.
  • the comparison module and determination module may each comprise a processor, a memory accessible by the processor to perform an application.
  • the methods described herein can be useful for determining any nucleotide differences (single or multiple) in any organism (viral, prokaryotic and eukaryotic). Specific examples are presented below for multiplex detection of mutations associated with antibiotic resistance in the bacteria Campylobacter jejuni, subtyping of influenza A virus with simultaneous detection of the mutation associated oseltamivir resistance across three highly divergent neuraminidase genes, and bacterial classification and typing by identifying nucleotide differences in the 16S ribosomal RNA gene.
  • Campylobacter jejuni is the leading reported cause of bacterial enteritis in developed countries. In 2004 in Canada, it was the leading notifiable enteric food- and waterborne disease, with 9345 reported cases. In Quebec province alone, nearly 3000 cases of diarrheal illness are attributed annually to Campylobacter enteritis, more than the combined total caused by Salmonella and Shigella species, E. coli 0157:1-17 and Yersinia enterocolitica. It was recently concluded that even these numbers appear to represent a substantial underestimate of the public health burden of this enteric pathogen and that for every case of Campylobacter infection reported in Canada each year, there are an additional unreported 23 to 49 cases.
  • Influenza is a major public health problem because of its considerable mortality in elderly people and morbidity in all. It appears as an epidemic during the winter months usually lasting 6 to 8 weeks in each particular location. Spread of the virus is by contact with respiratory secretions on infected hands and aerosols created by sneezing. As the much less severe common cold virus has similar symptoms, it is important to rapidly and accurately diagnosis influenza infections, especially in hospitalized patients as well as in nursing and retirement home residents and staff in order to prevent spread. Appropriate treatment should be given hospitalized patients and prophylaxis used to curtail spread. An improved method is presented here to rapidly diagnose influenza virus, to identify whether it is a pandemic strain or not, and to identify whether it is susceptible or not to Oseltamivir (TamifluTM) the most common influenza antiviral agent.
  • TamifluTM Oseltamivir
  • SNP single nucleotide polymorphisms
  • Example III an application of TaqTm probing to identify bacteria by their signature sequences in 16S ribosomal RNA genes. Infections are usually caused by a single bacterial species and only a limited number of different bacteria cause over 95% of infections at particular sites.
  • identity of bacteria present in vaginosis was determined based on the assumption that probes will react with highest melting temperatures for their exact intentional target, but will give lower melting temperatures for other organisms, allowing us to detect their presence. A large number of samples was analysed to explore the frequency of variation and its possible impact on this technology.
  • TaqTm probing strategies as shown herein could be employed to identify bacteria by their signature sequences in 16S ribosomal RNA genes. Alternatively, cpn60 genes could also have been employed. This procedure could be used for urinary tract infections as they are the most frequently tested in clinical laboratories.
  • Urinary tract infection A variety of possible primer pairs were analyzed for their ability to efficiently amplify enterobacteria and staphylococci and should amplify most other bacteria as well.
  • five different colored probes can be designed, one each for Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, group B streptococci and the fifth for a positive control. These probes are expected to react with highest melting temperatures for their intentional target, but give lower melting temperatures for related organisms, allowing us to detect their presence.
  • confirmation of these other organisms and antibiotic sensitivity determination would require culture or subsequent PCR reactions.
  • rapid results for most infections could be obtained. Slight adaptation of these sequences could be applied to other infection types like wounds, blood cultures, pneumonia or meningitis.
  • kits are in reference to a combination of articles that facilitate a process, method, assay, analysis or manipulation of a sample.
  • Kits can contain written instructions describing how to use the kit (e.g., instructions describing the methods), chemical reagents or enzymes required for the method, primers and probes, as well as any other components.
  • kits can include, for example but not limited to, reagents for sample collection, reagents for the collection and purification of RNA from blood, a reverse transcriptase, primers suitable for reverse transcription and first strand and second strand cDNA synthesis to produce an amplicon, probes, a thermostable DNA-dependent DNA polymerase and buffers containing deoxyribonucleotide triphosphates.
  • the enzyme comprising reverse transcriptase activity and thermostable DNA-dependent DNA polymerase activity are the same enzyme.
  • kits are diagnostic kits, where the information obtained from performing the methods enabled by the kits is used to identify the presence or absence of a microorganism or virus including drug resistance in or the subtype of the microorganism or virus.
  • the kits can provide any or all of the synthetic oligonucleotides used in methods described herein.
