Compositions and methods for detecting gastrointestinal pathogen nucleic acid

1. A multiplex method for determining the presence or absence of gastrointestinal pathogens comprising Campylobacter jejuni and Campylobacter coli in a sample, the method comprising:

(1) contacting a sample, the 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 and second set of amplification oligomers are, respectively:

(a) 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 with sequences consisting of a target-hybridizing region, wherein the target-hybridizing regions of the first and second oligomers respectively consist of the nucleotide sequences of: (i) SEQ ID NO:78 and SEQ ID NO:79; (ii) SEQ ID NO:51 and SEQ ID NO:52; (ii) 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; (vii) SEQ ID NO:71 and SEQ ID NO:72; or (viii) SEQ ID NO:75 and SEQ ID NO:76; and

(b) at least two C. coli -specific amplification oligomers for amplifying a target region of a C. coli target nucleic acid, wherein the at least two C. coli -specific amplification oligomers comprise first and second oligomers with sequences consisting of a target-hybridizing region, wherein the target-hybridizing regions of the first and second oligomers respectively consist of the nucleotide sequences of: (i) SEQ ID NO:91 and SEQ ID NO:92; (ii) SEQ ID NO:82 and SEQ ID NO:83; or (iii) SEQ ID NO:86 and SEQ ID NO:87;

(2) performing an in vitro nucleic acid amplification reaction, wherein any target nucleic acid, if present in the sample, is used as a template for generating one or more amplification products corresponding to the first and/or second target regions; and

(3) 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, wherein the first detection probe comprises a first detectable label and the second detection probe comprises a second detectable label,

thereby determining the presence or absence of C. jejuni and C. coli in the sample.

2. The multiplex method of claim 1 , wherein:

(I) the first detection probe comprises a target-hybridizing sequence substantially corresponding to the nucleotide sequence of

SEQ ID NO:80 or SEQ ID NO:81 if the first and second C. jejuni -specific oligomers are the oligomers of (a)(i);

SEQ ID NO:53 or SEQ ID NO:54 if the first and second C. jejuni -specific oligomers are the oligomers of (a)(ii);

SEQ ID NO:57 or SEQ ID NO:58 if the first and second C. jejuni -specific oligomers are the oligomers of (a)(iii);

SEQ ID NO:61 if the first and second C. jejuni -specific oligomers are the oligomers of (a)(iv);

SEQ ID NO:64 or SEQ ID NO:65 if the first and second C. jejuni -specific oligomers are the oligomers of (a)(v);

SEQ ID NO:68, SEQ ID NO:69, or SEQ ID NO:70 if the first and second C. jejuni -specific oligomers are the oligomers of (a)(vi);

SEQ ID NO:73 or SEQ ID NO:74 if the first and second C. jejuni -specific oligomers are the oligomers of (a)(vii); or

SEQ ID NO:77 if the first and second C. jejuni -specific oligomers are the oligomers of (a)(viii); and

(II) the second detection probe comprises a target-hybridizing sequence substantially corresponding to the nucleotide sequence of

SEQ ID NO:93 or SEQ ID NO:94 if the first and second C. coli -specific oligomers are the oligomers of (b)(i);

SEQ ID NO:84 or SEQ ID NO:85 if the first and second C. coli -specific oligomers are the oligomers of (b)(ii); or

SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90 if the first and second C. coli -specific oligomers are the oligomers of (b)(iii).

3. The multiplex method of claim 2 , wherein

the first and second C. jejuni -specific oligomers are the first and second oligomers of (a)(i), and the first detection probe has a target-hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:81; and

the first and second C. coli -specific oligomers are the first and second oligomers of (b)(i), and the second detection probe has a target-hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:93.

4. The multiplex method of claim 1 , wherein each of the first and second detection probes comprises a fluorescent dye compound, a non-fluorescent quenching dye compound, or both.

