基于DPO引物构建的六重PCR检测食品中常见致病菌的方法研究  

Study on multiplex-PCR for detecting 6 kinds of common pathogenic bacteria with DPO system

作　　者：沈飚 王忠发 胡兴娟 杨赛军 

机构地区：[1]舟山出入境检验检疫局,浙江舟山316021

出　　处：《中国卫生检验杂志》2015年第22期3855-3857,3860,共4页Chinese Journal of Health Laboratory Technology

基　　金：国家质检总局科技计划项目(2014IK278)

摘　　要：目的基于DPO引物在多重PCR检测中的优点,构建一种六重DPO-PCR方法用于检测食品及食源性疾病样本中常见的6种致病菌。方法以霍乱弧菌的vcc、志贺菌的ipa H、单增李斯特菌的iap、副溶血性弧菌的gyr B、金黄色葡萄球菌的nuc以及沙门菌的omp C为靶基因,设计6对DPO引物,经过反应体系优化,建立了六重DPO-PCR方法。结果六重PCR反应体系内部DPO引物之间干扰小;扩增后除靶基因外的其他15种细菌DNA无扩增条带出现;对6种致病菌的最低检出限分别为:霍乱弧菌,220 cfu/ml;志贺菌,150 cfu/ml;单增李斯特菌,190 cfu/ml;副溶血性弧菌,190 cfu/ml;金黄色葡萄球菌,700 cfu/ml;沙门菌,960 cfu/ml。结论用DPO引物构建的六重PCR方法具有特异性强,退火温度范围宽,引物之间干扰小,电泳图谱背景清晰,可以达到一管6检的目的,为食品及食源性疾病样本快速准确检测提供了一种新的高通量的快速检测方法。Objective According to advantage of the primers DPO system, a multiplex - DPO - PCR system for detecting 6 kinds of common pathogenic bacteria in food and food - borne disease samples were established. Methods Six pairs of DPO primers were designed to target the species ＇ specific gene including vcc gene of Vibrio cholerae for the multiplex PCR assay. Af- ter the optimization of the reaction system, six DPO - PCR method was established. Results Little interference among DPO primers within multiplex -PCR system. There were also no banding or belt in the DNA of other 15 bacterial out of the target gene. The lowest detection limits of the method for the 6 pathogen were 220 cfu/ml （ Vibrio cholerae）, 150 cfu/ml （ ipaH Shigella ） , 190 cfu/ml（ iap Listeria monocytogenes ） , 190 cfu/ml（gyrB Vibrio parahemolyticus ） , 700 cfu/ml（ nuc Staphylococcus au- reus）, 960 cfu/ml（ompC Salmonella）, respectively. Conclusion This assay had advantage of high specificity, wide range of annealing temperature, little interference between primers, with clear gel electrophoresis background, so as to finish 6 test in one, which provides a high - throughput and fast detection method for food and food - borne diseases sample.

关 键 词：DPO引物 多重PCR 常见致病菌 食源性疾病 

分 类 号：R446.5[医药卫生—诊断学]