Multiplex assay for detection of bacterial species in biological samples
View Patent ↗The invention provides a rapid, accurate, sensitive, and low-cost detection method for screening a biological sample for one or more desired bacterial species. The inventive method employs a two-step multiplex real-time PCR assay that comprises an internal amplification control and specific primer sets to detect and discriminate bacterial species based the unique melting temperatures of specific DNA sequences of each strain.
1. A method for detection of at least one bacterial species in a biological sample comprising real-time PCR amplification, said method comprising:
(i) enriching the bacterial concentration of the biological sample to result in an enriched biological sample;
(ii) isolating DNA from said enriched biological sample to produce an isolated DNA sample; and
(iii) detecting a sequence from said at least one bacterial species in said isolated DNA sample via real-time PCR, wherein said real-time PCR comprises:
(a) an internal amplification control comprising at least 90% identity to the internal amplification control set forth in SEQ ID NO:23 and more than one primer pair, wherein each primer of the primer pairs comprises at least 90% sequence identity to a primer set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:22;
(b) an internal amplification control comprising at least 90% identity to the internal amplification control set forth in SEQ ID NO:31 and more than one primer pair, wherein each primer of the primer pairs comprises at least 90% sequence identity to a primer set forth in SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:28, SEQ ID NO:29 or SEQ ID NO:30;
(c) an internal amplification control comprising at least 90% identity to the internal amplification control set forth in SEQ ID NO:42 and more than one primer pair, wherein each primer of the primer pairs comprises at least 90% sequence identity to a primer set forth in SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40 or SEQ ID NO:41; or
(d) an internal amplification control comprising at least 90% identity to the internal amplification control set forth in SEQ ID NO:53 and more than one primer pair, wherein each primer of the primer pairs comprises at least 90% sequence identity to a primer set forth in SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51 or SEQ ID NO:52.
2. The method of claim 1 , wherein said real-time PCR comprises an internal amplification control comprising at least 90% identity to the internal amplification control set forth in SEQ ID NO:23 and more than one primer pair, wherein each primer of the primer pairs comprises at least 90% sequence identity to a primer set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:22.
3. The method of claim 1 , wherein said real-time PCR comprises an internal amplification control comprising at least 90% identity to the internal amplification control set forth in SEQ ID NO:31 and more than one primer pair, wherein each primer of the primer pairs comprises at least 90% sequence identity to a primer set forth in SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:28, SEQ ID NO:29 or SEQ ID NO:30.
4. The method of claim 1 , wherein said real-time PCR comprises an internal amplification control comprising at least 90% identity to the internal amplification control set forth in SEQ ID NO:42 and more than one primer pair, wherein each primer of the primer pairs comprises at least 90% sequence identity to a primer set forth in SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40 or SEQ ID NO:41.
5. The method of claim 1 , wherein said real-time PCR comprises an internal amplification control comprising at least 90% identity to the internal amplification control set forth in SEQ ID NO:53 and more than one primer pair, wherein each primer of the primer pairs comprises at least 90% sequence identity to a primer set forth in SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51 or SEQ ID NO:52.
6. The method of claim 1 , wherein said real-time PCR comprises:
(a) the internal amplification control set forth in SEQ ID NO:23 and more than one primer pair selected from: (i) SEQ ID NO:1 and SEQ ID NO:2, (ii) SEQ ID NO:3 and SEQ ID NO:4, (ii) SEQ ID NO:7 and SEQ ID NO:8, (iv) SEQ ID NO:19 and SEQ ID NO:20, and (v) SEQ ID NO:21 and SEQ ID NO:22;
(b) the internal amplification control set forth in SEQ ID NO:31 and more than one primer pair selected from: (i) SEQ ID NO:24 and SEQ ID NO:25, (ii) SEQ ID NO:26 and SEQ ID NO:27, (iii) SEQ ID NO:14 and SEQ ID NO:15, (iv) SEQ ID NO:16 and SEQ ID NO:28, and (v) SEQ ID NO:29 and SEQ ID NO:30;
(c) the internal amplification control set forth in SEQ ID NO:42 and more than one primer pair selected from: (i) SEQ ID NO:32 and SEQ ID NO:33, (ii) SEQ ID NO:34 and SEQ ID NO:35, (iii) SEQ ID NO:36 and SEQ ID NO:37, (iv) SEQ ID NO:38 and SEQ ID NO:39, and (v) SEQ ID NO:40 and SEQ ID NO:41; or
(d) the internal amplification control set forth in SEQ ID NO:53 and more than one primer pair selected from: (i) SEQ ID NO:43 and SEQ ID NO:44, (ii) SEQ ID NO:45 and SEQ ID NO:46, (iii) SEQ ID NO:47 and SEQ ID NO:48, (iv) SEQ ID NO:49 and SEQ ID NO-50, and (v) SEQ ID NO:51 and SEQ ID NO:52.
7. The method of claim 6 , wherein said real-time PCR comprises the internal amplification control set forth in SEQ ID NO:23 and more than one primer pair selected from: (i) SEQ ID NO:1 and SEQ ID NO:2, (ii) SEQ ID NO:3 and SEQ ID NO:4, (iii) SEQ ID NO:7 and SEQ ID NO:8, (iv) SEQ ID NO:19 and SEQ ID NO:20, and (v) SEQ ID NO:21 and SEQ ID NO:22.
8. The method of claim 6 , wherein said real-time PCR comprises the internal amplification control set forth in SEQ ID NO:31 and more than one primer pair selected from: (i) SEQ ID NO:24 and SEQ ID NO:25, (ii) SEQ ID NO:26 and SEQ ID NO:27, (iii) SEQ ID NO:14 and SEQ ID NO:15, (iv) SEQ ID NO:16 and SEQ ID NO:28, and (v) SEQ ID NO:29 and SEQ ID NO:30.