5. The multiplex method of claim 1 , wherein the method is for determining the presence or absence of each of Salmonella, Shigella, C. jejuni , and C. coli in the sample; step (1) further comprises contacting the sample with at least a third set of amplification oligomers for amplifying a target region of a Salmonella target nucleic acid and a fourth set of amplification oligomers for amplifying a target region of a Shigella target nucleic acid, wherein:

(aa) the third set comprises first and second Salmonella -specific oligomers, wherein the at least two Salmonella -specific amplification oligomers comprise first and second oligomers with sequences consisting of a target-hybridizing region, wherein the target-hybridizing regions of the first and second oligomers respectively consist of the nucleotide sequences of: (i) SEQ ID NO:1 and SEQ ID NO:2; (ii) SEQ ID NO:4 and SEQ ID NO:5; (iii) SEQ ID NO:8 and SEQ ID NO:9; (iv) SEQ ID NO:12 and SEQ ID NO:13; (v) SEQ ID NO:16 and SEQ ID NO:17; or (vi) SEQ ID NO:18 and SEQ ID NO:2; and

(bb) the fourth set comprises first and second Shigella -specific oligomers wherein the at least two Shigella -specific amplification oligomers comprise first and second oligomers with sequences consisting of a target-hybridizing region, wherein the target-hybridizing regions of the first and second oligomers respectively consist of the nucleotide sequences of: (i) SEQ ID NO:45 and SEQ ID NO:46; (ii) SEQ ID NO:20 and SEQ ID NO:21; (iii) SEQ ID NO:26 and SEQ ID NO:21; (iv) SEQ ID NO:20 and SEQ ID NO:28; (v) SEQ ID NO:30 and SEQ ID NO:31; (vi) SEQ ID NO:36 and SEQ ID NO:37; or (vii) SEQ ID NO:41 and SEQ ID NO:42;

wherein any Salmonella, Shigella, C. jejuni , and C. coli target nucleic acid, if present in the sample, is used as a template in the nucleic acid amplification reaction of step (2) for generating one or more amplification products corresponding to the Salmonella, Shigella, C. jejuni , and C. coli target regions; and

step (3) further comprises detecting the presence or absence of the one or more amplification products using a third detection probe specific for the Salmonella target region and a fourth detection probe specific for the Shigella target region,

thereby determining the presence or absence of Salmonella, Shigella, C. jejuni , and C. coli in the sample.

6. The multiplex method of claim 5 , wherein

(I) the first detection probe comprises a target-hybridizing sequence substantially corresponding to the nucleotide sequence of

SEQ ID NO:3 if the first and second Salmonella -specific oligomers are the oligomers of (a)(i);

SEQ ID NO:6 or SEQ ID NO:7 if the first and second Salmonella -specific oligomers are the oligomers of (a)(ii);

SEQ ID NO:10 or SEQ ID NO:11 if the first and second Salmonella -specific oligomers are the oligomers of (a)(iii) or (a)(v);

SEQ ID NO:14 or SEQ ID NO:15 if the first and second Salmonella -specific oligomers are the oligomers of (a)(iv); or

SEQ ID NO:19 or SEQ ID NO:3 if the first and second Salmonella -specific oligomers are the oligomers of (a)(vi);

(II) the second detection probe comprises a target-hybridizing sequence substantially corresponding to the nucleotide sequence of

SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, or SEQ ID NO:50 if the first and second Shigella -specific oligomers are the oligomers of (b)(i);

SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25 if the first and second Shigella -specific oligomers are the oligomers of (b)(ii);

SEQ ID NO:27 or SEQ ID NO:23 if the first and second Shigella -specific oligomers are the oligomers of (b)(iii);

SEQ ID NO:29 or SEQ ID NO:22 if the first and second Shigella -specific oligomers are the oligomers of (b)(iv);

SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, or SEQ ID NO:35 if the first and second Shigella -specific oligomers are the oligomers of (b)(v);

SEQ ID NO:38, SEQ ID NO:39, or SEQ ID NO:40 if the first and second Shigella -specific oligomers are the oligomers of (b)(vi); or

SEQ ID NO:38, SEQ ID NO:43, or SEQ ID NO:44 if the first and second Shigella -specific oligomers are the oligomers of (b)(vii);

(III) the third detection probe comprises a target-hybridizing sequence substantially corresponding to the nucleotide sequence of

SEQ ID NO:80 or SEQ ID NO:81 if the first and second C. jejuni -specific oligomers are the oligomers of (c)(i);

SEQ ID NO:53 or SEQ ID NO:54 if the first and second C. jejuni -specific oligomers are the oligomers of (c)(ii);

SEQ ID NO:57 or SEQ ID NO:58 if the first and second C. jejuni -specific oligomers are the oligomers of (c)(iii);

SEQ ID NO:61 if the first and second C. jejuni -specific oligomers are the oligomers of (c)(iv);

