一种可快速检测食品中酿脓链球菌和无乳链球菌的RPA方法的建立  

A Rapid Detection of Streptococcus pyogenes and Streptococcus agalactiae in Food Based on RPA Method

作　　者：蔡妙森 许少坚 兰全学 Cai Miaosen;Xu Shaojian;Lan Quanxue(Shenzhen Longhua District Center for Disease Control and Prevention,Shenzhen,518109;Shenzhen Longgang District Center for Disease Control and Prevention,Shenzhen,518172)

机构地区：[1]深圳市龙华区疾病预防控制中心,深圳518109 [2]深圳市龙岗区疾病预防控制中心,深圳518172

出　　处：《基因组学与应用生物学》2021年第2期663-670,共8页Genomics and Applied Biology

摘　　要：为了实现食品中酿脓链球菌(Streptococcus pyogenes)和无乳链球菌(S.agalactiae)快速、高效检测,本研究建立了一种同时快速检测食品中这两种细菌的方法。本研究基于重组酶聚合酶等温扩增技术(recombinase polymerase amplification,RPA)原理,选择酿脓链球菌致热外毒素B基因(speB基因)和无乳链球菌表面免疫蛋白基因(SIP基因)的保守序列分别设计RPA引物和探针,分析检测方法的特异性和灵敏性,采用模拟污染实验验证检测效果。结果表明,检测时间为20 min;酿脓链球菌和无乳链球菌分别对speB和SIP基因检测结果为阳性,其他链球菌和非链球菌检测结果为阴性;方法对speB和SIP基因的检出限均可达100copies/反应;酿脓链球菌和无乳链球菌低菌量污染的样品增菌液,在经过短暂几小时的增菌培养后可被检出阳性信号。本研究建立的检测方法快速、准确、灵敏,可同时作为食品中酿脓链球菌、无乳链球菌的快速检测方法,具有较强的应用价值。In order to achieve rapid and efficient simultaneous detection of Streptococcus pyogenes and S.agalactiae,this study developed a rapid and simultaneous method in food for testing the two bacteria mentioned above.Based on recombinase polymerase amplification(RPA)method,streptococcal thermogenic exotoxin B gene(speB gene)of S.pyogenes and surface immunoprotein gene(SIP gene)of S.agalactiae were selected to design primes and probes.The specificity and sensitivity of RPA system were verified,and its performance was evaluated by simulant contaminated samples tests.The results showed that the detection time was 20 minutes,that S.pyogenes and S.agalactiae strains were positive for speB or SIP genes,respectively,and the other kinds of bacteria were negative,that the low limit of detection for the two genes was both 100 copies per reaction,and that the positive single could be obtained from the food sample contaminated by small amount of S.pyogenes and S.agalactiae after enrichment for a few hours.The RPA assay in this study is a rapid,accurate and sensitive method for screening S.pyogenes and S.agalactiae in food samples and has a great application value.

关 键 词：酿脓链球菌 无乳链球菌 RPA 快速检测 

分 类 号：TS207.4[轻工技术与工程—食品科学]