关键词: 多重PCR, 沙门氏菌, 单增李斯特菌, 金黄色葡萄球菌, 副溶血性弧菌

Abstract: A multiplex PCR method was presented to detect Salmonella, Listeria monocytogenes, Staphylococcus aureus and Vibrio parahaemolyticus from Penaeus vannamei, which are main food-borne pathogens. Specific primers were designed according to the invasion protein A gene (invA), invasion associated protein gene (iap), thermo-stable nuclease gene (nuc) and thermo-stable direct hemolysin gene (tdh) from Salmonella, L. monocytogenes, S. aureus and V. parahaemolyticus, respectively. A fragment of conserved sequence of 16S rRNA gene was used as the internal control of amplifiable bacterial DNA. The optimal PCR reaction system was identified as a 20-μL mixture composed of 10 × PCR buffer (+Mg2+) 2.0μL, dNTP 200μmol/L, DNA template 1μL and Taq DNA polymerase 2.5 U, with primer concentrations of 200 nmol/L for 16S rRNA, 250 nmol/L for tdh, 300 nmol/L for nuc, 350 nmol/L for iap and 250 nmol/L for invA, respectively. One reaction cycle consisted of denaturation at 94 ℃ for 30 s, annealing at 57 ℃for 40 s, and extension at 72 ℃ for 1 min followed by 10 min, and this cycle was repeated 30 times. The method was validated using Penaeus vannamei artificially inoculated with tested pathogens. The results showed that various target DNA fragments could be amplified by the multiplex PCR method in both non-enriched and pre-enriched samples. However, the detection sensitivity was significantly increased by enrichment at 37 ℃. Thus, the multiplex PCR method developed in this study offers an efficient and rapid way for highly sensitive detection of pathogens in aquatic food products.

Key words: multiplex PCR, Salmonella, Listeria monocytogenes, Staphylococcus aureus, Vibrio parahaemolyticus