  • the kits can provide oligonucleotide primer(s) suitable for priming reverse transcription from a viral RNA molecule to produce a viral cDNA.
  • the kits can provide amplification primers suitable for amplification of any suitable portion of a genome.
  • the invention provides suitable amplification primers that can be included in kits of the invention. It is understood that the invention is not limited to the primers recited herein, as any other suitable amplification primers also find use with the invention.
  • kits of the invention can include oligonucleotide probes suitable for the determination of the melting temperature as well as amplification curves. It is understood, however, that the kits of the invention are not limited to the primers and probes described in the experimental section, as the invention also provides guidance for the identification and synthesis of additional suitable primers and probes.
  • the probes provided in kits of the invention are preferably labeled (e.g. via a covalent link).
  • kits of the present invention can also include, for example but not limited to, apparatus and reagents for sample collection and/or sample purification (e.g., isolation of RNA from a blood sample), sample tubes, holders, trays, racks, dishes, plates, instructions to the kit user, solutions, buffers or other chemical reagents, suitable samples to be used for standardization, normalization, and/or control samples (e.g., positive controls, negative controls or calibration controls). Kits of the present invention can also be packaged for convenient storage and shipping, for example, in a container having a lid. The components of the kits may be provided in one or more containers within the kit, and the components may be packaged in separate containers or may be combined in any fashion. In some embodiments, kits of the invention can provide materials to facilitate high-throughput analysis of multiple samples, such as multiwell plates that can be read in a suitable real time PCR thermal cycler or fluorescence spectrophotometer.
  • C. jejuni was isolated from humans, bovines, birds and water. Crude bacterial DNA was extracted with a hot NaOH lysis procedure from bacterial colonies. Normally 1 ⁇ of bacterial sample DNA was added to 14 ⁇ of PCR reaction mix. Primers and probes preparation. The sequence of the primers and probes used are presented in Tables 1 and 2.
  • the nucleic acids were synthesized by IDT-DNA, Coralville, IA. The nucleic acids were dissolved in water to make 50 ⁇ stock solutions. Stock solutions were then diluted to 5 ⁇ working solutions.
  • PCR CampGyrWtHex Cip s 54.3°C 63.0 U C PCR protocol and apparatus.
  • PCR consisted of an initial denaturation step at 95°C [10 min], followed by 40 cycles (95°C [10 s] for denaturation, 60°C [30 s] for annealing and 72°C [15 s] for elongation). Fluorescent levels were acquired at 72°C after each cycle.
  • Real-time PCR reactions were carried out in a 96-well plate in a Roche LightCyclerTM II 480 real-time PCR system apparatus.
  • Melting temperature determinations Melting temperature determinations. Melting temperature determinations were measured theoretically using the DINAmeltTM program (http://dinamelt.bioinfo.rpi.edu/). A temperature melting curve (Tm) was generated by denaturing products at 95°C [1 min] and followed by a renaturation step at 40°C [1 min], then temperature was ramped from 45°C to 95°C at 0.03°C/sec, 5 acquisitions/°C. Fluorescence data generated by Tm curves were plotted and analyzed by the LightCyclerTM 480 Tm calling Software.
  • HRM High Resolution Melting
  • the reaction mix contained 0.2 ⁇ of the primers, 7.5 ⁇ of 2X Roche LightCyclerTM 480 High Resolution Melting Master Kit that containing ResolightTM fluorescent dye and 3 mM of MgCI 2 .
  • HRM program consisting of a denaturation step at 95°C [1 min] and renaturation step at 40°C [1 min].
  • Melting curves were obtained by gradually heating from 65°C to 95°C at 0.02°C/sec, with 25 acquisitions/°C. Fluorescence data generated by HRM curves were normalized, plotted and analyzed by the LightCyclerTM 480 Gene Scanning Software.
  • the reaction mix contained 0.2 ⁇ of forward and reverse primers, 0.1 ⁇ of fluorescent probes and 7.5 ⁇ of 2X Roche LightCyclerTM 480 Probe Master Kit.
  • the mix contained 0.1 ⁇ of unlabelled probe. Primer concentrations were modified to 0.7 ⁇ for the forward primers and to 0.07 ⁇ for the reverse primers.
  • the HRM reaction mix and conditions were used except that the melting temperature determination curve rather than an HRM analysis was performed.
