防污染PCR检测食源性沙门菌方法的建立  

Establishment of a Contaminant-Preventing PCR for Detection of Foodborne Salmonella

作　　者：黄名英[1] 傅安静[1] 王远微[2] HUANG Ming-ying;FU An-jing;WANG Yuan-wei(Chengdu Agricultural College,Chengdu 611130,China;College of Animal&Veterinary Sciences,Southwest Minzu University,Chengdu 610041,China)

机构地区：[1]成都农业科技职业学院,四川成都611130 [2]西南民族大学畜牧兽医学院,四川成都610041

出　　处：《中国兽医杂志》2021年第12期37-43,共7页Chinese Journal of Veterinary Medicine

基　　金：中央高校基本科研业务费专项基金项目(2020NYB23);成都农业科技职业学院院级孵化项目(自然科学)(22ZR107)。

摘　　要：本试验基于尿嘧啶糖基化酶(UNG)能特异地识别并切割DNA分子中出现的尿嘧啶核苷的原理,以构建一种防污染的食源性沙门菌(Salmonella)PCR检测方法。经过酶量、抑制剂(UGI)用量、反应时间的优化,最终确立的防污染条件为:在PCR反应前,扩增体系所用的dNTP中以3倍浓度的dUTP代替dTTP;在阳性对照的反应体系中加入1.5U UNG、2U UGI以及含有尿嘧啶的DNA(U-DNA)阳性模板;其他检测样品的反应体系中加入1.5U UNG;50℃保温10 min,再25℃保温15 min,然后进行常规的PCR扩增。结果显示,即使在高浓度U-DNA污染的反应体系里(所有PCR扩增产物均为含有尿嘧啶的U-DNA),UNG也可有效地消除污染,且对PCR的特异性和敏感性无影响。因此,本试验建立的防污染PCR检测方法高效快速,操作简便,对提高食源性沙门菌检测的准确性以及防止假阳性的出现具有实际意义。This study aimed to develop a contaminant-preventing PCR for detection of Salmonella based on the ability of uridine-n-glycosylase(UNG)to specifically identify and eliminate uridine nucleosides in DNA.After optimization of the parameters related to enzyme and inhibitor quantity,and reaction time,parameters for a contaminant-preventing PCR included:three times of dUTP instead of dTTP was used in dNTP;1.5 unit UNG,2 unit UGI and U-DNA positive template containing uridine nucleosides were added to the positive control;and 1.5 unit UNG was added into the test sample;after keeping at 50℃for 10 min,and then at 25℃for 15 min,these agents were used in the PCR.The results showed that UNG effectively prevented PCR contamination by high concentration of U-DNA and had no adverse effect on PCR specificity and sensitivity.Thus,the contaminant-preventing PCR described in this study significantly improves the accuracy of foodborne Salmonella detection.

关 键 词：沙门菌 PCR 尿嘧啶糖基化酶 防污染 

分 类 号：S852.612[农业科学—基础兽医学]