WO2015103710A1 - Methods, reagents and kits for the assessment of bacterial infection - Google Patents

Methods, reagents and kits for the assessment of bacterial infection Download PDF

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Publication number
WO2015103710A1
WO2015103710A1 PCT/CA2015/050020 CA2015050020W WO2015103710A1 WO 2015103710 A1 WO2015103710 A1 WO 2015103710A1 CA 2015050020 W CA2015050020 W CA 2015050020W WO 2015103710 A1 WO2015103710 A1 WO 2015103710A1
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Prior art keywords
nucleic acid
sequence
rrna
seq
difficile
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PCT/CA2015/050020
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French (fr)
Inventor
Yves LONGTIN
Matthew OUGHTON
Ivan Brukner
André DASCAL
Vincenzo FORGETTA
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The Royal Institution For The Advancement Of Learning/Mcgill University
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Publication of WO2015103710A1 publication Critical patent/WO2015103710A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria

Definitions

  • the present invention generally relates to the assessment of total bacterial load in a sample, as well as the assessment of infection status of opportunistic infections such as Clostridium difficile infection (CDI) in clinical settings.
  • CDI Clostridium difficile infection
  • Clostridium difficile (C. difficile) is a Gram-positive, anaerobic, spore-forming bacterium that has been identified as the major cause of nosocomial antibiotic-associated diarrhea, which in some cases leads to pseudomembranous colitis.
  • C. difficile has been reported to be carried asymptomatically by 0% to 15% of healthy adults and by 13% to 26% of hospitalized patients (Matsuda et al., Applied and Environmental Microbiology, 78(15): 51 1 1-51 18). Most of the hospitalized patients remain asymptomatic carriers, while some develop infections that vary from mild, watery diarrhea to fatal pseudomembranous colitis. The causes of this high variation in clinical presentation are not fully understood.
  • CDI C. difficile infection
  • ToxB toxin(s)
  • these methods could also increase the detection of colonization in healthy people, up to 55.5% of which are colonized (Naaber et al., J Clin. Microbiol, 49(10): 3656-3658).
  • CDI Crohn's disease
  • the present invention provides the following items 1 to 65:
  • nucleic acid molecule of 50 nucleotides or less comprising a sequence of at least 10 contiguous nucleotides of one of the following nucleotide sequences (I) to (VII):
  • the isolated nucleic acid molecule of item 1 which comprises a sequence of at least 15 contiguous nucleotides of one of the nucleotide sequences (I) to (VII). 3. The isolated nucleic acid molecule of item 1 or 2, which is of 35 nucleotides or less.
  • nucleic acid molecule of any one of items 1 to 3, the nucleotide sequence of which comprises the sequence 5'-TATTACCGCGGCTGCT-3' (SEQ ID NO: 1 ).
  • the isolated nucleic acid molecule of item 4 the nucleotide sequence of which comprises the sequence 5'-TACGTATTACCGCGGCTGCT-3' (SEQ ID NO: 8).
  • the isolated nucleic acid molecule of item 5 the nucleotide sequence of which consists of the sequence 5'-TACGTATTACCGCGGCTGCT-3' (SEQ ID NO: 8).
  • nucleic acid molecule of item 7 the nucleotide sequence of which consists of the sequence 5'-CGGCTAACTMCGTGCCAG-3' (SEQ ID NO: 2).
  • the isolated nucleic acid molecule of item 1 which comprises the sequence 5'- AAGSVMCGGCTAACTMCGTGCC-3' (SEQ ID NO: 3).
  • the isolated nucleic acid molecule of item 9 the nucleotide sequence of which consists of the sequence 5'-AAGSVMCGGCTAACTMCGTGCC-3' (SEQ ID NO: 3).
  • 1 The isolated nucleic acid molecule of item 1 , which comprises the sequence 5'- AATGTTGGCATGAGTAGCGAGATGT-3' (SEQ ID NO: 4).
  • the isolated nucleic acid molecule of item 15 the nucleotide sequence of which consists of the sequence 5'-AATGTTGGCATGAGTAGCGAGATGT-3' (SEQ ID NO: 4). 13. The isolated nucleic acid molecule of item 1 , which comprises the sequence 5'- TCTGAAGGATTACCTRTAATTGCAA-3' (SEQ ID NO: 5).
  • the isolated nucleic acid molecule of item 17 the nucleotide sequence of which consists of the sequence 5'-TCTGAAGGATTACCTRTAATTGCAA-3' (SEQ ID NO: 5).
  • the isolated nucleic acid molecule of item 1 which comprises the sequence 5'- TGCAGCCAAAGTTGTTGAAT-3' (SEQ ID NO: 6).
  • the isolated nucleic acid molecule of item 19 the nucleotide sequence of which consists of the sequence 5'-TGCAGCCAAAGTTGTTGAAT-3' (SEQ ID NO: 6).
  • the isolated nucleic acid molecule of item 1 which comprises the sequence 5'- GCTCTTTGATTGCTGCACCT-3' (SEQ ID NO: 7). 18. The isolated nucleic acid molecule of item 1 , the nucleotide sequence of which consists of the sequence 5'-GCTCTTTGATTGCTGCACCT-3' (SEQ ID NO: 7).
  • a method for amplifying and/or detecting a bacterial 16S rRNA nucleic acid in a sample comprising (a) contacting said bacterial 16S rRNA nucleic acid with a first primer and a second primer under conditions suitable for nucleic acid amplification, and (b) performing a nucleic acid amplification reaction, thereby generating an amplification product if bacterial 16S rRNA nucleic acid is present in said sample; wherein said first primer has a length of 50 nucleotides or less and comprises a sequence of at least 10 contiguous nucleotides of the sequence ACTCCTAYGGGRBGCWSCA (SEQ ID NO: 13), and said second primer comprises a sequence of at least 10 contiguous nucleotides of nucleotide sequence (I) or (VII) defined in any one of items 1 to 6.
  • said first primer comprises the sequence ACTCCTAYGGGRBGCWSCA (SEQ ID NO: 13).
  • said first primer comprises the sequence ACTCCTAYGGGRBGCASCAGT (SEQ ID NO: 14), ACTCCTAYGGGRGGCWGCAGT (SEQ ID NO: 15), ACTCCTACGGGRGGCWGCAGT (SEQ ID NO: 16), ACTCCTAYGGGRGGCWGCA (SEQ ID NO: 17), ACTCCTACGGGRGGCWGCA (SEQ ID NO: 18), ACTCCTACG G G RG G CAG C A (SEQ ID NO: 19), ACTCCTAYGGGRBGCAGCA (SEQ ID NO: 20), or ACTCCTACGGGRBGCWGCA (SEQ ID NO: 21 ).
  • nucleotide sequence of said first primer consists of the sequence ACTCCTAYGGGRBGCASCAGT (SEQ ID NO: 14).
  • a combination of nucleic acid molecules for amplifying or detecting a bacterial 16S rRNA nucleic acid comprising the first primer defined in any one of items 19 to 27 and the second primer defined in any one of items 19 to 27.
  • a method for determining whether a sample comprises a bacterial 16S rRNA nucleic acid comprising performing the method for amplifying or detecting a bacterial 16S rRNA nucleic acid defined in any one of items 19 to 36, determining the presence or absence of said amplification product, wherein the presence of the amplification product is indicative that said sample comprises a bacterial 16S rRNA nucleic acid.
  • a method for determining the amount of bacteria in a sample comprising (a) performing the method for amplifying or detecting a bacterial 16S rRNA nucleic acid defined in any one of items 19 to 36, (b) comparing the amount of amplification product present after said amplification reaction to a reference; and (c) determining the amount of bacteria in the sample based on said comparison.
  • a method for detecting a C. difficile-specific 16S rRNA nucleic acid in a sample comprising (a) contacting said C. difficile-specific 16S rRNA nucleic acid with a first primer and a second primer under conditions suitable for nucleic acid amplification, (b) performing a nucleic acid amplification reaction, thereby generating an amplification product if C.
  • nucleic acid amplification reaction with a probe that hybridizes to said amplification product if present, wherein said probe comprises a sequence of at least 10 contiguous nucleotides of nucleotide sequence (III) or (VII) defined in any one of items 1 to 3, 1 1 and 12.
  • a method for detecting a C. difficile toxin B nucleic acid in a sample comprising (a) contacting said C. difficile toxin B nucleic acid with a first primer and a second primer under conditions suitable for nucleic acid amplification, (b) performing a nucleic acid amplification reaction, thereby generating an amplification product if C.
  • said probe comprises a sequence of at least 10 contiguous nucleotides of nucleotide sequence (IV) or (VII) defined in any one of items 1 to 3, 13 and 14;
  • said first primer comprises a sequence of at least 10 contiguous nucleotides of nucleotide sequence (V) or (VII) defined in any one of items 1 to 3, 15 and 16; and/or said second primer comprises a sequence of at least 10 contiguous nucleotides of nucleotide sequence (VI) or (VII) defined in any one of items 1 to 3, 17 and 18.
  • nucleic acid molecules comprising a sequence of at least 10 contiguous nucleotides of sequences (IV), (V), (VI) and/or (VII) defined in any one of items 1 to 3 and 13 to 18 for amplifying and/or detecting a nucleic acid encoding a C. difficile toxin B in a sample.
  • RNaseP and/or a nucleic acid encoding a fungal 18S rRNA thereby obtaining a bacterial 16S rRNA signal, a human RNaseP signal and/or a fungal 18S rRNA signal; normalizing the C. difficile toxin signal using the bacterial 16S rRNA and/or human RNaseP signal, thereby obtaining a normalized C. difficile toxin signal;
  • a method for predicting the severity or acuteness of Clostridium difficile infection (CDI) in a subject comprising:
  • the method of item 52 further comprising performing an amplification reaction on a nucleic acid encoding a C. difficile-specific 16S nucleic acid, thereby obtaining a C. difficile-specific 16S amplified product, contacting the C. difficile-specific 16S amplified product with a probe hybridizing to said nucleic acid encoding a C. difficile-specific 16S, thereby obtaining a C.
  • AOI-CD score (1/Cpt)/(Cpu-Cpt) or (1/Cpt)/(Cpg-Cpt) wherein
  • Cpu bacterial 16S rRNA signal value
  • Cpg human RNaseP signal value. 55. The method of any one of items 50 to 54, wherein said amplification reaction is performed by polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • any one of items 50 to 56 wherein said sample is stool, feces, anal swab or rectal swab.
  • said method comprises performing an amplification reaction on a nucleic acid encoding a bacterial 16S rRNA, thereby obtaining a bacterial 16S rRNA signal.
  • a method for normalizing the amount of a pathogen of interest in a sample comprising:
  • pathogen is a bacterial pathogen.
  • opportunistic pathogen is Clostrodium difficile, Helicobacter pylori, Haemophilus influenza or Streptococcus pneumonia.
  • nucleic acid specific for said pathogen of interest is a C. difficile toxin nucleic acid.
  • FIG. 1 shows a nucleotide sequence from the rrsB operon (containing a 16S rRNA gene) of Escherichia coli strain K-12 substr. MG1655 (corresponding to nucleotides 4166659 to
  • Labeled Y-axis values from bottom to top: 14, 16, 18 and 20; labeled X-axis values, from left to right: 0, 0.01 , 0.02, 0.03 and 0.04.
  • Labeled Y-axis values from bottom to top: 13, 15, 17, 19 and 21 ; labeled X-axis values, from left to right: 16, 18 and 20.
  • FIG. 3 shows a comparison of the total bacterial load determined by the novel 16S rDNA qPCR assay described herein and anaerobic culture plating/counting method. Labeled Y- axis values, from bottom to top: 16, 18 and 20; labeled X-axis values, from left to right: 0-10 2 ,
  • FIG. 4 shows the distribution of Ct values using the novel 16S rDNA qPCR assay described herein amongst 500 stool swabs (from a collection of C. difficile positive samples).
  • FIG. 5 shows a representative relative fluorescence units signal (RFU) versus cycle diagram for stool sample.
  • REU relative fluorescence units signal
  • isolated nucleic acid molecule refers to a polymer of nucleotides, and includes, but should not limited to DNA and RNA.
  • isolated nucleic acid molecule is purified from its natural in vivo state, obtained by cloning or chemically synthesized.
  • Amplification refers to any in vitro procedure for obtaining multiple copies
  • amplicons or “amplification products” of a target nucleic acid sequence or its complement or fragments thereof.
  • In vitro amplification refers to production of an amplified nucleic acid that may contain less than the complete target region sequence or its complement.
  • In vitro amplification methods include, e.g., transcription-mediated amplification, replicase-mediated amplification, polymerase chain reaction (PCR) amplification, ligase chain reaction (LCR) amplification and strand-displacement amplification (SDA including multiple strand-displacement amplification method (MSDA)).
  • Replicase-mediated amplification uses self-replicating nucleic acid molecules, and a replicase such as G ⁇ -replicase (e.g., Kramer et al., U.S. Pat. No. 4,786,600).
  • G ⁇ -replicase e.g., Kramer et al., U.S. Pat. No. 4,786,600
  • PCR amplification is well known and uses DNA polymerase, primers and thermal cycling to synthesize multiple copies of the two complementary strands of DNA or cDNA (e.g., Mullis et al., U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159).
  • LCR amplification uses at least four separate oligonucleotides to amplify a target and its complementary strand by using multiple cycles of hybridization, ligation, and denaturation (e.g., EP Pat. App. Pub. No. 0 320 308).
  • SDA is a method in which a primer contains a recognition site for a restriction endonuclease that permits the endonuclease to nick one strand of a hemimodified DNA duplex that includes the target sequence, followed by amplification in a series of primer extension and strand displacement steps (e.g., Walker et al., U.S. Pat. No. 5,422,252).
  • oligonucleotide primer sequences of the present invention may be readily used in any in vitro amplification method based on primer extension by a polymerase, (see generally Kwoh et al., 1990, Am. Biotechnol. Lab. 8:14 25 and (Kwoh et al., 1989, Proc. Natl. Acad. Sci.
  • oligos are designed to bind to a complementary sequence under selected conditions.
  • Nucleic acid hybridization refers generally to the hybridization of two single stranded nucleic acid molecules having complementary base sequences, which under appropriate conditions will form a thermodynamically favored double stranded structure. Examples of hybridization conditions can be found in the two laboratory manuals referred above (Sambrook et al., 2000, and Ausubel et al., 1994, or further in Higgins and Hames (Eds.) "Nucleic acid hybridization, a practical approach” IRL Press Oxford, Washington DC, (1985)) and are commonly known in the art.
  • a nitrocellulose filter incubated overnight at a temperature representative of the desired stringency condition (e.g., about 60-65°C for high stringency, about 50-60°C for moderate stringency and about 40-50°C for low stringency conditions) with a labeled probe in a solution containing high salt (6x SSC or 5x SSPE), 5x Denhardt's solution, 0.5% SDS, and 100 ⁇ g ml denatured carrier DNA (e.g., salmon sperm DNA).
  • a temperature representative of the desired stringency condition e.g., about 60-65°C for high stringency, about 50-60°C for moderate stringency and about 40-50°C for low stringency conditions
  • a labeled probe in a solution containing high salt (6x SSC or 5x SSPE), 5x Denhardt's solution, 0.5% SDS, and 100 ⁇ g ml denatured carrier DNA (e.g., salmon sperm DNA).
  • the non-specifically binding probe can then be washed off the filter by several washes in 0.2 x SSC/0.1 % SDS at a temperature which is selected in view of the desired stringency: room temperature (low stringency), 42°C (moderate stringency) or 65°C (high stringency).
  • the salt and SDS concentration of the washing solutions may also be adjusted to accommodate for the desired stringency.
  • the selected temperature and salt concentration is based on the melting temperature (Tm) of the DNA hybrid.
  • Tm melting temperature
  • RNA-DNA hybrids can also be formed and detected.
  • the conditions of hybridization and washing can be adapted according to well-known methods by the person of ordinary skill. Stringent conditions will be preferably used (Sambrook et al., 2000, supra).
  • hybridization kits e.g., ExpressHybTM from BD Biosciences Clonetech®
  • annealing and washing solutions can also be used as well known in the art.
  • the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions. Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
  • Hybridizing nucleic acid molecules also comprise fragments of the above described molecules.
  • nucleic acid molecules which hybridize with any of the aforementioned nucleic acid molecules also include complementary fragments, derivatives and allelic variants of these molecules.
  • a hybridization complex refers to a complex between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration.
  • a hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., membranes, filters, chips, pins or glass slides to which, e.g., cells have been fixed).
  • a solid support e.g., membranes, filters, chips, pins or glass slides to which, e.g., cells have been fixed.
  • isolated it is meant that a sample containing a target nucleic acid is taken from its natural milieu, but the term does not connote any degree of purification.
  • the present invention provides an isolated nucleic acid molecule of 50 nucleotides or less comprising a sequence of at least 10 contiguous nucleotides of one of the following sequences (I) to (VI):
  • the present invention also provides an isolated nucleic acid molecule of 50 nucleotides or less comprising a sequence of at least 10 contiguous nucleotides of one of the following nucleotide sequences (I) to (VII):
  • the isolated nucleic acid molecule comprises a sequence of at least 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleotides of sequences (I) to (VI), or a sequence having at least 80%, 85%, 90% or 95% identity with said sequences.
  • the isolated nucleic acid molecule comprises at least 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or 25 nucleotides from one of sequences (I) to (VI).
  • the isolated nucleic acid molecule has a length of at least 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides.
  • the isolated nucleic acid molecule has a length of no more than 49, 48, 47, 46, 45, 44, 43, 42, 41 , 40, 39, 38, 37, 36, 35, 34, 33, 32, 31 or 30 nucleotides.
  • the isolated nucleic acid molecule has a length comprises within any of the minimal and maximal lengths defined above, for example a length of 12 to 40 nucleotides, 15 to 40 nucleotides, 12 to 35 nucleotides, 15 to 35 nucleotides, 12 to 30 nucleotides, 15 to 30 nucleotides, 12 to 30 nucleotides, 15 to 30 nucleotides, 12 to 25 nucleotides or 15 to 25 nucleotides.
  • nucleic acid molecules defined herein may comprise, at the 5' end, an AT clamp (e.g., an AT-rich nucleotide sequence).
  • the AT clamp comprises, or consists of, the nucleotide sequence AATAAATCATAA (SEQ ID NO: 10), or a 5'- deleted fragment thereof.
  • the AT clamp comprises, or consists of, the nucleotide sequence AATAAATCATAA (SEQ ID NO: 10).
  • Primers/probes for detecting a nucleic acid encoding a bacterial 16S rRNA (bacterial 16S rDNA or gene)
  • a “probe” is meant to include a nucleic acid oligomer that hybridizes specifically to a target sequence in a nucleic acid or its complement, under conditions that promote hybridization, thereby allowing detection of the target sequence or its amplified nucleic acid. Detection may either be direct (i.e., resulting from a probe hybridizing directly to the target or amplified sequence) or indirect (i.e., resulting from a probe hybridizing to an intermediate molecular structure that links the probe to the target or amplified sequence).
  • a probe's "target” generally refers to a sequence within an amplified nucleic acid sequence (i.e., a subset of the amplified sequence) that hybridizes specifically to at least a portion of the probe sequence by standard hydrogen bonding or "base pairing." Sequences that are "sufficiently complementary” allow stable hybridization of a probe sequence to a target sequence, even if the two sequences are not completely complementary.
  • a probe may be labeled or unlabeled.
  • a probe can be produced by molecular cloning of a specific DNA sequence or it can also be synthesized.
  • the probe defined herein is a hydrolysis probe (e.g., TaqMan® probe) and comprises a fluorophore and a quencher attached thereto.
  • a "primer” defines an oligonucleotide which is capable of annealing to a target sequence, thereby creating a double stranded region which can serve as an initiation point for nucleic acid synthesis under suitable conditions.
  • the primer's 5' region may be non- complementary to the target nucleic acid sequence and include additional bases, such as a promoter sequence (which is referred to as a "promoter primer”).
  • promoter primer which is referred to as a "promoter primer”
  • any oligomer that can function as a primer can be modified to include a 5' promoter sequence, and thus function as a promoter primer.
  • any promoter primer can serve as a primer, independent of its functional promoter sequence.
  • Size ranges for primers include those that are about 10 to about 50 nt long and contain at least about 10 contiguous bases, or even at least 12 contiguous bases that are complementary to a region of the target nucleic acid sequence (or a complementary strand thereof).
  • the contiguous bases are at least 80%, or at least 90%, or completely complementary to the target sequence to which the amplification oligomer binds.
  • An amplification oligomer may optionally include modified nucleotides or analogs, or additional nucleotides that participate in an amplification reaction but are not complementary to or contained in the target nucleic acid, or template sequence.
  • the isolated nucleic acid molecules comprising at least 10 contiguous nucleotides of the sequences (I) and/or (II) defined herein may be used as primers and/or probes (hereinafter referred to as bacterial 16S rRNA nucleic acid primers and/or probes) for amplifying and/or detecting a nucleic acid encoding a bacterial 16S rRNA (a bacterial 16S rDNA, such as a bacterial 16S rRNA gene) in a sample, and more particularly for detecting 16S rRNA nucleic acids (16S rDNA) derived from a plurality of bacteria (broad-coverage amplification/detection), which may be useful for example for quantifying 16S rRNA gene copy number, and in turn estimating bacterial load, in a sample.
  • primers and/or probes for amplifying and/or detecting a nucleic acid encoding a bacterial 16S rRNA (a bacterial 16S rDNA, such
  • the nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (I) comprises the sequence 5'-TATTACCGCGGCTGCT-3' (SEQ ID NO: 1 ), or a sequence having at least 80, 85, 90 or 95% identity therewith. In a further embodiment, the nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (I) comprises the sequence 5'-TATTACCGCGGCTGCT-3' (SEQ ID NO: 1 ).
  • the nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (I) further comprises, at its 5' end, the sequence GCITCC or TAGC, or 5'-deleted fragment thereof (e.g., CITCC, ITCC, AGC, GC, C, etc.).
  • the nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (I) comprises, or consists of, the sequence 5'-GCITCCTATTACCGCGGCTGCT-3' (SEQ ID NO: 1 1 ), or a sequence having at least 80, 85, 90 or 95% identity therewith.
  • the nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (I) comprises, or consists of, the sequence 5'-TACGTATTACCGCGGCTGCT-3' (SEQ ID NO: 8), or a sequence having at least 80, 85, 90 or 95% identity therewith.
  • the nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (I) comprises or consists of the sequence 5'-TACGTATTACCGCGGCTGCT-3' (SEQ ID NO: 8).
  • the term "consists of the sequence” means that the nucleic acid molecule does not comprise additional nucleotides, but does not exclude the presence of a moiety, label or tag (e.g., fluorescent tag) attached to the nucleic acid molecule.
  • a "label” refers to a molecular moiety or compound that can be detected or can lead to a detectable signal. A label is joined, directly or indirectly, to a nucleic acid probe or the nucleic acid to be detected (e.g., an amplification product).
  • Direct labeling can occur through bonds or interactions that link the label to the nucleic acid (e.g., covalent bonds or non-covalent interactions), whereas indirect labeling can occur through the use of a "linker” or bridging moiety, such as additional oligonucleotide(s), which is either directly or indirectly labeled.
  • Bridging moieties may amplify a detectable signal.
  • Labels can include any detectable moiety (e.g., a radionuclide, ligand such as biotin or avidin, enzyme or enzyme substrate, reactive group, chromophore such as a dye or colored particle, luminescent compound including a bioluminescent, phosphorescent or chemiluminescent compound, and fluorescent compound).
  • the nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (I) as defined herein is used as a primer (e.g., reverse primer) for the amplification and/or detection of a bacterial 16S rRNA nucleic acid, more particularly a fragment of a bacterial 16S rRNA nucleic acid.
  • the nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (I) may be used in combination with any suitable primer (e.g., forward primer) for the amplification and/or detection of a bacterial 16S rRNA nucleic acid, more particularly a fragment of a bacterial 16S rRNA nucleic acid.
  • the nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (I) is used in combination with another primer (e.g., forward primer) for the amplification of a fragment of about 150 to about 250 nucleotides, of about 175 to about 225 nucleotides, of about 190 to about 210 nucleotides, or of about 200 to 205 nucleotides, from a bacterial 16S rRNA nucleic acid.
  • another primer e.g., forward primer
  • the nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (I) is used in combination with another primer (e.g., forward primer) having a length of 50, 45, 40 or 35 nucleotides or less and comprises a sequence of at least 10, 1 1 , 12, 13, 14, 15, 16, 17, 18 or 19 contiguous nucleotides of the sequence ACTCCTAYGGGRBGCWSCA (SEQ ID NO: 13), or a sequence having at least 80, 85, 90 or 95% identity therewith.
  • the other primer e.g., forward primer
  • nucleic acid molecules defined by a sequence comprising degenerate nucleotides (e.g., Y, B, W, S) defined herein
  • nucleic acid molecule or “primer”, “probe”
  • nucleic acid molecule may either correspond to a nucleic acid molecule comprising one of the sequences encompassed by the sequence comprising one or more degenerate nucleotides, or any combination of nucleic acids, each of which comprising one of the sequences encompassed by the sequence comprising one or more degenerate nucleotides.
  • the nucleic acid molecule defined by a sequence comprising one or more degenerate nucleotides corresponds to a mixture/combination (e.g., an equimolar mixture) of all the nucleic acids encompassed by the sequence.
  • the present invention provides a nucleic acid molecule mixture comprising a plurality of nucleic acid molecules comprising a plurality of sequences encompassed by any of the sequences comprising one or more degenerate nucleotides defined herein.
  • the other primer comprises the sequence ACTCCTAYGGGRBGCASCAGT (SEQ ID NO: 14), ACTCCTAYGGGRGGCWGCAGT (SEQ ID NO: 15), ACTCCTACGGGRGGCWGCAGT (SEQ ID NO: 16), ACTCCTAYGGGRGGCWGCA (SEQ ID NO: 17), ACTCCTACGGGRGGCWGCA (SEQ ID NO: 18), ACTCCTACG G G RG G CAG C A (SEQ ID NO: 19), ACTCCTAYGGGRBGCAGCA (SEQ ID NO: 20), or ACTCCTACGGGRBGCWGCA (SEQ ID NO: 21 ) or ACTCCTACGGGAGGCAGCAGT (SEQ ID NO: 22), or a sequence having at least 80, 85, 90 or 95% identity therewith.
  • the other primer e.g., forward primer
  • the other primer further comprises, at its 5' end, the nucleotide sequence 5'-AATAAATCATAA-3' (SEQ ID NO: 10) or a 5'-deleted fragment thereof (e.g., TAAATCATAA (SEQ ID NO: 26), ATAAATCATAA (SEQ ID NO: 27), AAATCATAA, etc.).
  • the other primer comprises or consists of the sequence ACTCCTAYGGGRBGCASCAGT (SEQ ID NO: 14).
