4种食源性致病菌多重PCR检测体系的建立及其在乳品检测中的应用

Establishment and application of a multiplex PCR assay for detection of four foodborne pathogenic bacteria in dairy products

食源性致病菌是通过食物进入人体引起食物中毒和诱发疾病的有害微生物,严重威胁着人类健康和环境安全,为了预防和控制食源性疾病的发生,有效对策之一是对食品中的病原微生物进行安全检测。本研究采用一种多重聚合酶链式反应法(multiplex polymerase chain reaction,MPCR)快速检测乳品中4种食源性致病菌,结果表明,选取单核细胞增生李斯特氏菌prfA基因、蜡样芽胞杆菌gyrB基因、鼠伤寒沙门氏菌invA基因、大肠埃希氏菌O157∶H7 stx2A基因的保守序列设计4对引物,在优化的PCR反应体系和退火温度58℃下,PCR扩增表现出良好的特异性,分别扩增出274、221、482、108 bp条带,无非特异性扩增,4种病原菌检出限达到10~100 CFU/mL;对17份人工染菌牛奶样品进行检测,检出结果与国标培养法完全一致。该研究结果为快速、高效、准确地检测出乳品中单核细胞增生李斯特氏菌、蜡样芽胞杆菌、鼠伤寒沙门氏菌、大肠埃希氏菌O157∶H7提供了一种实用方法,也为其他食源性致病菌的快速检测提供了思路。

Food-borne pathogens are harmful bacteria that can cause food poisoning and induce diseases inside the body,and seriously threaten human health and environmental safety.In order to prevent and control the occurrences of food-borne diseases,the effective countermeasure is to carry out safety detection of pathogenic bacteria in foods.In this paper,a multiplex polymerase chain reaction(MPCR)method was used to rapidly detect four food-borne pathogenic bacteria in dairy products.The results showed the four bands of 274,221,482 and 108 bp were amplified without non-specific amplification,optimized PCR reaction and annealing temperature of 58℃,using the four pairs of primers that were designed according to the conserved sequences of the prfA gene of Listeria monocytogenes,the gyrB gene of Bacillus cereus,the invA gene of Salmonella,and the stx2A gene of Escherichia coli O157∶H7 respectively..The minimum detection limits of the four pathogens reached up to 101-102 CFU/mL,respectively.The detection results of the MPCR method for 17 artificial milk samples contaminated with pathogens were completely consistent with those of the national standard method.It provided a practical method for the rapid,efficient and accurate detection of Listeria monocytogenes,Bacillus cereus,Salmonella and Escherichia coli O157∶H7 in dairy products and a good idea for the rapid detection of other pathogenic bacteria.