食品与发酵工业 >

2021 , Vol. 47 >Issue 2: 241 - 246

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.024936

分析与检测

二重环介导等温扩增法快速检测乳粉中沙门氏菌和金黄色葡萄球菌

  • 徐文文 ,
  • 梁玉林 ,
  • 王云霞 ,
  • 刘秀 ,
  • 尹建军 ,
  • 周广军 ,
  • 宋全厚 ,
  • 丁梦璇 ,
  • 周鹏飞
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  • 1(中国食品发酵工业研究院有限公司,北京,100015)
    2(内蒙古伊利实业集团股份有限公司,内蒙古 呼和浩特,010110)
    3(山东稻香村食品工业有限公司,山东 菏泽,274100)
硕士研究生(刘秀高级工程师为通讯作者,E-mail:xiuliu1979@163.com)

收稿日期: 2020-07-03

  修回日期: 2020-09-01

  网络出版日期: 2021-02-07

基金资助

国家重点研发计划(2019YFC1606502)

收起

Rapid detection of Salmonella and Staphylococcus aureus in milk powder by double loop-mediated isothermal amplification

  • XU Wenwen ,
  • LIANG Yulin ,
  • WANG Yunxia ,
  • LIU Xiu ,
  • YIN Jianjun ,
  • ZHOU Guangjun ,
  • SONG Quanhou ,
  • DING Mengxuan ,
  • ZHOU Pengfei
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  • 1(China National Research Institute of Food & Fermentation industries Co.,Ltd.,Beijing 100015,China)
    2(Inner Mongolia Yili Industrial Group Limited by Share Ltd.,Hohhot 010110,China)
    3(Shandong daoxiangcun Food Industry Co.,Ltd.,Heze 274100,China)

Received date: 2020-07-03

  Revised date: 2020-09-01

  Online published: 2021-02-07

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摘要

为建立能够同时检测乳粉中沙门氏菌和金黄色葡萄球菌的二重环介导等温扩增(loop-mediated isothermal amplification,LAMP)方法。针对沙门氏菌invA基因、金黄色葡萄球菌nuc基因保守区域设计LAMP引物,优化引物浓度,并通过熔解曲线分析判断扩增靶标来源,建立同时检测沙门氏菌和金黄色葡萄球菌的二重LAMP检测方法。结果表明,当沙门氏菌与金黄色葡萄球菌引物浓度比为3∶1时,建立的二重检测体系扩增效率最佳;该方法特异性强、灵敏度高,对6株目标菌株和9株非目标菌株进行检测,未产生任何假阳性和假阴性结果,对2种食源性致病菌的检测灵敏度均达到102 fg/μL;根据熔解曲线中的退火温度可准确判断目标菌株,实现不同菌株的有效区分。建立的二重LAMP方法可用于乳制品企业大规模乳粉样品中沙门氏菌和金黄色葡萄球菌的现场快速筛查。

关键词： 等温扩增; 沙门氏菌; 金黄色葡萄球菌; 熔解曲线; 快速检测

本文引用格式

徐文文 , 梁玉林 , 王云霞 , 刘秀 , 尹建军 , 周广军 , 宋全厚 , 丁梦璇 , 周鹏飞 . 二重环介导等温扩增法快速检测乳粉中沙门氏菌和金黄色葡萄球菌[J]. 食品与发酵工业, 2021 , 47(2) : 241 -246 . DOI: 10.13995/j.cnki.11-1802/ts.024936

Abstract

The aim of this research is to establish a loop-mediated isothermal amplification (LAMP) method that can simultaneously detect Salmonella and Staphylococcus aureus in milk powder.LAMP primers were designed based on the conserved region of invA gene in Salmonella and the nuc gene in S.aureus.After primer concentration optimization and melting curve analysis to determine the source of amplification target,a double LAMP detection method for simultaneous detection of Salmonella and S.aureus was established.The results showed that when the primer concentration ratio of Salmonella to S.aureus was 3∶1,the established double detection system had the best amplification efficiency.The detection system had good specificity and high sensitivity.Six target strains and nine non-target strains were tested,resulting in no false positive and false negative result.The sensitivity of the two foodborne pathogens reached 102 fg/μL.In addition,according to the annealing temperature in the melting curve,the target strain can be accurately judged to complete the strain differentiation.The established detection method can be used for rapid on-site screening of Salmonella and S.aureus in milk powder samples of dairy companies.

Key words： isothermal amplification; Salmonella; Staphylococcus aureus; melting curve; rapid detection

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