  • the other primer e.g., forward primer
  • corresponds to a mixture/combination e.g., an equimolar mixture of all the nucleic acid molecules encompassed by the sequence of SEQ ID NO: 14.
  • One or both of the bacterial 16S rRNA nucleic acid primers may further comprise a moiety, label or tag (e.g., fluorescent tag) attached thereto.
  • a moiety, label or tag e.g., fluorescent tag
  • the bacterial 16S rRNA nucleic acid primers defined above may be used in combination with a probe (a bacterial 16S rRNA nucleic acid probe) that is capable of hybridizing to the amplification product generated by an amplification reaction with said bacterial 16S rRNA nucleic acid primers (i.e. capable of hybridizing to a sequence located between the primers in the sequence of the bacterial 16S rRNA nucleic acid).
  • a probe a bacterial 16S rRNA nucleic acid probe
  • the bacterial 16S rRNA nucleic acid probe is the isolated nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (II) defined above.
  • the bacterial 16S rRNA nucleic acid probe comprises, or consists of, the sequence 5'-CGGCTAACTMCGTGCCAG-3' (SEQ ID NO:2), or a sequence having at least 80, 85, 90 or 95% identity therewith.
  • the bacterial 16S rRNA nucleic acid probe further comprises the sequence 5'-AAGSVM-3', a 3'-deleted fragment thereof (e.g., AGSVM, GSVM, etc.) at its 5'-terminal.
  • the bacterial 16S rRNA nucleic acid probe comprises the sequence 5'-AAGSVMCGGCTAACTMCGTGCC-3' (SEQ ID NO: 3), or a sequence having at least 80, 85, 90 or 95% identity therewith.
  • the bacterial 16S rRNA nucleic acid probe comprises, or consists of, the sequence 5'- CGGCTAACTMCGTGCCAG-3' (SEQ ID NO: 2).
  • a moiety, a label or a tag e.g., fluorescent tag attached to the bacterial 16S rRNA nucleic acid probe, for example for detecting the complexes formed between the probe and the amplification product (bacterial 16S rRNA nucleic acid fragment).
  • the probe is a hydrolysis probe (e.g., TaqMan® probe) and comprises a fluorophore and a quencher attached thereto.
  • the nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (II) defined above comprises or consists of (a) the sequence 5'-CGGCTAACTMCGTGCCAG-3' (SEQ ID NO:2) and (b) one or more moiety, label or tag (e.g. a fluorescent tag and/or a quencher) attached thereto.
  • the present invention also provides a combination of nucleic acid molecules for amplifying or detecting a bacterial 16S rRNA nucleic acid, the combination comprising the bacterial 16S rRNA nucleic acid primers defined above.
  • the combination further comprises the bacterial 16S rRNA nucleic acid probe defined above.
  • the combination of nucleic acid molecules comprises a first primer comprising, or consisting of, the sequence ACTCCTAYGGGRBGCASCAGT (SED ID NO: 14), and a second primer comprising, or consisting of, the sequence TACGTATTACCGCGGCTGCT-3' (SEQ ID NO: 8).
  • the combination of nucleic acid molecules further comprises a probe comprising, or consisting of, the sequence CGGCTAACTMCGTGCCAG (SEQ ID NO: 2).
  • the nucleic acid molecules may be in solid form (e.g., lyophilized) or present in a solution (e.g., a suitable buffer).
  • the present invention provides a container (e.g., a tube, a vessel, a plate) comprising the nucleic acid molecules.
  • the present invention provides a bacterial 16S rRNA nucleic acid amplification or detection mixture, the mixture comprising the combination of nucleic acid molecules defined above and one or more reagents for performing an amplification reaction, for example a suitable DNA polymerase (e.g., a Tag polymerase, a Pfu polymerase or any other thermostable polymerase suitable for nucleic acid amplification), deoxynucleotide triphosphates (dNTPs), and a suitable buffer.
  • a suitable DNA polymerase e.g., a Tag polymerase, a Pfu polymerase or any other thermostable polymerase suitable for nucleic acid amplification
  • dNTPs deoxynucleotide triphosphates
  • the isolated nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (III) defined herein may be used as primers and/or probes (hereinafter referred to as C. Difficile 16S rRNA nucleic acid primers and/or probes) for amplifying and/or detecting a nucleic acid encoding a C. Difficile 16S rRNA (C. Difficile 16S rDNA, such as a C. Difficile 16S rRNA gene) in a sample, which may be useful for example for quantifying C. Difficile 16S rRNA gene copy number, and in turn estimating C. Difficile bacterial load, in a sample.
  • C. Difficile 16S rRNA nucleic acid primers and/or probes for amplifying and/or detecting a nucleic acid encoding a C. Difficile 16S rRNA (C. Difficile 16S rDNA, such as a C
  • the isolated nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (III) comprises, or consists of, the sequence 5'- AATGTTGGCATGAGTAGCGAGATGT-3' (SEQ ID NO: 4), or a sequence having at least 80, 85, 90 or 95% identity therewith.
  • the isolated nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (III) comprises, or consists of, the sequence 5'-AATGTTGGCATGAGTAGCGAGATGT-3' (SEQ ID NO: 4).
  • the nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (III) is used as a C.
  • Difficile 16S rRNA nucleic acid probe for the amplification and/or detection of a C. Difficile 16S rRNA nucleic acid, more particularly a fragment of a C. Difficile 16S rRNA nucleic acid.
  • a moiety, a label or a tag (e.g., fluorescent tag) attached to the C. Difficile 16S rRNA nucleic acid probe for example for detecting the complexes formed between the probe and the amplification product (C. Difficile 16S rRNA nucleic acid fragment).
  • the probe is a hydrolysis probe (e.g., TaqMan® probe) and comprises a fluorophore and a quencher attached thereto.
  • the C. Difficile 16S rRNA nucleic acid probe may be used in combination with suitable
  • C. Difficile 16S rRNA nucleic acid primers capable of generating an amplification product that may be detected by the probe (i.e. primers hybridizing to sequences located upstream and downstream of the sequence to which the probe hybridizes in the C. Difficile 16S rRNA nucleic acid).
  • Example of a suitable C. Difficile 16S rRNA nucleic acid primer pair for generating an amplification product that may be detected by the C. Difficile 16S rRNA nucleic acid probe comprises the following sequences: GGGAGCTTCCCATACGGGTTG (SEQ ID NO: 29) and TTGACTGCCTCAATGCTTGGGC (SEQ ID NO: 30).
  • the present invention also provides a combination of nucleic acid molecules for amplifying or detecting a C. Difficile 16S rRNA nucleic acid, the combination comprising the C. Difficile 16S rRNA nucleic acid probe defined above and suitable C. Difficile 16S rRNA nucleic acid primers capable of generating an amplification product that may be detected by the probe, for example the C. Difficile 16S rRNA nucleic acid primers defined above.
  • the nucleic acid molecules may be in solid form (e.g., lyophilized) or present in a solution (e.g., a suitable buffer).
  • the present invention provides a container (e.g., a tube, a vessel, a plate) comprising the nucleic acid molecules.
  • the present invention provides a C. Difficile 16S rRNA nucleic acid amplification or detection mixture, the mixture comprising the combination of nucleic acid molecules defined above and one or more reagents for performing an amplification reaction, for example a suitable DNA polymerase (e.g., a Tag polymerase, a Pfu polymerase or any other thermostable polymerase suitable for nucleic acid amplification), deoxynucleotide triphosphates (dNTPs), and a suitable buffer.
  • a suitable DNA polymerase e.g., a Tag polymerase, a Pfu polymerase or any other thermostable polymerase suitable for nucleic acid amplification
  • dNTPs deoxynucleotide triphosphates
  • the isolated nucleic acid molecule comprising at least 10 contiguous nucleotides of sequences (IV), (V) and/or (VI) defined herein may be used as primers and/or probes (hereinafter referred to as C. Difficile ToxB primers and/or probes) for amplifying and/or detecting a nucleic acid encoding a C. Difficile ToxB nucleic acid in a sample, which may be useful for example for quantifying toxigenic C. Difficile bacterial load in a sample.
  • the isolated nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (IV) comprises, or consists of, the sequence 5'- TCTGAAGGATTACCTRTAATTGCAA-3' (SEQ ID NO: 5), or a sequence having at least 80, 85, 90 or 95% identity therewith.
  • the isolated nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (IV) comprises, or consists of, the sequence 5'- TCTGAAGGATTACCTRTAATTGCAA-3' (SEQ ID NO: 5).
  • the nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (IV) is used as a C. Difficile ToxB probe for the amplification and/or detection of a C. Difficile ToxB nucleic acid (e.g. , C. Difficile ToxB RNA, cDNA or gene), more particularly a fragment of a C. Difficile ToxB nucleic acid.
  • a moiety, a label and/or a tag e.g., fluorescent tag
  • the probe is a hydrolysis probe (e.g. , TaqMan® probe) and comprises a fluorophore and a quencher attached thereto.
  • the C. Difficile ToxB probe may be used in combination with suitable C. Difficile ToxB primers capable of generating an amplification product that may be detected by the probe (i.e. primers hybridizing to sequences located upstream and downstream of the sequence to which the C. Difficile ToxB probe hybridizes in the C. Difficile ToxB nucleic acid).
  • the isolated nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (V) comprises, or consists of, the sequence 5'- TGCAGCCAAAGTTGTTGAAT-3' (SEQ ID NO: 6), or a sequence having at least 80, 85, 90 or 95% identity therewith.
  • the isolated nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (V) comprises, or consists of, the sequence 5'- TGCAGCCAAAGTTGTTGAAT-3' (SEQ ID NO: 6).
  • the nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (VI) comprises, or consists of, the sequence 5'- GCTCTTTGATTGCTGCACCT-3' (SEQ ID NO: 7), or a sequence having at least 80, 85, 90 or 95% identity therewith.
  • the nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (VI) comprises, or consists of, the sequence 5'- GCTCTTTGATTGCTGCACCT-3' (SEQ ID NO: 7).
  • the nucleic acid molecules comprising at least 10 contiguous nucleotides of sequences (V) and/or (VI) are used as C.
  • Difficile ToxB primers for the amplification and/or detection of a C. Difficile ToxB nucleic acid, more particularly a fragment of a C. Difficile ToxB nucleic acid.
  • the nucleic acid molecules comprising at least 10 contiguous nucleotides of sequences (V) and (VI) may be used together, or in combination with any suitable C. Difficile ToxB primer, for the amplification and/or detection of a C. Difficile ToxB nucleic acid, more particularly a fragment of a C. Difficile ToxB nucleic acid.
  • the nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (V) is used in combination with the nucleic acid molecule comprising at least 10 contiguous nucleotides of sequences (VI).
  • the present invention also provides a combination of nucleic acid molecules for amplifying or detecting a C. Difficile ToxB nucleic acid, the combination comprising a pair (e.g., first and second, or forward and reverse (or vice versa), primers) of the C. Difficile ToxB primers defined above.
  • the combination further comprises the C. Difficile ToxB probe defined above.
  • the present invention provides a combination of nucleic acid molecules for amplifying or detecting a C. Difficile ToxB nucleic acid, the combination comprising the C. Difficile ToxB probe defined above and suitable C.
  • the nucleic acid molecules may be in solid form (e.g., lyophilized) or present in a solution (e.g., a suitable buffer).
  • a container e.g., a tube, a vessel, a plate
  • nucleic acid molecules may be in solid form (e.g., lyophilized) or present in a solution (e.g., a suitable buffer).
  • the present invention provides a container (e.g., a tube, a vessel, a plate) comprising the nucleic acid molecules.
  • the present invention provides a C. Difficile ToxB nucleic acid amplification or detection mixture, the mixture comprising the combination of nucleic acid molecules defined above and one or more reagents for performing an amplification reaction, for example a suitable DNA polymerase (e.g., a Tag polymerase, a Pfu polymerase or any other thermostable polymerase suitable for nucleic acid amplification), deoxynucleotide triphosphates (dNTPs), and a suitable buffer.
  • a suitable DNA polymerase e.g., a Tag polymerase, a Pfu polymerase or any other thermostable polymerase suitable for nucleic acid amplification
  • dNTPs deoxynucleotide triphosphates
  • suitable buffer e.g., a bacterial 16S rRNA nucleic acid (bacterial 16S rDNA or gene)
  • the present invention provides the use of the isolated nucleic acid molecule comprising at least 10 contiguous nucleotides of sequences (I) and/or (II) (bacterial 16S rRNA nucleic acid primers and/or probes) for detecting a bacterial 16S rRNA nucleic acid in a sample.
  • the present invention provides a method for detecting a nucleic acid encoding a bacterial 16S rRNA nucleic acid) in a sample, the method comprising (i) contacting the sample with the bacterial 16S rRNA nucleic acid primers and/or probes defined above under conditions permitting hybridization of said bacterial 16S rRNA nucleic acid primers and/or probes with said bacterial 16S rRNA nucleic acid, if present, and (ii) detecting the hybridization signal.
  • the bacterial 16S nucleic acid is an amplification product from a biological sample from a subject.
  • the present invention provides a method for amplifying or detecting a bacterial 16S rRNA nucleic acid in a sample, said method comprising (a) contacting said bacterial 16S rRNA nucleic acid with a first primer and a second primer under conditions suitable for nucleic acid amplification, and (b) performing a nucleic acid amplification reaction, thereby generating an amplification product if bacterial 16S rRNA nucleic acid is present in said sample; wherein said first and second primers are the bacterial 16S rRNA nucleic acid primers defined herein.
  • the present invention provides a method for determining whether a sample comprises a bacterial 16S rRNA nucleic acid, the method comprising (i) contacting the sample with the bacterial 16S rRNA nucleic acid primers and/or probes defined herein under conditions permitting hybridization of said isolated nucleic acid molecule with said bacterial 16S nucleic acid, if present, and (ii) determining the presence or absence of an hybridization signal, wherein the presence of an hybridization signal is indicative that the sample comprises a bacterial 16S nucleic acid.
  • the present invention provides a method for detecting a nucleic acid encoding a bacterial 16S rRNA nucleic acid in a biological sample, the method comprising (i) performing an amplification reaction for said bacterial 16S nucleic acid on the biological sample; (ii) contacting the amplified sample with the bacterial 16S rRNA nucleic acid probes defined herein under conditions permitting hybridization of said isolated nucleic acid molecule with the amplified bacterial 16S nucleic acid, if present, and (iii) detecting the hybridization signal.
  • the present invention provides a method for determining whether a biological sample comprises a nucleic acid encoding a bacterial 16S rRNA (bacterial 16S rRNA nucleic acid, such as a bacterial 16S rDNA or gene), the method comprising (i) performing an amplification reaction for said bacterial 16S nucleic acid on the biological sample; (ii) contacting the amplified sample with the bacterial 16S rRNA nucleic acid probes defined herein under conditions permitting hybridization of said probes with the amplified bacterial 16S nucleic acid, if present, and (iii) determining the presence or absence of a hybridization signal, wherein the presence of a hybridization signal is indicative that the biological sample comprises a bacterial 16S nucleic acid.
  • bacterial 16S rRNA nucleic acid such as a bacterial 16S rDNA or gene
  • the bacterial 16S rRNA nucleic acid primers and/or probes are used in the contacting step.
  • the amplification reaction is performed using the following bacterial 16S rRNA nucleic acid primer pairs: a first primer comprising, or consisting of, the sequence ACTCCTAYGGGRBGCASCAGT (SED ID NO: 14), and a second primer which comprises, or consists of, the sequence TACGTATTACCGCGGCTGCT-3' (SEQ ID NO: 8).
  • the bacterial 16S rRNA nucleic acid and/or amplification product is detected using the following bacterial 16S rRNA nucleic acid probe: CGGCTAACTMCGTGCCAG (SEQ ID NO: 2).
  • the present invention provides a method for determining whether a sample comprises a bacterial 16S rRNA nucleic acid, said method comprising performing the method for amplifying or detecting a bacterial 16S rRNA nucleic acid defined above, determining the presence or absence of an amplification product, wherein the presence of an amplification product is indicative that said sample comprises a bacterial 16S rRNA nucleic acid.
  • the present invention provides a method for determining the amount of bacteria in a sample, said method comprising (a) performing the method for amplifying or detecting a bacterial 16S rRNA nucleic acid defined above, (b) comparing the amount of amplification product present after said amplification reaction to a reference; and (c) determining the amount of bacteria in the sample based on said comparison.
  • the reference may be the amount of amplification product present in one or more sample comprising a known or predetermined amount of bacteria.
  • the present invention provides the use of an isolated oligonucleotide comprising a sequence within sequence (VII) below for detecting a nucleic acid encoding a fungal 18S rRNA (fungal 18S rRNA nucleic acid, such as a fungal 18S rDNA gene) in a sample.
  • Sequence VII :
  • the present invention provides a method for detecting a nucleic acid encoding a fungal 18S rRNA (fungal 18S nucleic acid) in a sample, the method comprising (i) contacting the sample with an isolated oligonucleotide comprising a sequence within the sequence of SEQ ID NO: 31 under conditions permitting hybridization of said isolated nucleic acid molecule with said fungal 18S nucleic acid, if present, and (ii) detecting the hybridization signal.
  • the present invention provides a method for determining whether a sample comprises a nucleic acid encoding a fungal 18S rRNA (fungal 18S nucleic acid), the method comprising (i) contacting the sample with an isolated oligonucleotide comprising a sequence within the sequence of SEQ ID NO: 31 under conditions permitting hybridization of said isolated nucleic acid molecule with said fungal 18S nucleic acid, if present, and (ii) determining the presence or absence of an hybridization signal, wherein the presence of an hybridization signal is indicative that the sample comprises a fungal 18S nucleic acid.
  • the fungal 18S nucleic acid is an amplification product from a biological sample from a subject.
  • the present invention provides a method for detecting a nucleic acid encoding a fungal 18S rRNA (fungal 18S nucleic acid) in a biological sample, the method comprising (i) performing an amplification reaction for said fungal 18S nucleic acid on the biological sample; (ii) contacting the amplified sample with an isolated oligonucleotide (probe) comprising a sequence within the sequence of SEQ ID NO: 31 under conditions permitting hybridization of said isolated nucleic acid molecule with the amplified fungal 18S nucleic acid, if present, and (iii) detecting the hybridization signal.
  • an isolated oligonucleotide probe
  • the present invention provides a method for determining whether a biological sample comprises a nucleic acid encoding a fungal 18S rRNA (fungal 18S nucleic acid), the method comprising (i) performing an amplification reaction for said fungal 18S nucleic acid on the biological sample; (ii) contacting the amplified sample with an isolated oligonucleotide (probe) comprising a sequence within the sequence of SEQ ID NO: 31 under conditions permitting hybridization of said isolated nucleic acid molecule with the amplified fungal 18S nucleic acid, if present, and (iii) determining the presence or absence of a hybridization signal, wherein the presence of a hybridization signal is indicative that the biological sample comprises a fungal 18S nucleic acid.
  • an isolated oligonucleotide probe
  • determining the presence or absence of a hybridization signal wherein the presence of a hybridization signal is indicative that the biological sample comprises a fungal 18S nucleic acid.
  • the isolated oligonucleotide comprises one of the sequences underlined within sequence (VII), as noted above.
  • the amplification reaction is performed using a pair of primers comprising the following sequences: 5'-GGGAAACTTACCAGGTCCAG-3' (SEQ ID NO: 32) and 5'-TCGTCGTGATGGGGATAGATT-3' (SEQ ID NO: 33).
  • the fungal 18S nucleic acid (or amplified product) is detected using a probe comprising the sequence 5'- TGGTGCATGGCCGTT-3' (SEQ ID NO: 34).
  • the above-mentioned method does not comprise a step of isolating the nucleic acid from the sample (e.g., clinical sample) prior to performing the amplification reaction.
  • the above-mentioned method is compatible with hydrolysis probe technology (e.g., TaqMan). Detection of a nucleic acid encoding a C. d/ff/c/7e-specific 16S rRNA (C. difficile-specific 16S nucleic acid)
  • the present invention provides a use of the C. difficile-specific 16S rRNA primers and/or probes defined herein for amplifying or detecting a nucleic acid encoding a C. difficile-specific 16S rRNA (a C. difficile-specific 16S nucleic acid, such as a C. difficile-specific 16S rDNA or gene) in a sample.
  • a C. difficile-specific 16S rRNA a C. difficile-specific 16S nucleic acid, such as a C. difficile- specific 16S rDNA or gene
  • the present invention provides a method for detecting a nucleic acid encoding a C. difficile-specific 16S rRNA (a C. difficile-specific 16S nucleic acid) in a sample, the method comprising (i) contacting the sample with the C. difficile-specific 16S rRNA primers and/or probes under conditions permitting hybridization of said isolated nucleic acid molecule with said C. difficile-specific 16S nucleic acid, if present, and (ii) detecting the hybridization signal.
  • the present invention provides a method for determining whether a sample comprises a C. difficile-specific 16S nucleic acid, the method comprising (i) contacting the sample with the C. difficile-specific 16S rRNA primers and/or probes under conditions permitting hybridization of said isolated nucleic acid molecule with said C. difficile-specific 16S nucleic acid, if present, and (ii) determining the presence or absence of an hybridization signal, wherein the presence of an hybridization signal is indicative that the sample comprises a C. difficile-specific 16S nucleic acid.
  • the C. difficile-specific 16S rRNA primers and/or probes is contacted with an amplification product obtained from the sample.
  • the present invention provides a method for detecting a C. difficile-specific 16S nucleic acid in a biological sample, the method comprising (i) performing an amplification reaction for said C. difficile-specific 16S nucleic acid on the biological sample; (ii) contacting the amplified sample with the C. difficile-specific 16S rRNA primers and/or probes defined herein under conditions permitting hybridization of said primers and/or probes with the amplified C. difficile-specific 16S nucleic acid, if present, and (iii) detecting the hybridization signal.
  • the present invention provides a method for determining whether a biological sample comprises a C. difficile-specific 16S nucleic acid, the method comprising (i) performing an amplification reaction for said C. difficile-specific 16S nucleic acid on the biological sample; (ii) contacting the amplified sample with the C. difficile-specific 16S rRNA primers and/or probes defined herein under conditions permitting hybridization of said primers and/or probes with the amplified C. difficile-specific 16S nucleic acid, if present, and (iii) determining the presence or absence of a hybridization signal, wherein the presence of a hybridization signal is indicative that the biological sample comprises a C. difficile-specific 16S nucleic acid.
  • the amplification reaction is performed using the following primer pairs:
  • a first primer which comprises, or consists of, the sequence
  • GGGAGCTTCCCATACGGGTTG (SEQ ID NO: 29)
  • a second primer which comprises, or consists of, the sequence TTGACTGCCTCAATGCTTGGGC (SEQ ID NO: 30).
  • detection of the amplification product is performed using a probe which comprises, or consists of, the sequence: AATGTTGGCATGAGTAGCGAGATGT (SEQ ID NO: 4).
  • C. difficile toxin B C. difficile ToxB nucleic acid
  • the present invention provides the use of the C. difficile ToxB primers and/or probes defined herein for amplifying and/or detecting a nucleic acid encoding a C. difficile toxin B (C. difficile ToxB nucleic acid, such as a C. difficile ToxB DNA or gene) in a sample.
  • C. difficile toxin B C. difficile ToxB nucleic acid, such as a C. difficile ToxB DNA or gene
  • the present invention provides a method for detecting a nucleic acid encoding a C. difficile ToxB nucleic acid in a sample, the method comprising (i) contacting the sample with the C. difficile ToxB primers and/or probes defined herein under conditions permitting hybridization of said primers and/or probes with said C. difficile ToxB nucleic acid, if present, and (ii) detecting the hybridization signal.
  • the present invention provides a method for determining whether a sample comprises a C. difficile ToxB nucleic acid, the method comprising (i) contacting the sample with the C. difficile ToxB primers and/or probes defined herein under conditions permitting hybridization of said primers and/or probes with said C. difficile ToxB nucleic acid, if present, and (ii) determining the presence or absence of an hybridization signal, wherein the presence of a hybridization signal is indicative that the sample comprises a C. difficile ToxB nucleic acid.
  • the C. difficile ToxB primers and/or probes is contacted with an amplification product obtained from the sample.
  • the present invention provides a method for detecting a C. difficile ToxB nucleic acid in a biological sample comprising (i) performing an amplification reaction for said C. difficile ToxB nucleic acid on the biological sample; (ii) contacting the amplified sample with the C. difficile ToxB primers and/or probes defined herein under conditions permitting hybridization of said primers and/or probes with the amplified C. difficile ToxB nucleic acid, if present, and (iii) detecting the hybridization signal.
  • the present invention provides a method for determining whether a biological sample comprises a C. difficile ToxB nucleic acid, the method comprising (i) performing an amplification reaction for said C.
  • the C. difficile ToxB probe defined herein is used in the step of contacting, in an embodiment using the C. difficile ToxB probe comprising, or consisting of, the sequence AATGTTGGCATGAGTAGCGAGATGT (SEQ ID NO: 4).
  • the amplification reaction is performed using the C. difficile ToxB primers defined herein, in an embodiment using a first primer comprising, or consisting of, the sequence TGCAGCCAAAGTTGTTGAAT (SEQ ID NO: 6), and a second primer which comprises, or consists of, the sequence G CTCTTTG ATTG CTG C ACCT (SEQ ID NO: 7).
  • the amplification reaction is a primer-dependent nucleic acid amplification reaction.
  • the amplification reaction is allowed to proceed for a duration (e.g., number of cycles) and under conditions that generate a sufficient amount of amplification product.
  • PCR polymerase chain reaction
  • PCR has been developed, for instance Real Time PCR (also known as quantitative PCR, qPCR), hot-start PCR, competitive PCR, and so on, and these may all be employed where appropriate to the needs of the skilled person.
  • Real Time PCR also known as quantitative PCR, qPCR
  • hot-start PCR hot-start PCR
  • competitive PCR competitive PCR
  • the oligonucleotide primers are contacted with a reaction mixture containing the target sequence and free nucleotides in a suitable buffer. Thermal cycling of the resulting mixture in the presence of a DNA polymerase results in amplification of the sequence between the primers.
  • Optimal performance of the PCR process is influenced by choice of temperature, time at temperature, and length of time between temperatures for each step in the cycle.
  • a typical cycling profile for PCR amplification is (a) about 5 minutes of DNA melting (denaturation) at about 95°C; (b) about 30 seconds of DNA melting (denaturation) at about 95°C; (c) about 30 seconds of primer annealing at about 50-65°C; (d) about 30 seconds of primer extension at about 68°C-72°C, preferably 72°C; and steps (b)-(d) are repeated as many times as necessary to obtain the desired level of amplification.
  • a final primer extension step may also be performed.
  • the final primer extension step may be performed at about 68°C-72°C, preferably about 72°C.