  • VentRTM Exo-
  • KlenTaqDV ReadyMixTM Sigma Aldrich, Oakville ON
  • Taq Qiagen, Mississauga ON
  • KlenTaqTM, Pfu TurboTM Taq and Roche Taq supplied in the 2X Roche LightCyclerTM 480 Probe Master Kit (Roche Diagnostics, Laval, QC).
  • Fig. 3 Different polymerases were also evaluated (Fig. 3) to determine which one produces the more suited amplification profiles and melting temperature profiles.
  • the Roche Taq polymerase in the Roche LightCyclerTM 480 Probe Master Kit (Fig. 3A and 3E), KlenTaq (Fig. 3B and 3F), Taq (Fig. 3C and 3G) and Vent (exo-) (Fig. 3D and 3H) were tested.
  • the KlenTaqDV ReadyMixTM, PFU Turbo and Taq provided by Qiagen were also tested.
  • Roche Taq and Taq provided the two highest fluorescent intensities (Fig. 3).
  • the Roche enzyme in kit form proved to be the most robust for a large number of C. jejuni isolates.
  • the primer concentration ratio that provided a large quantity of the DNA strand complementary to the probe and minimized production of the DNA strand co- linear with the probe was determined.
  • Amplification profiles (Fig. 6A to 6D) followed by a melting curve analysis (Fig. 6.E to H) for SNP detection using asymmetric PCR with fluorescent probes, were obtained after simultaneous amplification of a 131 bp amplicon from the 23S rRNA gene and a 232 bp amplicon from the gyrA gene using erythromycin sensitive and a resistant C. jejuni isolates and ciprofloxacine sensitive and resistant C. jejuni isolates (Ery R /Cip s or Ery s /Cip R ). Erythromycin and ciprofloxacine sensitive or resistant strains were detected by four different fluorescent probes specific for the sensitive or resistant sequence (refer to table 1 B and 2B).
  • the mutated nucleotide conferring erythromycin resistance (A2075G) or ciprofloxacin resistance (C258T) produced a mismatch between probes specific for the sensitive sequence and resistant DNA strand (Fig. 6E, Ery R or Fig. 6G, Cip R ) or between probes specific for the resistance sequence and sensitive DNA strand (Fig. 6F, Ery s or Fig. 6H, Cip s ). Mismatches decreased the annealing temperature. Perfect matches between sensitive specific probes and sensitive DNA strand (Fig. 6E, Ery s or Fig. 6G, Cip s ), or perfect match between resistance specific probes and resistance DNA strand (Fig. 6.F, Ery R or Fig. 6H, Cip R ), showed higher annealing temperatures. Observation of these differences in annealing temperature, improves our ability to distinguish between sensitive and resistant C. jejuni isolates.
  • the 23S rRNA gene in C. jejuni is present in 3 copies, which may be identical, or occasionally, different. Correctly identifying bacteria with heterologous allelic sequences can be challenging.
  • Ery R /Ery R /Ery s DNA from bacteria sensitive to erythromycin were artificially mixed with resistant bacteria.
  • PCR was performed on these two artificial 23S bacteria mixes and on natural bacteria with all of three 23S gene copies that are resistant (Ery R /Ery R /Ery R ); or sensitive (Ery s /Ery s /Ery s ). Mixes produced ambiguous amplification curves where both sensitive and resistant specific probes gave partial reactions, but it was clear form the melting temperature analysis that both sensitive and resistant sequences were present and could be detected (Fig. 7).
  • RESISTANCE Viral RNA Nasopharyngeal aspirates containing either pandemic influenza A/H1 N1 , seasonal influenza A/H1 N1 , or influenza A H3N2 were obtained in 0.25 ml or 0.5 ml aliquots. Nucleic acids were extracted using either the MagnaPure CompactTM (Roche Diagnostics, Laval, Quebec) or the EasyMagTM apparatus (BioMerieux Canada, Montreal, Quebec).
  • RNA bacteriophage P7 RNA bacteriophage P7
  • RT-PCR One ⁇ aliquot of viral RNA or dilutions in pooled extracts of viral RNA samples was added to 14 ⁇ of reaction mix containing 0.7 ⁇ of both primers or, for asymmetric PCR, usually 1 ⁇ of the primer producing the DNA strand complementary to the probe and 0.07 ⁇ of the primer co-linear with the probe, usually 0.2 ⁇ probes (see Table 3A and 3B for primer and probe sequences synthesized by IDT-DNA), 3 ⁇ of QuantiTecTM Virus Master 5X (Qiagen), and 0.15 ⁇ QuantiTecTM RT mix (Qiagen).
