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

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。

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

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.