3种食源性致病菌共增菌及三重PCR检测

Co-enrichment and triple PCR detection of three foodborne pathogens

目的建立沙门菌、金黄色葡萄球菌和副溶血性弧菌三重PCR检测方法。方法3株菌在本实验室自行研制的共增菌培养基(SSV培养基)混合培养16h,分别对沙门菌invA基因、金黄色葡萄球菌nuc基因、副溶血性弧菌collagenase基因设计特异性引物,建立三重PCR体系,通过对引物浓度、退火温度及循环次数进行优化,从而建立最优体系,并从特异性、重复性、灵敏性3个方面对体系进行评价。结果三重PCR体系中,invA、nuc、collagenase引物浓度分别为10、12.5和12.5 nmol/L;最佳退火温度为55.6℃;目标菌的引物特异性好,且体系的检测灵敏度高,3株菌在三重PCR体系中的灵敏度都在10^(2) CFU/mL以上。结论建立的三重PCR方法特异性强、灵敏度高、重复性好。

OBJECTIVE A triple PCR method was established for the detection of Salmonella enteritidis,Staphylococcus aureus and Vibrio parahaemolyticus.METHODS The three strains were mixed cultured in SSV enrichment medium for 16 h,specific primers were designed for Salmonellaenteritidis invA gene,Staphylococcus aureus nuc gene and Vibrio parahaemolyticus collagenase gene,respectively,and the triple PCR system was established.By optimizing the primer concentration,annealing temperature and cycle times of the triple PCR system,thus optimal triple PCR system was established.The system was evaluated from three aspects:specificity,repeatability and sensitivity.RESULTS The optimized result showed that,in the triple PCR system,the primers concentration of invA,nuc and collagenase were 10 nmol/L,12.5 nmol/L and 12.5 nmol/L,respectively.The optimum annealing temperature was 55.6℃.The primers of the target bacteria had good specificity,and the detection sensitivity of the system was high.The sensitivity of the three strains in the triple PCR system was above 10^(2) CFU/mL.CONCLUSION The established triple PCR method has high specificity,high sensitivity and good repeatability,which provided a new technical support for the rapid detection of Salmonella enteritidis,Staphylococcus aureus and Vibrio parahaemolyticus.