核苷类药与慢性HBV感染者病毒P基因准种及变异特点的关系

目的观察核苷类药治疗不同时间段慢性HBV感染患者血清乙肝病毒聚合酶(HBV P)基因准种变化及变异特点。方法运用焦磷酸测序仪器配套的软件Assay Design SW在目的区域两端保守区域设计PCR引物及测序引物,采用套式PCR方法检测血清中HBV DNA,按照Pyro-Mark ID遗传分析系统用SNP模式进行PCR产物(HBV P基因相关位点)的焦磷酸测序和突变频率检测。108例慢性HBV感染患者中用药史明确者同期测定HBV DNA、HBV标志物、ALT。结果108例患者中,61例发生变异,变异模式以经典突变L180M、M204V/I、A181V/T、N236T突变为主,有少量V173L、S202G、T184G突变;其中40例用药史明确患者中,18例发生变异,发生变异者耐药突变型在样本中所占比例为20%-100%,HBV P基因变异可以在HBV DNA、ALT发生突变前、中、后检出。结论焦磷酸测序可以快速检测HBV P基因变异;变异株突变频率变化可初步反映HBVP基因准种异质性;HBV P基因变异模式、突变频率与核苷类药物敏感性密切相关。

Relationship between nucleoside drugs and the diversify and variation about quasispecies of P gene in patients with HBV infection

Objective To investigate the characteristics of diversify and variation about quasispecies of serum hepatitis B virus(HBV) P gene in HBV infection patients treated with nucleoside drugs at different time periods.Methods The PCR primer and sequencing primer were designed by software "Assay Design SW" supported by pyrophosphoric acid sequenator.The nest-PCR was used to detect serum HBV DNA.Then SNP mode according to PyroMark ID of genetic analysis system was used to analyze the DNA sequencing and mutation frequency in PCR products(related with HBV P gene sites).Meanwhile,HBV DNA,HBVM and ALT were analyzed in 40 of 108 HBV infection patients,who had a definite history of medication.Results Of 108 patients with HBV infection,61 cases were found to have the variation with the main classic mutations of L180M,M204V/I,A181V/T and N236T,in which a few of V173L,S202G and T184G mutations was detected as well.Of 40 HBV infected patients who had definite history of medication,the variation was detected in 18 cases.The proportion of drug resistance mutant was from 20% to 100% in HBV mutationed patients.Variation of HBV P gene was detected before,during and after the mutantation of HBV DNA and ALT.Conclusion DNA sequencing can quickly detect the variation of HBV P gene by pyrophosphoric acid sequenator.The mutation frequency of mutant can preliminary reflect the heterogeneity of quasispecies about HBV P gene.The variation mode and mutation frequency are closely related to the sensitivity of nucleoside drugs.