  • the annealing step is performed at about 50-60°C, e.g. about 50-58°C, 52-58°C, 54-58°C, 53-57°C, or 53-55°C.
  • the annealing step is performed at about 55°C (e.g. 55°C ⁇ 4°C, 55°C ⁇ 3°C, 55°C ⁇ 2°C 55 0 C ⁇ 1 °C or 55°C ⁇ 0.5°C).
  • the annealing step is performed at about 40-60°C, e.g.
  • the annealing step is performed at about 50°C (e.g. 50°C ⁇ 4°C, 50°C ⁇ 3°C, 50°C ⁇ 2°C 50°C ⁇ 1 °C or 50°C ⁇ 0.5°C).
  • the annealing step of other amplification reactions may also be performed at any of these temperatures.
  • the detection method of the present invention may be performed with any of the standard master mixes and enzymes available.
  • commercially available PCR mix may be used, such as the QUANTITEC® PCR Master Mix (QIAGEN®) or the MAXIMA® qPCR master mix (Thermo-Scientific®).
  • any conventional PCR (qPCR) instrument/system may be used, such as for example the LightCycler® systems (Roche), SLAN® Real-Time PCR Detection Systems (Daan Diagnostics® Ltd.), Bio-Rad® real-time PCR systems, and the like.
  • Double-stranded DNA-binding fluorescent dyes for instance SYBR Green, associate with the amplification product as it is produced and when associated the dye fluoresces. Accordingly, by measuring fluorescence after every PCR cycle, the relative amount of amplification product can be monitored in real time. Through the use of internal standards and controls, this information can be translated into quantitative data on the amount of template at the start of the reaction.
  • the fluorescent reporter probes used in qPCR are sequence-specific oligonucleotides, typically RNA or DNA, that have a fluorescent reporter molecule at one end and a quencher molecule at the other (e.g., the reporter molecule is at the 5' end and a quencher molecule at the 3' end or vice versa).
  • the probe is designed so that the reporter is quenched by the quencher.
  • the probe is also designed to hybridize selectively to particular regions of complementary sequence which might be in the template. If these regions are between the annealed PCR primers the polymerase, if it has exonuclease activity, will degrade (depolymerise) the bound probe as it extends the nascent nucleic acid chain it is polymerizing. This will relieve the quenching and fluorescence will rise. Accordingly, by measuring fluorescence after every PCR cycle, the relative amount of amplification product can be monitored in real time. Through the use of internal standard and controls, this information can be translated into
  • the amplification product may be detected, and amounts of amplification product can be determined by any convenient means.
  • a vast number of techniques are routinely employed as standard laboratory techniques and the literature has descriptions of more specialized approaches.
  • the amplification product may be detected by visual inspection of the reaction mixture at the end of the reaction or at a desired time point.
  • the amplification product will be resolved with the aid of a label that may be preferentially bound to the amplification product.
  • a dye substance e.g. a colorimetric, chromomeric fluorescent or luminescent dye (for instance ethidium bromide or SYBR green) is used.
  • a labelled oligonucleotide probe that preferentially binds the amplification product is used.
  • the amplification reaction is a multiplex amplification reaction (e.g., multiplexed PCR).
  • Multiplexed PCR means a PCR wherein multiple target sequences (or a single target sequence and one or more reference sequences) are simultaneously carried out in the same reaction mixture. Usually, distinct sets of primers are employed for each sequence being amplified. Typically, the number of target sequences in a multiplex PCR is in the range of from 2 to 10, or from 2 to 8, or more typically, from 3 to 6. Quantitative detection and normalization of the signal for a pathogen of interest using the bacterial 16S rRNA signal
  • the present invention provides a method for normalizing and/or quantitatively detecting the amount of a pathogen of interest in a sample, said method comprising:
  • the pathogen of interest is a bacterial pathogen.
  • the pathogen of interest is an opportunistic pathogen, for example Clostrodium difficile, Helicobacter pylori, Haemophilus influenza or Streptococcus pneumonia.
  • the opportunistic pathogen is Clostrodium difficile and the nucleic acid specific for the pathogen of interest is a C. difficile toxin nucleic acid, e.g., a C. difficile toxB nucleic acid.
  • the normalized pathogen of interest signal threshold(s) which could be converted to a scale, for example
  • Low normalized pathogen of interest signals (similar or below the threshold) being indicative that the subject has a low likelihood of being infected by the pathogen of interest or of developing an acute infection and exhibiting symptoms, and vice-versa.
  • CDI Clostridium difficile infection
  • the present invention provides a method for quantitatively detecting Clostridium difficile infection (CDI) in a sample (e.g., a biological sample from a subject), said method comprising
  • nucleic acid encoding a bacterial 16S rRNA (ii) a nucleic acid encoding a bacterial 16S rRNA, a nucleic acid encoding a human RNaseP and/or a nucleic acid encoding a fungal 18S rRNA, thereby obtaining a bacterial 16S rRNA signal, a human RNaseP signal and/or a fungal 18S rRNA signal;
  • the present invention provides a method for predicting the severity/acuteness of Clostridium difficile infection (CDI) in a subject, said method comprising performing, on a sample from the subject, an amplification reaction on CDI.
  • the above-mentioned method further comprises performing an amplification reaction on a nucleic acid encoding a C. d/7/ic//e-specific 16S nucleic acid, thereby obtaining a C. difficile-spec ⁇ ⁇ c 16S signal, establishing a ratio of the C. difficile toxin signal to the C. d/7/ic//e-specific 16S signal or vice-versa, and predicting the severity/acuteness of CDI in the subject on the basis of said normalized C. difficile toxin signal and said ratio.
  • Any suitable primer may be used to perform the amplification reaction on the nucleic acid encoding the C. difficile toxin, bacterial 16S rRNA, fungal 18S rRNA, human RNaseP, and C. difficile-specific 16S rRNA.
  • one or more of the primers and/or probes defined herein are used.
  • one or more oligonucleotides comprising one or more of the following sequences are used: GATTTGGACCTGCGAGCG (SEQ ID NO: 35); GAGCGGCTGTCTCCACAAGT (SEQ ID NO: 36) and/or TTCTGACCTGAAGGCTCTGCGCG (SEQ ID NO: 37).
  • the following primer pair is used to perform the amplification reaction on the RNAseP nucleic acid: a first primer comprising, or consisting of, the sequence GATTTGGACCTGCGAGCG (SEQ ID NO: 35) and a second primer comprising, or consisting of, the sequence GAGCGGCTGTCTCCACAAGT (SEQ ID NO: 36).
  • the present invention provides a method for quantitatively detecting Clostridium difficile infection (CDI) in a sample (e.g., a biological sample from a subject), said method comprising:
  • the present invention provides a method for predicting the severity of Clostridium difficile infection (CDI) in a subject, said method comprising:
  • the above-mentioned method further comprises performing an amplification reaction on a nucleic acid encoding a C. difficile-specific 16S nucleic acid, thereby obtaining a C. difficile-specific 16S amplified product, contacting the C. difficile-specific 16S amplified product with a probe hybridizing to said nucleic acid encoding a C. difficile-specific 16S, thereby obtaining a C. difficile-specific 16S signal, establishing a ratio of the C. difficile toxin signal to the C. difficile-specific 16S signal or vice-versa, and predicting the severity of CDI in the subject on the basis of said normalized C. difficile toxin signal and said ratio.
  • Any suitable probe may be used to perform the hybridization reaction on the amplified products.
  • one or more of the primers and/or probes defined above are used.
  • the method comprises performing an amplification reaction on a nucleic acid encoding a bacterial 16S rRNA according to the method defined herein.
  • the normalized value is compared to a corresponding "control” or “reference” normalized value.
  • Control value or “reference value” or “standard value” are used interchangeably herein and broadly refers to a separate value measured in a comparable control sample, which is generally from a subject not suffering from CDI or having a known CDI severity (or a sample known to not contain C. difficile).
  • the corresponding control value may be a value corresponding to an average or median value calculated based of the values obtained in samples from several reference or control subjects (e.g., a pre-determined or established standard value).
  • the control value may be a pre-determined "cut-off" value recognized in the art or established based on values measured in one or a group of control subjects.
  • control value can thus be a single number/value, equally applicable to every patient individually, or the control value can vary, according to specific subpopulations of patients, types of samples. Thus, for example, older men might have a different control value than younger men, and women might have a different control value than men.
  • the predetermined standard value can be arranged, for example, where a tested population is divided equally (or unequally) into groups, such as a low-severity group, a medium-severity group and a high-severity group or into quadrants or quintiles, the lowest quadrant or quintile being individuals with the lowest severity (i.e., lowest normalized C. difficile toxin value) and the highest quadrant or quintile being individuals with the highest severity (i.e., highest normalized C. difficile toxin value).
  • groups such as a low-severity group, a medium-severity group and a high-severity group or into quadrants or quintiles, the lowest quadrant or quintile being individuals with the lowest severity (i.e., lowest normalized C. difficile toxin value) and the highest quadrant or quintile being individuals with the highest severity (i.e., highest normalized C. difficile toxin value).
  • control value according to the invention may be, in addition to predetermined values, values measured in other samples (e.g. from healthy/normal subjects or subjects having a known CDI severity) tested in parallel with the experimental sample.
  • the normalization comprises dividing the C. difficile toxin signal value (Cpt) by the bacterial 16S rRNA signal value (Cpu), fungal 18S RNA signal value (Cpf) and/or human RNaseP signal value (Cpg) or vice-versa. In an embodiment, the normalization comprises subtracting the C.
  • the above-mentioned method comprises determining an acuteness of infection C. difficile (AOI-CD) score using the following formula:
  • AOI-CD score (1/Cpt)/(Cpu-Cpt) or (1/Cpt)/(Cpg-Cpt)
  • the AOI-CD score may be compared to a reference or control AOI-CD score to quantify CDI and/or determine/predict the acuteness or severity of CDI in the sample/subject tested.
  • the present invention provides a method for the follow-up of a C. difficile-infected patient's condition (over time), the method comprising obtaining a first normalized C. difficile toxin signal from a sample using the above-defined method at a first time point; obtaining a second normalized C. difficile toxin signal from a sample using the above- defined method at a second, later time point; and comparing the first and second normalized C. difficile toxin signals, wherein a second normalized C. difficile toxin signal that is higher than the first normalized C. difficile toxin signal is indicative that said patient's condition is deteriorating, and wherein a second normalized C. difficile toxin signal that is lower than the first normalized C. difficile toxin signal is indicative that said patient's condition is improving.
  • the C. difficile-infected patient is undergoing treatment for CDI between said first and second time points, and said method permits to determine whether the patient is responding to the treatment.
  • the sample which is tested according to the methods described herein is any sample suspected of containing bacteria such as C. difficile, for example a body fluid, swab or other cellular or non-cellular sample from a subject, e.g. a human.
  • samples include, but are not limited to, bodily fluids which contain cellular materials and may or may not contain cells, e.g., blood, plasma, serum, urine, conjunctival secretions, seminal fluid, saliva, ocular lens fluid, lymphatic fluid, amniotic fluid, feces/stool and the like; endocervical, urethral, rectal, vaginal, vulva-vaginal, nasopharyngeal and pulmonary samples; and archival samples with known diagnosis.
  • Test samples may also be sections of tissues such as frozen sections.
  • the sample may be any sample taken from the gastrointestinal Gl tract.
  • a Gl tract sample of use in the invention may include, but is not limited to, any fluid or solid taken from the lumen or surface of the Gl tract or any sample of any of the tissues that form the organs of the Gl tract.
  • the sample may be any luminal content of the Gl tract (e.g. stomach contents, intestinal contents, mucus and feces/stool, or combinations thereof) as well as samples obtained mechanically from the Gl tract e.g., by swab, rinse, aspirate or scrape of a Gl tract cavity or surface or by biopsy of a Gl tract tissue/organ.
  • the sample can also be obtained from part of a Gl tract tissue/organ which has been removed surgically.
  • the sample may be a portion of the excised tissue/organ.
  • the sample may comprise a part of the mucosa, the submucosa, the muscularis, the adventitia and/or the serosa of the Gl tract tissue/organ.
  • tissue samples may be obtained by biopsy during an endoscopic procedure.
  • Samples for use in the invention may also include environmental samples, preferably samples from a hospital or other clinical setting.
  • environmental samples include samples obtained from surfaces (e.g., floors), samples obtained from clothing, samples obtained from toilets, commodes, bedpans and the like, samples obtained from clinical devices (e.g. endoscopes), samples of the water supply, or air treatment apparatus of the hospital or other clinical setting, and samples obtained from the hands of healthcare workers.
  • sample also encompasses any material derived by processing a biological sample.
  • Derived materials include, but are not limited to, cells (or their progeny) isolated from the sample (e.g., clinical isolates of bacteria such as Clostridium difficile), cell components, proteins/peptides and nucleic acid molecules (DNA or RNA) extracted from the sample.
  • Processing of biological samples to obtain a test sample may involve one or more of: filtration, distillation, centrifugation, extraction, concentration, dilution, purification, inactivation of interfering components, addition of reagents, and the like.
  • the sample has not been subjected to a nucleic acid enrichment/isolation/purification step, e.g., the sample does not comprise isolated/purified nucleic acids (isolated/purified DNA).
  • the subject may be any human or non-human animal subject, but more particularly may be a vertebrate, e.g., an animal selected from mammals, birds, amphibians, fish and reptiles.
  • the animal may be a livestock or a domestic animal or an animal of commercial value, including laboratory animals or an animal in a zoo or game park.
  • the subject is a human.
  • the subject may be of any age, e.g. an infant, a child, a juvenile, an adolescent or an adult.
  • the present invention provides a method comprising:
  • treating said subject against said bacterial infection for example by administering an effective amount of one or more suitable antibiotics to said subject.
  • the bacterial infection is an opportunistic infection, such as
  • Clostrodium difficile Helicobacter pylori, Haemophilus influenza or Streptococcus pneumonia.
  • the present invention provides a method comprising:
  • CDI Clostridium difficile infection
  • Example 1 Materials and Methods for Examples 2 to 4
  • PCR assay for Clostridium difficile in stool samples This assay was performed using the primers and probe combinations listed below, together with an inhibition Control (IC), for detection of PCR inhibitors in the stool.
  • IC inhibition Control
  • the 6FAM/fluorescing channel was used for monitoring the real-time signal from the C. difficile toxin B gene and the Cy5 (555 red) channel was used for detection of the Inhibition Control signal.
  • the assay was compatible with all qPCR instruments. Lysis of bacterial cells prior to amplification was performed with glass beads, as described below. The analytical limits-of-detection are in the range of 1800 copies per swab sample.
  • genomic DNA of C. difficile strain ATCC 43255 was used as positive C. difficile control (PC).
  • NC negative control
  • IC inhibition control
  • PC and NC are used with each batch.
  • the inhibition control (IC) was a sample-spiked oligonucleotide which was able to generate PCR product with corresponding primers and probe. If a sample did contain an unknown PCR-inhibition component/substance which could influence the end-result (Ct value), it would be detected by comparing the inhibition control (IC) in the context of a water solution, with the IC in the context of the sample solution. The inhibition test is done in a separate reaction mix and does not compete with any other reaction.
  • the whole assay was repeated once more with a sample of the original dilution which has been subjected to a 10x dilution and/or one freeze/thaw cycle.
  • Specimen/sample preparation for the complete assay was compatible with/similar to all current commercial sample preparation protocols.
  • Specimens were vortexed at high speeds for 15 seconds and a sterile dry swab was dipped into the fecal material for testing. Excess stool was removed. The swab was placed in a sample buffer tube (SBT).
  • the 25 ⁇ _ of stool suspension was transferred to one labeled LT (containing 100 ⁇ of 1XTE buffer and 50uL equivalent volume of the glass beads). Each lysis tube was vortexed for five 5 minutes at high speed. Tubes were lysed by heat treatment at 95 ⁇ 2°C for five minutes, and cooled by transferring onto ice or a cooling block. Preparation of Master Mixes, prepared in a PCR hood/workstation (same for C.difficile reaction mix and IC reaction mix):
  • Amplifications and detections were performed using a Light Cycler® 480 instrument, using Detection Format: 3-colour hydrolysis probe. Absolute quantification was performed with Filter combination FAM 465-510. The High sensitivity software package was used. Crossing point (Cp) values for each sample were recorded into an Excel® file. The same procedure was done for IC.
  • Results with a generated Cp value should be considered as positive if the RFU end- point values have RFU (Relative Fluorescence Unit) values higher than 1.
  • RFU Relative Fluorescence Unit
  • Results with no Cp value generated and with an RFU noise line below 1 and linear RFU vs. cycle slope should be estimated for the presence of PCR inhibition. If PCR is not inhibited, the sample should be considered as negative.
  • the Inhibition control (IC) is read at a separate reaction well. All Cp sample values are compared with an IC Cp value of the negative control (which has water instead of sample).
  • 16S-Probe1 CGGCTAACTMCGTGCCAG (SEQ ID NO: 2);
  • 16S-Probe2 AAGSVMCGGCTAACTMCGTGCC (SEQ ID NO: 3);
  • 16SU-F ACTCCTACGGGAGGCAGCAGT (SEQ ID NO: 22); 16SU-R1 : TATTACCGCGGCTGCTGGC (SEQ ID NO: 38);
  • 16SU-R2 GCITCCTATTACCGCGGCTGCT (SEQ ID NO: 1 1 );
  • 16SU-R3 AATAAATCATAAGCITCCTATTACCGCGGCTGCT (SEQ ID NO: 12);
  • 16SU-R4 5'-AATAAATCATAACCTACGTATTACCGCGGCTGC-3' (SEQ ID NO: 39);
  • 16SCD-Probe AATGTTGGCATGAGTAGCGAGATGT (SEQ ID NO: 4);
  • 16SCD-F GGGAGCTTCCCATACGGGTTG (SEQ ID NO: 29);
  • 16SCD-R TTGACTGCCTCAATGCTTGGGC (SEQ ID NO: 30).
  • tcdB.RT2-Probe TCTGAAGGATTACCTRTAATTGCAA (SEQ ID NO: 5);
  • tcdB.RT2-F TGCAGCCAAAGTTGTTGAAT (SEQ ID NO: 6);
  • tcdB.RT.2-R GCTCTTTGATTGCTGCACCT (SEQ ID NO: 7).
  • RNaseP-Probe TTCTGACCTGAAGGCTCTGCGCG (SEQ ID NO: 37);
  • RNaseP-F GATTTGGACCTGCGAGCG (SEQ ID NO: 35);
  • RNaseP-R GAGCGGCTGTCTCCACAAGT (SEQ ID NO: 36).
  • the concentration of primers used were in the ranges of 0.5-2 ⁇ (preferably 0.6 ⁇ ), and the concentration of hydrolyzing probes were in the ranges of 0.25-1.2 ⁇ (preferably 0.3 ⁇ ).
  • the QUANTITEC® PCR Master Mix (QIAGEN®) or MAXIMA® qPCR master mix (Thermo-Scientific®) were used. PCR conditions were as follows: 95°C for 15 min, following by 45 cycles of annealing at 50°C, 60 sec and denaturation at 95°C, 10 sec.
  • Example 2 Linearity of the 16S rDNA signal
  • the numerical Cp values reflect total rDNA load in the sample.
  • the sample processing and PCR was performed under the same conditions as described above for C. difficile.
  • Numerical values of Cp spanned the range of 13 Cp units (indicating that bacterial fecal load will vary from sample to sample by minimum 2 power 13 unit values).
  • the "protective effect” was calculated using a ratio between Cp values of toxB and Cp value of 16S rDNA of C. difficile.
  • the protective effect was measured using the formula 100*(Cpt Cpc)-1 ), although other mathematical transformations could have been used.
  • the rationale for calculation is that if 2 signals (Cp of toxB (Cpt) and Cp of 16 rDNA of C. difficile (Cpc)) are similar, the value (ratio) will be close to 1. This means that the dominant strain is toxicogenic and that there is no protective effect of other, non-toxicogenic strains.
  • the parameter (1/Cpt)/(Cpu-Cpt) provides information about the level of toxB DNA signal (1/Cpt, a lower ratio 1/Cpt is indicative of low toxB DNA levels in the sample), but also provides information on the relative contribution/frequency of toxB DNA relative to the total bacterial microbial presence in the sample (Cpu-Cpt). If this relative frequency of toxicogenic C. difficile is high, the Cpu-Cpt number is close to zero and the parameter 1/Cpt/(Cpu-Cpt) is higher, indicative of more severe cases. If the relative frequency of toxicogenic C.
  • C. difficile is low, relative to the total bacterial microbial presence (microbiota), then (Cpu-Cpt) is higher, and the parameter 1/Cpt/(Cpu-Cpt) is lower, indicative of asymptomatic, or less severe cases.
  • Cpu-Cpt the final scoring of C. difficile infected patients will not only depend on the numerical value of Cpt related to number of toxB DNA molecules, but will be "normalized” based on the relative frequency of this DNA molecule among all bacteria present in the sample. There will be cases which have low toxB number of DNA molecules (low absolute numbers of toxicogenic C. difficile), but also low Cpu (low total microbiota sample load). This is indicative that these samples are depleted from the rest of the bacterial flora (i.e.
  • a completely analogous protocol can be used for the normalisation of qPCR signal using other normalisation "universal" genes, such as the RNaseP human gene (to normalise the signal based on the number of human cells in the sample), and assigning a different weighting parameter for severity of infection (for the contribution of each assay).
  • RNaseP human gene to normalise the signal based on the number of human cells in the sample
  • assigning a different weighting parameter for severity of infection for the contribution of each assay.
  • the dominant deterministic factor for normalising qPCR signal and estimating current status of bacterial infection in highly complex bacterial flora is obtained, using 16S rDNA assay.
  • Melting curve analysis is an assessment of the dissociation-characteristics of double- stranded DNA during heating. Juxtaposition of probes (one featuring a fluorophore and the other, a suitable quencher) can be used to determine the complementarity of the probe to the target sequence. Typically the user is using polymerase chain reaction (PCR) prior to melting curve analysis to amplify the DNA region in which species-specific sequence variability of interest lies. In the sample tube there are now many copies of the DNA region of interest. Region that is amplified is known as the amplicon. The process is a precise warming of the amplicon DNA from around 50°C up to around 95°C.
  • PCR polymerase chain reaction
  • the melting temperature of the amplicon is reached and the two strands of DNA separate or "melt" apart, while hybridization Tm-probes (which are in molar excess and which are visible via fluorescence) will hybridize with a single stranded DNA amplicon and "melt" proportionally to the thermal stability of probe-target region.
  • a perfectly matched probe(s) will melt at a higher temperature then the mismatched probe(s) bound to a target sequence.
  • the purpose of Melting Curve analysis in the present case is to determine the melting temperature spectrum changes characteristic to loss of bacterial diversity in fecal samples.
  • Example 5 Estimating of total bacterial load in stool samples using a short 16S rDNA
  • sample study group was represented by 500 liquid and 50 formed stools, submitted for screening of C. difficile and stool culture.
  • 49 stool samples were randomly chosen and processed as described below.
  • PCR assay and cycling conditions 3 ⁇ of nucleic acid elute or crude lysis material was added into 17 ⁇ I of total volume mix (QuantiNova® Probe PCR master, QIAGEN®), according to the concentration of primer/probe and cycling conditions recommended by the manufacturer 42 , except for the annealing temperature that was set at 50°C).
  • the Cp values were calculated using the default parameters of the LightCycler® 480 software provided with the realtime PCR system (LightCycler® 480 Instrument II, Roche).
  • F-V1-16S-NOV14 AATAAATCATAAACTCCTAYGGGRGGCWGCAGT 48 33
  • F-V2-16S-NOV14 AATAAATCATAAACTCCTACGGGRGGCWGCAGT 49 33
  • F-V6-16S-NOV14 AATAAATCATAAACTCCTAYGGGRBGCAGCA 53 31
  • Nadkarni (2002) Nadkarni MA et al. Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set. Microbiology 2002;148:257-66
  • Nadkarni Wang Y, Qian PY. Conservative fragments in bacterial 16S r genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies. PLoS one 2009;4:e7401
  • Nadkarni Liu CM, Aziz M, Kachur S, et al. BactQuant: an enhanced broad-coverage bacterial quantitative real-time PCR assay. BMC microbiology 2012;12:56
  • FIG. 2A shows that there is no correlation between the stool mass (mg of stool entering sample buffer) and the bacterial load, measured on different stool samples, using the novel 16S rDNA qPCR assay described herein.
  • FIG. 3 shows that among different samples, there is a correlation between the bacterial counts as measured by the culture plating method and the Ct values obtained using the novel 16S rDNA qPCR assay described herein. Ct values above 18 indicate low bacterial load and may be considered as samples with strongly reduced microflora. Comparable results were obtained using the forward primers comprising the sequences of SEQ ID NOs: 22 and 48-55.
  • FIG. 4 shows the variability of the total bacterial load among clinical samples of liquid stool. Distribution of Ct values using the novel 16S rDNA qPCR assay described herein amongst 500 stool swabs (from collection of C. difficile positive samples) indicates that the total bacterial load may vary by up to about 1000-fold (Ct value from 2 12 to 2 21 ) for the same stool mass equivalent. Comparable results were obtained using the forward primers comprising the sequences of SEQ ID NOs: 22 and 48-55.
  • Example 6 Comparison of Primer Pairs Using in silica PCR of the SILVA Database
  • sequence coverage sometimes defined as "depth of sequence coverage” is defined as number of events/times primer sequence match with targeted sequence within defined sequence data base, versus the total number of matching events.
  • depth of sequence coverage is defined as number of events/times primer sequence match with targeted sequence within defined sequence data base, versus the total number of matching events.
  • SILVA TestPrime 1.0 http://www.arb-silva.de/search/testprime/, Klindworth, A et ai, Nucl. Acids Res.
  • Table VINA and VIIIB below illustrate less-stringent (right, 4 nt. mismatch, in bold) versus more stringent (left, 2 nt. Mismatch) sequence matching of primers with target binding sequence motif/s.
  • the novel bacterial 16S rRNA primer pair described herein can have a broad ("universal") sequence coverage range under non-stringent conditions (3 or 4 nt. mismatchs, corresponding to lower temperature of annealing, e.g., about 50°C) thus tolerating up to 4 mismatches and perform same, if not better, than primers showing higher identity with sequence binding motifs such as those disclosed in Clifford (2012) and Nadkarni (2002).
  • first and last row show bacterial genera missed and identified by all primer pairs, respectively. Remaining rows show combinations thereof.
  • the first row of Tables VINA and VIIIB indicates the number of bacterial genera missed by the primer pair of the novel bacterial 16S rRNA assay described herein and identified by the primer pairs disclosed in Clifford (2012) and Nadkarni (2002).
  • Lyra A Forssten S, Rolny P, et al. Comparison of bacterial quantities in left and right colon biopsies and faeces. World journal of gastroenterology : WJG 2012;18:4404-1 1.

