[Rapid detection of Listeria monocytogenes in pork samples by real-time PCR with Taqman probe].

ABSTRACT

OBJECTIVE: To develop a real-time PCR method for detection Listeria monocytogenes in pork samples. METHODS: Listeria monocytogenes specific primers and Taqman probe were chosen on the basis of hlyA gene. Real-time PCR method was developed and its specificity was proved. Serial 10-fold diluted pure suspension culture of CMCC 540004 were detected by real-time PCR, and standard curve was constructed. Artificially contaminated experiment was done, six artificially-inoculated samples containing final concentration of Listeria monocytogenes CMCC 540004 (1.3 x 10(0), 1.3 x 10(1), 1.3 x 10(2), 1.3 x 10(3), 1.3 x 10(4), 1.3 x 10(5) and 1.3 x 10(6) CFU per 25 g pork samples) were preparated respectively, meanwhile one sample without inoculation was as control of background value. All the samples were incubated in LB1 enrichment for 24 h and then take 0.1 ml culture solutions to 10 ml LB2 enrichment for 18 - 24 h. All the samples were incubated for 0, 4, 8, 12, 18, 24, 30, 36 and 46 h, and detected Listeria monocytogenes bacteria by PCR, respectively. Twenty-four samples of retail pork were collected from markets in Beijing and detected by the above three methods. RESULTS: Real-time PCR method established was specific for the detection of Listeria monocytogenes. The sensitivity was 1.3 x 10(3) CFU/ml for pure culture without enrichment. Real-time PCR detection limit for artificially contaminated samples after enriching for 24 h was 1.3 CFU/ 25 g, which is the same with the limit of PCR and traditional method after enrichment for 46 h. Standard curve of sample after enrichment for 24 h was established. The positive rate out of total 24 samples was 70.83% (17/24) by real-time PCR, which is the same with the result of PCR and traditional method. The positive ones were quantitative analyzed using standard curve of sample and determined the initial Listeria monocytogenes numbers of CFU/25 g. CONCLUSION; The established real-time PCR technology was simple, rapid, sensitive and specific, which was suitable to rapid detect Listeria monocytogenes in pork samples and the process was finished in 27 h.