建立PCR熔解曲线法检测HBV基因型  

Establishment of melt curve analysis for genotyping of hepatitis B virus

作　　者：商红艳[1] 程祖建[1] 欧启水[1] 

机构地区：[1]福建医科大学附属第一医院检验科,福州350005

出　　处：《临床检验杂志》2011年第3期225-227,共3页Chinese Journal of Clinical Laboratory Science

基　　金：福建省卫生厅创新课题(2009CX-9);福建省科技厅重点课题(2010Y0022);福建医科大学教授基金(JS10014)

摘　　要：目的建立检测HBV基因型的PCR熔解曲线法,并验证该方法临床应用的可靠性。方法依据文献报道的HBV B、C型全基因组序列,分别设计B、C型的特异引物序列,建立HBV基因分型的PCR熔解曲线法。用该法检测186例HBV DNA阳性血清样本,并从中选取51例样本,用商品化的HBV基因分型试剂盒进行检测,用Kappa一致性检验对两法检测结果进行比较。结果 186例标本中,PCR熔解曲线法检测到B型133例(71.5%),C型37例(19.9%),B/C混合型16例(占8.6%)。选取的51例样本中,熔解曲线法与市售试剂盒检测B型符合率100%,C型符合率100%,B/C混合型符合率81.25%,总符合率94.1%(Kappa=0.909,P<0.05)。结论建立的PCR熔解曲线法操作简便,与商品试剂盒的检测结果有较高的符合率,可用于临床上HBV B、C以及B/C混合型的检测。Objective To develop a new method for the detection of HBV genotype with melt curve based on real-time PCR and investigate the reliability of HBV genotyping in clinical application.Methods The type-specific primers were designed according to the sequence of type B and C of hepatitis B virus genome published in GenBank and PCR assay with melt curve were developed.A total of 186 HBV DNA-positive sera were tested by the developed melt curve assay.Of 186 samples 51 were randomly selected to detect genotype with commercial kit.Kappa consistency test was used to compare the results from melt curve PCR assay and commercial kit respectively.Results Among the 186 HBV-positive samples,the number of genotype B,C and mixed B/C were 133（71.51%）,37（19.89%） and 16（8.60%） respectively.Among the 51 samples detected with the two assays,the accordance rates were 100% for both genotype B and C,81.25% for mixed B/C,and 94.12% for all the 51 samples（Kappa=0.909,P0.05）.Conclusions The real-time PCR melt curve assay should be a convenient method for the detection of HBV B,C genotyping and B/C mixed genotyping,and its results showed high accordance rate with those of commercial kit.

关 键 词：乙型肝炎病毒 基因型 熔解曲线法 聚合酶链反应 

分 类 号：R373.21[医药卫生—病原生物学]