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Abstract

Novel reagents and methods for the detection and quantification of total bacterial load in a sample are described. These reagents and methods are based on the amplification and detection of a bacterial 16S rRNA nucleic acid. Also described herein are novel reagents and methods for the detection and quantification of Clostrodium difficile infection (CDI). The reagents and assays allow for the normalization, based for example on the amount of bacterial 16S rRNA nucleic acid, of the bacterial load of bacteria of interest, including opportunistic bacteria such as pathogenic Clostrodium difficile, in various samples such as stool, feces, anal swab or rectal swab. Methods for analytical assessment of the acuteness of CDI based on the normalized bacterial load of pathogenic Clostrodium difficile are also described.

Description

METHODS, REAGENTS AND KITS FOR THE ASSESSMENT OF BACTERIAL
INFECTION
CROSS REFERENCE TO RELATED APPLICATIONS
The present application claims the benefit of U.S. Provisional Application Serial No. 61/926,480 filed on January 13, 2014, which is incorporated herein by reference in its entirety.
TECHNICAL FIELD
The present invention generally relates to the assessment of total bacterial load in a sample, as well as the assessment of infection status of opportunistic infections such as Clostridium difficile infection (CDI) in clinical settings.
SEQUENCE LISTING
This application contains a Sequence Listing in computer readable form entitled "1 1 168_404_SeqJist.txt", created January 13, 2015 and having a size of about 13 KB. The computer readable form is incorporated herein by reference. BACKGROUND ART
In clinical microbiology laboratories, traditional culture-based approaches remain the primary methodology used for quantifying bacterial load. However, these methods are inherently limited for assessing the complex bacterial communities that exist in many clinical and environmental samples. Likewise, standard culture-based methods are typically ineffective for quantifying many fastidious and uncultivable bacterial species. Even cultivable bacterial species typically need to be tested over several serial dilutions, due to the low operative assay range. That makes culture plating assay unpractical for quantifying species which frequently show logarithmic variability per same mass/volume unit of input sample. Among culture- independent approaches, quantitative real-time PCR (qPCR) is currently best suited for measuring bacterial load, because of its intrinsic quantitative capability, ease of use, and flexibility in assay design. In spite of exponential growth of sequencing data3, the design of suitable qPCR assays for assessing bacterial load, including those based on "universal" 16S rDNA primers (and probes), remains a challenge. The choice of 16S rDNA primers and probe is important for the correct estimation of relative abundance of bacterial species in the context of complex microbial community. This is particularly challenging for samples where opportunistic pathogens may be present, but below certain relative abundance, thus not causing any symptoms. These patients are typically referred as asymptomatic carriers, and may sometimes represent a significant proportion of the subjects (from 4% to up to 50% reported in the case of C. difficile). Current PCR-based assays for detecting bacterial 16S rDNA have some limitations, including non-optimal primer/probe coverage; amplicon length not suitable for routine qPCR- based clinical diagnostics and associated with inefficient amplification (leading to false negative results), as well as self-priming and/or mis-priming issues leading to false positive results11"32. Thus, there is a need for novel reagents and assays for the amplification, detection and/or quantification of bacterial 16S rDNA.
Clostridium difficile (C. difficile) is a Gram-positive, anaerobic, spore-forming bacterium that has been identified as the major cause of nosocomial antibiotic-associated diarrhea, which in some cases leads to pseudomembranous colitis. C. difficile has been reported to be carried asymptomatically by 0% to 15% of healthy adults and by 13% to 26% of hospitalized patients (Matsuda et al., Applied and Environmental Microbiology, 78(15): 51 1 1-51 18). Most of the hospitalized patients remain asymptomatic carriers, while some develop infections that vary from mild, watery diarrhea to fatal pseudomembranous colitis. The causes of this high variation in clinical presentation are not fully understood.
The diagnosis of C. difficile infection (CDI) is usually based on the detection of the microbe in feces by culture and the detection of its toxin(s) (e.g., ToxB) by enzyme immunoassays. Recently introduced molecular methods, such as real-time PCR, have greater sensitivity than rapid toxin tests and thus are able to detect significantly more positive cases. However, these methods could also increase the detection of colonization in healthy people, up to 55.5% of which are colonized (Naaber et al., J Clin. Microbiol, 49(10): 3656-3658).
The detection of C. difficile and its toxins in routine diagnostics and major clinical studies remains qualitative. However, a quantitative approach to measuring CDI may have some advantages in investigating relationships within the microbial micro ecosystem or in distinguishing between low-level colonization and clinical infection. Quantitative estimation of CDI involves proper normalization of the signal (e.g., ToxB signal) to improve its predictive clinical value, but such normalization is very challenging for certain types of samples rich in microbiota, such as oral, vaginal, stool, anal, and rectal samples, as well as skin swabs. Other sources of variations in the quality/quantity of sample include differential sampling techniques, pre-treatment of sampling surfaces, recent antibiotic/antifungal therapy, and dilution effects (especially relevant for stool samples).
There is thus a need for novel methods, assays, reagents and/or kits for the quantitative assessment of CDI in clinical settings.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety. SUMMARY OF THE INVENTION
The present invention provides the following items 1 to 65:
1. An isolated nucleic acid molecule of 50 nucleotides or less comprising a sequence of at least 10 contiguous nucleotides of one of the following nucleotide sequences (I) to (VII):
(I) 5'-TATTACCGCGGCTGCT-3' (SEQ ID NO: 1 ), wherein said isolated nucleic acid molecule does not comprise the sequence GGC at its 3' end;
(II) 5'-CGGCTAACTMCGTGCCAG-3' (SEQ ID NO: 2);
(III) 5'-AATGTTGGCATGAGTAGCGAGATGT-3' (SEQ ID NO: 4);
(IV) 5'-TCTGAAGGATTACCTRTAATTGCAA-3' (SEQ ID NO: 5);
(V) 5'-TGCAGCCAAAGTTGTTGAAT-3' (SEQ ID NO: 6);
(VI) 5'-GCTCTTTGATTGCTGCACCT-3' (SEQ ID NO: 7); or
(VII) a sequence having at least 80% identity with any of (I) to (VI).
2. The isolated nucleic acid molecule of item 1 , which comprises a sequence of at least 15 contiguous nucleotides of one of the nucleotide sequences (I) to (VII). 3. The isolated nucleic acid molecule of item 1 or 2, which is of 35 nucleotides or less.
4. The isolated nucleic acid molecule of any one of items 1 to 3, the nucleotide sequence of which comprises the sequence 5'-TATTACCGCGGCTGCT-3' (SEQ ID NO: 1 ).
5. The isolated nucleic acid molecule of item 4, the nucleotide sequence of which comprises the sequence 5'-TACGTATTACCGCGGCTGCT-3' (SEQ ID NO: 8). 6. The isolated nucleic acid molecule of item 5, the nucleotide sequence of which consists of the sequence 5'-TACGTATTACCGCGGCTGCT-3' (SEQ ID NO: 8).
7. The isolated nucleic acid molecule of any one of items 1 to 3, which comprises the sequence 5'-CGGCTAACTMCGTGCCAG-3' (SEQ ID NO: 2).
8. The isolated nucleic acid molecule of item 7, the nucleotide sequence of which consists of the sequence 5'-CGGCTAACTMCGTGCCAG-3' (SEQ ID NO: 2).
9. The isolated nucleic acid molecule of item 1 , which comprises the sequence 5'- AAGSVMCGGCTAACTMCGTGCC-3' (SEQ ID NO: 3).
10. The isolated nucleic acid molecule of item 9, the nucleotide sequence of which consists of the sequence 5'-AAGSVMCGGCTAACTMCGTGCC-3' (SEQ ID NO: 3). 1 1. The isolated nucleic acid molecule of item 1 , which comprises the sequence 5'- AATGTTGGCATGAGTAGCGAGATGT-3' (SEQ ID NO: 4).
12. The isolated nucleic acid molecule of item 15, the nucleotide sequence of which consists of the sequence 5'-AATGTTGGCATGAGTAGCGAGATGT-3' (SEQ ID NO: 4). 13. The isolated nucleic acid molecule of item 1 , which comprises the sequence 5'- TCTGAAGGATTACCTRTAATTGCAA-3' (SEQ ID NO: 5).
14. The isolated nucleic acid molecule of item 17, the nucleotide sequence of which consists of the sequence 5'-TCTGAAGGATTACCTRTAATTGCAA-3' (SEQ ID NO: 5).
15. The isolated nucleic acid molecule of item 1 , which comprises the sequence 5'- TGCAGCCAAAGTTGTTGAAT-3' (SEQ ID NO: 6).
16. The isolated nucleic acid molecule of item 19, the nucleotide sequence of which consists of the sequence 5'-TGCAGCCAAAGTTGTTGAAT-3' (SEQ ID NO: 6).
17. The isolated nucleic acid molecule of item 1 , which comprises the sequence 5'- GCTCTTTGATTGCTGCACCT-3' (SEQ ID NO: 7). 18. The isolated nucleic acid molecule of item 1 , the nucleotide sequence of which consists of the sequence 5'-GCTCTTTGATTGCTGCACCT-3' (SEQ ID NO: 7).
19. A method for amplifying and/or detecting a bacterial 16S rRNA nucleic acid in a sample, said method comprising (a) contacting said bacterial 16S rRNA nucleic acid with a first primer and a second primer under conditions suitable for nucleic acid amplification, and (b) performing a nucleic acid amplification reaction, thereby generating an amplification product if bacterial 16S rRNA nucleic acid is present in said sample; wherein said first primer has a length of 50 nucleotides or less and comprises a sequence of at least 10 contiguous nucleotides of the sequence ACTCCTAYGGGRBGCWSCA (SEQ ID NO: 13), and said second primer comprises a sequence of at least 10 contiguous nucleotides of nucleotide sequence (I) or (VII) defined in any one of items 1 to 6.
20. The method of item 19, wherein said first primer has a length of 35 nucleotides or less.
21. The method of item 19 or 20, wherein said first primer comprises a sequence of at least 15 contiguous nucleotides of the sequence ACTCCTAYGGGRBGCWSCA (SEQ ID NO: 13).
22. The method of any one of items 19 to 21 , wherein said first primer comprises the sequence ACTCCTAYGGGRBGCWSCA (SEQ ID NO: 13). 23. The method of item 22, wherein said first primer comprises the sequence ACTCCTAYGGGRBGCASCAGT (SEQ ID NO: 14), ACTCCTAYGGGRGGCWGCAGT (SEQ ID NO: 15), ACTCCTACGGGRGGCWGCAGT (SEQ ID NO: 16), ACTCCTAYGGGRGGCWGCA (SEQ ID NO: 17), ACTCCTACGGGRGGCWGCA (SEQ ID NO: 18), ACTCCTACG G G RG G CAG C A (SEQ ID NO: 19), ACTCCTAYGGGRBGCAGCA (SEQ ID NO: 20), or ACTCCTACGGGRBGCWGCA (SEQ ID NO: 21 ).
24. The method of any one of items 19 to 23, wherein said first primer further comprises, at its 5' end, the nucleotide sequence 5'-AATAAATCATAA-3' (SEQ ID NO: 10) or a 5'-deleted fragment thereof. 25. The method of item 24, wherein said first primer comprises the sequence ACTCCTAYGGGRBGCASCAGT (SEQ ID NO: 14).
26. The method of item 25, wherein the nucleotide sequence of said first primer consists of the sequence ACTCCTAYGGGRBGCASCAGT (SEQ ID NO: 14).
27. The method of any one of items 19 to 26, wherein said second primer is the nucleic acid molecule defined in item 6.
28. The method of any one of items 19 to 27, wherein said conditions suitable for nucleic acid amplification comprise an annealing step at about 45°C to about 55°C.
29. The method of item 29, wherein said conditions suitable for nucleic acid amplification comprise an annealing step at about 50°C. 30. The method of any one of items 19 to 29, wherein said method further comprises contacting said nucleic acid amplification reaction with a probe that hybridizes to said amplification product if present.
31. The method of item 30, wherein said probe comprises (i) a sequence of at least 10 contiguous nucleotides of nucleotide sequence (II) or (VII) defined in any one of items 1 to 3, 7 and 8.
32. The method of item 31 , wherein said probe is the nucleic acid molecule defined in item 8.
33. The method of any one of items 30 to 32, wherein said probe is tagged with a detectable label. 34. The method of item 33, wherein said detectable label is a fluorescent label. 35. The method of any one of items 19 to 34, wherein said amplification reaction is performed by polymerase chain reaction (PCR).
36. The method of item 35, wherein said PCR is quantitative PCR.
37. A combination of nucleic acid molecules for amplifying or detecting a bacterial 16S rRNA nucleic acid, said combination comprising the first primer defined in any one of items 19 to 27 and the second primer defined in any one of items 19 to 27.
38. The combination of item 37, further comprising the probe defined in any one of items 30 to 34.
39. A method for determining whether a sample comprises a bacterial 16S rRNA nucleic acid, said method comprising performing the method for amplifying or detecting a bacterial 16S rRNA nucleic acid defined in any one of items 19 to 36, determining the presence or absence of said amplification product, wherein the presence of the amplification product is indicative that said sample comprises a bacterial 16S rRNA nucleic acid.
40. The method of item 39, wherein said sample is a clinical sample. 41 . The method of item 39 or 40, wherein said sample is stool, feces, anal swab or rectal swab.
42. A method for determining the amount of bacteria in a sample, said method comprising (a) performing the method for amplifying or detecting a bacterial 16S rRNA nucleic acid defined in any one of items 19 to 36, (b) comparing the amount of amplification product present after said amplification reaction to a reference; and (c) determining the amount of bacteria in the sample based on said comparison.
43. Use of one or more of the isolated nucleic acid molecules comprising (i) a sequence of at least 10 contiguous nucleotides of sequence (I), (II) and/or (VII) defined in any one of items 1 to 10, for amplifying and/or detecting a bacterial 16S rRNA nucleic acid in a sample. 44. A method for detecting a C. difficile-specific 16S rRNA nucleic acid in a sample, said method comprising (a) contacting said C. difficile-specific 16S rRNA nucleic acid with a first primer and a second primer under conditions suitable for nucleic acid amplification, (b) performing a nucleic acid amplification reaction, thereby generating an amplification product if C. difficile-specific 16S rRNA nucleic acid is present in said sample; and (c) contacting said nucleic acid amplification reaction with a probe that hybridizes to said amplification product if present, wherein said probe comprises a sequence of at least 10 contiguous nucleotides of nucleotide sequence (III) or (VII) defined in any one of items 1 to 3, 1 1 and 12.
45. The method of item 44, wherein said first primer comprises the sequence 5'- GGGAGCTTCCCATACGGGTTG-3' (SEQ ID NO: 22). 46. The method of item 44 or 45, wherein said second primer comprises the sequence 5'- TTGACTGCCTCAATGCTTGGGC-3' (SEQ ID NO: 23).
47. Use of one or more of the isolated nucleic acid molecules comprising a sequence of at least 10 contiguous nucleotides of sequence (III) or (VII) defined in any one of items 1 to 3, 1 1 and 12 for amplifying and/or detecting a C. difficile-specific 16S rRNA nucleic acid in a sample. 48. A method for detecting a C. difficile toxin B nucleic acid in a sample, said method comprising (a) contacting said C. difficile toxin B nucleic acid with a first primer and a second primer under conditions suitable for nucleic acid amplification, (b) performing a nucleic acid amplification reaction, thereby generating an amplification product if C. difficile toxin B nucleic acid is present in said sample; and (c) contacting said nucleic acid amplification reaction with a probe that hybridizes to said amplification product if present, wherein (i) said probe comprises a sequence of at least 10 contiguous nucleotides of nucleotide sequence (IV) or (VII) defined in any one of items 1 to 3, 13 and 14; (ii) said first primer comprises a sequence of at least 10 contiguous nucleotides of nucleotide sequence (V) or (VII) defined in any one of items 1 to 3, 15 and 16; and/or said second primer comprises a sequence of at least 10 contiguous nucleotides of nucleotide sequence (VI) or (VII) defined in any one of items 1 to 3, 17 and 18.
49. Use of one or more of the isolated nucleic acid molecules comprising a sequence of at least 10 contiguous nucleotides of sequences (IV), (V), (VI) and/or (VII) defined in any one of items 1 to 3 and 13 to 18 for amplifying and/or detecting a nucleic acid encoding a C. difficile toxin B in a sample. 50. A method for predicting the severity or acuteness of Clostridium difficile infection (CDI) in a subject, said method comprising
performing, on a sample from the subject, an amplification reaction on
(i) a nucleic acid encoding a C. difficile toxin, thereby obtaining a C. difficile toxin signal; and
(ii) a nucleic acid encoding a bacterial 16S rRNA, a nucleic acid encoding a human
RNaseP and/or a nucleic acid encoding a fungal 18S rRNA, thereby obtaining a bacterial 16S rRNA signal, a human RNaseP signal and/or a fungal 18S rRNA signal; normalizing the C. difficile toxin signal using the bacterial 16S rRNA and/or human RNaseP signal, thereby obtaining a normalized C. difficile toxin signal;
predicting the severity or acuteness of CDI in the subject on the basis of said normalized C. difficile toxin signal. 51 . The method of item 50, further comprising performing an amplification reaction on a nucleic acid encoding a C. difficile-specific 16S nucleic acid, thereby obtaining a C. difficile- specific 16S signal, establishing a ratio of the C. difficile toxin signal to the C. difficile-specific 16S signal or vice-versa, and predicting the severity of CDI in the subject on the basis of said normalized C. difficile toxin signal and said ratio. 52. A method for predicting the severity or acuteness of Clostridium difficile infection (CDI) in a subject, said method comprising:
performing, on a sample from the subject, an amplification reaction on a nucleic acid encoding a C. difficile toxin, thereby obtaining a C. difficile toxin amplified product;
performing on said sample an amplification reaction on a nucleic acid encoding a bacterial 16S rRNA, a nucleic acid encoding a fungal 18S rRNA and/or a nucleic acid encoding a human RNaseP, thereby obtaining a bacterial 16S rRNA amplified product, a fungal 18S rRNA amplified product and/or a human RNaseP amplified product;
contacting the C. difficile toxin amplified product with a probe hybridizing to said nucleic acid encoding a C. difficile toxin, thereby obtaining a C. difficile toxin signal;
contacting the bacterial 16S rRNA amplified product, fungal 18S rRNA amplified product and/or human RNaseP amplified product with a probe hybridizing to said nucleic acid encoding a bacterial 16S rRNA, a probe hybridizing to said nucleic acid encoding fungal 18S rDNA and/or a probe hybridizing to said nucleic acid encoding a human RNaseP, thereby obtaining a bacterial 16S rRNA signal, a fungal 18S rRNA signal, fungal 18S rRNA signal and/or human RNaseP signal;
normalizing the C. difficile toxin signal using the bacterial 16S rRNA, fungal 18S rRNA and/or a human RNaseP signal, thereby obtaining a normalized C. difficile toxin signal; and predicting the severity or acuteness of CDI in the subject on the basis of said normalized C. difficile toxin signal. 53. The method of item 52, further comprising performing an amplification reaction on a nucleic acid encoding a C. difficile-specific 16S nucleic acid, thereby obtaining a C. difficile- specific 16S amplified product, contacting the C. difficile-specific 16S amplified product with a probe hybridizing to said nucleic acid encoding a C. difficile-specific 16S, thereby obtaining a C. difficile-specific 16S signal, establishing a ratio of the C. difficile toxin signal to the C. difficile- specific 16S signal or vice-versa, and predicting the severity of CDI in the subject on the basis of said normalized C. difficile toxin signal and said ratio.
54. The method of any one of items 50 to 53, wherein said method comprises determining an acuteness of infection C. difficile (AOI-CD) score using the following formula:
AOI-CD score = (1/Cpt)/(Cpu-Cpt) or (1/Cpt)/(Cpg-Cpt) wherein
Cpt = C. difficile toxin signal value;
Cpu = bacterial 16S rRNA signal value;
Cpg = human RNaseP signal value. 55. The method of any one of items 50 to 54, wherein said amplification reaction is performed by polymerase chain reaction (PCR).
56. The method of item 55, wherein said PCR is quantitative PCR.
57. The method of any one of items 50 to 56, wherein said sample is stool, feces, anal swab or rectal swab. 58. The method of any one of items 50 to 56, wherein said method comprises performing an amplification reaction on a nucleic acid encoding a bacterial 16S rRNA, thereby obtaining a bacterial 16S rRNA signal.
59. The method of item 58, wherein said amplification reaction is performed according to the method of any one of items 19 to 36. 60. A method for normalizing the amount of a pathogen of interest in a sample, said method comprising:
performing, on said sample, an amplification reaction on a nucleic acid specific for said pathogen of interest to obtain a pathogen of interest signal;
performing, on said sample, an amplification reaction on a bacterial 16S rRNA nucleic acid according to the method of 19 to 36 to obtain a bacterial 16S rRNA signal;
normalizing the first signal using the bacterial 16S rRNA signal, thereby obtaining a normalized pathogen of interest signal.
61 The method of item 60, wherein said pathogen of interest is an opportunistic pathogen.
62. The method of item 60 or 61 , wherein said pathogen is a bacterial pathogen. 63. The method of item 61 , wherein said opportunistic pathogen is Clostrodium difficile, Helicobacter pylori, Haemophilus influenza or Streptococcus pneumonia.
64. The method of item 63, wherein said opportunistic pathogen is Clostrodium difficile.
65. The method of item 64, wherein said nucleic acid specific for said pathogen of interest is a C. difficile toxin nucleic acid.
Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
In the appended drawings:
FIG. 1 shows a nucleotide sequence from the rrsB operon (containing a 16S rRNA gene) of Escherichia coli strain K-12 substr. MG1655 (corresponding to nucleotides 4166659 to
4167658 of NCBI Reference Sequence: NC_000913.3; SEQ ID NO: 25). Also shown are the sequences of the primers of SEQ ID NOs: 15 and 9, and the probe of SEQ ID NO: 2, and their corresponding regions in SEQ ID NO: 25.
FIG. 2A shows the correlation between stool sample mass and Ct values as determined using the novel 16S rDNA qPCR assay described herein (R2 = -0.12, P= 0.58).
Labeled Y-axis values, from bottom to top: 14, 16, 18 and 20; labeled X-axis values, from left to right: 0, 0.01 , 0.02, 0.03 and 0.04.
FIG. 2B shows the correlation between Ct values obtained from a lysate of stool samples (using glass beads, vortex, and heating at 95°C for 5 min) versus Ct values obtained from DNA isolated from the same mass equivalent of input stool material (R2 = 0.90, P= 6.4e-8).
Labeled Y-axis values, from bottom to top: 13, 15, 17, 19 and 21 ; labeled X-axis values, from left to right: 16, 18 and 20.
FIG. 3 shows a comparison of the total bacterial load determined by the novel 16S rDNA qPCR assay described herein and anaerobic culture plating/counting method. Labeled Y- axis values, from bottom to top: 16, 18 and 20; labeled X-axis values, from left to right: 0-102,
102-104, and 104-106.
FIG. 4 shows the distribution of Ct values using the novel 16S rDNA qPCR assay described herein amongst 500 stool swabs (from a collection of C. difficile positive samples).
FIG. 5 shows a representative relative fluorescence units signal (RFU) versus cycle diagram for stool sample. 6 serial dilutions (10-fold) of DNA isolated from stool were subjected to qPCR performed according to Natkarni et al (2012) (right) or the novel 16S rDNA qPCR assay described herein (left). The graphs show that there is a good correlation for both assays in range of 12-24 Ct units. 10 stool samples were pooled to get average microbiota diversity representation.
DISCLOSURE OF INVENTION
Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. In general, the terminology "about" is meant to designate a possible variation of up to 10%. Therefore, a variation of 1 , 2, 3, 4, 5, 6, 7, 8, 9 and 10% of a value is included in the term "about". Nucleotide sequences are presented herein by single strand, in the 5' to 3' direction, from left to right, using the one-letter nucleotide symbols as commonly used in the art and in accordance with the recommendations of the lUPAC IUB Biochemical Nomenclature Commission. An "isolated nucleic acid molecule", as is generally understood and used herein, refers to a polymer of nucleotides, and includes, but should not limited to DNA and RNA. The "isolated" nucleic acid molecule is purified from its natural in vivo state, obtained by cloning or chemically synthesized.
"Amplification" refers to any in vitro procedure for obtaining multiple copies
("amplicons" or "amplification products") of a target nucleic acid sequence or its complement or fragments thereof. In vitro amplification refers to production of an amplified nucleic acid that may contain less than the complete target region sequence or its complement. In vitro amplification methods include, e.g., transcription-mediated amplification, replicase-mediated amplification, polymerase chain reaction (PCR) amplification, ligase chain reaction (LCR) amplification and strand-displacement amplification (SDA including multiple strand-displacement amplification method (MSDA)). Replicase-mediated amplification uses self-replicating nucleic acid molecules, and a replicase such as G^-replicase (e.g., Kramer et al., U.S. Pat. No. 4,786,600). PCR amplification is well known and uses DNA polymerase, primers and thermal cycling to synthesize multiple copies of the two complementary strands of DNA or cDNA (e.g., Mullis et al., U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159). LCR amplification uses at least four separate oligonucleotides to amplify a target and its complementary strand by using multiple cycles of hybridization, ligation, and denaturation (e.g., EP Pat. App. Pub. No. 0 320 308). SDA is a method in which a primer contains a recognition site for a restriction endonuclease that permits the endonuclease to nick one strand of a hemimodified DNA duplex that includes the target sequence, followed by amplification in a series of primer extension and strand displacement steps (e.g., Walker et al., U.S. Pat. No. 5,422,252). Two other known strand- displacement amplification methods do not require endonuclease nicking (Dattagupta et al., U.S. Patent No. 6,087,133 and U.S. Patent No. 6,124,120 (MSDA)). Those skilled in the art will understand that the oligonucleotide primer sequences of the present invention may be readily used in any in vitro amplification method based on primer extension by a polymerase, (see generally Kwoh et al., 1990, Am. Biotechnol. Lab. 8:14 25 and (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86, 1 173 1 177; Lizardi et al., 1988, BioTechnology 6:1 197 1202; Malek et al., 1994, Methods Mol. Biol., 28:253 260; and Sambrook et al., 2000, Molecular Cloning - A Laboratory Manual, Third Edition, CSH Laboratories). As commonly known in the art, the oligos are designed to bind to a complementary sequence under selected conditions.
