DNA sequences for the detection of and differentiation amongst pathogenic
Oligonucleotide sequences and methods for specifically detecting and differentiating amongst pathogenic E. coli in a complex sample. The complex sample can be a food sample, water sample, or selectively enriched food matrix. The methods of detection may utilize PCR amplification with, or without, an internal positive control, and appropriate primer pairs. Reagents for performing the methods can be supplied as a kit and/or in tablet form.
1. A method for detecting the presence of pathogenic E. coli in a sample, the method comprising:
(a) performing PCR amplification of the sample using a primer pair selected from the group consisting of:
(i) SEQ ID NOs:2 and 3,
(ii) SEQ ID NOs:5 and 6,
(iii) SEQ ID NOs:8 and 9,
(iv) SEQ ID NOs:10 and 11,
(v) SEQ ID NOs:13 and 14, and
(vi) SEQ ID NOs:16 and 17,
to produce a PCR amplification result; and
(b) examining the PCR amplification result of step (a) to detect for an amplification product of the primer pair, whereby a positive detection of the amplification product of the primer pair indicates the presence of pathogenic E. coli in the sample.
2. The method of claim 1 , wherein the primer pair is selected from the group consisting of (a)(ii), (a)(iii), and (a)(iv).
3. The method of claim 1 , wherein the primer pair comprises (a)(ii).
4. The method of claim 1 , wherein the primer pair comprises (a)(iii).
5. The method of claim 1 , wherein the primer pair comprises (a)(iv).
6. A method for detecting the presence of pathogenic E. coli in a sample, the method comprising:
(a) performing PCR amplification of the sample using two different primer pairs selected from the group consisting of:
(i) SEQ ID NOs:2 and 3,
(ii) SEQ ID NOs:5 and 6,
(iii) SEQ ID NOs:8 and 9,
(iv) SEQ ID NOs:10 and 11,
(v) SEQ ID NOs:13 and 14, and
(vi) SEQ ID NOs:16 and 17,
to produce a PCR amplification result; and
(b) examining the PCR amplification result of step (a) to detect for amplification products of both of the two different primer pairs, whereby a positive detection of the amplification products of both of the two different primer pairs indicates the presence of pathogenic E. coli in the sample.
7. The method of claim 6 , wherein the two primer pairs comprise (a)(ii) and (a)(iv).
8. The method of claim 6 , wherein the two primer pairs comprise (a)(ii) and (a)(iii).
9. The method of claim 1 or 2 , wherein in step (b) a melting curve analysis is used to detect for amplification product.
10. The method of claim 1 or 2 , further comprising a step of preparing the sample for PCR amplification prior to said step (a).
11. The method of claim 10 , wherein said preparing step comprises at least one of the following processes: (1) bacterial enrichment, (2) separation of bacterial cells from the sample, (3) cell lysis, and (4) total DNA extraction.
12. The method of claim 1 or 2 , wherein the sample comprises a food sample or a water sample.
13. The method of claim 1 or 2 , wherein the sample comprises a selectively enriched food matrix.
14. A primer for use in primer directed amplification for detection of pathogenic E. coli consisting essentially of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:17.
15. A kit for detection of pathogenic E. coli , comprising:
(a) at least one primer pair selected from the group consisting of:
(i) SEQ ID NOs:2 and 3,
(ii) SEQ ID NOs:5 and 6,
(iii) SEQ ID NOs:8 and 9,
(iv) SEQ ID NOs:10 and 11,
(v) SEQ ID NOs:13 and 14, and
(vi) SEQ ID NOs:16 and 17; and
(b) thermostable DNA polymerase.
16. The kit of claim 15 , wherein component (a) comprises both (a)(ii) and (a)(iv).
17. The kit of claim 15 , wherein component (a) comprises both (a)(ii) and (a)(iii).
18. A replication composition for use in performance of PCR, comprising:
(a) at least one primer pair selected from the group consisting of:
(i) SEQ ID NOs:2 and 3,
(ii) SEQ ID NOs:5 and 6,
(iii) SEQ ID NOs:8 and 9,
(iv) SEQ ID NOs:10 and 11,
(v) SEQ ID NOs:13 and 14, and
(vi) SEQ ID NOs:16 and 17; and
(b) thermostable DNA polymerase.
19. The replication composition of claim 18 , wherein component (a) comprises both (a)(ii) and (a)(iv).
20. The replication composition of claim 18 , wherein component (a) comprises both (a)(ii) and (a)(iii).
21. A tablet comprising the replication composition of claim 18 , 19 , or 20 .
22. A kit for detection of pathogenic E. coli in a sample, comprising the tablet of claim 21 .