产志贺毒素大肠杆菌O26多重双启动寡核苷酸引物-PCR检测方法的建立  

Establishment of Multiplex DPO-PCR Method for Detection of Shiga Toxin-producing Escherichia coli O26

作　　者：杨广珠 张淑红[1] 黄远斌 吴清平[1] 张菊梅[1] YANG Guang-zhu;ZHANG Shu-hong;HUANG Yuan-bin;WU Qing-ping;ZHANG Ju-mei(State Key Laboratory of Applied Microbiology Southern China,Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application,Guangdong Open Laboratory of Applied Microbiology,Guangdong Institute of Microbiology,Guangdong Academy of Sciences,Guangzhou 510070,China)

机构地区：[1]华南应用微生物国家重点实验室,广东省菌种保藏与应用重点实验室,广东省微生物应用新技术公共实验室,广东省微生物研究所,广东省科学院,广东广州510070

出　　处：《现代食品科技》2020年第4期290-295,24,共7页Modern Food Science and Technology

基　　金：国家重点研发计划专项(2018YFD0400901);广东省科技计划项目(2017B090904004);广州市科技计划项目(201704020089);广东省科学院实施创新驱动发展能力建设专项(2019GDASYL-0201001)。

摘　　要：为建立一种三重DPO-PCR方法用于食品样品中的产志贺毒素大肠杆菌O26的检测。以志贺毒素stx1和stx2、O抗原基因wzx O26特异性和实际检测效果,设计引物,构建三重DPO-PCR反应体系,进行特异性、灵敏度、模拟样品验证和实际样品验证。结果表明,三对DPO引物对退火温度不敏感,在49~69℃之间均能发生扩增,且引物之间干扰较小,具有较高的特异性,除目的基因外非目标细菌均无扩增条带出现,纯菌灵敏度检测表明,三重DPO-PCR方法对O26的最低检测限为3.8×10^3 cfu/g。在模拟样品和实际样品中具有良好的检测效果。本研究基于DPO引物构建的三重DPO-PCR方法具有效率高,特异性强,不受退火温度限制等优点,可用于食品样品中产志贺毒素大肠杆菌O26的快速准确检测提供一种高效的辅助检测方法。In order to develop a triple DPO-PCR assay for the detection of Shiga toxin-producing Escherichia coli O26 in foods based DPO primers, three sets of DPO primers were designed according to shiga toxin stx1, stx2 and O antigen wzx O26 genes. Their specificity, sensitivity and detection effect were evaluated. The results showed that an optimized system was established, which showed a high specificity and sensitivity. The amplification could product at 49~69 ℃. The detection limit of this assay for Escherichia coli O26 was 3.8×10^3 cfu/g. Further, this method demonstrated good detection results in artifical contaminated samples and natural food samples. The triple DPO-PCR assay in this work based on DPO primers possessed highly advantages of high efficiency, specificity and wide annealing temperature range, which can provide a reliable and efficient molecule assay for the rapid and accurate detection of Shiga toxin-producing E. coli O26 in food samples method.

关 键 词：产志贺毒素大肠杆菌O26 DPO引物 三重PCR 

分 类 号：TS207.5[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]