"Nucleic acid hybridization" refers generally to the hybridization of two single stranded nucleic acid molecules having complementary base sequences, which under appropriate conditions will form a thermodynamically favored double stranded structure. Examples of hybridization conditions can be found in the two laboratory manuals referred above (Sambrook et al., 2000, and Ausubel et al., 1994, or further in Higgins and Hames (Eds.) "Nucleic acid hybridization, a practical approach" IRL Press Oxford, Washington DC, (1985)) and are commonly known in the art. In the case of a hybridization to a nitrocellulose filter (or other such support like nylon), as for example in the well-known Southern blotting procedure, a nitrocellulose filter can be incubated overnight at a temperature representative of the desired stringency condition (e.g., about 60-65°C for high stringency, about 50-60°C for moderate stringency and about 40-50°C for low stringency conditions) with a labeled probe in a solution containing high salt (6x SSC or 5x SSPE), 5x Denhardt's solution, 0.5% SDS, and 100 μg ml denatured carrier DNA (e.g., salmon sperm DNA). The non-specifically binding probe can then be washed off the filter by several washes in 0.2 x SSC/0.1 % SDS at a temperature which is selected in view of the desired stringency: room temperature (low stringency), 42°C (moderate stringency) or 65°C (high stringency). The salt and SDS concentration of the washing solutions may also be adjusted to accommodate for the desired stringency. The selected temperature and salt concentration is based on the melting temperature (Tm) of the DNA hybrid. Of course, RNA-DNA hybrids can also be formed and detected. In such cases, the conditions of hybridization and washing can be adapted according to well-known methods by the person of ordinary skill. Stringent conditions will be preferably used (Sambrook et al., 2000, supra). Other protocols or commercially available hybridization kits (e.g., ExpressHyb™ from BD Biosciences Clonetech®) using different annealing and washing solutions can also be used as well known in the art. As is well known, the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions. Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility. Hybridizing nucleic acid molecules also comprise fragments of the above described molecules. Furthermore, nucleic acid molecules which hybridize with any of the aforementioned nucleic acid molecules also include complementary fragments, derivatives and allelic variants of these molecules. Additionally, a hybridization complex refers to a complex between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration. A hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., membranes, filters, chips, pins or glass slides to which, e.g., cells have been fixed).
By "isolated" it is meant that a sample containing a target nucleic acid is taken from its natural milieu, but the term does not connote any degree of purification.
In a first aspect, the present invention provides an isolated nucleic acid molecule of 50 nucleotides or less comprising a sequence of at least 10 contiguous nucleotides of one of the following sequences (I) to (VI):
(I) 5'-TATTACCGCGGCTGCT-3' (SEQ ID NO: 1 ), wherein said isolated nucleic acid molecule does not comprise the sequence GGC at its 3' end;
(II) 5'-CGGCTAACTMCGTGCCAG-3' (SEQ ID NO: 2);
(III) 5'-AATGTTGGCATGAGTAGCGAGATGT-3' (SEQ ID NO: 4);
(IV) 5'-TCTGAAGGATTACCTRTAATTGCAA-3' (SEQ ID NO: 5);
(V) 5'-TGCAGCCAAAGTTGTTGAAT-3' (SEQ ID NO: 6); or
(VI) 5'-GCTCTTTGATTGCTGCACCT-3' (SEQ ID NO: 7),
or a sequence having at least 80%, 85%, 90% or 95% identity with said sequences. The present invention also provides an isolated nucleic acid molecule of 50 nucleotides or less comprising a sequence of at least 10 contiguous nucleotides of one of the following nucleotide sequences (I) to (VII):
(I) 5'-TATTACCGCGGCTGCT-3' (SEQ ID NO: 1 ), wherein said isolated nucleic acid molecule does not comprise the sequence GGC at its 3' end;
(II) 5'-CGGCTAACTMCGTGCCAG-3' (SEQ ID NO: 2);
(III) 5'-AATGTTGGCATGAGTAGCGAGATGT-3' (SEQ ID NO: 4);
(IV) 5'-TCTGAAGGATTACCTRTAATTGCAA-3' (SEQ ID NO: 5);
(V) 5'-TGCAGCCAAAGTTGTTGAAT-3' (SEQ ID NO: 6);
(VI) 5'-GCTCTTTGATTGCTGCACCT-3' (SEQ ID NO: 7); or
(VII) a sequence having at least 80% identity with any of (I) to (VI).
In embodiments, the isolated nucleic acid molecule comprises a sequence of at least 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleotides of sequences (I) to (VI), or a sequence having at least 80%, 85%, 90% or 95% identity with said sequences. In an embodiment, the isolated nucleic acid molecule comprises at least 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or 25 nucleotides from one of sequences (I) to (VI). In an embodiment, the isolated nucleic acid molecule has a length of at least 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides. In another embodiment, the isolated nucleic acid molecule has a length of no more than 49, 48, 47, 46, 45, 44, 43, 42, 41 , 40, 39, 38, 37, 36, 35, 34, 33, 32, 31 or 30 nucleotides. In embodiments, the isolated nucleic acid molecule has a length comprises within any of the minimal and maximal lengths defined above, for example a length of 12 to 40 nucleotides, 15 to 40 nucleotides, 12 to 35 nucleotides, 15 to 35 nucleotides, 12 to 30 nucleotides, 15 to 30 nucleotides, 12 to 30 nucleotides, 15 to 30 nucleotides, 12 to 25 nucleotides or 15 to 25 nucleotides.
One or more of the nucleic acid molecules defined herein may comprise, at the 5' end, an AT clamp (e.g., an AT-rich nucleotide sequence). In an embodiment, the AT clamp comprises, or consists of, the nucleotide sequence AATAAATCATAA (SEQ ID NO: 10), or a 5'- deleted fragment thereof. In a further embodiment, the AT clamp comprises, or consists of, the nucleotide sequence AATAAATCATAA (SEQ ID NO: 10).
Primers/probes for detecting a nucleic acid encoding a bacterial 16S rRNA (bacterial 16S rDNA or gene)
A "probe" is meant to include a nucleic acid oligomer that hybridizes specifically to a target sequence in a nucleic acid or its complement, under conditions that promote hybridization, thereby allowing detection of the target sequence or its amplified nucleic acid. Detection may either be direct (i.e., resulting from a probe hybridizing directly to the target or amplified sequence) or indirect (i.e., resulting from a probe hybridizing to an intermediate molecular structure that links the probe to the target or amplified sequence). A probe's "target" generally refers to a sequence within an amplified nucleic acid sequence (i.e., a subset of the amplified sequence) that hybridizes specifically to at least a portion of the probe sequence by standard hydrogen bonding or "base pairing." Sequences that are "sufficiently complementary" allow stable hybridization of a probe sequence to a target sequence, even if the two sequences are not completely complementary. A probe may be labeled or unlabeled. A probe can be produced by molecular cloning of a specific DNA sequence or it can also be synthesized. In an embodiment, the probe defined herein is a hydrolysis probe (e.g., TaqMan® probe) and comprises a fluorophore and a quencher attached thereto.
As used herein, a "primer" defines an oligonucleotide which is capable of annealing to a target sequence, thereby creating a double stranded region which can serve as an initiation point for nucleic acid synthesis under suitable conditions. The primer's 5' region may be non- complementary to the target nucleic acid sequence and include additional bases, such as a promoter sequence (which is referred to as a "promoter primer"). Those skilled in the art will appreciate that any oligomer that can function as a primer can be modified to include a 5' promoter sequence, and thus function as a promoter primer. Similarly, any promoter primer can serve as a primer, independent of its functional promoter sequence. Size ranges for primers include those that are about 10 to about 50 nt long and contain at least about 10 contiguous bases, or even at least 12 contiguous bases that are complementary to a region of the target nucleic acid sequence (or a complementary strand thereof). The contiguous bases are at least 80%, or at least 90%, or completely complementary to the target sequence to which the amplification oligomer binds. An amplification oligomer may optionally include modified nucleotides or analogs, or additional nucleotides that participate in an amplification reaction but are not complementary to or contained in the target nucleic acid, or template sequence. It is understood that when referring to ranges for the length of an oligonucleotide, amplicon, or other nucleic acid, that the range is inclusive of all whole numbers (e.g., 19-25 contiguous nucleotides in length includes 19, 20, 21 , 22, 23, 24 and 25).
The isolated nucleic acid molecules comprising at least 10 contiguous nucleotides of the sequences (I) and/or (II) defined herein may be used as primers and/or probes (hereinafter referred to as bacterial 16S rRNA nucleic acid primers and/or probes) for amplifying and/or detecting a nucleic acid encoding a bacterial 16S rRNA (a bacterial 16S rDNA, such as a bacterial 16S rRNA gene) in a sample, and more particularly for detecting 16S rRNA nucleic acids (16S rDNA) derived from a plurality of bacteria (broad-coverage amplification/detection), which may be useful for example for quantifying 16S rRNA gene copy number, and in turn estimating bacterial load, in a sample.
In an embodiment, the nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (I) comprises the sequence 5'-TATTACCGCGGCTGCT-3' (SEQ ID NO: 1 ), or a sequence having at least 80, 85, 90 or 95% identity therewith. In a further embodiment, the nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (I) comprises the sequence 5'-TATTACCGCGGCTGCT-3' (SEQ ID NO: 1 ). In an embodiment, the nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (I) further comprises, at its 5' end, the sequence GCITCC or TAGC, or 5'-deleted fragment thereof (e.g., CITCC, ITCC, AGC, GC, C, etc.). In an embodiment, the nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (I) comprises, or consists of, the sequence 5'-GCITCCTATTACCGCGGCTGCT-3' (SEQ ID NO: 1 1 ), or a sequence having at least 80, 85, 90 or 95% identity therewith. In an embodiment, the nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (I) comprises, or consists of, the sequence 5'-TACGTATTACCGCGGCTGCT-3' (SEQ ID NO: 8), or a sequence having at least 80, 85, 90 or 95% identity therewith. In a further embodiment, the nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (I) comprises or consists of the sequence 5'-TACGTATTACCGCGGCTGCT-3' (SEQ ID NO: 8). As used herein, the term "consists of the sequence" means that the nucleic acid molecule does not comprise additional nucleotides, but does not exclude the presence of a moiety, label or tag (e.g., fluorescent tag) attached to the nucleic acid molecule. A "label" refers to a molecular moiety or compound that can be detected or can lead to a detectable signal. A label is joined, directly or indirectly, to a nucleic acid probe or the nucleic acid to be detected (e.g., an amplification product). Direct labeling can occur through bonds or interactions that link the label to the nucleic acid (e.g., covalent bonds or non-covalent interactions), whereas indirect labeling can occur through the use of a "linker" or bridging moiety, such as additional oligonucleotide(s), which is either directly or indirectly labeled. Bridging moieties may amplify a detectable signal. Labels can include any detectable moiety (e.g., a radionuclide, ligand such as biotin or avidin, enzyme or enzyme substrate, reactive group, chromophore such as a dye or colored particle, luminescent compound including a bioluminescent, phosphorescent or chemiluminescent compound, and fluorescent compound).
In an embodiment, the nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (I) as defined herein is used as a primer (e.g., reverse primer) for the amplification and/or detection of a bacterial 16S rRNA nucleic acid, more particularly a fragment of a bacterial 16S rRNA nucleic acid. The nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (I) may be used in combination with any suitable primer (e.g., forward primer) for the amplification and/or detection of a bacterial 16S rRNA nucleic acid, more particularly a fragment of a bacterial 16S rRNA nucleic acid. In an embodiment, the nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (I) is used in combination with another primer (e.g., forward primer) for the amplification of a fragment of about 150 to about 250 nucleotides, of about 175 to about 225 nucleotides, of about 190 to about 210 nucleotides, or of about 200 to 205 nucleotides, from a bacterial 16S rRNA nucleic acid. In an embodiment, the nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (I) is used in combination with another primer (e.g., forward primer) having a length of 50, 45, 40 or 35 nucleotides or less and comprises a sequence of at least 10, 1 1 , 12, 13, 14, 15, 16, 17, 18 or 19 contiguous nucleotides of the sequence ACTCCTAYGGGRBGCWSCA (SEQ ID NO: 13), or a sequence having at least 80, 85, 90 or 95% identity therewith. In an embodiment, the other primer (e.g., forward primer) comprises the sequence ACTCCTAYGGGRBGCWSCA (SEQ ID NO: 13), or a sequence having at least 80, 85, 90 or 95% identity therewith.
For nucleic acid molecules defined by a sequence comprising degenerate nucleotides (e.g., Y, B, W, S) defined herein, it should be understood that the term "nucleic acid molecule" (or "primer", "probe") may either correspond to a nucleic acid molecule comprising one of the sequences encompassed by the sequence comprising one or more degenerate nucleotides, or any combination of nucleic acids, each of which comprising one of the sequences encompassed by the sequence comprising one or more degenerate nucleotides. In an embodiment, the nucleic acid molecule defined by a sequence comprising one or more degenerate nucleotides corresponds to a mixture/combination (e.g., an equimolar mixture) of all the nucleic acids encompassed by the sequence. Thus, in another aspect, the present invention provides a nucleic acid molecule mixture comprising a plurality of nucleic acid molecules comprising a plurality of sequences encompassed by any of the sequences comprising one or more degenerate nucleotides defined herein.
In an embodiment, the other primer (e.g., forward primer) comprises the sequence ACTCCTAYGGGRBGCASCAGT (SEQ ID NO: 14), ACTCCTAYGGGRGGCWGCAGT (SEQ ID NO: 15), ACTCCTACGGGRGGCWGCAGT (SEQ ID NO: 16), ACTCCTAYGGGRGGCWGCA (SEQ ID NO: 17), ACTCCTACGGGRGGCWGCA (SEQ ID NO: 18), ACTCCTACG G G RG G CAG C A (SEQ ID NO: 19), ACTCCTAYGGGRBGCAGCA (SEQ ID NO: 20), or ACTCCTACGGGRBGCWGCA (SEQ ID NO: 21 ) or ACTCCTACGGGAGGCAGCAGT (SEQ ID NO: 22), or a sequence having at least 80, 85, 90 or 95% identity therewith. In a further embodiment, the other primer (e.g., forward primer) further comprises, at its 5' end, the nucleotide sequence 5'-AATAAATCATAA-3' (SEQ ID NO: 10) or a 5'-deleted fragment thereof (e.g., TAAATCATAA (SEQ ID NO: 26), ATAAATCATAA (SEQ ID NO: 27), AAATCATAA, etc.).
In an embodiment, the other primer (e.g., forward primer) comprises or consists of the sequence ACTCCTAYGGGRBGCASCAGT (SEQ ID NO: 14). In a further embodiment, the other primer (e.g., forward primer) corresponds to a mixture/combination (e.g., an equimolar mixture) of all the nucleic acid molecules encompassed by the sequence of SEQ ID NO: 14.
One or both of the bacterial 16S rRNA nucleic acid primers may further comprise a moiety, label or tag (e.g., fluorescent tag) attached thereto.
The bacterial 16S rRNA nucleic acid primers defined above may be used in combination with a probe (a bacterial 16S rRNA nucleic acid probe) that is capable of hybridizing to the amplification product generated by an amplification reaction with said bacterial 16S rRNA nucleic acid primers (i.e. capable of hybridizing to a sequence located between the primers in the sequence of the bacterial 16S rRNA nucleic acid).
In an embodiment, the bacterial 16S rRNA nucleic acid probe is the isolated nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (II) defined above. In an embodiment, the bacterial 16S rRNA nucleic acid probe comprises, or consists of, the sequence 5'-CGGCTAACTMCGTGCCAG-3' (SEQ ID NO:2), or a sequence having at least 80, 85, 90 or 95% identity therewith. In an embodiment, the bacterial 16S rRNA nucleic acid probe further comprises the sequence 5'-AAGSVM-3', a 3'-deleted fragment thereof (e.g., AGSVM, GSVM, etc.) at its 5'-terminal. In an embodiment, the bacterial 16S rRNA nucleic acid probe comprises the sequence 5'-AAGSVMCGGCTAACTMCGTGCC-3' (SEQ ID NO: 3), or a sequence having at least 80, 85, 90 or 95% identity therewith. In an embodiment, the bacterial 16S rRNA nucleic acid probe comprises, or consists of, the sequence 5'- CGGCTAACTMCGTGCCAG-3' (SEQ ID NO: 2). In an embodiment, a moiety, a label or a tag (e.g., fluorescent tag) attached to the bacterial 16S rRNA nucleic acid probe, for example for detecting the complexes formed between the probe and the amplification product (bacterial 16S rRNA nucleic acid fragment). In an embodiment, the probe is a hydrolysis probe (e.g., TaqMan® probe) and comprises a fluorophore and a quencher attached thereto. In embodiments, the nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (II) defined above comprises or consists of (a) the sequence 5'-CGGCTAACTMCGTGCCAG-3' (SEQ ID NO:2) and (b) one or more moiety, label or tag (e.g. a fluorescent tag and/or a quencher) attached thereto.
In another aspect, the present invention also provides a combination of nucleic acid molecules for amplifying or detecting a bacterial 16S rRNA nucleic acid, the combination comprising the bacterial 16S rRNA nucleic acid primers defined above. In an embodiment, the combination further comprises the bacterial 16S rRNA nucleic acid probe defined above. In an embodiment, the combination of nucleic acid molecules comprises a first primer comprising, or consisting of, the sequence ACTCCTAYGGGRBGCASCAGT (SED ID NO: 14), and a second primer comprising, or consisting of, the sequence TACGTATTACCGCGGCTGCT-3' (SEQ ID NO: 8). In a further embodiment, the combination of nucleic acid molecules further comprises a probe comprising, or consisting of, the sequence CGGCTAACTMCGTGCCAG (SEQ ID NO: 2). The nucleic acid molecules may be in solid form (e.g., lyophilized) or present in a solution (e.g., a suitable buffer). In another aspect, the present invention provides a container (e.g., a tube, a vessel, a plate) comprising the nucleic acid molecules.
In another aspect, the present invention provides a bacterial 16S rRNA nucleic acid amplification or detection mixture, the mixture comprising the combination of nucleic acid molecules defined above and one or more reagents for performing an amplification reaction, for example a suitable DNA polymerase (e.g., a Tag polymerase, a Pfu polymerase or any other thermostable polymerase suitable for nucleic acid amplification), deoxynucleotide triphosphates (dNTPs), and a suitable buffer. Primers/probes for detecting C. Difficile 16S rRNA nucleic acid (C. Difficile 16S rDNA or gene)
The isolated nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (III) defined herein may be used as primers and/or probes (hereinafter referred to as C. Difficile 16S rRNA nucleic acid primers and/or probes) for amplifying and/or detecting a nucleic acid encoding a C. Difficile 16S rRNA (C. Difficile 16S rDNA, such as a C. Difficile 16S rRNA gene) in a sample, which may be useful for example for quantifying C. Difficile 16S rRNA gene copy number, and in turn estimating C. Difficile bacterial load, in a sample. In an embodiment, the isolated nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (III) comprises, or consists of, the sequence 5'- AATGTTGGCATGAGTAGCGAGATGT-3' (SEQ ID NO: 4), or a sequence having at least 80, 85, 90 or 95% identity therewith. In an embodiment, the isolated nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (III) comprises, or consists of, the sequence 5'-AATGTTGGCATGAGTAGCGAGATGT-3' (SEQ ID NO: 4). In an embodiment, the nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (III) is used as a C. Difficile 16S rRNA nucleic acid probe for the amplification and/or detection of a C. Difficile 16S rRNA nucleic acid, more particularly a fragment of a C. Difficile 16S rRNA nucleic acid. In an embodiment, a moiety, a label or a tag (e.g., fluorescent tag) attached to the C. Difficile 16S rRNA nucleic acid probe, for example for detecting the complexes formed between the probe and the amplification product (C. Difficile 16S rRNA nucleic acid fragment). In an embodiment, the probe is a hydrolysis probe (e.g., TaqMan® probe) and comprises a fluorophore and a quencher attached thereto.
The C. Difficile 16S rRNA nucleic acid probe may be used in combination with suitable
C. Difficile 16S rRNA nucleic acid primers capable of generating an amplification product that may be detected by the probe (i.e. primers hybridizing to sequences located upstream and downstream of the sequence to which the probe hybridizes in the C. Difficile 16S rRNA nucleic acid). Example of a suitable C. Difficile 16S rRNA nucleic acid primer pair for generating an amplification product that may be detected by the C. Difficile 16S rRNA nucleic acid probe comprises the following sequences: GGGAGCTTCCCATACGGGTTG (SEQ ID NO: 29) and TTGACTGCCTCAATGCTTGGGC (SEQ ID NO: 30).
In another aspect, the present invention also provides a combination of nucleic acid molecules for amplifying or detecting a C. Difficile 16S rRNA nucleic acid, the combination comprising the C. Difficile 16S rRNA nucleic acid probe defined above and suitable C. Difficile 16S rRNA nucleic acid primers capable of generating an amplification product that may be detected by the probe, for example the C. Difficile 16S rRNA nucleic acid primers defined above. The nucleic acid molecules may be in solid form (e.g., lyophilized) or present in a solution (e.g., a suitable buffer). In another aspect, the present invention provides a container (e.g., a tube, a vessel, a plate) comprising the nucleic acid molecules.
In another aspect, the present invention provides a C. Difficile 16S rRNA nucleic acid amplification or detection mixture, the mixture comprising the combination of nucleic acid molecules defined above and one or more reagents for performing an amplification reaction, for example a suitable DNA polymerase (e.g., a Tag polymerase, a Pfu polymerase or any other thermostable polymerase suitable for nucleic acid amplification), deoxynucleotide triphosphates (dNTPs), and a suitable buffer. Probes/primers for detecting a C. difficile toxin B (ToxB) nucleic acid
The isolated nucleic acid molecule comprising at least 10 contiguous nucleotides of sequences (IV), (V) and/or (VI) defined herein may be used as primers and/or probes (hereinafter referred to as C. Difficile ToxB primers and/or probes) for amplifying and/or detecting a nucleic acid encoding a C. Difficile ToxB nucleic acid in a sample, which may be useful for example for quantifying toxigenic C. Difficile bacterial load in a sample.
In an embodiment, the isolated nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (IV) comprises, or consists of, the sequence 5'- TCTGAAGGATTACCTRTAATTGCAA-3' (SEQ ID NO: 5), or a sequence having at least 80, 85, 90 or 95% identity therewith. In an embodiment, the isolated nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (IV) comprises, or consists of, the sequence 5'- TCTGAAGGATTACCTRTAATTGCAA-3' (SEQ ID NO: 5).
In an embodiment, the nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (IV) is used as a C. Difficile ToxB probe for the amplification and/or detection of a C. Difficile ToxB nucleic acid (e.g. , C. Difficile ToxB RNA, cDNA or gene), more particularly a fragment of a C. Difficile ToxB nucleic acid. In an embodiment, a moiety, a label and/or a tag (e.g., fluorescent tag) attached to the C. Difficile ToxB probe, for example for detecting the complexes formed between the probe and the amplification product (C. Difficile ToxB nucleic acid fragment). In an embodiment, the probe is a hydrolysis probe (e.g. , TaqMan® probe) and comprises a fluorophore and a quencher attached thereto.
The C. Difficile ToxB probe may be used in combination with suitable C. Difficile ToxB primers capable of generating an amplification product that may be detected by the probe (i.e. primers hybridizing to sequences located upstream and downstream of the sequence to which the C. Difficile ToxB probe hybridizes in the C. Difficile ToxB nucleic acid).
In an embodiment, the isolated nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (V) comprises, or consists of, the sequence 5'- TGCAGCCAAAGTTGTTGAAT-3' (SEQ ID NO: 6), or a sequence having at least 80, 85, 90 or 95% identity therewith. In an embodiment, the isolated nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (V) comprises, or consists of, the sequence 5'- TGCAGCCAAAGTTGTTGAAT-3' (SEQ ID NO: 6).
In an embodiment, the nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (VI) comprises, or consists of, the sequence 5'- GCTCTTTGATTGCTGCACCT-3' (SEQ ID NO: 7), or a sequence having at least 80, 85, 90 or 95% identity therewith. In an embodiment, the nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (VI) comprises, or consists of, the sequence 5'- GCTCTTTGATTGCTGCACCT-3' (SEQ ID NO: 7). In an embodiment, the nucleic acid molecules comprising at least 10 contiguous nucleotides of sequences (V) and/or (VI) are used as C. Difficile ToxB primers for the amplification and/or detection of a C. Difficile ToxB nucleic acid, more particularly a fragment of a C. Difficile ToxB nucleic acid. The nucleic acid molecules comprising at least 10 contiguous nucleotides of sequences (V) and (VI) may be used together, or in combination with any suitable C. Difficile ToxB primer, for the amplification and/or detection of a C. Difficile ToxB nucleic acid, more particularly a fragment of a C. Difficile ToxB nucleic acid. In an embodiment, the nucleic acid molecule comprising at least 10 contiguous nucleotides of sequence (V) is used in combination with the nucleic acid molecule comprising at least 10 contiguous nucleotides of sequences (VI).
In another aspect, the present invention also provides a combination of nucleic acid molecules for amplifying or detecting a C. Difficile ToxB nucleic acid, the combination comprising a pair (e.g., first and second, or forward and reverse (or vice versa), primers) of the C. Difficile ToxB primers defined above. In an embodiment, the combination further comprises the C. Difficile ToxB probe defined above. In another aspect, the present invention provides a combination of nucleic acid molecules for amplifying or detecting a C. Difficile ToxB nucleic acid, the combination comprising the C. Difficile ToxB probe defined above and suitable C. Difficile ToxB primers capable of generating an amplification product that may be detected by the probe, for example the C. Difficile ToxB primers defined above. The nucleic acid molecules may be in solid form (e.g., lyophilized) or present in a solution (e.g., a suitable buffer). In another aspect, the present invention provides a container (e.g., a tube, a vessel, a plate) comprising the nucleic acid molecules.
In another aspect, the present invention provides a C. Difficile ToxB nucleic acid amplification or detection mixture, the mixture comprising the combination of nucleic acid molecules defined above and one or more reagents for performing an amplification reaction, for example a suitable DNA polymerase (e.g., a Tag polymerase, a Pfu polymerase or any other thermostable polymerase suitable for nucleic acid amplification), deoxynucleotide triphosphates (dNTPs), and a suitable buffer. Detection of a bacterial 16S rRNA nucleic acid (bacterial 16S rDNA or gene)
In another aspect, the present invention provides the use of the isolated nucleic acid molecule comprising at least 10 contiguous nucleotides of sequences (I) and/or (II) (bacterial 16S rRNA nucleic acid primers and/or probes) for detecting a bacterial 16S rRNA nucleic acid in a sample.
In another aspect, the present invention provides a method for detecting a nucleic acid encoding a bacterial 16S rRNA nucleic acid) in a sample, the method comprising (i) contacting the sample with the bacterial 16S rRNA nucleic acid primers and/or probes defined above under conditions permitting hybridization of said bacterial 16S rRNA nucleic acid primers and/or probes with said bacterial 16S rRNA nucleic acid, if present, and (ii) detecting the hybridization signal. In an embodiment, the bacterial 16S nucleic acid is an amplification product from a biological sample from a subject.
