初步比较6种荧光PCR试剂盒对肠道病毒核酸的检测效果  

Preliminary comparison of six commercially available fluorescence PCR kits in detecting nucleic acid of enterovirus

作　　者：梁志超[1] 林长缨[1] 杨扬[1] 崔淑娟[2] 高志勇[1] 黄瑛[1] 张新[1] 李仁清[1] Liang Zhichao;Lin Changying;Yang Yang;Cui Shujuan;Gao Zhiyong;Huang Ying;Zhang Xin;Li Renqing(Institute for Infectious Disease Prevention and Control,Beijing Center for Disease Prevention and Control,Beijing 100013,China;Department of STD/AIDS Prevention and Control,Beijing Center for Disease Prevention and Control,Beijing 100013,China)

机构地区：[1]北京市疾病预防控制中心传染病地方病控制所,100013 [2]北京市疾病预防控制中心性病艾滋病防治所,100013

出　　处：《国际病毒学杂志》2024年第3期206-210,共5页International Journal of Virology

基　　金：北京市自然科学基金-海淀原始创新联合基金(L192012);首都卫生发展科研专项(首发2020-2-1012);国家重点研发计划(2023YFC2308705)。

摘　　要：目的初步比较6种品牌肠道病毒EV-A71/CVA16/肠道通用核酸检测试剂盒CVA6/CVA10核酸检测试剂盒的灵敏度和特异性,为检测试剂的选择及疾病监测提供参考。方法选择EV-A71、CVA16、CVA6和CVA10四种血清型毒株作为样本盘,分别10倍梯度稀释。使用数字PCR对梯度稀释的病毒进行核酸定量后,制备混合样本盘并进行倍比稀释,使用实时荧光PCR方法检测。分析各试剂盒的检出限及灵敏度;检测非肠道病原的核酸,分析其特异度;通过分组打分和量化评分法评价扩增曲线特征及Ct值结果,进行综合评分。结果对灵敏度的比较分析中,试剂盒E、A和F检测较为灵敏;在扩增曲线及Ct值分析中,按分值排名检测EV通用的试剂前3为A、C、E;检测EV-A71中前3名为E、A、F;检测CVA16的试剂中,前3名为A、D、E;检测CVA6的试剂前3名为E、D、F;检测CVA10的试剂中,前3名的为E、A、C。各品牌的特异性均较好。结论在EV-A71/CVA16/EV通用3重荧光PCR检测试剂盒中,A、E、C和D综合评分较高,在CVA6/CVA10双重荧光PCR检测试剂盒中E、F综合评分较高,建议在检测肠道病毒相关的监测及疫情样本时,常备两种品牌及以上试剂盒对检测结果相互比较验证,并对扩增曲线和Ct值结果综合判断。本研究的量化评分方法可为其他荧光PCR检测试剂盒评价提供参考。Objective To compare preliminarily the sensitivity and specificity of EV-A71/CVA16/enterovirus(EV)nucleic acid detection kits and CVA6/CVA10 nucleic acid detection kits of six commercial brands,so as to provide reference for selection of detection reagents as well as disease prevention and surveillance.Methods The virus strains of four serotypes,including EV-A71,CVA16,CVA6 and CVA10 were selected as the sample pool.The sample strains were serially diluted in ten fold.The nucleic acids of serial diluted samples were quantified by digital PCR.The samples were then mixed to created a pool of mixture and diluted in serial.The real-time fluorescence PCR was then used for detection.The detection limit and sensitivity were analyzed.The kits were used to analyze nucleic acid from virus other than enterovirus to evaluate the specificity.Group scoring and quantity scoring were used to evaluate the characters of amplification curves and Ct values and obtain the generalized scores.Results In the comparison of sensitivities,kit A,D and E were more sensitive.In the analysis of amplification curve and Ct values,the top three kits in universal detection of enteroviruses were kits A,C and E and kits for detection of EV-A-71,CVA16,CVA6 and CVA10 were kits E,A and F,kits A,D and E,kits E,D and F,and kits E,A and C,respectively.All kits had good specificity.Conclusions In the triplex fluorescence PCR kits for detecting nucleic acid of EV-A71/CVA16/EVs,the kits A,E,C and D had higher general scores.For duplex fluorescence PCR kits for detecting nucleic acid of CVA6/CVA10,kits E and F had higher general scores.It is recommended to have kits of two or more brands and compare the detection results.The quantitative scoring method could be the reference for the evaluation of other fluorescence PCR kits.

关 键 词：手足口病 肠道病毒 实时荧光PCR 

分 类 号：R512.5[医药卫生—内科学] R440[医药卫生—临床医学]