SEQUENCES AND THEIR USE FOR DETECTION OF SALMONELLA ENTERITIDIS AND/OR SALMONELLA TYPHIMURIUM
This invention relates to a rapid, accurate method for detection and characterization of Salmonella enteritidis and/or Salmonella typhimurium based on the presence of nucleic acid sequences, in particular, to a PCR-based method for detection, and to oligonucleotide molecules and reagents and kits useful therefore. This method is preferably employed to detect S. enteritidis and/or S. typhimurium in an environmental sample. The present invention further relates to replication compositions and kits for carrying out the method of the present invention.
1 . A method for detecting the presence of Salmonella enteritidis and/or Salmonella typhimurium in a sample, said sample comprising nucleic acids, said method comprising:
(a) providing a reaction mixture comprising suitable primer pairs for amplification of at least a portion of
(i) a Salmonella enteritidis SEN0908A/SEN0909/SEN0910 region, and/or
(ii) a Salmonella typhimurium type II restriction enzyme methylase region;
(b) performing PCR amplification of said nucleic acids of said sample using the reaction mixture of step (a); and
(c) detecting the amplification of step (b), whereby a positive detection of amplification indicates the presence of Salmonella enteritidis and/or Salmonella typhimurium in the sample.
2 . The method of claim 1 , wherein step (a)(i) comprises suitable primer pairs for amplification of SEQ ID NO:1.
3 . The method of claim 2 , wherein said primer pair for amplification of the nucleic acid region of SEQ ID NO:1 comprises SEQ ID NO:3 and SEQ ID NO:4.
4 . The method of claim 1 , wherein step (a)(ii) comprises suitable primers pairs for amplification of SEQ ID NO:2.
5 . The method of claim 4 , wherein said primer pair for amplification of the nucleic acid region of SEQ ID NO:2 comprises a first nucleic acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, and SEQ ID NO:13 and a second nucleic acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, and SEQ ID NO:14.
6 . The method of claim 3 , wherein said reaction mixture further comprises at least one nucleic acid probe for each nucleic acid region to be detected.
7 . The method of claim 6 , wherein said at least one nucleic acid probe comprises one or more of SEQ ID NO:5, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, and SEQ ID NO:24.
8 . The method of claim 7 , wherein each of said at least one nucleic acid probe comprises a detectable label.
9 . The method of claim 8 , wherein said reaction mixture further comprises a blocking oligonucleotide capable of quenching said detectable label of said at least one nucleic acid probe comprising SEQ ID NO:6, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:26.
10 . The method of claim 1 , wherein the sample comprises a food sample or an environmental sample.
11 . A primer comprising a polynucleotide sequence having at least 95% sequence identity based on the BLASTN method of alignment to the polynucleotide sequence set forth in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:33.
12 . The primer of claim 11 , wherein the primer comprises the polynucleotide sequence set forth in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:33.
13 . A probe/quencher pair comprising polynucleotide sequences having at least 95% sequence identity based on the BLASTN method of alignment to the polynucleotide sequences set forth in SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:15 and SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, SEQ ID NO:21 and SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25, or SEQ ID NO:24 and SEQ ID NO:26.
14 . The probe/quencher pair of claim 13 , wherein the probe/quencher pair comprises the polynucleotide nucleotide sequences set forth in SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:15 and SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, SEQ ID NO:21 and SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25, or SEQ ID NO:24 and SEQ ID NO:26.
15 . A Salmonella enteritidis or Salmonella typhimurium detection sequence comprising a polynucleotide sequence having at least 95% sequence identity based on the BLASTN method of alignment to the polynucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.
16 . The Salmonella enteritidis or Salmonella typhimurium detection sequence of claim 15 comprising the polynucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.
17 - 18 . (canceled)
19 . A replication composition for use in performance of PCR, comprising:
(a) a set of primer pairs selected from the group consisting of:
(i) one or more primer pairs comprising nucleic acid sequences comprising:
(A) SEQ ID NO:3 and SEQ ID NO:4;
(B) SEQ ID NO:28 and SEQ ID NO:29; and/or
(C) SEQ ID NO:30 and 31;
(ii) one or more primer pairs comprising nucleic acid sequences comprising:
(A) SEQ ID NO:7 and SEQ ID NO:8;
(B) SEQ ID NO:9 and SEQ ID NO:10;
(C) SEQ ID NO:11 and SEQ ID NO:12;
(D) SEQ ID NO:13 and SEQ ID NO:14; and/or
(E) SEQ ID NO:32 and SEQ ID NO:33; and
(iii) a combination thereof; and
(b) thermostable DNA polymerase.
20 . The replication composition of claim 19 further comprising at least one probe/quencher pair selected from the group consisting of SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:15 and SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, SEQ ID NO:21 and SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25, or SEQ ID NO:24 and SEQ ID NO:26, and a combination thereof.
21 . A kit for detection of Salmonella enteritidis in a sample, comprising the replication composition of claim 20 , wherein the kit comprises the primer pair of (a)(i) and the probe/quencher pair of SEQ ID NO:5 and SEQ ID NO:6.
22 . A kit for detection of Salmonella typhimurium in a sample, comprising the replication composition of claim 20 , wherein the kit comprises the one or more primer pairs of (a)(ii) and at least one probe/quencher pair selected from the group consisting of SEQ ID NO:15 and SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, SEQ ID NO:21 and SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25, or SEQ ID NO:24 and SEQ ID NO:26, and a combination thereof.
23 . A kit for the detection of Salmonella enteritidis and/or Salmonella typhimurium in a sample, comprising the replication composition of claim 20 , wherein the kit comprises the primer pair of (a)(i); the one or more primer pairs of (a)(ii); the probe/quencher pair of SEQ ID NO:5 and SEQ ID NO:6; and at least one probe/quencher pair selected from the group consisting of SEQ ID NO:15 and SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, SEQ ID NO:21 and SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25, or SEQ ID NO:24 and SEQ ID NO:26, and a combination thereof.