基于探针熔解曲线分析的多重PCR同步鉴定6种腹泻病原菌

Simultaneous identification of 6 diarrhea pathogens based on probe melting curve analysis and multiple

目的基于多重聚合酶链反应(PCR)和探针熔解曲线分析技术,建立可同时鉴定6种常见急性感染性腹泻病原菌的高灵敏度诊断方法,并验证其检测性能。方法采用熔解曲线分析6种常见急性感染性腹泻病原菌熔解温度(Tm)值,建立基于探针熔解曲线分析的多重PCR检测方法,并评价其灵敏度、特异性和重复性。采用该方法检测175例疑似感染性腹泻患者粪便样本,比较该方法鉴定结果与细菌培养法和荧光PCR法鉴定结果的差异,不一致结果通过测序进行确认。结果建立的多重PCR检测方法可实现单次PCR同步检测6种肠道病原菌,检测限为1×10^(3)~1×10^(4)拷贝·mL^(-1),不同病原菌间无交叉反应,特异性高。175例腹泻患者粪便样本中共检出91例(52%)阳性样本。6种病原菌的检测内变异系数(CV)均<0.1%。与细菌培养法相比,该方法检测沙门菌、志贺菌的符合率分别为94.85%(kappa=0.894)、100.00%(kappa=1.000);与荧光PCR相比,该方法检测艰难梭菌的符合率为98.28%(kappa=0.900)。结论建立的基于探针熔解曲线分析的多重PCR方法同时鉴定6种腹泻病原菌具有较高的灵敏度和特异性,可为急性感染性腹泻的临床诊断及时提供实验室依据。

Objective To establish a rapid,accurate and highly sensitive diagnostic method for simultaneous identification of 6 common acute infectious diarrhea pathogens based on multiple polymerase chain reaction(PCR)and probe melting curve analysis,and to verify its determination performance.Methods The melting temperature(Tm)values of 6 common acute infectious diarrhea pathogens were analyzed by probe melting curve analysis,and the multiple PCR combined probe melting curve analysis system was established.The sensitivity,specificity and repeatability were evaluated.The method was used to determine fecal samples from 175 patients with suspected infectious diarrhea.The difference between the method results and the results of bacterial culture and fluorescent PCR was compared.The inconsistent results were confirmed by sequencing.Results The established method could determine 6 kinds of pathogens simultaneously by single PCR,the determination limit was 1×10^(3)-1×10^(4) copies/mL,and there was no cross-reaction among different pathogens.The specificity was high.A total of 91(52%)fecal samples from 175 diarrhea patients were positive.The coefficients of variation(CV)within determination for 6 pathogens were<0.1%.The consistency rates of Salmonella,Shigella and Clostridium difficile were 94.85%(kappa=0.894),100.00%(kappa=1.000)and 98.28%(kappa=0.900),respectively.Conclusions The established multiple PCR based on probe melting curve analysis has high sensitivity and specificity for determining 6 kinds of diarrhea pathogens,which can provide a reference for clinical diagnosis of acute infectious diarrhea in time.