DNA测序与线性反向探针杂交法检测HBV P基因耐药突变的比较

Drug-resistant mutation of hepatitis B viral P gene in patients with chronic hepatitis B receiving nucleotides

目的建立和优化扩增慢性乙型肝炎患者血清HBV P基因的PCR方法,比较DNA测序法与线性反向探针杂交法检测HBV基因突变情况,并对耐药相关基因突变及其意义进行分析。方法采用巢式PCR法扩增93例慢性乙型肝炎患者血清HBV P基因反转录酶区,对PCR产物进行DNA测序,采用Bioedit6.0.5、SeqmanTMⅡ、EditSeq TM和Me-gAlign软件分析结果;同时随机抽取40份血清进行线性反向探针杂交,比较两者的检测结果,并对11个已知耐药相关突变位点进行分析。结果在93份标本中DNA序列测定法检测到碱基突变113个,其中与LAM相关突变50份(53.78%),与ADV相关突变6份(6.46%),与LdT相关突变3份(3.23%),与ETV相关突变3份(3.23%);在40份血清中与线性反向探针杂交检测结果一致的突变为87.5%(35/40),氨基酸位点一致为98.0%(431/440)。此外,还检测到V84I、S85C、A181S、L229W、N238T等与耐药相关的突变位点。结论应用线性反向探针杂交检测虽然比较快捷,但其价格昂贵且只能检测已知常见的变异位点;建立在巢式PCR基础上的DNA序列测定法敏感性与线性反向探针杂交法相当,两者检测结果有较高的符合率,但似可检出更多的耐药相关突变。

Objective To compare hepatitis B viral P gene mutations detected by direct DNA sequencing and INNO-LiPA HBV DR line probe assay. Methods Sera of 93 patients with chronic hepatitis B were collected and the HBV P gene mutation were detected by DNA sequencing and by INNO-LiPA HBV DR line probe assay. Remits 113 amino acid mutations were detected in 93 sera,out of which 50 （53.78%） related to LAM,6 （6.46%） to ADV,3（3.23%） to LdT,and 3 （3.23%） to ETV;The same P gene mutation by line probe and by sequencing was observed in 87.5% of samples （35/40） and the same amino acid mutation was found in 98.0% （431/440）;We also detected some other mutation positions such as V84I,S85C,A181S,L229W and N238T. Conclusions Direct DNA sequencing basing on nested PCR products has a similar sensitivity with INNO-LiPA HBV DR line probe assay.