  • Amplification started with 20 min at 50°C for reverse transcription, 5 minutes at 95°C for denaturation of DNA/RNA products and activation of the enzyme, followed by 45 cycles of 15 sec at 95°C, 30 sec at 60°C with a single acquisition, and 15 sec at 72°C.
  • a melting curve was generated by denaturing products for 10 sec at 95°C, renaturing at 30°C or 40°C for 30 sec, then gradually heating to 80°C or 95°C and measuring fluorescence at each temperature.
  • Melting temperature determinations Melting temperature determinations. Melting temperature determinations were measured theoretically using the DINAmeltTM program (http://dinamelt.bioinfo.rpi.edu/) for 0.1 ⁇ DNA strands in 50 ttiM NaCI and 3 mM MgCI 2 . Melting curves were also performed using fluorescent or unlabelled probes together with synthetic unlabelled DNA with the sequence of pandemic H1 N1 , seasonal H1 N1 , seasonal H3N2 or avian H5N1 from the genome portion complementary to the probes (see Table 3C).
  • Both the probe and the synthetic DNA were used at 0.1 ⁇ in RT- PCR hybridization buffer (50 mM NaCI, 10 mM Tris-HCI, pH 8.8, 3 mM MgCI 2 ) or in 1X QuantiTecTM Virus Master. SyBrTM green (Molecular Probes) was added to 1X when unlabelled probes were used.
  • Matrix FluAM52C CTTCTAACCGAGGTCGAAACG SEQ ID NO: 10
  • Matrix FluAM253R AGGGCATTYTGGACAAAGCGTCTA SEQ ID NO: 11
  • Neuraminidase ACCACA (SEQ ID NO: 27)
  • Neuraminidase ACCACA (SEQ ID NO: 28)
  • Neuraminidase ACCACA (SEQ ID NO: 29)
  • Matrix gene for detection and subtyping. Most influenza genes are highly variable between subtypes with the gene coding for the Matrix protein being the most highly conserved. It was reported that primers in this gene would amplify all influenza sub-types. Within this amplicon, there are regions conserved among most influenza subtypes where probes to allow RT-PCR detection have been designed (see Figure 8). Within the amplified sequence used for detection, there is also a region where many influenza subtypes differ (see Figure 8, subtype specific probes are identified) that is not necessarily amplified by other techniques. Using the 3 sub-type specific probes, it was determined that, with an annealing temperature of 60°C, each probe would be specific for its sub-type within the 3 most common human sub-types.
  • pandemic probe might react with the avian H5N1 influenza amplicon.
  • RT-PCR using these probes with pandemic H1 N1 , seasonal H1 N1 , seasonal H3N2 was performed. Briefly, purified RNA was extracted from patient samples containing different sub-types of influenza A virus.
  • pandemic H1 N1 , seasonal H1 N1 , and H3N2 were amplified in reaction mixes containing matrix primers (Forward primer at 0.07 ⁇ and reverse primer at 0.7 ⁇ ) that amplify all subtypes as well as four probes (all in Forward orientation at 0.2 ⁇ ) with different fluorophores: one specific for each of pandemic H1 N1 , seasonal H1 N1 , H3N2 and a universal probe that should detect all subtypes.
  • Representative amplification curves are shown in Fig 9A, 9C, and 9E and confirm the specificity of the method.
  • the melting temperature of the various amplicons was determined. Briefly, “observed” values were obtained by mixing synthetic targets (Table 3C) with the individual probe (Table 3B) separately and performing a melting curve. The “predicted” value was obtained using the DINAmeltTM software as described above. Hybridizing the sub-type specific probes with synthetic unlabelled DNA with the sequence of pandemic H1 N1 , seasonal H1 N1 , seasonal H3N2 or avian H5N1 from the genome portion complementary to the probes indicated that avian H5N1 would cross-react with the pandemic probe (see Table 4), but with a melting temperature lower than that obtained for the pandemic target.
  • pandemic H1 N1 complementary sequence hybridized with the H3N2 probe with a melting temperature of 55°C, but did not produce an amplification curve when RT-PCR was performed with an annealing temperature of 60°C (see Fig. 9A).
  • 0.1 ⁇ primer and 0.2 ⁇ probe might leave intact probe at the end of the PCR reaction in order for a melting curve to be established.
  • a PCR enzyme that does not have a 5' endonuclease activity and would not degrade the probe could be employed to preserve intact probe. It would not generate a typical PCR curve however because it would not degrade the probe, so signal acquisition would have to be determined rapidly at the annealing temperature before the polymerase displaced the probe in the course of synthesis of a new DNA strand.
