基于多重荧光定量PCR技术快速检测桶装水中大肠埃希氏菌和铜绿假单胞菌  

Rapid Detection of Escherichia coli and Pseudomonas aeruginosa in Barreled Water Based on Multiple Fluorescence Quantitative PCR

作　　者：龙慧[1] 帅淑芬 龙永艳[1] 王伟[1] 吴葵 钟淙[1] LONG Hui;SHUAI Shufen;LONG Yongyan;WANG Wei;WU Kui;ZHONG Zong(Nanchang Center for Disease Control and Prevention,Nanchang 330038,China)

机构地区：[1]南昌市疾病预防控制中心,江西南昌330038

出　　处：《现代食品》2022年第23期180-184,188,共6页Modern Food

基　　金：南昌市科技支撑计划项目(洪科字[2021]129号);江西省科技厅重点研发项目(20203BBG73064)。

摘　　要：目的:建立大肠埃希氏菌(Escherichia coli,E.coli)和铜绿假单胞菌(Pseudomonas aeruginosa,P.aeruginosa)多重荧光定量PCR检测方法,用于桶装水中E.coli和P.aeruginosa的同时检测。方法:根据国家生物技术信息中心(National Center for Biotechnology Information,NCBI)数据库中uid A和gyr B基因序列设计引物和探针,优化体系退火温度,构建多重荧光定量PCR检测体系,评价方法特异性、灵敏度、重复性和回收率,同时将该方法用于检测桶装水中的E.coli和P.aeruginosa。结果:多重荧光定量PCR能够同时检测E.coli和P.aeruginosa,方法检测周期短,从核酸提取到完成PCR扩增只需要2.0 h。方法检测灵敏度高,E.coli和P.aeruginosa的检测限均为102CFU·mL^(-1),且扩增效率高、线性范围宽、标准曲线具有良好的线性相关性;组内重复性好,16组扩增数据的变异系数在0.58%~2.34%,均小于5%;方法特异性强,能够准确区分检测各种非目标菌,且引物和探针之间不存在交叉扩增;对高浓度目标菌回收率均在70%以上,且能够用于桶装水目标菌的加标检测。结论:该方法检测性能优越,能够同时、快速、灵敏、准确的检测桶装水中的E.coli和P.aeruginosa。Objective:To establish a multiplex fluorescence quantitative PCR(Polymerase chain reaction,PCR)method for the simultaneous detection of Escherichia coli and Pseudomona aeruginosa in barreled water.Methods:Design primers and probes according to the uidA and gyrB gene sequences in the NCBI database,and then optimize the annealing temperature to construct a multiplex fluorescence quantitative PCR detection system.Evaluate the specificity,sensitivity,repeatability and recovery rate of the method,and use the method to detect E.coli and P.aeruginosa in barreled water.Result:Multiplex fluorescence quantitative PCR can simultaneously detect E.coli and P.aeruginosa.This method has a short detection cycle,and it only takes 2.0 hours from nucleic acid extraction to completion of PCR amplification.The method has high detection sensitivity,the detection limits of E.coli and P.aeruginosa are both 10~2 CFU·mL^(-1),and the amplification efficiency is high,the linear range was wide,and the standard curve has good linear correlation;The repeatability within the group was good,the coefficient of variation of the 16 groups of amplification data was between 0.58%~2.34%,all less than 5%;The method has strong specificity,can accurately distinguish and detect various non-target bacteria,and there was no cross-amplification between primers and probes;the recovery rate of high-concentration target bacteria was more than 70%,and it can be used for the detection of target bacteria in barreled water.Conclusion:This method has superior detection performance and can simultaneously,quickly,sensitively and accurately detect E.coli and P.aeruginosa in barreled water.

关 键 词：桶装水 大肠埃希氏菌 铜绿假单胞菌 多重检测 

分 类 号：R123.1[医药卫生—环境卫生学]