人疱疹病毒6型三重芯片式数字PCR方法的建立  

Establishment of triple chip digital PCR method for human herpesvirus 6

作　　者：王文军 宋娟[1] 王瑞芳 万以秋 魏泽 姚海兰[4] 韩俊[1] Wang Wenjun;Song Juan;Wang Ruifang;Wan Yiqiu;Wei Ze;Yao Hailan;Han Jun(State Key Laboratory of Infectious Disease Prevention and Control,National Institute of Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;School of Medicine,Anhui University of Science and Technology,Huainan 232001,China;Baotou Medical School,College of Public Health,Baotou 014040,China;Department of Biochemistry and Immunology,Capital Institute of Pediatrics,Beijing 100020,China)

机构地区：[1]中国疾病预防控制中心病毒病预防控制所传染病预防控制国家重点实验室,北京102206 [2]安徽理工大学医学院,淮南232001 [3]包头医学院公共卫生学院,014040 [4]首都儿科研究所生化免疫研究室,北京100020

出　　处：《中华实验和临床病毒学杂志》2021年第5期570-574,共5页Chinese Journal of Experimental and Clinical Virology

基　　金：国家传染病重大专项(2018ZX10102001,2018ZX10711001,2018ZX10734404);传染病预防控制国家重点实验室发展基金(2011SKLID104)。

摘　　要：目的建立同时检测疱疹病毒(human herpesvirus,HHV)-6A、B和核糖核酸酶P/MRP 30 kDa亚基(RPP30)的三重芯片式数字PCR(chip digital PCR,cdPCR)方法确定HHV-6A与HHV-6B感染的病毒载量,及高病毒载量的HHV-6是否由病毒整合到染色体导致。方法根据已建立的HHV-6A、HHV-6B实时荧光定量PCR(real-time fluorescence quantitative PCR,RT-qPCR)方法,建立HHV-6三重cdPCR方法。分别使用HHV-6A、HHV-6B病毒培养物进行敏感性检测,并与其他疱疹病毒进行特异性检测。随后,使用127份全血样本进行三重cdPCR方法验证。结果HHV-6 cdPCR与RT-qPCR方法检测结果的相关性良好(R2>0.97),且与其他疱疹病毒无交叉反应。对经RT-qPCR与cdPCR检测均为阳性的14份样本,经三重cdPCR方法检测,得到HHV-6A和HHV-6B的最低检测病毒载量分别为50拷贝/ml和105拷贝/ml。并且14份样本中HHV-6病毒载量与(RPP30拷贝数/2)的比值均小于1。结论建立的三重cdPCR具有较好的敏感性和特异性,且HHV-6三重cdPCR方法可以定量检测HHV-6A、HHV-6B的病毒载量以及RPP30的拷贝数。通过检测样本中HHV-6病毒载量与(RPP30拷贝数/2)的比值,可以确定高病毒载量的HHV-6是否存在染色体整合。Objective To determine the viral load of human herpesvirus 6 A(HHV-6A),HHV-6B and chromosomal integrated HHV-6(ciHHV-6)simultaneously through a triple chip digital PCR(tcdPCR)method for detection of HHV-6A/6B and ribonuclease P-30(RPP30).Methods According to optimal reaction conditions of real-time fluorescence quantitative PCR(RT-qPCR)method,the tcdPCR mehod of HHV-6A,HHV-6B and RPP30 was established.The sensitivity of tcdPCR was determined by virus cultures and the specificity of tcdPCR was detected with other herpesviruses.Subsequently,the tcdPCR of HHV-6A,HHV-6B and RPP30 was verified through 127 whole blood samples.Results The consistency between RT-qPCR and tcdPCR for HHV-6 detection was good(R2>0.97).And there was no cross-reaction with other herpesviruses.The 14 positive samples could be detected effectively by the tcdPCR of HHV-6A,HHV-6B and RPP30.The lowest detectable viral load of HHV-6A and HHV-6B was 50 copies/ml and 105 copies/ml,respectively.And the ratio of HHV-6/(RPP30/2)in 14 positive samples was less than 1.Conclusions The tcdPCR has good sensitivity and specificity.And HHV-6 tcdPCR method can quantitatively detect the viral load of HHV-6 infection and the copy number of RPP30,and ciHHV-6 can be judged by ratio of HHV-6/(RPP30/2)in clinical samples.

关 键 词：人疱疹病毒6型 三重芯片式数字PCR 染色体整合HHV-6 

分 类 号：R373.11[医药卫生—病原生物学]