SEQ ID NO:64 or SEQ ID NO:65 if the first and second C. jejuni -specific oligomers are the oligomers of (c)(v);

SEQ ID NO:68, SEQ ID NO:69, or SEQ ID NO:70 if the first and second C. jejuni -specific oligomers are the oligomers of (c)(vi);

SEQ ID NO:73 or SEQ ID NO:74 if the first and second C. jejuni -specific oligomers are the oligomers of (c)(vii); or

SEQ ID NO:77 if the first and second C. jejuni -specific oligomers are the oligomers of (c)(viii); and

(IV) the fourth detection probe comprises a target-hybridizing sequence substantially corresponding to the nucleotide sequence of

SEQ ID NO:93 or SEQ ID NO:94 if the first and second C. coli -specific oligomers are the oligomers of (d)(i);

SEQ ID NO:84 or SEQ ID NO:85 if the first and second C. coli -specific oligomers are the oligomers of (d)(ii); or

SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90 if the first and second C. coli -specific oligomers are the oligomers of (d)(iii).

7. The multiplex method of claim 6 , wherein

the first and second Salmonella -specific oligomers are the first and second oligomers of (a)(i), and the first detection probe comprises the target-hybridizing sequence substantially corresponding to the nucleotide sequence of SEQ ID NO:3;

the first and second Shigella -specific oligomers are the first and second oligomers of (b)(i), and the second detection probe comprises the target-hybridizing sequence substantially corresponding to the nucleotide sequence of SEQ ID NO:50;

the first and second C. jejuni -specific oligomers are the first and second oligomers of (c)(i), and the third detection probe comprises the target-hybridizing sequence substantially corresponding to the nucleotide sequence of SEQ ID NO:81; and

the first and second C. coli -specific oligomers are the first and second oligomers of (d)(i), and the fourth detection probe comprises the target-hybridizing sequence substantially corresponding to the nucleotide sequence of SEQ ID NO:93.

8. The multiplex method of claim 5 , wherein the first detectable label and the second detectable label comprise a fluorescent dye compound, a non-fluorescent quenching dye compound, or both; and the third and fourth detection probes comprise a fluorescent dye compound, a non-fluorescent quenching dye compound, or both.

9. The multiplex method of claim 5 , wherein the third detection probe comprises a target-hybridizing sequence substantially corresponding to the nucleotide sequence of:

SEQ ID NO:3 if the first and second Salmonella -specific oligomers are the oligomers of (aa)(i);

SEQ ID NO:6 or SEQ ID NO:7 if the first and second Salmonella -specific oligomers are the oligomers of (aa)(ii);

SEQ ID NO:10 or SEQ ID NO:11 if the first and second Salmonella -specific oligomers are the oligomers of (aa)(iii) or (aa)(v);

SEQ ID NO:14 or SEQ ID NO:15 if the first and second Salmonella -specific oligomers are the oligomers of (aa)(iv); or

SEQ ID NO:19 or SEQ ID NO:3 if the first and second Salmonella -specific oligomers are the oligomers of (aa)(vi); and

the fourth detection probe comprises a target-hybridizing sequence substantially corresponding to the nucleotide sequence of:

SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, or SEQ ID NO:50 if the first and second Shigella -specific oligomers are the oligomers of (bb)(i);

SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25 if the first and second Shigella -specific oligomers are the oligomers of (bb)(ii);

SEQ ID NO:27 or SEQ ID NO:23 if the first and second Shigella -specific oligomers are the oligomers of (bb)(iii);

SEQ ID NO:29 or SEQ ID NO:22 if the first and second Shigella -specific oligomers are the oligomers of (bb)(iv);

SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, or SEQ ID NO:35 if the first and second Shigella -specific oligomers are the oligomers of (bb)(v);

SEQ ID NO:38, SEQ ID NO:39, or SEQ ID NO:40 if the first and second Shigella -specific oligomers are the oligomers of (bb)(vi); or

SEQ ID NO:38, SEQ ID NO:43, or SEQ ID NO:44 if the first and second Shigella -specific oligomers are the oligomers of (bb)(vii).

10. The multiplex method of claim 5 , wherein:

the first and second Salmonella -specific oligomers are the first and second oligomers of (aa)(i), and the first detection probe has a target-hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:3; and

the first and second Shigella -specific oligomers are the first and second oligomers of (bb)(i), and the second detection probe has a target-hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:50.