In another aspect, the present invention provides a method for amplifying or detecting a bacterial 16S rRNA nucleic acid in a sample, said method comprising (a) contacting said bacterial 16S rRNA nucleic acid with a first primer and a second primer under conditions suitable for nucleic acid amplification, and (b) performing a nucleic acid amplification reaction, thereby generating an amplification product if bacterial 16S rRNA nucleic acid is present in said sample; wherein said first and second primers are the bacterial 16S rRNA nucleic acid primers defined herein.
In another aspect, the present invention provides a method for determining whether a sample comprises a bacterial 16S rRNA nucleic acid, the method comprising (i) contacting the sample with the bacterial 16S rRNA nucleic acid primers and/or probes defined herein under conditions permitting hybridization of said isolated nucleic acid molecule with said bacterial 16S nucleic acid, if present, and (ii) determining the presence or absence of an hybridization signal, wherein the presence of an hybridization signal is indicative that the sample comprises a bacterial 16S nucleic acid.
In another aspect, the present invention provides a method for detecting a nucleic acid encoding a bacterial 16S rRNA nucleic acid in a biological sample, the method comprising (i) performing an amplification reaction for said bacterial 16S nucleic acid on the biological sample; (ii) contacting the amplified sample with the bacterial 16S rRNA nucleic acid probes defined herein under conditions permitting hybridization of said isolated nucleic acid molecule with the amplified bacterial 16S nucleic acid, if present, and (iii) detecting the hybridization signal.
In another aspect, the present invention provides a method for determining whether a biological sample comprises a nucleic acid encoding a bacterial 16S rRNA (bacterial 16S rRNA nucleic acid, such as a bacterial 16S rDNA or gene), the method comprising (i) performing an amplification reaction for said bacterial 16S nucleic acid on the biological sample; (ii) contacting the amplified sample with the bacterial 16S rRNA nucleic acid probes defined herein under conditions permitting hybridization of said probes with the amplified bacterial 16S nucleic acid, if present, and (iii) determining the presence or absence of a hybridization signal, wherein the presence of a hybridization signal is indicative that the biological sample comprises a bacterial 16S nucleic acid.
In an embodiment, the bacterial 16S rRNA nucleic acid primers and/or probes are used in the contacting step.
In an embodiment, the amplification reaction is performed using the following bacterial 16S rRNA nucleic acid primer pairs: a first primer comprising, or consisting of, the sequence ACTCCTAYGGGRBGCASCAGT (SED ID NO: 14), and a second primer which comprises, or consists of, the sequence TACGTATTACCGCGGCTGCT-3' (SEQ ID NO: 8). In an embodiment, the bacterial 16S rRNA nucleic acid and/or amplification product is detected using the following bacterial 16S rRNA nucleic acid probe: CGGCTAACTMCGTGCCAG (SEQ ID NO: 2).
In another aspect, the present invention provides a method for determining whether a sample comprises a bacterial 16S rRNA nucleic acid, said method comprising performing the method for amplifying or detecting a bacterial 16S rRNA nucleic acid defined above, determining the presence or absence of an amplification product, wherein the presence of an amplification product is indicative that said sample comprises a bacterial 16S rRNA nucleic acid.
In another aspect, the present invention provides a method for determining the amount of bacteria in a sample, said method comprising (a) performing the method for amplifying or detecting a bacterial 16S rRNA nucleic acid defined above, (b) comparing the amount of amplification product present after said amplification reaction to a reference; and (c) determining the amount of bacteria in the sample based on said comparison. The reference may be the amount of amplification product present in one or more sample comprising a known or predetermined amount of bacteria.
Detection of a fungal 18S rDNA nucleic acid
In another aspect, the present invention provides the use of an isolated oligonucleotide comprising a sequence within sequence (VII) below for detecting a nucleic acid encoding a fungal 18S rRNA (fungal 18S rRNA nucleic acid, such as a fungal 18S rDNA gene) in a sample. Sequence VII:
CGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTT ACCAGGTCCAGACATAGTAAGGATTGACAGATTGAGAGCTCTTTCTTGATTCTATGGGTGG TGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTCAACGAACGA GACCTCAGCCTGCTAAATAGTTGGACCCTACTCTTAGGGCCACAACTTCTTAGAGGGACTA TGTGCGTGTAGCACGTGGAAGTTTGAGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCT GGGCCGCACGCGCGCTACACTGACGAATTCAACGAGCTTATAACCTTGGCCGAAAGGTCT GGGTAATCTCCAAAATTCGTCGTGATGGGGATAGATTATTGCAATTATTAATCTTC (SEQ ID NO: 31 ).
In another aspect, the present invention provides a method for detecting a nucleic acid encoding a fungal 18S rRNA (fungal 18S nucleic acid) in a sample, the method comprising (i) contacting the sample with an isolated oligonucleotide comprising a sequence within the sequence of SEQ ID NO: 31 under conditions permitting hybridization of said isolated nucleic acid molecule with said fungal 18S nucleic acid, if present, and (ii) detecting the hybridization signal. In another aspect, the present invention provides a method for determining whether a sample comprises a nucleic acid encoding a fungal 18S rRNA (fungal 18S nucleic acid), the method comprising (i) contacting the sample with an isolated oligonucleotide comprising a sequence within the sequence of SEQ ID NO: 31 under conditions permitting hybridization of said isolated nucleic acid molecule with said fungal 18S nucleic acid, if present, and (ii) determining the presence or absence of an hybridization signal, wherein the presence of an hybridization signal is indicative that the sample comprises a fungal 18S nucleic acid.
In an embodiment, the fungal 18S nucleic acid is an amplification product from a biological sample from a subject.
In another aspect, the present invention provides a method for detecting a nucleic acid encoding a fungal 18S rRNA (fungal 18S nucleic acid) in a biological sample, the method comprising (i) performing an amplification reaction for said fungal 18S nucleic acid on the biological sample; (ii) contacting the amplified sample with an isolated oligonucleotide (probe) comprising a sequence within the sequence of SEQ ID NO: 31 under conditions permitting hybridization of said isolated nucleic acid molecule with the amplified fungal 18S nucleic acid, if present, and (iii) detecting the hybridization signal.
In another aspect, the present invention provides a method for determining whether a biological sample comprises a nucleic acid encoding a fungal 18S rRNA (fungal 18S nucleic acid), the method comprising (i) performing an amplification reaction for said fungal 18S nucleic acid on the biological sample; (ii) contacting the amplified sample with an isolated oligonucleotide (probe) comprising a sequence within the sequence of SEQ ID NO: 31 under conditions permitting hybridization of said isolated nucleic acid molecule with the amplified fungal 18S nucleic acid, if present, and (iii) determining the presence or absence of a hybridization signal, wherein the presence of a hybridization signal is indicative that the biological sample comprises a fungal 18S nucleic acid.
In an embodiment, the isolated oligonucleotide comprises one of the sequences underlined within sequence (VII), as noted above.
In an embodiment, the amplification reaction is performed using a pair of primers comprising the following sequences: 5'-GGGAAACTTACCAGGTCCAG-3' (SEQ ID NO: 32) and 5'-TCGTCGTGATGGGGATAGATT-3' (SEQ ID NO: 33). In another embodiment, the fungal 18S nucleic acid (or amplified product) is detected using a probe comprising the sequence 5'- TGGTGCATGGCCGTT-3' (SEQ ID NO: 34).
In an embodiment, the above-mentioned method does not comprise a step of isolating the nucleic acid from the sample (e.g., clinical sample) prior to performing the amplification reaction.
In an embodiment, the above-mentioned method is compatible with hydrolysis probe technology (e.g., TaqMan). Detection of a nucleic acid encoding a C. d/ff/c/7e-specific 16S rRNA (C. difficile-specific 16S nucleic acid)
In another aspect, the present invention provides a use of the C. difficile-specific 16S rRNA primers and/or probes defined herein for amplifying or detecting a nucleic acid encoding a C. difficile-specific 16S rRNA (a C. difficile-specific 16S nucleic acid, such as a C. difficile- specific 16S rDNA or gene) in a sample.
In another aspect, the present invention provides a method for detecting a nucleic acid encoding a C. difficile-specific 16S rRNA (a C. difficile-specific 16S nucleic acid) in a sample, the method comprising (i) contacting the sample with the C. difficile-specific 16S rRNA primers and/or probes under conditions permitting hybridization of said isolated nucleic acid molecule with said C. difficile-specific 16S nucleic acid, if present, and (ii) detecting the hybridization signal.
In another aspect, the present invention provides a method for determining whether a sample comprises a C. difficile-specific 16S nucleic acid, the method comprising (i) contacting the sample with the C. difficile-specific 16S rRNA primers and/or probes under conditions permitting hybridization of said isolated nucleic acid molecule with said C. difficile-specific 16S nucleic acid, if present, and (ii) determining the presence or absence of an hybridization signal, wherein the presence of an hybridization signal is indicative that the sample comprises a C. difficile-specific 16S nucleic acid.
In an embodiment, the C. difficile-specific 16S rRNA primers and/or probes is contacted with an amplification product obtained from the sample.
In another aspect, the present invention provides a method for detecting a C. difficile- specific 16S nucleic acid in a biological sample, the method comprising (i) performing an amplification reaction for said C. difficile-specific 16S nucleic acid on the biological sample; (ii) contacting the amplified sample with the C. difficile-specific 16S rRNA primers and/or probes defined herein under conditions permitting hybridization of said primers and/or probes with the amplified C. difficile-specific 16S nucleic acid, if present, and (iii) detecting the hybridization signal.
In another aspect, the present invention provides a method for determining whether a biological sample comprises a C. difficile-specific 16S nucleic acid, the method comprising (i) performing an amplification reaction for said C. difficile-specific 16S nucleic acid on the biological sample; (ii) contacting the amplified sample with the C. difficile-specific 16S rRNA primers and/or probes defined herein under conditions permitting hybridization of said primers and/or probes with the amplified C. difficile-specific 16S nucleic acid, if present, and (iii) determining the presence or absence of a hybridization signal, wherein the presence of a hybridization signal is indicative that the biological sample comprises a C. difficile-specific 16S nucleic acid.
In an embodiment, the amplification reaction is performed using the following primer pairs:
a first primer which comprises, or consists of, the sequence
GGGAGCTTCCCATACGGGTTG (SEQ ID NO: 29), and a second primer which comprises, or consists of, the sequence TTGACTGCCTCAATGCTTGGGC (SEQ ID NO: 30).
In an embodiment, detection of the amplification product is performed using a probe which comprises, or consists of, the sequence: AATGTTGGCATGAGTAGCGAGATGT (SEQ ID NO: 4).
Detection of a nucleic acid encoding a C. difficile toxin B (C. difficile ToxB nucleic acid)
In another aspect, the present invention provides the use of the C. difficile ToxB primers and/or probes defined herein for amplifying and/or detecting a nucleic acid encoding a C. difficile toxin B (C. difficile ToxB nucleic acid, such as a C. difficile ToxB DNA or gene) in a sample.
In another aspect, the present invention provides a method for detecting a nucleic acid encoding a C. difficile ToxB nucleic acid in a sample, the method comprising (i) contacting the sample with the C. difficile ToxB primers and/or probes defined herein under conditions permitting hybridization of said primers and/or probes with said C. difficile ToxB nucleic acid, if present, and (ii) detecting the hybridization signal.
In another aspect, the present invention provides a method for determining whether a sample comprises a C. difficile ToxB nucleic acid, the method comprising (i) contacting the sample with the C. difficile ToxB primers and/or probes defined herein under conditions permitting hybridization of said primers and/or probes with said C. difficile ToxB nucleic acid, if present, and (ii) determining the presence or absence of an hybridization signal, wherein the presence of a hybridization signal is indicative that the sample comprises a C. difficile ToxB nucleic acid.
In an embodiment, the C. difficile ToxB primers and/or probes is contacted with an amplification product obtained from the sample.
In another aspect, the present invention provides a method for detecting a C. difficile ToxB nucleic acid in a biological sample comprising (i) performing an amplification reaction for said C. difficile ToxB nucleic acid on the biological sample; (ii) contacting the amplified sample with the C. difficile ToxB primers and/or probes defined herein under conditions permitting hybridization of said primers and/or probes with the amplified C. difficile ToxB nucleic acid, if present, and (iii) detecting the hybridization signal. In another aspect, the present invention provides a method for determining whether a biological sample comprises a C. difficile ToxB nucleic acid, the method comprising (i) performing an amplification reaction for said C. difficile ToxB nucleic acid on the biological sample; (ii) contacting the amplified sample with the C. difficile ToxB primers and/or probes under conditions permitting hybridization of said primers and/or probes with the amplified C. difficile ToxB nucleic acid, if present, and (iii) determining the presence or absence of a hybridization signal, wherein the presence of an hybridization signal is indicative that the biological sample comprises a C. difficile ToxB nucleic acid.
In an embodiment, the C. difficile ToxB probe defined herein is used in the step of contacting, in an embodiment using the C. difficile ToxB probe comprising, or consisting of, the sequence AATGTTGGCATGAGTAGCGAGATGT (SEQ ID NO: 4).
In an embodiment, the amplification reaction is performed using the C. difficile ToxB primers defined herein, in an embodiment using a first primer comprising, or consisting of, the sequence TGCAGCCAAAGTTGTTGAAT (SEQ ID NO: 6), and a second primer which comprises, or consists of, the sequence G CTCTTTG ATTG CTG C ACCT (SEQ ID NO: 7).
In an embodiment, the amplification reaction is a primer-dependent nucleic acid amplification reaction. The amplification reaction is allowed to proceed for a duration (e.g., number of cycles) and under conditions that generate a sufficient amount of amplification product. Most conveniently, polymerase chain reaction (PCR) will be used, although the skilled person would be aware of other techniques.
Many variations of PCR have been developed, for instance Real Time PCR (also known as quantitative PCR, qPCR), hot-start PCR, competitive PCR, and so on, and these may all be employed where appropriate to the needs of the skilled person.
In one basic embodiment using a PCR based amplification, the oligonucleotide primers are contacted with a reaction mixture containing the target sequence and free nucleotides in a suitable buffer. Thermal cycling of the resulting mixture in the presence of a DNA polymerase results in amplification of the sequence between the primers.
Optimal performance of the PCR process is influenced by choice of temperature, time at temperature, and length of time between temperatures for each step in the cycle. A typical cycling profile for PCR amplification is (a) about 5 minutes of DNA melting (denaturation) at about 95°C; (b) about 30 seconds of DNA melting (denaturation) at about 95°C; (c) about 30 seconds of primer annealing at about 50-65°C; (d) about 30 seconds of primer extension at about 68°C-72°C, preferably 72°C; and steps (b)-(d) are repeated as many times as necessary to obtain the desired level of amplification. A final primer extension step may also be performed. The final primer extension step may be performed at about 68°C-72°C, preferably about 72°C. In certain embodiments the annealing step is performed at about 50-60°C, e.g. about 50-58°C, 52-58°C, 54-58°C, 53-57°C, or 53-55°C. In other embodiments the annealing step is performed at about 55°C (e.g. 55°C±4°C, 55°C±3°C, 55°C±2°C 550C±1 °C or 55°C±0.5°C). In other embodiments the annealing step is performed at about 40-60°C, e.g. about 45-55°C, 46-54°C, 47-53°C, 48-52°C, or 49-51 °C. In other embodiments the annealing step is performed at about 50°C (e.g. 50°C ± 4°C, 50°C ± 3°C, 50°C ± 2°C 50°C ± 1 °C or 50°C ± 0.5°C). The annealing step of other amplification reactions may also be performed at any of these temperatures.
The detection method of the present invention may be performed with any of the standard master mixes and enzymes available. For example, commercially available PCR mix may be used, such as the QUANTITEC® PCR Master Mix (QIAGEN®) or the MAXIMA® qPCR master mix (Thermo-Scientific®). Furthermore, any conventional PCR (qPCR) instrument/system may be used, such as for example the LightCycler® systems (Roche), SLAN® Real-Time PCR Detection Systems (Daan Diagnostics® Ltd.), Bio-Rad® real-time PCR systems, and the like.
Modifications of the basic PCR method such as qPCR (Real-Time PCR) have been developed that can provide quantitative information on the template being amplified. Numerous approaches have been taken although the two most common techniques use double-stranded DNA binding fluorescent dyes or selective fluorescent reporter probes.
Double-stranded DNA-binding fluorescent dyes, for instance SYBR Green, associate with the amplification product as it is produced and when associated the dye fluoresces. Accordingly, by measuring fluorescence after every PCR cycle, the relative amount of amplification product can be monitored in real time. Through the use of internal standards and controls, this information can be translated into quantitative data on the amount of template at the start of the reaction.
The fluorescent reporter probes used in qPCR are sequence-specific oligonucleotides, typically RNA or DNA, that have a fluorescent reporter molecule at one end and a quencher molecule at the other (e.g., the reporter molecule is at the 5' end and a quencher molecule at the 3' end or vice versa). The probe is designed so that the reporter is quenched by the quencher. The probe is also designed to hybridize selectively to particular regions of complementary sequence which might be in the template. If these regions are between the annealed PCR primers the polymerase, if it has exonuclease activity, will degrade (depolymerise) the bound probe as it extends the nascent nucleic acid chain it is polymerizing. This will relieve the quenching and fluorescence will rise. Accordingly, by measuring fluorescence after every PCR cycle, the relative amount of amplification product can be monitored in real time. Through the use of internal standard and controls, this information can be translated into quantitative data.
The amplification product may be detected, and amounts of amplification product can be determined by any convenient means. A vast number of techniques are routinely employed as standard laboratory techniques and the literature has descriptions of more specialized approaches. At its most simple the amplification product may be detected by visual inspection of the reaction mixture at the end of the reaction or at a desired time point. Typically the amplification product will be resolved with the aid of a label that may be preferentially bound to the amplification product. Typically a dye substance, e.g. a colorimetric, chromomeric fluorescent or luminescent dye (for instance ethidium bromide or SYBR green) is used. In other embodiments a labelled oligonucleotide probe that preferentially binds the amplification product is used.
In an embodiment, the amplification reaction is a multiplex amplification reaction (e.g., multiplexed PCR). "Multiplexed PCR" means a PCR wherein multiple target sequences (or a single target sequence and one or more reference sequences) are simultaneously carried out in the same reaction mixture. Usually, distinct sets of primers are employed for each sequence being amplified. Typically, the number of target sequences in a multiplex PCR is in the range of from 2 to 10, or from 2 to 8, or more typically, from 3 to 6. Quantitative detection and normalization of the signal for a pathogen of interest using the bacterial 16S rRNA signal
For more reliable quantification of a pathogen of interest in a sample and/or comparison of results obtained in different samples for a pathogen of interest, it is desirable to normalize the signal obtained with the pathogen of interest with the signal obtained for the total amount of bacteria present in the sample, which may be determined/estimated using the method of amplifying/detecting a bacterial 16S rRNA nucleic acid defined above.
Accordingly, in another aspect, the present invention provides a method for normalizing and/or quantitatively detecting the amount of a pathogen of interest in a sample, said method comprising:
performing, on said sample, an amplification reaction on a nucleic acid specific for said pathogen of interest to obtain a pathogen of interest signal;
performing, on said sample, an amplification reaction on a bacterial 16S rRNA nucleic acid according to the method of amplifying a bacterial 16S rRNA nucleic acid defined above to obtain a bacterial 16S rRNA signal;
normalizing the first signal using the bacterial 16S rRNA signal, thereby obtaining a normalized pathogen of interest signal.
In an embodiment, the pathogen of interest is a bacterial pathogen.
In an embodiment, the pathogen of interest is an opportunistic pathogen, for example Clostrodium difficile, Helicobacter pylori, Haemophilus influenza or Streptococcus pneumonia.
In an embodiment, the opportunistic pathogen is Clostrodium difficile and the nucleic acid specific for the pathogen of interest is a C. difficile toxin nucleic acid, e.g., a C. difficile toxB nucleic acid. Based on the normalized pathogen of interest signals obtained in asymptomatic subjects and patients exhibiting mild, medium or severe symptoms of an infection with the pathogen of interest, it is possible to define normalized pathogen of interest signal threshold(s) (which could be converted to a scale, for example) that may be used to determine the likelihood that a subject is infected by the pathogen of interest (or the risk of developing an acute infection and exhibiting symptoms), and/or the acuteness/severity of the infection. Low normalized pathogen of interest signals (similar or below the threshold) being indicative that the subject has a low likelihood of being infected by the pathogen of interest or of developing an acute infection and exhibiting symptoms, and vice-versa.
Quantitative detection and prediction of the acuteness/severitv of Clostridium difficile infection (CDI)
In another aspect, the present invention provides a method for quantitatively detecting Clostridium difficile infection (CDI) in a sample (e.g., a biological sample from a subject), said method comprising
performing, on said sample, an amplification reaction on
(i) a nucleic acid encoding a C. difficile toxin, thereby obtaining a C. difficile toxin signal; and
(ii) a nucleic acid encoding a bacterial 16S rRNA, a nucleic acid encoding a human RNaseP and/or a nucleic acid encoding a fungal 18S rRNA, thereby obtaining a bacterial 16S rRNA signal, a human RNaseP signal and/or a fungal 18S rRNA signal;
(iii) normalizing the C. difficile toxin signal using the bacterial 16S rRNA, human RNaseP, and/or fungal 18S rRNA signal, there by obtaining a normalized C. difficile toxin signal; and
(iv) quantitatively detecting CDI in the sample on the basis of said normalized C. difficile toxin signal.
In another aspect, the present invention provides a method for predicting the severity/acuteness of Clostridium difficile infection (CDI) in a subject, said method comprising performing, on a sample from the subject, an amplification reaction on
(i) a nucleic acid encoding a C. difficile toxin, thereby obtaining a C. difficile toxin signal; and
(ii) a nucleic acid encoding a bacterial 16S rRNA, a nucleic acid encoding a human RNaseP and/or a nucleic acid encoding a fungal 18S rRNA, thereby obtaining a bacterial 16S rRNA signal, a human RNaseP signal and/or a fungal 18S rRNA signal; (iii) normalizing the C. difficile toxin signal using the bacterial 16S rRNA, human RNaseP, and/or fungal 18S rRNA signal, thereby obtaining a normalized C. difficile toxin signal; and
(iv) predicting the severity/acuteness of CDI in the subject on the basis of said normalized C. difficile toxin signal.
In an embodiment, the above-mentioned method further comprises performing an amplification reaction on a nucleic acid encoding a C. d/7/ic//e-specific 16S nucleic acid, thereby obtaining a C. difficile-spec\ \c 16S signal, establishing a ratio of the C. difficile toxin signal to the C. d/7/ic//e-specific 16S signal or vice-versa, and predicting the severity/acuteness of CDI in the subject on the basis of said normalized C. difficile toxin signal and said ratio.
Any suitable primer may be used to perform the amplification reaction on the nucleic acid encoding the C. difficile toxin, bacterial 16S rRNA, fungal 18S rRNA, human RNaseP, and C. difficile-specific 16S rRNA. In an embodiment, one or more of the primers and/or probes defined herein are used.
In an embodiment, for the amplification of the RNAseP nucleic acid, one or more oligonucleotides comprising one or more of the following sequences are used: GATTTGGACCTGCGAGCG (SEQ ID NO: 35); GAGCGGCTGTCTCCACAAGT (SEQ ID NO: 36) and/or TTCTGACCTGAAGGCTCTGCGCG (SEQ ID NO: 37). In a further embodiment, the following primer pair is used to perform the amplification reaction on the RNAseP nucleic acid: a first primer comprising, or consisting of, the sequence GATTTGGACCTGCGAGCG (SEQ ID NO: 35) and a second primer comprising, or consisting of, the sequence GAGCGGCTGTCTCCACAAGT (SEQ ID NO: 36).
In another aspect, the present invention provides a method for quantitatively detecting Clostridium difficile infection (CDI) in a sample (e.g., a biological sample from a subject), said method comprising:
performing, on said sample, an amplification reaction on a nucleic acid encoding a C. difficile toxin, thereby obtaining a C. difficile toxin amplified product; performing on said sample an amplification reaction on a nucleic acid encoding a bacterial 16S rRNA, a nucleic acid encoding a fungal 18S rRNAand/or a nucleic acid encoding a human RNaseP, thereby obtaining a bacterial 16S rRNA amplified product, a fungal 18S rRNA amplified product and/or a human RNaseP amplified product;
contacting the C. difficile toxin amplified product with a probe hybridizing to said nucleic acid encoding a C. difficile toxin, thereby obtaining a C. difficile toxin signal; contacting the bacterial 16S rRNA amplified product, fungal 18S rRNA amplified product and/or human RNaseP amplified product with a probe hybridizing to said nucleic acid encoding a bacterial 16S rRNA, a probe hybridizing to said nucleic acid encoding fungal 18S rDNA and/or a probe hybridizing to said nucleic acid encoding a human RNaseP, thereby obtaining a bacterial 16S rRNA signal, a fungal 18S rRNA signal and/or a human RNaseP signal;
normalizing the C. difficile toxin signal using the bacterial 16S rRNA, fungal 18S rRNA signal and/or a human RNaseP signal, thereby obtaining a normalized C. difficile toxin signal; and
quantitatively detecting CDI in the subject on the basis of said normalized C. difficile toxin signal.
In another aspect, the present invention provides a method for predicting the severity of Clostridium difficile infection (CDI) in a subject, said method comprising:
performing, on a sample from the subject, an amplification reaction on a nucleic acid encoding a C. difficile toxin, thereby obtaining a C. difficile toxin amplified product;
performing on said sample an amplification reaction on a nucleic acid encoding a bacterial 16S rRNA, a nucleic acid encoding a fungal 18S rRNA and/or a nucleic acid encoding a human RNaseP, thereby obtaining a bacterial 16S rRNA amplified product, a fungal 18S rRNA amplified product and/or a human RNaseP amplified product;
contacting the C. difficile toxin amplified product with a probe hybridizing to said nucleic acid encoding a C. difficile toxin, thereby obtaining a C. difficile toxin signal; contacting the bacterial 16S rRNA amplified product, fungal 18S rRNA amplified product and/or human RNaseP amplified product with a probe hybridizing to said nucleic acid encoding a bacterial 16S rRNA, a probe hybridizing to said nucleic acid encoding fungal 18S rDNA and/or a probe hybridizing to said nucleic acid encoding a human RNaseP, thereby obtaining a bacterial 16S rRNA signal, a fungal 18S rRNA signal and/or a human RNaseP signal;
normalizing the C. difficile toxin signal using the bacterial 16S rRNA, fungal 18S rRNA signal and/or a human RNaseP signal, thereby obtaining a normalized C. difficile toxin signal;
predicting the severity of CDI in the subject on the basis of said normalized C. difficile toxin signal.
