Detection of stx1 and stx2 and subtyping of Shiga toxin-producing Escherichia coli using asymmetric PCR combined with lateral flow immunoassay

亚型 放大器 STX2 志贺毒素 大肠杆菌 聚合酶链反应 生物 微生物学 化学 基因 遗传学 计算机科学 程序设计语言
作者
Shan Shan,Yanmei Huang,Zhao-hong Huang,Zhong-er Long,Chengwei Li,Xuelong Zhao,Keyu Xing,Xiaoyue Xiao,Jintao Liu,Yunhong Huang,Weihua Lai,Daofeng Liu
出处
期刊:Food Control [Elsevier BV]
卷期号:126: 108051-108051 被引量:5
标识
DOI:10.1016/j.foodcont.2021.108051
摘要

Shiga toxin-producing Escherichia coli (STEC) is known as a kind of foodborne pathogens that can produce Shiga toxin type1, type 2, or both, encoded by stx1 and stx2 genes, respectively. Rapid and accurate detection of STEC and stx1/stx2 subtyping assay in food and environment is essential for foodborne outbreak investigation. In this study, a method for the accurate and rapid subtyping of STEC single colony using asymmetric polymerase chain reaction (asPCR) combined with lateral flow immunoassay (LFIA) was established. AsPCR was conducted using biotinylated stx1 and digoxin-labeled stx2 primers to obtain several single stranded DNA amplicons labeled with biotin or/and digoxin. Subsequently, these amplicons were tagged with specific probes labeled with carboxylate-modified red polystyrene microspheres using sequential hybridization. Finally, STEC with different Shiga toxin gene types could be distinguished depending on the appearance of different test lines on an LFIA strip. Thus, the STEC single colony could be identified and subtyped accurately and conveniently using this method. This method showed high specificity. Twenty-four strains of STEC in milk were detected and subtyped using our method, which combines asPCR with LFIA.
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