Rapid enumeration of Listeria monocytogenes in pork meat using competitive PCR

초록

Competitive polymerase chain reaction (cPCR) was used to develop a direct enumeration method of Listeria monocytogenes in pork meat. Pork meat was artificially inoculated with L. monocytogenes and DNA was extracted using guanidine thiocyanate-phenol-chloroform and subjected to PCR amplification. Sixteen primer sets for L. monocytogenes hlyA gene were tested for sensitive detection and the DG69/DG74 primer set was selected. The detection limit achieved with this primer set was as low as 860 colony-forming units (cfu) per 0.1 g of pork meat. When the samples were cultured at 30 degrees C for 16 hr in Brain Heart Infusion (BHI) medium, even a single bacterium could be detected with this primer set by PCR. For cPCR, the hlyA gene, which features a 148 bp-deletion, was cloned in the pGEM-4Z vector. A known amount of competitor DNA which has the same primer binding sites was co-amplified with L. monocytogenes total DNA from the artificially inoculated pork meat. The cell-number determined by cPCR was approximately equal to cfu from the Most Probable Number (MPN) method. The whole procedure took only 5 hr.

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