3种致病菌多重real-time PCR检测方法的建立及其在散装即食肉制品中的应用  

Establishment and Application of Multiple Real-time PCR Method for Detection of Three Kinds of Pathogenic Bacteria in Bulk Ready-to-eat Meat Products

作　　者：范维[1] 高晓月[1] 李贺楠[1] 董雨馨 李宇轩 刘虹宇 郭文萍[1] FAN Wei;GAO Xiaoyue;LI Henan;DONG Yuxin;LI Yuxuan;LIU Hongyu;GUO Wenping(China Meat Research Center,Beijing Academy of Food Sciences,Beijing 100068,China)

机构地区：[1]中国肉类食品综合研究中心,北京食品科学研究院,北京100068

出　　处：《食品科学》2022年第2期332-338,共7页Food Science

基　　金：“十三五”国家重点研发计划重点专项(2018YFC1603400);北京市优秀人才培养资助青年骨干个人项目(2017754154700G101)。

摘　　要：建立一种同时快速检测沙门氏菌、金黄色葡萄球菌和蜡样芽孢杆菌的三重实时聚合酶链式反应(realtime polymerase chain reaction,real-time PCR)方法。以沙门氏菌invA基因、金黄色葡萄球菌Sa442基因和蜡样芽孢杆菌Cereolysin AB基因为靶基因设计引物和TaqMan探针,建立多重real-time PCR方法,对该方法进行方法学验证,并对实际的散装即食肉制品样品进行检测。结果表明,该方法特异性强、灵敏度高、重复性好。对15株非目标菌进行检测,结果均为阴性;3种致病菌的定量线性浓度范围为10^(2)~10^(8) CFU/mL,且定量检测批内和批间的变异系数均小于2%;沙门氏菌、金黄色葡萄球菌和蜡样芽孢杆菌在未增菌的即食肉制品样品中检出限分别为3.8×10^(2)、4.9×10^(2) CFU/mL和5.7×10^(2) CFU/mL,经增菌5 h后,检出限提高到3.8、4.9 CFU/mL和5.7 CFU/mL。对100份实际样品进行检测,结果与标准方法一致,说明建立的多重real-time PCR法可以用于散装即食肉制品中3种致病菌的检测。A triplex real-time polymerase chain reaction(real-time PCR)method for rapid simultaneous detection of Salmonella(SAL),Staphylococcus aureus(SA)and Bacillus cereus(BC)was established.Primers and TaqMan probes were designed targeting the invA gene of SAL,the Sa442 gene of SA,and the Cereolysin AB gene of BC.The performance indicators of the developed method were evaluated,and actual samples of ready-to-eat meat products were detected by it.The results showed that the real-time PCR method was highly reproducible,specific and sensitive.Fifteen non-target reference strains were tested by this method and the results were all negative.The standard curves for the three pathogenic bacteria were linear over the concentration range of 10^(2)–10^(8) CFU/mL,and the intra-assay and inter-assay coefficients of variation(CVs)were less than 2%in the quantitative tests.Without bacterial enrichment,the limits of detection(LOD)for SAL,SA,and BC were 3.8×10^(2),4.9×10^(2),and 5.7×10^(2) CFU/mL,respectively.After 5 h enrichment,they increased to 3.8,4.9,and 5.7 CFU/mL,respectively.The results of the real-time PCR method for 100 actual samples were consistent with those of the national standard method,indicating that the real-time PCR method can be used for the detection of the three pathogenic bacteria in bulk ready-to-eat meat products.

关 键 词：沙门氏菌 金黄色葡萄球菌 蜡样芽孢杆菌 多重实时聚合酶链式反应 即食肉制品 

分 类 号：TS251.7[轻工技术与工程—农产品加工及贮藏工程]