人巨细胞病毒糖蛋白B基因的分型

Glycoprotein B genotype of human cytomegalovirus analysis by nPCR-RFLP

目的建立人巨细胞病毒(HCMV)糖蛋白B(gB)基因的套式聚合酶链反应(nPCR)加限制性长度多态性分析(RFLP)的分型方法。方法34例HCMV感染患儿的11份全血标本和23份尿标本,用nPCR扩增gB基因片段,RFLP对gB基因进行分型,并通过测序进行验证。结果34例HCMV感染患儿中,检出gBⅠ型13例,gBⅡ型12例,gBⅢ型9例,未发现gBⅣ型。测序结果与基因库中相应的标准株序列进行比较,Ⅰ、Ⅱ和Ⅲ型的同源性分别为98.1%～99.6%、98.9%～100%、97.3%～98.9%。结论nPCR检测HCMV感染敏感特异,RFLP对HCMV的gB基因分型明确可靠。

Objective Aim to set up the method of glycoprotein B genotype of human cytomegalovirus (HCMV) analysis by using nested polymerase chain reaction(nPCR) and restriction fragment length polymorphism(RFLP). Methods Eleven blood samples and twenty-three urine samples were obtained from thirty-four HCMV-infected patients. A fragment of gB gene was amplified by nPCR. HCMV gB genotyping was carried out by RFLP, and the amplified DNA fragments were verified by DNA sequencing. Results Of the 34 patients, gB type Ⅰ was found in 13 patients, gB type Ⅱ in 12 patients, gB type Ⅲ in 9 patients, and none had the gB type Ⅳ sequence. The similarities of PCR products of HCMV gB Ⅰ, Ⅱ and Ⅲ amplified compared with the sequences of prototype strains in GenBank were 98.1%～99.6%, 98.9%～100%, 97.3%～98.9%. Conclusions The nPCR assay developed in this study was sensitive and specific for detection of HCMV, and RFLP analysis of HCMV gB genotype was definite and reliable.