为建立能够同时检测食品中单核细胞增生李斯特氏菌、沙门氏菌属、克罗诺杆菌属、志贺氏菌属、金黄色葡萄球菌、副溶血性弧菌、大肠埃希氏菌O157:H7、铜绿假单胞菌、粪链球菌、产气荚膜梭菌等10种食源性致病菌的多重PCR方法,通过特异性引物的设计、引物特异性验证、引物灵敏度验证和多重PCR检测体系主要反应条件优化,建立多重PCR检测体系,并对其特异性、灵敏度以及人工感染样品应用进行评价。结果表明,建立的多重PCR检测体系可以扩增出10种食源性致病菌的特异目标条带,无非特异扩增。测序结果与目的基因序列进行比对,其序列同源性高达98%,体系灵敏度可达10-1 ng/μL。人工污染样品应用结果表明,采用多重PCR方法简单快速,仅需简单增菌,检出限可达10 CFU/mL,整个检测时间在48 h以内。建立的多重PCR检测体系特异性强、灵敏度较高,与检验机构实际检测工作联系紧密,具有推广应用价值。
To establish a multiplex PCR method for the simultaneous detection of 10 kinds of foodborne pathogens in food, including Listeria monocytogenes, Salmonella spp., Cronobacteria spp., Shigella spp., Staphylococcus aureus, Vibrio parahaemolyticus, Escherichia coli O157: H7, Pseudomonas aeruginosa, Streptococcus faecalis and Clostridium perfringens, the multiplex PCR detection system was established by specific primer design, primer specificity verification, primer sensitivity verification and optimization of the main reaction conditions. Furthermore, the specificity, sensitivity and application of artificially infected samples of the multiplex PCR system were evaluated. The results showed that the specific target bands of 10 foodborne pathogens could be amplified by the multiplex PCR system without nonspecific amplification. The sequencing results showed 98% homology with the target gene sequence, and the sensitivity of the system was up to 10-1 ng/μL. The application results of artificially contaminated samples showed that the multiplex PCR method was simple and rapid, requiring only simple multiplication, with a detection limit of up to 10 CFU/mL, and the detection time was less than 48 h. The multiplex PCR detection system have strong specificity and high sensitivity, which is closely related to actual detection work of inspection institutions, and have great promotion and application value.
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