用PCR方法快速检测用拉米夫定治疗的乙型肝炎病人中HBV P基因区codon 550的变异

Use of PCR method for rapid detection of codon 550 mutation in HBV polymerase gene region of chronic hepatitis B patients treated by lamivudine

目的 建立一种快速特异的乙型肝炎病毒 (HBV)YMDD变异的检测方法。方法 针对HBVcodon 5 5 0位置设计 2条高度特异的的引物 ,用PCR方法进行DNA扩增。根据电泳结果可分析下列 2种情况 :(1)A741G变异型(YVDD) ;(2 )G743T变异型 (YIDD)。用该方法对 32例病人在拉米夫定治疗前 (0周 ) ,治疗中 (5 2周和 10 4周 )共 6 1份血清进行YMDD变异检测。结果 特异性 :10例病人分别做 0周和 5 2周标本codon 5 5 0特异性PCR和DNA测序。 0周血清检测结果全部为野生型 ,5 2周血清中 5例为A741G ,2例为G743T ,两种方法的检测结果完全相同。敏感性 :6 1份血清中 2例血清PCR未能检出阳性 ,codon 5 5 0变异为 2 9份 ,野生型为 30份 ,和葛兰素威康公司检测结果的符合率为98.3 %。混合变异株检测 3例病人 10 4周血清用PCR测定发现同时存在A741G和G743T两种变异株 ,其中 2例结果经多克隆测序的方法得到确证。结论 HBVcodon 5 5 0特异性PCR方法具有快速、简便和经济实用的优点 ,适用于临床拉米夫定治疗病人HBVYMDD变异的跟踪测定。

Objective To develop a rapid specific method to identify the presence of HBV codon 550 mutation which is the methionine residue of the conserved YMDD motif of HBV polymerase gene. Methods Two pairs of highly specific primers were designed to detect codon 550 mutation,another pair of primer was used in PCR as HBV DNA control.Each sample was done 3 PCRs in 3 eppendorf tubes simultaneously.After PCR,the mutation at codon 550 were then analyzed as 2 types by electrophoresis:(1)A741G mutant(YVDD);(2)G743T mutant(YIDD).61 serum samples from 32 patients with CHB during lamivudine treatment for 52w and 104w were analyzed.Results 1.Specificity:(1) Detection of HBV plasmid:the result showed that there was only HBV DNA control band on electrophoresis.(2)Detection of HBV DNA clone No.10 and NO.11 of sample 214# which were sequenced and proven that codon 550 were ATT and GTG,the results also showed at the responding sites there were specific DNA bands.(3)Comparison with the DNA sequencing:the samples from 10 patients at 0w and 52w respectively were tested with codon 550 PCR and DNA sequencing simultaneously,the same results were obtained:the 10 samples at 0w were all of HBV wild type;of the 10 samples at 52w,7 were HBV codon 550 mutation including 5 A741G(YVDD) and 2 G743T(YIDD).2.Sensitivity:Of the total 61 serum samples from 32 cases ,besides the 2 samples at 104w were PCR( ),29 were determined to have HBV codon 550 mutants,the remaining were of HBV wild type.These results were concordant with those detedted in Glaxo Wellcome Company.3. Detection of mixed mutants:Both HBV A741G and G743T mutants were found in each serum samples from 3 patients at 104w respectively,among them,the results of 2 patients were coincident with that of DNA sequencing after multiple cloning.Conclusion This specific PCR method for HBV codon 550 mutation is rapid,simple and specific and useful for the clinical monitoring the emergence of HBV YMDD mutation during lamivudine therapy.