HBVDNA定量与基因分型双重分子信标实时PCR方法的建立及初步应用  

Quantification and genotyping of hepatitis B virus in a single reaction based on dual molecular beacon real-time PCR

作　　者：王炜[1] 陈晓[1] 江凌[2] 刘灿[2] 商红艳[2] 欧启水[2] 杨滨[2] 

机构地区：[1]福建医科大学第一临床医学院,福州350005 [2]福建医科大学附属第一医院检验科福建医科大学基因诊断研究室

出　　处：《中华检验医学杂志》2013年第4期333-338,共6页Chinese Journal of Laboratory Medicine

基　　金：基金项目：福建省医学创新课题资助项目（2009.CX-9）;福建省科技计划重点项目资助课题（2010Y0022,2011Y0023）;福建医科大学教授发展基金资助项目（2010JS0014）

摘　　要：目的研究建立同时进行HBVDNA定量与基因分型检测的双重分子信标实时PCR法（以下简称“定量与分型PCR法”），并探讨其应用价值。方法构建B、c基因型重组质粒作为标准品，通过设计B、c基因型特异性引物和分子信标（B、c基因型分子信标5’端分别标记FAN和Hex荧光基团），建立在1个反应体系中同时定量与分型PER法。然后，用10倍梯度稀释的B、c基因型标准品（10。～1011 kIU／L）评价其检测线性范围和灵敏度；用丙型肝炎病毒感染者、单纯疱疹病毒感染者、人类乳头瘤病毒感染者、健康志愿者血清各5份评价其检测特异性；用高、中、低3份不同浓度的B、c基因型质粒标准品（10、106、104 kIU／L）进行同批次和不同批次各10次重复检测，分别计算批内及批间ct值的变异系数（CV）评价其检测重复性。然后，以湖南圣湘生物科技有限公司HBV核酸定量测定试剂盒为HBVDNA定量参照方法，申友公司HBV基因分型试剂盒为HBV基因分型参照方法，同定量与分型PCR法平行检测132例HBV感染者血清标本，评价自建定量和分型PCR法的准确性。最后，将132例HBV感染者分为无症状携带组（21例）、慢性乙型肝炎组（77例）、肝硬化组（25例）、肝癌组（9例），分析评价基因型、疾病进展的不同阶段与DNA载量间的相关性。结果用自建定量与分型PCR法检测B、c基因型的灵敏度均为103 kIU／L；线性范围均为103一1011 kIU／L；检测B基因型批内CV为1．51％～1．80％，批间CV为2．11％～3．03％，检测C基因型批内CV为1．79％～1．95％，批问CV为2．53％～2．91％；对其他病毒感染者和健康志愿者血清检测结果均为阴性。定量与分型PCR法检On．0132例HBV感染者的基因分型结果与HBV测序分型试剂盒总符合率为90．9％（120／132，Kappa=0．832，P〈0．05）；其DNA定量结果[5．07（3．89～6．33）lgkIU／L]与HBVObjective To develop a new method for simultaneous quantifying and genotyping of HBV in a single reaction based on dual molecular beacon real-time PCR. Methods Genotype B and C recombinant plasmids were constructed as the standards and genotype-specific primers and molecular beacons were designed for each genotype. The molecular beacons of genotype B and C were labeled with FAM and Hex respectively. In this way, a simultaneous qualification and genotyping method for HBV DNA in a single real-time PCR reaction system was developed. Firstly, 10-fold gradient dilution of genotype B and C standard plasmids （103-10H kIU/L） were utilized to evaluate the linear ranges and sensitivity of this approach. The clinical specificity was tested with twenty different serum specimens （5 cases with hepatitis C virus, 5 cases with herpes simplex virus and 5 cases with human papilloma virus as well as 5 healthy volunteers ） ; the reproducibility was assessed by intra-assay and inter-assay coefficient of variation （CV） of cycle threshold （Ct） value through 10 repeated detections within a batch and between batches of the B, C standard plasmids （l0s ,106 and 104 kIU/L）. Then the accuracy of qualifying and genotyping of the self-built method was evaluated by a parallel examination with 132 HBV infected patients by use of two commercial kits as the references. Finally, these HBV-positive patients were divided into 4 groups ： asymptomatic carrier （ n = 21 ） , chronic hepatitis （ n = 77 ）, liver cirrhosis （ n = 25 ） and hepatocellular carcinoma （ n = 9） to investigate the relationship of genotypes, stages of disease progression and HBV DNA load. Results A simultaneous qualification and genotyping assay was successfully built and its genotyping sensitivity was 103 kIU/L and the linear range was 103-10II kIU/L. The intra-assay CV of B genotyping was 1.51% to 1.80% and the inter- assay CV was 2. 11% to 3.03%, while the intra-assay CV of C genotyping was 1.79% to 1.95% and the inter-assay CV was 2.53% to 2.

关 键 词：肝炎病毒 乙型 DNA 病毒 基因型 聚合酶链反应 

分 类 号：R512.62[医药卫生—内科学]