In an embodiment, the above-mentioned method further comprises performing an amplification reaction on a nucleic acid encoding a C. difficile-specific 16S nucleic acid, thereby obtaining a C. difficile-specific 16S amplified product, contacting the C. difficile-specific 16S amplified product with a probe hybridizing to said nucleic acid encoding a C. difficile-specific 16S, thereby obtaining a C. difficile-specific 16S signal, establishing a ratio of the C. difficile toxin signal to the C. difficile-specific 16S signal or vice-versa, and predicting the severity of CDI in the subject on the basis of said normalized C. difficile toxin signal and said ratio.
Any suitable probe may be used to perform the hybridization reaction on the amplified products. In an embodiment, one or more of the primers and/or probes defined above are used.
In an embodiment, the method comprises performing an amplification reaction on a nucleic acid encoding a bacterial 16S rRNA according to the method defined herein.
In an embodiment, the normalized value is compared to a corresponding "control" or "reference" normalized value. "Control value" or "reference value" or "standard value" are used interchangeably herein and broadly refers to a separate value measured in a comparable control sample, which is generally from a subject not suffering from CDI or having a known CDI severity (or a sample known to not contain C. difficile). The corresponding control value may be a value corresponding to an average or median value calculated based of the values obtained in samples from several reference or control subjects (e.g., a pre-determined or established standard value). The control value may be a pre-determined "cut-off" value recognized in the art or established based on values measured in one or a group of control subjects. The corresponding reference/control/value may be adjusted or normalized for age, gender, race, type of sample, or other parameters. The "control value" can thus be a single number/value, equally applicable to every patient individually, or the control value can vary, according to specific subpopulations of patients, types of samples. Thus, for example, older men might have a different control value than younger men, and women might have a different control value than men. The predetermined standard value can be arranged, for example, where a tested population is divided equally (or unequally) into groups, such as a low-severity group, a medium-severity group and a high-severity group or into quadrants or quintiles, the lowest quadrant or quintile being individuals with the lowest severity (i.e., lowest normalized C. difficile toxin value) and the highest quadrant or quintile being individuals with the highest severity (i.e., highest normalized C. difficile toxin value).
It will also be understood that the control value according to the invention may be, in addition to predetermined values, values measured in other samples (e.g. from healthy/normal subjects or subjects having a known CDI severity) tested in parallel with the experimental sample.
Any suitable statistical transformation or algorithm may be used to normalize the C. difficile toxin signal, quantitatively detecting CDI in the sample and/or predicting the severity of CDI in the subject based on said normalized C. difficile toxin signal. In an embodiment, the normalization comprises dividing the C. difficile toxin signal value (Cpt) by the bacterial 16S rRNA signal value (Cpu), fungal 18S RNA signal value (Cpf) and/or human RNaseP signal value (Cpg) or vice-versa. In an embodiment, the normalization comprises subtracting the C. difficile toxin signal value (Cpt) from the bacterial 16S rRNA signal value (Cpu), fungal 18S rRNA signal value (Cpf) and/or human RNaseP signal value (Cpg), or vice-versa. In an embodiment, the above-mentioned method comprises determining an acuteness of infection C. difficile (AOI-CD) score using the following formula:
AOI-CD score = (1/Cpt)/(Cpu-Cpt) or (1/Cpt)/(Cpg-Cpt)
The AOI-CD score may be compared to a reference or control AOI-CD score to quantify CDI and/or determine/predict the acuteness or severity of CDI in the sample/subject tested.
In another embodiment, the present invention provides a method for the follow-up of a C. difficile-infected patient's condition (over time), the method comprising obtaining a first normalized C. difficile toxin signal from a sample using the above-defined method at a first time point; obtaining a second normalized C. difficile toxin signal from a sample using the above- defined method at a second, later time point; and comparing the first and second normalized C. difficile toxin signals, wherein a second normalized C. difficile toxin signal that is higher than the first normalized C. difficile toxin signal is indicative that said patient's condition is deteriorating, and wherein a second normalized C. difficile toxin signal that is lower than the first normalized C. difficile toxin signal is indicative that said patient's condition is improving. In an embodiment, the C. difficile-infected patient is undergoing treatment for CDI between said first and second time points, and said method permits to determine whether the patient is responding to the treatment.
The sample which is tested according to the methods described herein is any sample suspected of containing bacteria such as C. difficile, for example a body fluid, swab or other cellular or non-cellular sample from a subject, e.g. a human. Such samples include, but are not limited to, bodily fluids which contain cellular materials and may or may not contain cells, e.g., blood, plasma, serum, urine, conjunctival secretions, seminal fluid, saliva, ocular lens fluid, lymphatic fluid, amniotic fluid, feces/stool and the like; endocervical, urethral, rectal, vaginal, vulva-vaginal, nasopharyngeal and pulmonary samples; and archival samples with known diagnosis. Test samples may also be sections of tissues such as frozen sections.
The sample may be any sample taken from the gastrointestinal Gl tract. A Gl tract sample of use in the invention may include, but is not limited to, any fluid or solid taken from the lumen or surface of the Gl tract or any sample of any of the tissues that form the organs of the Gl tract. Thus the sample may be any luminal content of the Gl tract (e.g. stomach contents, intestinal contents, mucus and feces/stool, or combinations thereof) as well as samples obtained mechanically from the Gl tract e.g., by swab, rinse, aspirate or scrape of a Gl tract cavity or surface or by biopsy of a Gl tract tissue/organ.
The sample can also be obtained from part of a Gl tract tissue/organ which has been removed surgically. The sample may be a portion of the excised tissue/organ. In embodiments where the sample is a sample of a Gl tract tissue/organ the sample may comprise a part of the mucosa, the submucosa, the muscularis, the adventitia and/or the serosa of the Gl tract tissue/organ. Such tissue samples may be obtained by biopsy during an endoscopic procedure.
Samples for use in the invention may also include environmental samples, preferably samples from a hospital or other clinical setting. Examples of such environmental samples include samples obtained from surfaces (e.g., floors), samples obtained from clothing, samples obtained from toilets, commodes, bedpans and the like, samples obtained from clinical devices (e.g. endoscopes), samples of the water supply, or air treatment apparatus of the hospital or other clinical setting, and samples obtained from the hands of healthcare workers.
The term "sample" also encompasses any material derived by processing a biological sample. Derived materials include, but are not limited to, cells (or their progeny) isolated from the sample (e.g., clinical isolates of bacteria such as Clostridium difficile), cell components, proteins/peptides and nucleic acid molecules (DNA or RNA) extracted from the sample. Processing of biological samples to obtain a test sample may involve one or more of: filtration, distillation, centrifugation, extraction, concentration, dilution, purification, inactivation of interfering components, addition of reagents, and the like. In an embodiment, the sample has not been subjected to a nucleic acid enrichment/isolation/purification step, e.g., the sample does not comprise isolated/purified nucleic acids (isolated/purified DNA).
The subject may be any human or non-human animal subject, but more particularly may be a vertebrate, e.g., an animal selected from mammals, birds, amphibians, fish and reptiles. The animal may be a livestock or a domestic animal or an animal of commercial value, including laboratory animals or an animal in a zoo or game park. Preferably the subject is a human. The subject may be of any age, e.g. an infant, a child, a juvenile, an adolescent or an adult. Methods for treating a bacterial infection such as CDI in a subject
In another aspect, the present invention provides a method comprising:
quantitatively detecting a bacterial infection in a subject using the methods defined above to identify a subject in need of treatment against a bacterial infection;
treating said subject against said bacterial infection, for example by administering an effective amount of one or more suitable antibiotics to said subject.
In an embodiment, the bacterial infection is an opportunistic infection, such as
Clostrodium difficile, Helicobacter pylori, Haemophilus influenza or Streptococcus pneumonia.
In another aspect, the present invention provides a method comprising:
quantitatively detecting and/or predicting the severity of Clostridium difficile infection (CDI) in a subject using the methods defined above to identify a subject in need of treatment against CDI; treating said subject against CDI, for example by administering an effective amount of one or more suitable antibiotics to said subject.
MODE(S) FOR CARRYING OUT THE INVENTION
The present invention is illustrated in further details by the following non-limiting examples.
Example 1 : Materials and Methods for Examples 2 to 4
PCR assay for Clostridium difficile in stool samples: This assay was performed using the primers and probe combinations listed below, together with an inhibition Control (IC), for detection of PCR inhibitors in the stool. In this assay, the 6FAM/fluorescing channel was used for monitoring the real-time signal from the C. difficile toxin B gene and the Cy5 (555 red) channel was used for detection of the Inhibition Control signal. The assay was compatible with all qPCR instruments. Lysis of bacterial cells prior to amplification was performed with glass beads, as described below. The analytical limits-of-detection are in the range of 1800 copies per swab sample. For assay quality control, genomic DNA of C. difficile strain ATCC 43255 was used as positive C. difficile control (PC). Water (from QUANTITEC® kit) was used as the negative control (NC). PC and NC are used with each batch. The inhibition control (IC) was a sample-spiked oligonucleotide which was able to generate PCR product with corresponding primers and probe. If a sample did contain an unknown PCR-inhibition component/substance which could influence the end-result (Ct value), it would be detected by comparing the inhibition control (IC) in the context of a water solution, with the IC in the context of the sample solution. The inhibition test is done in a separate reaction mix and does not compete with any other reaction. A negative IC result for a particular sample, or a sample Cp value which is increased by more than 4Cp unit values relative to the negative control (NC), was indicative of inhibition. In such a case, the whole assay was repeated once more with a sample of the original dilution which has been subjected to a 10x dilution and/or one freeze/thaw cycle.
Specimen/sample preparation for the complete assay was compatible with/similar to all current commercial sample preparation protocols. In an embodiment, there is one (1 ) sample buffer tube (1 ml_ of TE buffer, SBT) and one (1 ) lysis tube (LT), (50 uL volume of glass beads + 100uL of TE buffer) which were used for each specimen to be tested. Specimens were vortexed at high speeds for 15 seconds and a sterile dry swab was dipped into the fecal material for testing. Excess stool was removed. The swab was placed in a sample buffer tube (SBT). The 25 μΙ_ of stool suspension was transferred to one labeled LT (containing 100 μί of 1XTE buffer and 50uL equivalent volume of the glass beads). Each lysis tube was vortexed for five 5 minutes at high speed. Tubes were lysed by heat treatment at 95 ± 2°C for five minutes, and cooled by transferring onto ice or a cooling block. Preparation of Master Mixes, prepared in a PCR hood/workstation (same for C.difficile reaction mix and IC reaction mix):
C. difficile Master Mix (units: μΙ)
Figure imgf000038_0001
Amplifications and detections were performed using a Light Cycler® 480 instrument, using Detection Format: 3-colour hydrolysis probe. Absolute quantification was performed with Filter combination FAM 465-510. The High sensitivity software package was used. Crossing point (Cp) values for each sample were recorded into an Excel® file. The same procedure was done for IC.
Results with a generated Cp value should be considered as positive if the RFU end- point values have RFU (Relative Fluorescence Unit) values higher than 1. In special and rare cases where a linear vs. sigmoidal shape is dominant, the sample was processed on an alternative instrument. Results with no Cp value generated and with an RFU noise line below 1 and linear RFU vs. cycle slope should be estimated for the presence of PCR inhibition. If PCR is not inhibited, the sample should be considered as negative. The Inhibition control (IC) is read at a separate reaction well. All Cp sample values are compared with an IC Cp value of the negative control (which has water instead of sample). If there is no Cp value, or the operator observes an increase in Cp by more than 4 Cp values from the negative control or if RFU units are less than one, inhibition of the PCR reaction was suspected. In such cases, the sample processing was repeated by subjecting the original sample to freezing/thawing and a 10x dilution. If the PCR run is repeated and the results appear the same, it is indicative of PCR inhibition.
An intra-laboratory positive quality control of C. difficile is used in each run (the internal PCR positive control which was used is the genomic DNA of C. difficile strain ATCC 43255). The synthetic (IDT) RNaseP target (in the concentration of 100 molecules per reaction mix) was used as the inhibition control of PCR reaction.
Primers and probes used for amplification
16S Universal rDNA
16S-Probe1 : CGGCTAACTMCGTGCCAG (SEQ ID NO: 2);
16S-Probe2: AAGSVMCGGCTAACTMCGTGCC (SEQ ID NO: 3);
16SU-F: ACTCCTACGGGAGGCAGCAGT (SEQ ID NO: 22); 16SU-R1 : TATTACCGCGGCTGCTGGC (SEQ ID NO: 38);
16SU-R2: GCITCCTATTACCGCGGCTGCT (SEQ ID NO: 1 1 );
16SU-R3: AATAAATCATAAGCITCCTATTACCGCGGCTGCT (SEQ ID NO: 12);
16SU-R4:5'-AATAAATCATAACCTACGTATTACCGCGGCTGC-3' (SEQ ID NO: 39);
16SU-R5:5'-CCTAGCTATTACCGCGGCTGCT-3' (SEQ ID NO: 40)
16S C. difficile-specific rDNA
16SCD-Probe: AATGTTGGCATGAGTAGCGAGATGT (SEQ ID NO: 4);
16SCD-F: GGGAGCTTCCCATACGGGTTG (SEQ ID NO: 29);
16SCD-R: TTGACTGCCTCAATGCTTGGGC (SEQ ID NO: 30).
ToxB:
tcdB.RT2-Probe: TCTGAAGGATTACCTRTAATTGCAA (SEQ ID NO: 5);
tcdB.RT2-F: TGCAGCCAAAGTTGTTGAAT (SEQ ID NO: 6);
tcdB.RT.2-R: GCTCTTTGATTGCTGCACCT (SEQ ID NO: 7).
Human RNaseP:
RNaseP-Probe: TTCTGACCTGAAGGCTCTGCGCG (SEQ ID NO: 37);
RNaseP-F: GATTTGGACCTGCGAGCG (SEQ ID NO: 35);
RNaseP-R: GAGCGGCTGTCTCCACAAGT (SEQ ID NO: 36).
The concentration of primers used were in the ranges of 0.5-2 μΜ (preferably 0.6 μΜ), and the concentration of hydrolyzing probes were in the ranges of 0.25-1.2 μΜ (preferably 0.3 μΜ). To perform the qPCR experiments, the QUANTITEC® PCR Master Mix (QIAGEN®) or MAXIMA® qPCR master mix (Thermo-Scientific®) were used. PCR conditions were as follows: 95°C for 15 min, following by 45 cycles of annealing at 50°C, 60 sec and denaturation at 95°C, 10 sec. Example 2: Linearity of the 16S rDNA signal
The linearity of the 16S rDNA signal was assessed using 3 stool samples and corresponding 10x serial dilutions of "control" stool samples. Both Cp values and number of colonies from these 10 stool samples (and corresponding serial dilutions) showed good linearity, as shown in Table I.
Table I
10X serial dilution of stool swab vs. CFU of anaerobic culture Parameter 1.00E-01 1.00E-02 1.00E-03 1.00E-04 1.00E-05 1.00E-06
Av. Cp 12.4 15.9 18.1 21.5 25 28.7
Av. CFU n.d. n.d. n.d. n.d. n.d. -10,000
The numerical Cp values reflect total rDNA load in the sample. The sample processing and PCR was performed under the same conditions as described above for C. difficile. Numerical values of Cp spanned the range of 13 Cp units (indicating that bacterial fecal load will vary from sample to sample by minimum 2 power 13 unit values). Cp values of 16S universal rDNA, over 300 positive samples, have the following distribution (mean Cp=16.9, Standard Deviation of Cp values=2.34; Min=1 1.72; Max=25.3). Negative samples have similar distribution of bacterial load (on average), characterised by a mean of 17.4, Min=1 1 , Max=25 and Standard Deviation=3.8 Cp units. This indicates that total bacterial load is an inherently variable parameter of samples, which is not having a significant predictive power per se.
Example 3: Predictive value of normalized toxB signal in CDI
All experiments (including all orders/numerically coded samples on the left side) were performed under identical qPCR conditions and using the same simple sample processing protocol. In other words, the numerical value of toxB presence was measured (Cp value reflects the number of toxicogenic C. difficile bacteria in sample material); the inhibition controls were recorded from same sample material. The rest of the sample was used for measuring 16s rDNA specific for C. difficile and 16s rDNA reflecting all bacterial (Universal 16S rDNA). All measurements were done using the processing/measurement conditions described above.
All data was grouped into order number(s) and that for each order number (equivalent to particular clinical samples), toxB (Cpt) values, 16S rDNA of C. difficile (Cpc) values, and universal 16S rDNA (Cpu) values were obtained. Using these basic qPCR parameters for 2 (or more) different gene families, new values including (Cpu-Cpt) or (1/Cpt)/(Cpu-Cpt) were calculated, to determine whether these new values have more significant or stronger clinical meaning or predictive power relative to each value taken independently.
For example, the "protective effect" was calculated using a ratio between Cp values of toxB and Cp value of 16S rDNA of C. difficile. The protective effect was measured using the formula 100*(Cpt Cpc)-1 ), although other mathematical transformations could have been used. The rationale for calculation is that if 2 signals (Cp of toxB (Cpt) and Cp of 16 rDNA of C. difficile (Cpc)) are similar, the value (ratio) will be close to 1. This means that the dominant strain is toxicogenic and that there is no protective effect of other, non-toxicogenic strains. However, if 16S rDNA is a few logarithmic units in excess over the toxicogenic strain (toxB), the ratio must be a number higher than 1 , indicating that a high number of non-toxicogenic strain(s) of C. difficile is present in the sample. Subtracting unit 1 (when there is no difference among Cp values) and multiplying by 100 permits to obtain a spectrum of values which are higher only if non toxicogenic strains are in larger excess (protective effect) over the toxicogenic strains. The same ranking can be done by subtracting Cpt (toxB) from Cpc (16S rDNA C. difficile). There are a number of similar mathematical transformations which would keep the rank of samples based on potential protective scoring (presence of non-toxicogenic C. difficile).
The parameter (1/Cpt)/(Cpu-Cpt) provides information about the level of toxB DNA signal (1/Cpt, a lower ratio 1/Cpt is indicative of low toxB DNA levels in the sample), but also provides information on the relative contribution/frequency of toxB DNA relative to the total bacterial microbial presence in the sample (Cpu-Cpt). If this relative frequency of toxicogenic C. difficile is high, the Cpu-Cpt number is close to zero and the parameter 1/Cpt/(Cpu-Cpt) is higher, indicative of more severe cases. If the relative frequency of toxicogenic C. difficile is low, relative to the total bacterial microbial presence (microbiota), then (Cpu-Cpt) is higher, and the parameter 1/Cpt/(Cpu-Cpt) is lower, indicative of asymptomatic, or less severe cases. Overall, the final scoring of C. difficile infected patients will not only depend on the numerical value of Cpt related to number of toxB DNA molecules, but will be "normalized" based on the relative frequency of this DNA molecule among all bacteria present in the sample. There will be cases which have low toxB number of DNA molecules (low absolute numbers of toxicogenic C. difficile), but also low Cpu (low total microbiota sample load). This is indicative that these samples are depleted from the rest of the bacterial flora (i.e. almost a monoculture of C. difficile in the sample), and provides evidence of a possible severe CDI (e.g., needing a rapid/aggressive therapeutic intervention). In contrast, a medium-to-low Cpt, but very high Cpu, suggests mild clinical symptoms in the context of still preserved non C. difficile related microbiota.
A completely analogous protocol can be used for the normalisation of qPCR signal using other normalisation "universal" genes, such as the RNaseP human gene (to normalise the signal based on the number of human cells in the sample), and assigning a different weighting parameter for severity of infection (for the contribution of each assay). However, the dominant deterministic factor for normalising qPCR signal and estimating current status of bacterial infection in highly complex bacterial flora (like stool) is obtained, using 16S rDNA assay. Table II: (CDI positive toxB=27-40) analytical prediction: "carriers/asymptomatic"
Figure imgf000041_0001
H8012495 36.8 28.54 14.72 28.94183602 0.02717 22.08 1.2307
H8021934 37.07 28.06 15.47 32.10976479 0.02698 21 .6 1.24889 i0180652 34.83 30.34 12.93 14.79894529 0.02871 21 .9 1 .31 1
I6273268 27.5 25.2 9.126984127 0.03636 27.5 1.32231 i1240925 35.57 28.33 14.73 25.55594776 0.0281 1 20.84 1.34902 i1 181179 40 34.07 21 .48 17.40534194 0.025 18.52 1.34989 i1092140 36 — 15.54 — 0.02778 20.46 1.35766
10041487 35.34 — 14.56 — 0.0283 20.78 1.36172
19130592 36.19 — 16 — 0.02763 20.19 1.3686 i0210713 34.2 26.88 13.08 27.23214286 0.02924 21 .12 1.38446
H8191050 35.61 26.92 15.35 32.2808321 0.02808 20.26 1.38608
Table III: (CDI positive toxB=27-32) analytical prediction "no symptoms/ mild symptoms"
Figure imgf000042_0002
Table IV: 4 (CDI positive CP toxB=27-31 ) analytically prediction "medium cases"
Figure imgf000042_0001
i1060519 28.8 25.21 17.86 14.2403808 0.0347 10.94 3.1739
H8252671 27.36 24.56 15.88 11 .4006515 0.0365 1 1 .48 3.1838
I6250682 26.8 23.8 15.09 12.605042 0.0373 1 1 .71 3.1865
14060641 30.17 27.75 19.89 8.72072072 0.0331 10.28 3.2243
I8280845 32 31 .24 22.31 2.43277849 0.0313 9.69 3.225
12140765 31 .19 24.61 21 .27 26.7370987 0.0321 9.92 3.232
18110789 28.9 29.02 18.22 -0.41350793 0.0346 10.68 3.2399
Table V: (CDO positive CptoxB=27-30, analytical prediction "severe cases")
Figure imgf000043_0001
Example 4: Assessment of melting profiles of universal 16S and 18s to measure loss of biota diversity
Melting curve analysis is an assessment of the dissociation-characteristics of double- stranded DNA during heating. Juxtaposition of probes (one featuring a fluorophore and the other, a suitable quencher) can be used to determine the complementarity of the probe to the target sequence. Typically the user is using polymerase chain reaction (PCR) prior to melting curve analysis to amplify the DNA region in which species-specific sequence variability of interest lies. In the sample tube there are now many copies of the DNA region of interest. Region that is amplified is known as the amplicon. The process is a precise warming of the amplicon DNA from around 50°C up to around 95°C. At some point during this process, the melting temperature of the amplicon is reached and the two strands of DNA separate or "melt" apart, while hybridization Tm-probes (which are in molar excess and which are visible via fluorescence) will hybridize with a single stranded DNA amplicon and "melt" proportionally to the thermal stability of probe-target region. A perfectly matched probe(s) will melt at a higher temperature then the mismatched probe(s) bound to a target sequence. The purpose of Melting Curve analysis in the present case (C. difficile PCR assay) is to determine the melting temperature spectrum changes characteristic to loss of bacterial diversity in fecal samples. All the assays described herein are adaptable to Tm (melting profile analysis) in the regions of the described 16S and 18S rDNA genes, covering 50bp plus or minus for the presently listed position of hybridization probe. The species-dependent changes in bacterial/fungal diversity are recorded in routine melting profile analysis qPCR program. The result is compared with clinical cases of severe CDI.
Example 5: Estimating of total bacterial load in stool samples using a short 16S rDNA
PCR assay
Materials and Methods
Stool samples. The sample study group was represented by 500 liquid and 50 formed stools, submitted for screening of C. difficile and stool culture. For measurements of wet stool mass entering the PCR reaction, 49 stool samples were randomly chosen and processed as described below.
Stool swabbing and wet mass measurements. Swabs (n=49) were uniquely labelled and measured before use, with an analytical precision in low milligram range (< 1 mg). After stool swabbing (performed according to the method disclosed in GeneOhm™ Cdiff Assay instructions Manual37), swabs were closed and re-measured. The difference in mass, before and after swabbing, was recorded for each sample.
Sample processing and Quantitative anaerobic culture plating. Swabs were transferred into 1 ml_ TE buffer (tube 1 ), and mixed by vortexing. For quantitative anaerobic culture, samples are serially diluted (1-fold, 100-fold and 10,000-fold) from sample tube, until the number of anaerobic bacteria was countable. 50 μΙ was plated on Columbia Blood agar and after 2 days incubation under anaerobic conditions at 37°C, submitted to colony counts. The original number of bacteria in sample was calculated considering the dilution factor and compared with the 16S rDNA qPCR, which has a higher Linear Operative Range of quantification relative to culture. For crude lysate directly entering qPCR reaction, the method described in BD GeneOhm™ Cdiff Assay instructions Manual37 was used.
For measuring comparative qPCR efficacy, operative range and average limits of detection (LoD) among the assay described herein and referenced 16S rDNA qPCR assay (according to Natkarni et al.19), nucleic acids were isolated from the stool using the NucliSENS® EasyMag® system (Biomerieux)38, according to the manufacturer's "stool isolation protocol" (100μΙ input, 100μΙ elute output). Serial dilution (10-fold) of the nucleic acid elute was performed to illustrate differential performance of different assays. The referenced assay is not suitable for direct PCR assay due to the length of the amplicon.
Primers and probes for qPCR. The Natkarni 16S universal assay was performed using the originally described primers/probes19, as well as newly described primers derived therefrom24,39, referred to herein as Forward variant 2: CCTAYGGGRBGCASCAG (SEQ ID NO: 41 ); Forward variant 3: CCTACGGGDGGCWGCAGT (SEQ ID NO: 42); Reverse variant 2; GGACTACHVGGGTWTCTAAT (SEQ ID NO: 43); Reverse Variant 3: GGACTACHVGGGTMTCTAAT (SEQ ID NO: 44) and (Nadkarni 516probe) TGCCAGCAGCCGCGGTAATAC (SEQ ID NO: 45).
The sequences of the primer/probe used in the novel 16S rDNA qPCR assay described herein are depicted in Table VI below (in bold).
PCR assay and cycling conditions. 3 μΙ of nucleic acid elute or crude lysis material was added into 17 μ I of total volume mix (QuantiNova® Probe PCR master, QIAGEN®), according to the concentration of primer/probe and cycling conditions recommended by the manufacturer42, except for the annealing temperature that was set at 50°C). The Cp values were calculated using the default parameters of the LightCycler® 480 software provided with the realtime PCR system (LightCycler® 480 Instrument II, Roche).
