分型鉴别HPIV1、HPIV2及HPIV3多重荧光定量RT-PCR方法的建立及验证

Development and verification of multiplex real-time RT-PCR assay for classing and identification of HPIV1,HPIV2 and HPIV3

目的建立可同时鉴别人副流感病毒1型(human parainfluenza virus type 1,HPIV1)、2型(HPIV2)、3型(HPIV3)的多重荧光定量RT-PCR方法,并进行验证。方法通过数据库下载HPIV1、HPIV2和HPIV3全基因组序列进行比对分析,选择保守区域,针对这3种病毒分别设计特异性引物及探针,以人核糖核酸酶P(RNase P)为内质控,建立多重荧光定量RT-PCR检测方法,并对方法的灵敏度、特异性和精密度进行验证。采用建立的方法对192份临床样本进行检测。结果经优化后,确定多重荧光定量RT-PCR反应体系为30μL,其中10×NeoscriptRTase/UNG Multi mix 3μL,5×Neoscript RT Premix Multi Buffer 6μL,HPIV1、HPIV2、HPIV3(100μmol/L)上下游引物各0.1μL,HPIV1、HPIV2、HPIV3探针(100μmol/L)各0.05μL,RNase P上下游引物(50μmol/L)各0.06μL,RNase P探针(50μmol/L)各0.03μL,模板15μL,ddH2O补至30μL。反应条件:50℃20 min,95℃3 min;95℃15 s,54℃30 s,共45个循环,每个循环退火时收集荧光信号。建立的多重荧光定量RT-PCR方法对HPIV1、HPIV2和HPIV3的最低检测限均达到500 copies/mL;与甲型流感病毒、乙型流感病毒、呼吸道合胞病毒、新型冠状病毒无交叉反应;3种不同浓度的重组质粒标准品混合物组内和组间变异系数(CV)均小于3%。192份临床样本中,HPIV1、HPIV2和HPIV3均检测出,阳性率分别为7.81%、0.05%和3.1%。结论本研究建立的HPIV1、HPIV2和HPIV3多重荧光定量RT-PCR检测方法具有良好的灵敏度、特异性和精密度,在HPIV的快速诊断和鉴别领域具有较高的临床应用前景。

Objective To develop and verify a multiplex real-time RT-PCR assay for simultaneous identification of human parainfluenza virus type 1(HPIV1),type 2(HPIV2)and type 3(HPIV3).MethodsThe whole genome sequences of HPIV1,HPIV2 and HPIV3 were downloaded from the database for alignment analysis,and the conserved regions were selected.Specific primers and probes were designed for the three viruses respectively to develop a multiplex real-time RTPCR assay with human ribonuclease P(RNase P)as theinternal quality control.The method was verified for the sensitivity,specificity and precision and used to detect 192 clinical samples.ResultsAfter optimization,the multiplex real-time RTPCR reaction system was determined to be 30μL,including 10×NeoscriptRTase/UNG Multi mix 3μL,5×Neoscript RT Premix Multi Buffer 6μL,upstream and downstream primers of HPIV1,HPIV2 and HPIV3(100μmol/L)0.1μL respectively,HPIV1,HPIV2,HPIV3 probes(100μmol/L)0.05μL respectively,RNase P upstream and downstream primers(50μmol/L)0.06μL respectively,RNase P probe(50μmol/L)0.03μL respectively,template 15μL,and ddH2O supplemented to 30μL.The reaction conditions were 50℃20 min,95℃3 min and 45 cycles of 95℃15 s and54℃30 s.Fluorescence signals were collected during annealing in each cycle.The minimum detection limits of HPIV1,HPIV2 and HPIV3 were all 500 copies/mL by the multiplex real-time RT-PCR assay;The method showed no cross-reaction with influenza A virus,influenza B virus,respiratory syncytial virus andnovel coronaviruses.The coefficients of variation(CVs)in intra-and inter-groups of the recombinant plasmid standard mixture with three different concentrations were all less than 3%.HPIV1,HPIV2 and HPIV3 were detected in 192 clinical samples,and the positive rates were7.81%,0.05%and 3.1%,respectively.ConclusionThe multiplex real-time RT-PCR assay for detection of HPIV1,HPIV2 and HPIV3 developed in this study has good sensitivity,specificity and precision,which has a high clinical application prospect in the field of rapid diagnosis and identification of HPIV.