Method for real-time detection of Salmonella in food using a cleavable chimeric probe
AssigneeSamsung Techwin Co., Ltd.
InventorsJason Opdyke, Win Den Cheung, Jun Li
Life of this patent
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Abstract
A method is described for the real-time detection of Salmonella species in foods and on surfaces. Salmonella are enriched in media to increase their cell density prior to analysis. DNA is recovered by lysis in the presence of azide, proteinase K, and detergent. Real-time detection of Salmonella species is performed in a PCR reaction using gene specific primers and a cleavable chimeric fluorescent probe. The method also describes an internal control to confirm the efficiency of nucleic acid amplification and detection. The method is amenable to medium and high throughput analysis.
Claims54
A method for the real-time detection of Salmonella in a sample, comprising the steps of:
- a) providing a sample to be tested for the presence of a Salmonella invA gene target DNA;
- b) providing a pair of amplification primers that can anneal to the Salmonella invA gene target DNA, wherein the pair of amplification primers is the pair of primers of SEQ ID NOs:
- 1 and 2;
- c) providing a probe comprising a detectable label and DNA and RNA nucleic acid sequences that are substantially complementary to the Salmonella target DNA;
- d) amplifying a PCR fragment between the first and second amplification primers in the presence of an amplifying polymerase activity, amplification buffer;
- an RNAse H activity and the probe and under conditions where the RNA sequences within the probe can form a RNA:DNA heteroduplex with the complementary DNA sequences in the PCR fragment of the Salmonella target DNA;
- and
- e) detecting a real-time increase in the emission of a signal from the label on the probe, wherein the increase in signal indicates the presence of the Salmonella target DNA in the sample.
A method for the real-time detection of Salmonella in a sample, comprising the steps of:
- a) providing a sample to be tested for the presence of a Salmonella invA target RNA;
- b) providing a pair of forward and reverse amplification primers that can anneal to the Salmonella invA target nucleic acid sequence, wherein the pair of amplification primers is the pair of primers of SEQ ID NOs:
- 1 and 2;
- c) providing a probe comprising a detectable label and DNA and RNA nucleic acid sequences that are substantially complementary to the cDNA of the Salmonella target RNA;
- d) reverse transcribing the Salmonella invA target RNA in the presence of a reverse transcriptase activity and the reverse amplification primer to produce a target cDNA sequence;
- e) amplifying a PCR fragment between the forward and reverse amplification primers in the presence of the target cDNA sequence, an amplifying polymerase activity, an amplification buffer;
- an RNAse H activity and the probe under conditions where the RNA sequences within the probe can form a RNA:DNA heteroduplex with complementary sequences in the PCR fragment;
- and
- f) detecting a real-time increase in the emission of a signal from the label on the probe, wherein the increase in signal indicates the presence of the Salmonella target RNA in the sample.
A kit for the real-time detection of Salmonella in a sample, comprising:
- a) a pair of amplification primers that can anneal to a Salmonella invA target DNA sequence, wherein the pair of amplification primers is the pair of primers of SEQ ID NOs:
- 1 and 2;
- b) a probe comprising a detectable label and DNA and RNA nucleic acid sequences that are substantially complementary to the Salmonella invA target DNA sequence;
- c) an amplifying polymerase activity, and
- d) an RNAse H activity.
A kit for the real-time detection of Salmonella in a sample, comprising:
- a) a reverse transcriptase activity for the reverse transcription of a target Salmonella invA RNA sequence to produce a target cDNA sequence;
- b) a pair of amplification primers that can anneal to the target cDNA sequence, wherein the pair of amplification primers is the pair of primers of SEQ ID NOs:
- 1 and 2;
- c) an amplifying activity for the PCR amplification of the target cDNA sequence between the pair of amplification primers to produce a Salmonella invA PCR fragment;
- d) a probe comprising a detectable label and DNA and RNA nucleic acid sequences that are substantially complementary to DNA sequences within the PCR fragment;
- and
- e) an RNAse H activity.
Dependencies are inferred from the claim text.