Table VI: Sequences of primers and probes used in the studies described herein
NAME OF OLIGO/ASSAY SEQUENCE SEQ ID NO: LENGTH (NT)
Nadkarni (2002)-F TCCTACGGGAGGCAGCAGT 46 19
Nadkarni (2002)-R GGACTCCAGGGTATCTAATCCTGTT 47 25
Nadkarni Probe TGCCAGCAGCCGCGGTAATAC 45 21
Nadkarni (2014M1)-F CCTAYGGGRBGCASCAG 41 17
Nadkarni (2014M1 )-R GGACTACHVGGGTWTCTAAT 43 20
Nadkarni (2014M2)-F CCTACGGGDGGCWGCAGT 42 18
Nadkarni (2014M2)-R GGACTACHVGGGTMTCTAAT 44 20
Clifford (2012) R TATTACCGCGGCTGCTGGC 56 19
Clifford (2012) F ACTCCTACGGGAGGCAGCAGT 22 21
F-V1-16S-NOV14 AATAAATCATAAACTCCTAYGGGRGGCWGCAGT 48 33
F-V2-16S-NOV14 AATAAATCATAAACTCCTACGGGRGGCWGCAGT 49 33
F-V3-16S-NOV14 AATAAATCATAAACTCCTAYGGGRGGCWGCA 50 31
F-V4-16S-NOV14 AATAAATCATAAACTCCTACGGGRGGCWGCA 51 31
F-V5-16S-NOV14 AATAAATCATAAACTCCTACGGGRGGCAGCA 52 31
F-V6-16S-NOV14 AATAAATCATAAACTCCTAYGGGRBGCAGCA 53 31
F-V7-16S-NOV14 AATAAATCATAAACTCCTACGGGRBGCWGCA 54 31
JGH R-V8-16S-NOV14 ATAACCTAGCTATTACCGCGGCTGCT 55 26
JGH-3 (2014) R TAGCTATTACCGCGGCTGCT 9 20
JGH-PROBE CGGCTAACTMCGTGCCAG 1 18 JGH (2014) F ACTCCTAYGGGRBGCASCAGT 14 21
AT CLAMP AT 5' END AATAAATCATAA 10 12
Nadkarni (2002) = Nadkarni MA et al. Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set. Microbiology 2002;148:257-66
Nadkarni (2014M1) = Wang Y, Qian PY. Conservative fragments in bacterial 16S r genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies. PLoS one 2009;4:e7401
Nadkarni (2014M2) = Liu CM, Aziz M, Kachur S, et al. BactQuant: an enhanced broad-coverage bacterial quantitative real-time PCR assay. BMC microbiology 2012;12:56
Results
FIG. 2A shows that there is no correlation between the stool mass (mg of stool entering sample buffer) and the bacterial load, measured on different stool samples, using the novel 16S rDNA qPCR assay described herein. FIG. 2B shows that there is a good correlation (R2 = 0.90, P= 6.4e-8) between the Ct values (indicative of the bacterial load) measured in a stool lysate and in isolated DNA samples, using same mass equivalent of input stool material. Comparable results were obtained using the forward primers comprising the sequences of SEQ ID NOs: 22 and 48-55.
FIG. 3 shows that among different samples, there is a correlation between the bacterial counts as measured by the culture plating method and the Ct values obtained using the novel 16S rDNA qPCR assay described herein. Ct values above 18 indicate low bacterial load and may be considered as samples with strongly reduced microflora. Comparable results were obtained using the forward primers comprising the sequences of SEQ ID NOs: 22 and 48-55.
FIG. 4 shows the variability of the total bacterial load among clinical samples of liquid stool. Distribution of Ct values using the novel 16S rDNA qPCR assay described herein amongst 500 stool swabs (from collection of C. difficile positive samples) indicates that the total bacterial load may vary by up to about 1000-fold (Ct value from 212 to 221) for the same stool mass equivalent. Comparable results were obtained using the forward primers comprising the sequences of SEQ ID NOs: 22 and 48-55.
The results depicted in FIG. 5 show that the Linear Operative Range (LOR), Average Limit of Detection (LOD), efficiency and correlation coefficient (R2) obtained with the novel 16S rDNA qPCR assay described herein (left graph) are comparable to those obtained with the primers and probes described in Natkarni et a/.19 (right graph). The results are summarized in Table VII below. Comparable results were obtained using the primers of the sequences of SEQ ID NO: 41-44 (Nadkarni assay) and the forward primers comprising the sequences of SEQ ID NOs: 22 and 48-55. Table VII: Summary of the results depicted in FIG. 5
Average
LOR LOD R2
efficiency (SD) Nadkarniia 10b-108 1000 97% (2%) > 0.0994
Novel 16S rDNA
105-108 1000 94% (4%) > 0.0961 qPCR assay
LOR: Linear Operative Range LOD: Average Limit of Detection
R2: Correlation Coefficient SD: Standard Deviation
Example 6: Comparison of Primer Pairs Using in silica PCR of the SILVA Database The evaluation of sequence coverage, sometimes defined as "depth of sequence coverage" is defined as number of events/times primer sequence match with targeted sequence within defined sequence data base, versus the total number of matching events. In silico PCR was performed using SILVA TestPrime 1.0 (http://www.arb-silva.de/search/testprime/, Klindworth, A et ai, Nucl. Acids Res. (7 January 2013) 41 (1 ): eldoi: 10.1093/nar/gks808) with 2 different criteria: (a) absolute matching (100% sequence identity) among primers and targeted sequences in 16S rRNA sequence data base is restricted to 3 and/or 4 last nucleotides at 3' end and (b) permissive matching among primers and primer-binding region of 16S rRNA gene within data base allows 2, 3, or 4 mismatches, under conditions that criterion (a) is satisfied.
Bacterial 16S rRNA primer sequences from the novel bacterial 16S rRNA assay described herein (in bold in Table VI above), Clifford (2012) (see Table VI above), and Nadkarni (2002) (see Table VI above) were submitted separately to the online TestPrime program. In addition, each primer pair was assessed by varying the allowable number of perfect 3' matches (3 nt, Table VIIIB or 4 nt, Table VINA), as well as the allowable number of mismatches (2, 3 or 4 nt), resulting in a total of 6 TestPrime results for each primer pair. For each result from TestPrime the taxonomy list CSV file was saved locally (total 10,466 rows of coverage summaries for all taxonomic units in SILVA). These result files, 18 in total, were merged using a custom script written in the R statistical programming language. The resulting merged file was filtered for coverage summary statistics of non-bacterial entries (Archaea and Eukarya), as well as for non-genus taxonomic levels. After filtering, 2,475 rows of in silico PCR coverage summary statistics remained, reflecting all genus-level bacterial results from within the SILVA database. For each bacterial genus, the in silico PCR that had at least 70% coverage i.e., zero or more of the 18 total runs submitted to SILVA TestPrime, were identified. There results were tabulated across all 2,475 genera.
Table VINA and VIIIB below illustrate less-stringent (right, 4 nt. mismatch, in bold) versus more stringent (left, 2 nt. Mismatch) sequence matching of primers with target binding sequence motif/s. What may be concluded from this data is that the novel bacterial 16S rRNA primer pair described herein can have a broad ("universal") sequence coverage range under non-stringent conditions (3 or 4 nt. mismatchs, corresponding to lower temperature of annealing, e.g., about 50°C) thus tolerating up to 4 mismatches and perform same, if not better, than primers showing higher identity with sequence binding motifs such as those disclosed in Clifford (2012) and Nadkarni (2002). Thus, these analyses did not reveal any significant difference in sequence coverage between the novel bacterial 16S rRNA primer pair described herein and those disclosed in Clifford (2012) and Nadkarni (2002) under the following conditions: (a) a 3 or 4 nucleotides fixed full matching segment at the 3'region of primers and (b) permissive total number of mismatches in primer binding region is 3-4 nucleotides. It is important to note that the primer pairs disclosed in Clifford (2012) and Nadkarni (2002) are not designed to be used at low annealing temperatures, while the novel bacterial 16S rRNA primer pair described herein is. That feature makes the novel bacterial 16S rRNA assay described herein suitable to preserve proper 16S rDNA-specific priming under conditions in which perfect matching is not present. Thus, the specificity toward the bacterial 16S rDNA gene is preserved, while minimizing miss-priming with non-bacterial DNA or primer-induced self-priming.
Table VINA: Comparison of Bacterial Genera Detected by Three Tested Primer Pairs Using
SUVA TestPrime 1.0 (4 nt perfect 3' matches)
Total number (%) of genera detected
by primer pairs1 (of 2475 total)
Detected with primers:2 4 nt3
Novel
Clifford Nadkarni
16S
(2012) (2002)
rRNA 2 nt4 3 nt4 4 nt4
- - - 207 (8.4) 182 (7.4) 178 (7.2)
- - + 14 (0.6) 1 1 (0.4) 9 (0.4)
- + - 246 (9.9) 63 (2.5) 12 (0.5)
- + + 1 165 (47.1 ) 145 (5.9) 23 (0.9)
+ - - 1 (0) 2 (0.1 ) 3 (0.1)
+ - + 0 (0) 0 (0) 0 (0)
+ + - 122 (4.9) 321 (13) 371 (15)
+ + + 720 (29.1 ) 1751 (70.7) 1879 (75.9)
Table VI I IB: Comparison of Bacterial Genera Detected by Three Tested Primer Pairs Using
SUVA TestPrime 1.0 (3 nt perfect 3' matches)
Total number (%) of
genera detected by
primer pairs1 (of
2475 total)
Detected with primers:2 3 nt3
Novel
Clifford Nadkarni
rRNA <2012> (2002> 2 nt4 3 nt4 4 nt4
202 (8.2) 175 (7.1 ) 170 (6.9)
+ 16 (0.6) 14 (0.6) 11 (0.4)
+ - 152 (6.1 ) 36 (1.5) 8 (0.3)
1260
171 (6.9) 26 (1.1)
(50.9) + 0 (0) 2 (0.1 ) 2 (0.1)
+ + 1 (0) 1 (0) 2 (0.1)
+ + 52 (2.1 ) 143 (5.8) 167 (6.7)
+ + + 792 (32) 1933 (78.1 ) 2089 (84.4)
1 Number of bacterial genera with at least >= 70% of species detected.
2 + or - denotes detection or lack of genus-level detection for that primer pair. For instance, first and last row show bacterial genera missed and identified by all primer pairs, respectively. Remaining rows show combinations thereof. For example, the first row of Tables VINA and VIIIB indicates the number of bacterial genera missed by the primer pair of the novel bacterial 16S rRNA assay described herein and identified by the primer pairs disclosed in Clifford (2012) and Nadkarni (2002).
3 Length of 0-mismatch zone at 3' end.
4 Maximum number of mismatches.
Although the present invention has been described herein above by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims. In the claims, the word "comprising" is used as an open-ended term, substantially equivalent to the phrase "including, but not limited to". The singular forms "a", "an" and "the" include corresponding plural references unless the context clearly dictates otherwise.
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Claims

WHAT IS CLAIMED IS:
1. An isolated nucleic acid molecule of 50 nucleotides or less comprising a sequence of at least 10 contiguous nucleotides of one of the following nucleotide sequences (I) to (VII):
(I) 5'-TATTACCGCGGCTGCT-3' (SEQ ID NO: 1 ), wherein said isolated nucleic acid molecule does not comprise the sequence GGC at its 3' end;
(II) 5'-CGGCTAACTMCGTGCCAG-3' (SEQ ID NO: 2);
(III) 5'-AATGTTGGCATGAGTAGCGAGATGT-3' (SEQ ID NO: 4);
(IV) 5'-TCTGAAGGATTACCTRTAATTGCAA-3' (SEQ ID NO: 5);
(V) 5'-TGCAGCCAAAGTTGTTGAAT-3' (SEQ ID NO: 6);
(VI) 5'-GCTCTTTGATTGCTGCACCT-3' (SEQ ID NO: 7); or
(VII) a sequence having at least 80% identity with any of (I) to (VI).
2. The isolated nucleic acid molecule of claim 1 , which comprises a sequence of at least 15 contiguous nucleotides of one of the nucleotide sequences (I) to (VII).
3. The isolated nucleic acid molecule of claim 1 or 2, which is of 35 nucleotides or less.
4. The isolated nucleic acid molecule of any one of claims 1 to 3, which comprises the sequence 5'-TATTACCGCGGCTGCT-3' (SEQ ID NO: 1 ).
5. The isolated nucleic acid molecule of claim 4, which comprises the sequence 5'- TACGTATTACCGCGGCTGCT-3' (SEQ ID NO: 8).
6. The isolated nucleic acid molecule of claim 5, the nucleotide sequence of which consists of the sequence 5'-TACGTATTACCGCGGCTGCT-3' (SEQ ID NO: 8).
7. The isolated nucleic acid molecule of any one of claims 1 to 3, which comprises the sequence 5'-CGGCTAACTMCGTGCCAG-3' (SEQ ID NO: 2).
8. The isolated nucleic acid molecule of claim 7, the nucleotide sequence of which consists of the sequence 5'-CGGCTAACTMCGTGCCAG-3' (SEQ ID NO: 2).
9. The isolated nucleic acid molecule of claim 1 , which comprises the sequence 5'- AAGSVMCGGCTAACTMCGTGCC-3' (SEQ ID NO: 3).
10. The isolated nucleic acid molecule of claim 9, the nucleotide sequence of which consists of the sequence 5'-AAGSVMCGGCTAACTMCGTGCC-3' (SEQ ID NO: 3).
1 1. The isolated nucleic acid molecule of claim 1 , which comprises the sequence 5'- AATGTTGGCATGAGTAGCGAGATGT-3' (SEQ ID NO: 4).
12. The isolated nucleic acid molecule of claim 15, the nucleotide sequence of which consists of the sequence 5'-AATGTTGGCATGAGTAGCGAGATGT-3' (SEQ ID NO: 4).
13. The isolated nucleic acid molecule of claim 1 , which comprises the sequence 5'- TCTGAAGGATTACCTRTAATTGCAA-3' (SEQ ID NO: 5).
14. The isolated nucleic acid molecule of claim 17, the nucleotide sequence of which consists of the sequence 5'-TCTGAAGGATTACCTRTAATTGCAA-3' (SEQ ID NO: 5).
15. The isolated nucleic acid molecule of claim 1 , which comprises the sequence 5'- TGCAGCCAAAGTTGTTGAAT-3' (SEQ ID NO: 6).
16. The isolated nucleic acid molecule of claim 19, the nucleotide sequence of which consists of the sequence 5'-TGCAGCCAAAGTTGTTGAAT-3' (SEQ ID NO: 6).
17. The isolated nucleic acid molecule of claim 1 , which comprises the sequence 5'- GCTCTTTGATTGCTGCACCT-3' (SEQ ID NO: 7).
18. The isolated nucleic acid molecule of claim 1 , the nucleotide sequence of which consists of the sequence 5'-GCTCTTTGATTGCTGCACCT-3' (SEQ ID NO: 7).
19. A method for amplifying and/or detecting a bacterial 16S rRNA nucleic acid in a sample, said method comprising (a) contacting said bacterial 16S rRNA nucleic acid with a first primer and a second primer under conditions suitable for nucleic acid amplification, and (b) performing a nucleic acid amplification reaction, thereby generating an amplification product if bacterial 16S rRNA nucleic acid is present in said sample; wherein said first primer has a length of 50 nucleotides or less and comprises a sequence of at least 10 contiguous nucleotides of the sequence ACTCCTAYGGGRBGCWSCA (SEQ ID NO: 13), and said second primer comprises a sequence of at least 10 contiguous nucleotides of nucleotide sequence (I) or (VII) defined in any one of claims 1 to 6.
20. The method of claim 19, wherein said first primer has a length of 35 nucleotides or less.
21. The method of claim 19 or 20, wherein said first primer comprises a sequence of at least 15 contiguous nucleotides of the sequence ACTCCTAYGGGRBGCWSCA (SEQ ID NO: 13).
22. The method of any one of claims 19 to 21 , wherein said first primer comprises the sequence ACTCCTAYGGGRBGCWSCA (SEQ ID NO: 13).
23. The method of claim 22, wherein said first primer comprises the sequence ACTCCTAYGGGRBGCASCAGT (SEQ ID NO: 14), ACTCCTAYGGGRGGCWGCAGT (SEQ ID NO: 15), ACTCCTACGGGRGGCWGCAGT (SEQ ID NO: 16), ACTCCTAYGGGRGGCWGCA (SEQ ID NO: 17), ACTCCTACGGGRGGCWGCA (SEQ ID NO: 18), ACTCCTACG G G RG G CAG C A (SEQ ID NO: 19), ACTCCTAYGGGRBGCAGCA (SEQ ID NO: 20), or ACTCCTACGGGRBGCWGCA (SEQ ID NO: 21 ).
24. The method of any one of claims 19 to 23, wherein said first primer further comprises, at its 5' end, the nucleotide sequence 5'-AATAAATCATAA-3' (SEQ ID NO: 10) or a 5'-deleted fragment thereof.
25. The method of claim 24, wherein said first primer comprises the sequence ACTCCTAYGGGRBGCASCAGT (SEQ ID NO: 14).
26. The method of claim 25, wherein the nucleotide sequence of said first primer consists of the sequence ACTCCTAYGGGRBGCASCAGT (SEQ ID NO: 14).
27. The method of any one of claims 19 to 26, wherein said second primer is the nucleic acid molecule defined in claim 6.
28. The method of any one of claims 19 to 27, wherein said conditions suitable for nucleic acid amplification comprise an annealing step at about 45°C to about 55°C.
29. The method of claim 29, wherein said conditions suitable for nucleic acid amplification comprise an annealing step at about 50°C.
30. The method of any one of claims 19 to 29, wherein said method further comprises contacting said nucleic acid amplification reaction with a probe that hybridizes to said amplification product if present.
31. The method of claim 30, wherein said probe comprises (i) a sequence of at least 10 contiguous nucleotides of nucleotide sequence (II) or (VII) defined in any one of claims 1 to 3, 7 and 8.
32. The method of claim 31 , wherein said probe is the nucleic acid molecule defined in claim 8.
33. The method of any one of claims 30 to 32, wherein said probe is tagged with a detectable label.
34. The method of claim 33, wherein said detectable label is a fluorescent label.
35. The method of any one of claims 19 to 34, wherein said amplification reaction is performed by polymerase chain reaction (PCR).
36. The method of claim 35, wherein said PCR is quantitative PCR.
37. A combination of nucleic acid molecules for amplifying or detecting a bacterial 16S rRNA nucleic acid, said combination comprising the first primer defined in any one of claims 19 to 27 and the second primer defined in any one of claims 19 to 27.
38. The combination of claim 37, further comprising the probe defined in any one of claims 30 to 34.
39. A method for determining whether a sample comprises a bacterial 16S rRNA nucleic acid, said method comprising performing the method for amplifying or detecting a bacterial 16S rRNA nucleic acid defined in any one of claims 19 to 36, determining the presence or absence of said amplification product, wherein the presence of the amplification product is indicative that said sample comprises a bacterial 16S rRNA nucleic acid.
40. The method of claim 39, wherein said sample is a clinical sample.
41 . The method of claim 39 or 40, wherein said sample is stool, feces, anal swab or rectal swab.
42. A method for determining the amount of bacteria in a sample, said method comprising (a) performing the method for amplifying or detecting a bacterial 16S rRNA nucleic acid defined in any one of claims 19 to 36, (b) comparing the amount of amplification product present after said amplification reaction to a reference; and (c) determining the amount of bacteria in the sample based on said comparison.
43. Use of one or more of the isolated nucleic acid molecules comprising (i) a sequence of at least 10 contiguous nucleotides of sequence (I), (II) and/or (VII) defined in any one of claims 1 to 10, for amplifying and/or detecting a bacterial 16S rRNA nucleic acid in a sample.
44. A method for detecting a C. difficile-specific 16S rRNA nucleic acid in a sample, said method comprising (a) contacting said C. difficile-specific 16S rRNA nucleic acid with a first primer and a second primer under conditions suitable for nucleic acid amplification, (b) performing a nucleic acid amplification reaction, thereby generating an amplification product if C. difficile-specific 16S rRNA nucleic acid is present in said sample; and (c) contacting said nucleic acid amplification reaction with a probe that hybridizes to said amplification product if present, wherein said probe comprises a sequence of at least 10 contiguous nucleotides of nucleotide sequence (III) or (VII) defined in any one of claims 1 to 3, 1 1 and 12.
45. The method of claim 44, wherein said first primer comprises the sequence 5'- GGGAGCTTCCCATACGGGTTG-3' (SEQ ID NO: 22).
46. The method of claim 44 or 45, wherein said second primer comprises the sequence 5'- TTGACTGCCTCAATGCTTGGGC-3' (SEQ ID NO: 23).
47. Use of one or more of the isolated nucleic acid molecules comprising a sequence of at least 10 contiguous nucleotides of sequence (III) or (VII) defined in any one of claims 1 to 3, 1 1 and 12 for amplifying and/or detecting a C. difficile-specific 16S rRNA nucleic acid in a sample.
48. A method for detecting a C. difficile toxin B nucleic acid in a sample, said method comprising (a) contacting said C. difficile toxin B nucleic acid with a first primer and a second primer under conditions suitable for nucleic acid amplification, (b) performing a nucleic acid amplification reaction, thereby generating an amplification product if C. difficile toxin B nucleic acid is present in said sample; and (c) contacting said nucleic acid amplification reaction with a probe that hybridizes to said amplification product if present, wherein (i) said probe comprises a sequence of at least 10 contiguous nucleotides of nucleotide sequence (IV) or (VII) defined in any one of claims 1 to 3, 13 and 14; (ii) said first primer comprises a sequence of at least 10 contiguous nucleotides of nucleotide sequence (V) or (VII) defined in any one of claims 1 to 3, 15 and 16; and/or said second primer comprises a sequence of at least 10 contiguous nucleotides of nucleotide sequence (VI) or (VII) defined in any one of claims 1 to 3, 17 and 18.
49. Use of one or more of the isolated nucleic acid molecules comprising a sequence of at least 10 contiguous nucleotides of sequences (IV), (V), (VI) and/or (VII) defined in any one of claims 1 to 3 and 13 to 18 for amplifying and/or detecting a nucleic acid encoding a C. difficile toxin B in a sample.
50. A method for predicting the severity or acuteness of Clostridium difficile infection (CDI) in a subject, said method comprising
performing, on a sample from the subject, an amplification reaction on
(i) a nucleic acid encoding a C. difficile toxin, thereby obtaining a C. difficile toxin signal; and
(ii) a nucleic acid encoding a bacterial 16S rRNA, a nucleic acid encoding a human
RNaseP and/or a nucleic acid encoding a fungal 18S rRNA, thereby obtaining a bacterial 16S rRNA signal, a human RNaseP signal and/or a fungal 18S rRNA signal; normalizing the C. difficile toxin signal using the bacterial 16S rRNA and/or human RNaseP signal, thereby obtaining a normalized C. difficile toxin signal;
predicting the severity or acuteness of CDI in the subject on the basis of said normalized C. difficile toxin signal.
51 . The method of claim 50, further comprising performing an amplification reaction on a nucleic acid encoding a C. difficile-specific 16S nucleic acid, thereby obtaining a C. difficile- specific 16S signal, establishing a ratio of the C. difficile toxin signal to the C. difficile-specific 16S signal or vice-versa, and predicting the severity of CDI in the subject on the basis of said normalized C. difficile toxin signal and said ratio.
52. A method for predicting the severity or acuteness of Clostridium difficile infection (CDI) in a subject, said method comprising:
performing, on a sample from the subject, an amplification reaction on a nucleic acid encoding a C. difficile toxin, thereby obtaining a C. difficile toxin amplified product;
performing on said sample an amplification reaction on a nucleic acid encoding a bacterial 16S rRNA, a nucleic acid encoding a fungal 18S rRNA and/or a nucleic acid encoding a human RNaseP, thereby obtaining a bacterial 16S rRNA amplified product, a fungal 18S rRNA amplified product and/or a human RNaseP amplified product;
contacting the C. difficile toxin amplified product with a probe hybridizing to said nucleic acid encoding a C. difficile toxin, thereby obtaining a C. difficile toxin signal;
contacting the bacterial 16S rRNA amplified product, fungal 18S rRNA amplified product and/or human RNaseP amplified product with a probe hybridizing to said nucleic acid encoding a bacterial 16S rRNA, a probe hybridizing to said nucleic acid encoding fungal 18S rDNA and/or a probe hybridizing to said nucleic acid encoding a human RNaseP, thereby obtaining a bacterial 16S rRNA signal, a fungal 18S rRNA signal, fungal 18S rRNA signal and/or human RNaseP signal;
normalizing the C. difficile toxin signal using the bacterial 16S rRNA, fungal 18S rRNA and/or a human RNaseP signal, thereby obtaining a normalized C. difficile toxin signal; and predicting the severity or acuteness of CDI in the subject on the basis of said normalized C. difficile toxin signal.
53. The method of claim 52, further comprising performing an amplification reaction on a nucleic acid encoding a C. difficile-specific 16S nucleic acid, thereby obtaining a C. difficile- specific 16S amplified product, contacting the C. difficile-specific 16S amplified product with a probe hybridizing to said nucleic acid encoding a C. difficile-specific 16S, thereby obtaining a C. difficile-specific 16S signal, establishing a ratio of the C. difficile toxin signal to the C. difficile- specific 16S signal or vice-versa, and predicting the severity of CDI in the subject on the basis of said normalized C. difficile toxin signal and said ratio.
54. The method of any one of claims 50 to 53, wherein said method comprises determining an acuteness of infection C. difficile (AOI-CD) score using the following formula:
AOI-CD score = (1/Cpt)/(Cpu-Cpt) or (1/Cpt)/(Cpg-Cpt) wherein
Cpt = C. difficile toxin signal value;
Cpu = bacterial 16S rRNA signal value;
Cpg = human RNaseP signal value.
55. The method of any one of claims 50 to 54, wherein said amplification reaction is performed by polymerase chain reaction (PCR).
56. The method of claim 55, wherein said PCR is quantitative PCR.
57. The method of any one of claims 50 to 56, wherein said sample is stool, feces, anal swab or rectal swab.
58. The method of any one of claims 50 to 56, wherein said method comprises performing an amplification reaction on a nucleic acid encoding a bacterial 16S rRNA, thereby obtaining a bacterial 16S rRNA signal.
59. The method of claim 58, wherein said amplification reaction is performed according to the method of any one of claims 19 to 36.
60. A method for normalizing the amount of a pathogen of interest in a sample, said method comprising:
performing, on said sample, an amplification reaction on a nucleic acid specific for said pathogen of interest to obtain a pathogen of interest signal;
performing, on said sample, an amplification reaction on a bacterial 16S rRNA nucleic acid according to the method of 19 to 36 to obtain a bacterial 16S rRNA signal;
normalizing the first signal using the bacterial 16S rRNA signal, thereby obtaining a normalized pathogen of interest signal.
61. The method of claim 60, wherein said pathogen of interest is an opportunistic pathogen.
62. The method of claim 60 or 61 , wherein said pathogen is a bacterial pathogen.
63. The method of claim 61 , wherein said opportunistic pathogen is Clostrodium difficile, Helicobacter pylori, Haemophilus influenza or Streptococcus pneumonia.
64. The method of claim 63, wherein said opportunistic pathogen is Clostrodium difficile.
65. The method of claim 64, wherein said nucleic acid specific for said pathogen of interest is a C. difficile toxin nucleic acid.
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