重组酶聚合酶扩增检测产志贺毒素大肠埃希菌的微流控芯片技术

doi:10.7506/spkx1002-6630-20200323-349

食品科学 ›› 2021, Vol. 42 ›› Issue (10): 297-304.doi: 10.7506/spkx1002-6630-20200323-349

重组酶聚合酶扩增检测产志贺毒素大肠埃希菌的微流控芯片技术

范一灵，王淑娟，李琼琼，胡颖，宋明辉，秦峰，刘浩，杨美成

（1.国家药品监督管理局药品微生物检测技术重点实验室，上海市食品药品检验研究院，上海 201203；2.中国食品药品检定研究院，北京 102629；3.遵义医科大学公共卫生学院，贵州遵义 563006；4.复旦大学化学系，生物医学研究院，上海 200438）

出版日期:2021-05-25 发布日期:2021-06-02
基金资助:
“十三五”国家重点研发计划重点专项（2018YFC1603900）

Rapid Detection of Shiga Toxin-producing Escherichia coli by Recombinase Polymerase Amplification Combined with Centrifugal Compact Disc Microfluidic Chip

Fan Yiling, WANG Shujuan, LI Qiongqiong, HU Ying, SONG Minghui, Qin Feng, Liu Hao, YANG Meicheng

(1. Key Laboratory for Testing Technology of Pharmaceutical Microbiology, Shanghai Institute for Food and Drug Control, National Medical Products Administration, Shanghai 201203, China; 2. National Institutes for Food and Drug Control, Beijing 102629, China; 3. School of Public Health, Zunyi Medical University, Zunyi 563006, China; 4. Institutes of Biomedical Sciences, Department of Chemistry, Fudan University, Shanghai 200438, China)

Online:2021-05-25 Published:2021-06-02

摘要/Abstract

摘要： 采用重组酶聚合酶扩增技术（recombinase polymerase amplification，RPA），开发产志贺毒素大肠埃希菌（Shiga toxin-producing Escherichia coli，STEC）的等温检测方法，结合圆盘式微流控芯片快速检测目标微生物。以stx1和stx2基因为靶点，设计和筛选适宜的RPA检测引物和探针序列，验证了19 株STEC菌株（含9 种stx基因亚型）和21 株非STEC菌株，并采用人工污染的牛肉样品对集成化的微流控芯片进行评价。筛选出检测stx1和stx2基因的高特异性引物、探针组合，与美国农业部微生物实验室技术指南中STEC定量聚合酶链式反应（real-time polymerase chain reaction，real-time PCR）方法相比，目标基因检测的包容性和排他性均为100%。荧光RPA微流控芯片法在20 min内可同时进行32 个反应，可检测STEC菌体灵敏度为9.5×103 CFU/mL。根据GB 4789.6—2016《食品微生物学检验致泻大肠埃希氏菌检验》的规定经增菌培养后在人工污染牛肉样品中可检测1 CFU/25 g的STEC污染，方法的相对正确度和相对检出水平均为100%。本研究开发了一种荧光RPA微流控芯片检测方法，可检测常见stx基因亚型STEC菌株，操作简单、反应速度快，适用于STEC的高通量快速检测。

关键词: 重组酶聚合酶扩增；微流控芯片；产志贺毒素大肠埃希菌；stx基因；快速检测

Abstract: Recombinase polymerase amplification (RPA) combined with a centrifugal compact disc microfluidic chip system was used to develop a method for the detection of Shiga toxin-producing Escherichia coli (STEC). Specific primers and probes targeting the stx1 and stx2 genes were designed and evaluated. The efficiency of RPA was verified using 19 STEC strains including 9 stx gene subtypes and 21 non-STEC strains. The fluorescence RPA-integrating microfluidic chip was evaluated using artificially contaminated beef samples. The inclusivity and exclusivity of fluorescence RPA were 100% when compared to the real-time polymerase chain reaction (real-time PCR) from the Microbiology Laboratory Guidebook of USDA, namely the United States Department of Agriculture. The fluorescence RPA microfluidic chip method could conduct 32 reactions simultaneously within 20 min with a sensitivity of 9.5 × 103 CFU/mL for STEC strains. The STEC strains could be detected in artificially contaminated beef samples at an inoculation level of 1 CFU/25 g after enrichment culture by the GB 4789.6-2016 Food Microbiological Examination-Diarrheagenic Escherichia coli. The relative trueness and the relative detection level of the method were both 100%. The fluorescence RPA microfluidic chip method can detect 9 stx gene subtypes of STEC with simple operation and fast response, and it is suitable for rapid and high-throughput detection of STEC.

Key words: recombinase polymerase amplification; microfluidic chip; Shiga toxin-producing Escherichia coli; stx gene; rapid detection

中图分类号:

R917

范一灵，王淑娟，李琼琼，胡颖，宋明辉，秦峰，刘浩，杨美成. 重组酶聚合酶扩增检测产志贺毒素大肠埃希菌的微流控芯片技术[J]. 食品科学, 2021, 42(10): 297-304.

Fan Yiling, WANG Shujuan, LI Qiongqiong, HU Ying, SONG Minghui, Qin Feng, Liu Hao, YANG Meicheng. Rapid Detection of Shiga Toxin-producing Escherichia coli by Recombinase Polymerase Amplification Combined with Centrifugal Compact Disc Microfluidic Chip[J]. FOOD SCIENCE, 2021, 42(10): 297-304.

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重组酶聚合酶扩增检测产志贺毒素大肠埃希菌的微流控芯片技术

Rapid Detection of Shiga Toxin-producing Escherichia coli by Recombinase Polymerase Amplification Combined with Centrifugal Compact Disc Microfluidic Chip

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