Compositions and methods for detecting gastrointestinal pathogen nucleic acid
Disclosed are nucleic acid oligomers, including amplification oligomers, detection probes, and combinations thereof, for detection of one or more gastrointestinal pathogens selected from Salmonella, Shigella, Campylobacter jejuni , and Campylobacter coli . Also disclosed are methods of specific nucleic acid amplification and detection, including multiplex assays, using the disclosed oligomers, as well as corresponding reaction mixtures and kits.
1. A multiplex method for determining the presence or absence of gastrointestinal pathogens comprising Campylobacter jejuni and Shigella in a sample, said method comprising:
(1) contacting a sample, said sample suspected of containing at least one of the gastrointestinal pathogens, with at least a first set of amplification oligomers for amplifying a first nucleic acid target region and a second set of amplification oligomers for amplifying a second nucleic acid target region, wherein
the first set of amplification oligomers comprises at least two C. jejuni -specific amplification oligomers for amplifying a target region of a C. jejuni target nucleic acid, wherein the at least two C. jejuni -specific amplification oligomers comprise first and second oligomers respectively comprising target-hybridizing sequences consisting of the nucleotide sequences of: (i) SEQ ID NO:75 and SEQ ID NO:76; (ii) SEQ ID NO:51 and SEQ ID NO:52; (iii) SEQ ID NO: 55 and SEQ ID NO:56; (iv) SEQ ID NO:59 and SEQ ID NO:60; (v) SEQ ID NO:62 and SEQ ID NO:63; (vi) SEQ ID NO:66 and SEQ ID NO:67; or (vii) SEQ ID NO:71 and SEQ ID NO: 72; and
the second set of amplification oligomers comprises at least two Shigella -specific amplification oligomers for amplifying a target region of a Shigella target nucleic acid, wherein the at least two Shigella -specific amplification oligomers comprise first and second oligomers respectively comprising target-hybridizing sequences consisting of SEQ ID NO:45 and SEQ ID NO: 46;
(2) performing an in vitro nucleic acid amplification reaction, wherein any target nucleic acid, if present in said sample, is used as a template for generating one or more amplification products corresponding to the first and/or second target regions; and
(3) determining the sequences of the one or more amplification products, or detecting the presence or absence of the one or more amplification products using a first detection probe specific for the first target region and a second detection probe specific for the second target region,
thereby determining the presence or absence of C. jejuni and Shigella in said sample.
2. The method of claim 1 , where the amplification reaction is a polymerase chain reaction (PCR).
3. The method of claim 1 , wherein step (3) comprises use of the first and second detection probes.
4. The method of claim 3 , wherein the amplification reaction is a real-time polymerase chain reaction (RT-PCR).
5. The method of claim 1 , wherein each of the first and second detection probes comprises a fluorescent dye compound.
6. The method of claim 5 , wherein each of the first and second detection probes further comprises a non-fluorescent quenching dye compound.
7. The method of claim 1 , further comprising purifying the target nucleic acid from other sample components before step (1).
8. The method of claim 7 , wherein purifying the target nucleic acid comprises selectively separating the target nucleic acid from the other sample components using one or more target capture oligonucleotides each comprising a target hybridizing sequence joined at its 3′ end to a tail sequence.
9. The method of claim 8 , wherein the target hybridizing sequence of the one or more target capture oligonucleotides comprises a randomized or non-randomized poly-GU, poly-GT, or poly U sequence.