  • Fluorescence intensity and crossing points (cycle when fluorescence becomes first visible) in the fluorescent acquisition over cycles curve as well as the fluorescence intensity of melting temperature peaks for a fixed amount of the primer producing the strand complementary to the probe (0.7 ⁇ ) and progressively lower amounts of the other primer from 0.2 to 0.01 ⁇ was evaluated (see Table 5). With the exception of the lowest concentration of the reverse primer where no amplification curve was observed, the crossing points (Cp) did not vary with different amounts of the reverse primer. The maximum fluorescence intensity of the amplification curve decreased only slightly between 0.2 and 0.03 ⁇ . At all of the reverse primer concentrations tested, a melting temperature peak at the appropriate temperature was observed.
  • Neuraminidase probes to distinguish oseltamivir sensitivity A single FAM labeled probe with a sequence corresponding to that of the region surrounding the mutation causing resistance to oseltamivir in pandemic H1 N1 virus yielded melting temperatures with synthetic probes corresponding to sensitive and resistant pandemic H1 N1 , seasonal H1 N1 and H5N1 virus (see Figure 10 and Figure 11C).
  • this probe in a multiplex RT-PCR reaction with primers that amplify the matrix gene target as well as primers that amplify the neuraminidase gene, the matrix and neuraminidase targets can be efficiently amplified and probes used to detect them.
  • FIG 11 A & 11 B it is shown that an osteltamivir sensitive pandemic H1 N1 and oseltamivir sensitive and resistant seasonal H1 N1 virus amplified with matrix and neuraminidase primers with the universal probe for Influenza A matrix gene labeled with the fluorophore TEX615 and the neuraminidase probe labeled with FAM.
  • Amplification curves are detected for both subtypes by fluorescence at 615 nM (not shown), but only the pandemic H1 N1 virus yields an amplification curve with fluorescence at 510 (FAM). Both influenza sub-types give the same melting temperature peak at 615 nM (not shown).
  • pandemic H1 N1 isolate gives a melting temperature of 65°C typical of oseltamivir sensitivity whereas the seasonal H1 N1 isolates give melting temperature peaks at 42°C typical of oseltamivir sensitivity and at 37°C typical of oseltamivir resistance in this sub-type (Fig. 11 A), even though amplification curves were not observed (Fig. 11 B).
  • Neuraminidase probes with inosine bases The melting temperatures of the oseltamivir sensitive and resistant seasonal H1 N1 virus are near the limits of detection with most thermal cyclers.
  • the inclusion of inosine bases at some of the bases of mismatch would slightly reduce melting temperatures for the pandemic and H5N1 sub-types, but would significantly increase melting temperatures for the seasonal H1 N1 (see Figure 10 and Fig 11 D) making detection more straightforward.
  • Primers and probes preparation were synthesized by IDT- DNA, Coraiville, IA and were dissolved in water to make 50 ⁇ stock solutions. Stock solutions were then diluted to 5 ⁇ working solutions.
  • Dialister pneumosintes (16S rRNA Dial476F TGACGGTACCGGAAAAGC (SEQ ID gene) - 196 bp amplicon NO: 31 )
  • Dialister pneumosintes (16S rRNA Dial662R CTCTCCGATACTCCAGCTTC (SEQ ID gene) - 196 bp amplicon NO: 32)
  • Anaerococcus sp. (16S rRNA gene) AnCoc318F ATTGGGACTGAGACACGGC (SEQ ID - 334 bp amplicon NO: 34)
  • Anaerococcus sp. (16S rRNA gene) AnCoc642R CACTAGGAATTCCACTTTCCCT (SEQ - 334 bp amplicon ID NO: 35)
  • PCR protocol Reaction mixes in a final volume of 15 ⁇ contained Roche Fast Start RT-PCR master mix, were analysed on a Roche LC480TM thermal cycler. Two (2) ⁇ of a mixture of equal volumes of specimen and specimen diluent were added to a final volume of 12 ⁇ containing the LC480 Probe Master kitTM (Roche) and 0.2 ⁇ of labelled probe, 0.7 ⁇ of primers for the strand complementary to the probe, and 0.07 ⁇ of primers for the strand collinear to the probe. After 10 minutes at 95°C, 50 cycles of 10 sec at 93°C, 15 sec at 55°C and 15 sec at 72°C were performed.