9. The method of claim 6 , wherein said real-time PCR comprises the internal amplification control set forth in SEQ ID NO:42 and more than one primer pair selected from: (i) SEQ ID NO:32 and SEQ ID NO:33, (ii) SEQ ID NO:34 and SEQ ID NO:35, (iii) SEQ ID NO:36 and SEQ ID NO:37, (iv) SEQ ID NO:38 and SEQ ID NO:39, and (v) SEQ ID NO:40 and SEQ ID NO:41.
10. The method of claim 6 , wherein said real-time PCR comprises the internal amplification control set forth in SEQ ID NO:53 and more than one primer pair selected from: (i) SEQ ID NO:43 and SEQ ID NO:44, (ii) SEQ ID NO:45 and SEQ ID NO:46, (iii) SEQ ID NO:47 and SEQ ID NO:48, (iv) SEQ ID NO:49 and SEQ ID NO:50, and (v) SEQ ID NO:51 and SEQ ID NO:52.
11. The method of claim 1 , wherein said method further comprises a melt curve assay for visualization of individual amplicons.
12. The method of claim 11 , wherein said melt curve assay uses real-time PCR with SEQ ID NO: 54 and SEQ ID NO: 55.
13. The method of claim 1 , wherein detecting said at least one sequence comprises:
(1) a first real-time PCR with the more than one primer pair and the internal amplification control of part (a) and a second real-time PCR with the more than one primer pair and the internal amplification control of part (b); or
(2) a first real-time PCR with the more than one primer pair and the internal amplification control of part (c) and a second real-time PCR with the more than one primer pair and the internal amplification control of part (d).
14. The method of claim 1 , wherein said enriching step comprises incubating said biological sample aerobically at approximately 42° C. in an enrichment media.
15. The method of claim 14 , wherein said enrichment media is selected from the group consisting of brain heart infusion broth (BHI), tryptic soy broth (TSB), and Buffered Peptone Water (BPW).
16. The method of claim 15 , wherein said enrichment media is supplemented with an antibiotic.
17. The method of claim 16 , wherein said enrichment media is BPW supplemented with vancomycin.
18. The method of claim 1 , wherein said biological sample comprises a food or a beverage.
19. The method of claim 18 , wherein said food or beverage comprises meat, produce, or juice.
20. The method of claim 1 , wherein said biological sample comprises a clinical sample.
21. The method of claim 20 , wherein said clinical sample comprises stool, urine, or blood.
22. The method of claim 1 , wherein said sequence comprises:
(1) a sequence from at least one bacterial species selected from the group consisting of E. coli O121, E. coli O145, E. coli O157, E. coli O26, E. coli O111, E. coli O103, E. coli O45, and Salmonella ; or a stx 1 or stx 2 sequence; or
(2) a sequence selected from the group consisting of bla KPC-like , bla NDM-like , bla CTX-M-1/2group , bla CMY-like , bla VIM-like , bla IMP-like , bla OXA-like , bla SHV-like , bla TEM-like , and bla ACC-like.
23. A kit for detection of at least one bacterial species in a biological sample, comprising:
(i) an internal amplification control comprising at least 90% identity to the internal amplification control set forth in SEQ ID NO:23 and more than one primer pair, wherein each primer of the primer pairs comprises at least 90% sequence identity to a primer set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:22;
(ii) an internal amplification control comprising at least 90% identity to the internal amplification control set forth in SEQ ID NO:31 and more than one primer pair, wherein each primer of the primer pairs comprises at least 90% sequence identity to a primer set forth in SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:28, SEQ ID NO:29 or SEQ ID NO:30;
(iii) an internal amplification control comprising at least 90% identity to the internal amplification control set forth in SEQ ID NO:42 and more than one primer pair, wherein each primer of the primer pairs comprises at least 90% sequence identity to a primer set forth in SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO: 35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO: NO:39, SEQ ID NO:40 or SEQ ID NO:41; or
(iv) an internal amplification control comprising at least 90% identity to the internal amplification control set forth in SEQ ID NO:53 and more than one primer pair, wherein each primer of the primer pairs comprises at least 90% sequence identity to a primer set forth in SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51 or SEQ ID NO:52.
24. The kit of claim 23 , further comprising a primer pair of SEQ ID NO: 54 and SEQ ID NO: 55.
25. The kit of claim 23 , comprising an internal amplification control comprising at least 90% identity to the internal amplification control set forth in SEQ ID NO:23 and more than one primer pair, wherein each primer of the primer pairs comprises at least 90% sequence identity to a primer set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22.
26. The kit of claim 23 , comprising an internal amplification control comprising at least 90% identity to the internal amplification control set form in SEQ ID NO:31 and more than one primer pair, wherein each primer of the primer pairs comprises at least 90% sequence identity to a primer set forth in SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30.
27. The kit of claim 23 , comprising an internal amplification control comprising at least 90% identity to the internal amplification control set forth in SEQ ID NO:42 and more than one primer pair, wherein each primer of the primer pairs comprises at least 90% sequence identity to a primer set forth in SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, or SEQ ID NO:41.
28. The kit of claim 23 , comprising an internal amplification control comprising at least 90% identity to the internal amplification control set forth in SEQ ID NO:53 and more than one primer pair, wherein each primer of the primer pairs comprises at least 90% sequence identity to a primer set forth in SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, or SEQ ID NO:52.