Multiplex PCR Assay For Detection Of Four Bacterial Pathogens In Meat Sample

Food safety emergencies are high frequency in recent years. In these problems,the food poisoning result of Bacterial Pathogens was the most harmful. According to data came from hospitals and inspection-quarantine departments,the main bacterial pathogens were Staphylococcus aureus, Salmonella spp, Shigella spp, listeria monocytogenes and so on. Since contamination levels are generally low in foods, detection methods require lengthy culture enrichment steps to increase target bacterial numbers before isolation and identification using standard cultural procedures. These conventional food microbiological techniques often require several days to detect bacterial pathogens present in foods at low levels. With the advent of PCR, Individual PCR assays have been developed for detection and identification of the bacterial pathogens, but a large number of individual PCR assays would be necessary if single primer sets are used in separate reactions on a large number of food samples, which can be a relatively costly and time-consuming process. To reduce the number of tests needed for diagnosis of Individual PCR assays,the simultaneous detection of several pathogens with a multiplex PCR (m-PCR) approach would be relatively rapid and cost-effective.In this study, took use of Staphylococcus aureus of special sequences of conservative nuc gene, Salmonella spp ipaB gene, Shigella spp ipaH gene, listeria monocytogenes inlA gene, according to the result of BLAST to design four special primers. This m-PCR assay was developed for the simultaneous detection of Staphylococcus aureus, Salmonella spp, Shigella spp, and listeria monocytogenes from meat. In the PCR process, DNA concentration, Mg²⁺ concentration, template volume, annealing temperature and PCR circles are optimized to determine the optimal PCR. The reaction mixture consisted of 50μL: 5μL10×PCR buffer, 25 mmol/L Mg²⁺ 3.5μL,5.5μL mixture of dNTPs, 2μL of 10 uM forward primer (each) , 2μL of 10 uM reverse primer (each), 0.8μL (5UμL^-1) Taq enzyme, FTA cards as the template,ddH₂O 29.2μL . The reaction was run under the following conditions: Cool start; DNA pre-denaturation at 94℃for 5 min, DNA denaturation at 94℃for 1 min, primer annealing at 55.5℃for 45 s ,and DNA extention at 72℃for 1 min, run 35 cycles; the final extention was performed at 72℃for 5 min. The PCR products were examined by 1% agarose gel electrophoresis. PCR products were confirmed by DNA sequencing. The result of sequencing compared with the target gene sequence at gene bank showed that PCR amplified products were certified.There were 20 bacterial strains to be detected including 2 S. aureus standard strains, 4 Salmonella spp , 4 Shigella spp , 1 listeria monocytogenes and 9 other bacterial strains in order to determine specificity of amplification of primers. The results of 2 strains of Staphylococcus aureus, 4 Salmonella spp , 4 Shigella spp and 1 listeria monocytogenes were positive and those of 9 other strains were negative. The specificity of the m-PCR assay was evaluated by testing the four primer sets with the purified DNAs of all the strains (separately or in different combinations) indicated above. Positive PCR amplification of DNA templates from Staphylococcus aureus, Salmonella spp, Shigella spp, and listeria monocytogenes produced a single fragment, of the expected, for each pathogen. The four bacterial pathogens were simultaneously amplified.The sensitivity of m-PCR for the detection of one of four bacterial pathogens when using purified DNA of the bacterial type strains was 10¹cfu/ml for Staphylococcus aureus, Salmonella spp, Shigella spp, and listeria monocytogenes. The sensitivity of the simultaneous detection of the four bacterial pathogens with m-PCR when using purified DNA of the bacterial type strains was 10²cfu/ml for Staphylococcus aureus, 10²cfu/ml for Salmonella spp, 10¹cfu/ml for Shigella spp, and 10¹cfu/ml for listeria monocytogenes.In this study, real detection was made. The result indicates: the sensitivity of the m-PCR assay is 100%, the specificity is 95.5% for Staphylococcus aureus, 95.7% for Salmonella spp, 91.3% for Shigella spp, 94.1% for listeria monocytogenes. the coincidence rate is s 98% for Staphylococcus aureus, 98% for Salmonella spp, 96% for Shigella spp, 94 % for listeria monocytogenes.The bacterial pathogens DNA was directly extracted using FTA filter. The extracted DNA was suitable for m-PCR detection. The detection limit of the m-PCR assay for meat was 10²cfu/g for Staphylococcus aureus, 10²cfu/g for Salmonella spp, 10¹cfu/g for Shigella spp, and 10¹ cfu/g for listeria monocytogenes. The whole experimental procedure can be completed only 6 hours, shorter than the traditional biochemistry detection. The developed method in this assay allows detection of the pathogens in meat in less than 6h. This method is useful for the detection of Listeria monocytogenes in food.