大肠埃希氏菌、金黄色葡萄球菌、沙门氏菌、溶藻弧菌、副溶血弧菌、铜绿假单胞菌、产气荚膜梭菌、单增李斯特菌和创伤弧菌是贝类食品中常见的食源性致病菌。该文通过设计特异性引物,优化反应参数以建立多重实时荧光串联PCR(multiplexed tandem PCR,MT-PCR)方法,进一步评估方法的特异性和灵敏度,并用于检测生蚝样品。优化后的MT-PCR方法与常规qPCR具有相同定量能力,可同时检测上述9种致病菌。在对混合菌液的检测中,MT-PCR方法的检测灵敏度最低可达102 CFU/mL,且此法对其中6种致病菌的检测灵敏度高于qPCR。应用该技术检测人工染菌的生蚝样品,检测灵敏度为102~103CFU/g。该研究建立的MT-PCR方法可实现高通量、快速准确地检测生蚝中这9种常见的食源性致病菌,对保障食品安全和评估食源性致病菌污染风险具有重要意义。
Escherichia coli, Staphylococcus aureus, Salmonella, Vibrio alginolyticus, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Clostridium perfringens, Listeria monocytogenes and Vibrio vulnificus are common foodborne pathogens in shellfish such as oyster. To provide technical support for rapid screening of foodborne pathogens, a multiplexed tandem polymerase chain reaction (MT-PCR) assay was developed. By designing specific primers and optimizing reaction parameters, the specificity and sensitivity of MT-PCR was further evaluated and applied in oyster samples. The optimized MT-PCR has equal quantitative ability as qPCR and can simultaneously detect nine pathogenic bacteria. The sensitivity of MT-PCR for detecting nine pathogens was less to 102 CFU/mL in mixed culture, especially, the sensitivity of MT-PCR was 10 times higher than qPCR for detecting E. coli, S. aureus, P. aeruginosa, C. perfringens, L. monocytogenes and V. vulnificus. The MT-PCR can detect nine target strains at 102-103 CFU/g in spiked oyster samples. The MT-PCR assay could achieve high-throughput, rapid and accurate detection of these nine pathogens in oyster samples, which is of great significance to ensure food safety and evaluate the risk of foodborne pathogens contamination.
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