食品中克罗诺杆菌(原阪崎肠杆菌)双重PCR检测方法研究  

Duplex PCR assay against Cronobacter spp.(Enterobacteria sakazakii) in food samples

作　　者：王翔[1] 徐幸莲[1] 祝长青[1,2] 周光宏[1] 

机构地区：[1]南京农业大学肉品加工与质量控制教育部重点实验室,江苏南京210095 [2]江苏出入境检验检疫局,江苏南京210001

出　　处：《南京农业大学学报》2012年第1期113-118,共6页Journal of Nanjing Agricultural University

基　　金：科技部国际科技合作项目(2009DFA31770);农业部转基因生物新品种培育重大专项(2008ZX08011-004)

摘　　要：根据克罗诺杆菌(Cronobacter spp.)16S rRNA基因以及局部大分子合成(MMS)操纵子特异序列设计2对引物,经反应体系和条件优化,建立了双重PCR检测方法。特异性检测结果显示,Cronobacter spp.菌株PCR扩增均可见2条特异性条带,而其他菌株PCR扩增均为阴性。纯菌检测双重PCR的灵敏度为6.3×103 CFU.mL-1,而相对应单重PCR的灵敏度分别为6.3×101 CFU.mL-1和6.3×103 CFU.mL-1;人工污染的食品样品(奶粉、牛奶、鸡肉)在经过24 h增菌后,检测限均可达100 CFU.mL-1或100 CFU.g-1。在鼠伤寒沙门氏菌(Salmonella typhimurium)存在的条件下,双重PCR的检测限没有受到影响。表明本试验建立的双重PCR检测方法具有很好的特异性和灵敏度,能克服食品样品基质及杂菌的干扰,可应用于食品中Cronobacter spp.的检测。Duplex PCR assays were developed for the detection of Cronobacter spp.based on its 16S rRNA gene and partial macromolecular synthesis operon.Among all the bacterial strains used in this study,duplex PCR amplified two specific products from 4 strains of Cronobacter spp.but not from others.In pure culture,the sensitivity of single PCR for former gene was 6.3×101 CFU·mL-1,while for the latter was 6.3×103 CFU·mL-1;whereas for duplex PCR,it was 6.3×103 CFU·mL-1.The detection limit for duplex PCR assays was 100 CFU·mL-1 or 100 CFU·g-1 for different levels of Cronobacter spp.inoculated into food samples（milk powder,milk and chicken meat）after 24 h enrichment.The detection limit of the duplex PCR,however,remained unaffected in the presence of Salmonella typhimurium in milk powder.So,the methods described in our study can be adopted to detect Cronobacter spp.in food samples with higher sensitivity and specificity.

关 键 词：克罗诺杆菌(原阪崎肠杆菌) 双重PCR 食品检测 

分 类 号：TS207.4[轻工技术与工程—食品科学] R155.5[轻工技术与工程—食品科学与工程]