Sequences and their use for detection of salmonella
This invention relates to a rapid method for detection of Salmonella in a sample 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. In certain embodiments, the method is employed to detect Salmonella in a food or water sample. The present invention further relates to isolated polynucleotides, replication compositions, and kits for carrying out the method of the present invention.
1. A method for detecting the presence of Salmonella in a sample, said sample comprising nucleic acids, said method comprising
(a) providing a reaction mixture comprising at least one primer and probe, wherein said primer is at least 11 nucleotides in length and said probe is at least 14 nucleotides in length, and wherein
(i) said primer comprises a nucleic acid sequence capable of selectively hybridizing at a Tm higher than 55° C. to the sequence of SEQ ID NO: 1 or a sequence fully complementary thereto, and said probe comprises a nucleic acid sequence capable of selectively hybridizing at a Tm higher than 55° C. to the sequence of SEQ ID NO: 2 or a sequence fully complementary thereto; or
(ii) said primer comprises a nucleic acid sequence capable of selectively hybridizing at a Tm higher than 55° C. to the nucleic acid sequence of SEQ ID NO: 3 or a sequence fully complementary thereto, and said probe comprises a nucleic acid sequence capable of selectively hybridizing at a Tm higher than 55° C. to the sequence of SEQ ID NO: 4 or a sequence fully complementary thereto; and
(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 said amplification indicates the presence of Salmonella in said sample.
2. The method of claim 1 , wherein said reaction mixture comprises both the primer and probe of (i) and the primer and probe of (ii).
3. The method of claim 1 , wherein said primer and probe each possess a 3′ terminus and a 5′ terminus, and wherein the 3′ terminus of said probe is directly or indirectly attached to the 5′ terminus of the primer, thereby forming a primer-probe complex.
4. The method of claim 3 , wherein said primer-probe complex comprises a detectable label.
5. The method of claim 1 , wherein said primer capable of selectively hybridizing to SEQ ID NO: 1 comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 40-54, and wherein said probe capable of selectively hybridizing to SEQ ID NO: 2 comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 6-39.
6. The method of claim 3 , wherein said primer-probe complex further comprises a 5′ Stem Sequence and a 3′ Stem Sequence, wherein the 3′ terminus of the 5′ Stem Sequence is directly or indirectly attached to the 5′ terminus of the probe, wherein the 5′ terminus of the 3′ Stem Sequence is directly or indirectly attached to the 3′ terminus of the probe, and wherein the 3′ terminus of the 3′ Stem Sequence is directly or indirectly attached of the 5′ terminus of the primer.
7. The method of claim 6 , wherein said 5′ Stem Sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 112-119, and wherein said 3′ Stem Sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 120-130.
8. The method of claim 1 , wherein said reaction mixture further comprises a quencher oligonucleotide capable of selectively hybridizing at a Tm higher than 55° C. to the probe.