  • Tm melting temperature curve
  • Sequences of the target organisms were compared with other bacteria of similar genera in order to identify primers and probes that would be specific. Examples of the location of some of the primers and probes used are shown in Figure 12.
  • Figure 13 shows an example of the comparison of primers and probes within very similar genera. Differences within the sequence of the probes and primers for the genera Dialister are highlighted. The differences within the probe sequence between D. pseumosintes, D. invisus, and D. microaerophilus should yield maximal melting temperatures by TaqTm probing for the homologous D. pseumosintes, but much lower temperatures for D. invisus, and D. microaerophilus. With the PCR protocol presented herewith, it is not expect that species other than D.
  • pseumosintes would register a positive result.
  • Other species not shown, or indeed unknown, might have fewer mismatches and yield a positive result by conventional PCR with a re-association temperature of 55°C, but if they had even one mismatch would give a renaturation temperature lower than the temperature expected for D. pseumosintes and could thus be detected.
  • a variant D. pseumosintes strain with a slightly different 16S rRNA gene sequence would also give a lower melting temperature, but we would see this and be able to further characterize such very similar isolates.
  • melting temperatures can be obtained readily for rRNA genes amplified from a variety of bacteria found in vaginal fluid using TaqTm probing. Maximal melting temperatures probably indicate the precise bacteria or species whereas lower temperatures probably reveal related or variant organisms. It was presented that most of the positive reactions observed had an exact melting temperature match with the probe, except for Anaerococcus where two different targets were detected with quite different probe:amplicon melting temperatures. It will be important to sequence amplified DNA from some of these samples in order to identify more precisely the microorganism(s) present and the possible variations they carry.

Abstract

La présente demande concerne des procédés et des kits afférents pour la détection d'une différence nucléotidique entre une séquence cible et des séquences standards. Les procédés comprennent la détermination d'une température de fusion d'un complexe entre un amplicon de la séquence cible et une sonde qui est sensiblement identique à une des séquences standards. La sonde comprend un marqueur détectable, lié à son extrémité 5' ainsi qu'un fluorochrome suppresseur, lié à son extrémité 3'. Le procédé selon l'invention peut être facilement adapté à de multiples sondes et la PCR multiplex.
PCT/CA2011/050443 2010-07-20 2011-07-20 Procédés de détection de polymorphismes génétiques à l'aide de la température de fusion de complexes sonde—amplicon WO2012009813A1 (fr)

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US20140329763A1 (en) * 2011-11-09 2014-11-06 Nanjing Sen Nan Biotechnology Research Co., Ltd Anti-influenza nucleic acid, peptide nucleic acid and preparations thereof
US9279125B2 (en) * 2011-11-09 2016-03-08 Nanjing Sen Nan Biotechnology Research Co., Ltd. Anti-influenza nucleic acid, peptide nucleic acid and preparations thereof
WO2013113748A1 (fr) * 2012-02-02 2013-08-08 Primer Design Ltd Procédé pour détecter et génotyper un acide nucléique cible
CN103421892A (zh) * 2013-05-24 2013-12-04 华中农业大学 鉴别空肠弯曲菌及大环内酯类耐药突变的荧光pcr方法
CN103525929A (zh) * 2013-10-14 2014-01-22 中国农业大学 快速检测空肠弯曲菌gyrA基因突变位点的引物组及其应用
CN103525929B (zh) * 2013-10-14 2015-10-14 中国农业大学 快速检测空肠弯曲菌gyrA基因突变位点的引物组及其应用
CN103525930A (zh) * 2013-10-15 2014-01-22 中国农业大学 一种检测耐喹诺酮类抗生素空肠弯曲杆菌的多重pcr方法及试剂盒
CN103695559A (zh) * 2014-01-03 2014-04-02 中国农业大学 检测空肠弯曲菌喹诺酮类抗生素耐药突变位点的方法
CN103695559B (zh) * 2014-01-03 2015-06-17 中国农业大学 检测空肠弯曲菌喹诺酮类抗生素耐药突变位点的方法
WO2021138544A1 (fr) * 2020-01-03 2021-07-08 Visby Medical, Inc. Dispositifs et procédés d'essai de susceptibilité aux antibiotiques
US11352675B2 (en) 2020-01-03 2022-06-07 Visby Medical, Inc. Devices and methods for antibiotic susceptability testing
US11952636B2 (en) 2020-01-03 2024-04-09 Visby Medical, Inc. Devices and methods for antibiotic susceptibility testing

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