基质辅助激光解析电离飞行时间质谱法与PCR产物测序法检测乙型肝炎病毒耐药变异的比较  

Comparison study of HBV-P mutation detection by MALDI-TOF Ms and direct PCR sequencing

作　　者：刘菲[1] 肖婷[1] 汪玲[1] 谢建萍[1] 李国宏 梁巧玲 罗春慧 

机构地区：[1]中南大学湘雅医院感染病科,长沙410008 [2]深圳华大基因研究院

出　　处：《中华肝脏病杂志》2011年第6期436-439,共4页Chinese Journal of Hepatology

摘　　要：目的比较基质辅助激光解析电离飞行时间质谱法（MALDI-TOFMS）和PCR产物直接测序法检测HBV耐药基因位点的敏感性。方法共收集100例慢性乙型肝炎（CHB）患者血清其中90例服用拉米夫定（LAM）等核苷（酸）类似物治疗1年以上、HBVDNA〉500拷贝／ml10例肝功能正常、HBVDNA〉1×10^5拷贝／ml，未进行抗病毒治疗。对所有血清均采用MALDITOFMS法及PCR产物直接测序法（简称直接测序法）检测HBVP基因已知9个变异位点的变异。膳况，通过软件分析得出变异位点序列。根据样本量用相应的Pearsonx2检验与YatesX2检验。鲤果在服用LAM等核苷（酸）类似物的90份患者血清中，MALDITOFMS法测得53份阳性，耗出率58．89％，共发现86个变异位点；PCR直接测序法仅检测到19份阳性，检出率21．11％，伍发现28个变异位点，两者间检出率差异有统计学意义（P〈0．05）。10例从未接受过口服抗病毒药物治疗的患者血清用两种检测方法均没有检测出变异位点。检测90例接受核苷（酸）类似物治疗的患者血清，HBVDNA水平在500～1000拷贝／ml、10’^3～10^4拷贝／ml、10^4～10^5拷贝／ml时，质谱法阳性检出率分别为50．00％、52．08％和77．27％，而PCR直接测序法分别为0、8．33％和45．45％。在相同中、低滴度HBVDNA载量下，质谱法阳性检出率均高于测序法，两者差异有统计学意义（P〈0．05）。结论MALDI-TOFMS法检测已知耐药位点的敏感性高于PCR直接测序法。Objective To compare the sensitivities of MALDI-TOF MS and direct PCR sequencing on gene mutations detection of hepatitis B virus. Methods 100 serum samples from chronic hepatitis B patients were collected, which consisted of 90 serum samples （study group） from 90 chronic hepatitis B patients received nucleoside analogues （NA） therapy for more than 1 year and HBV DNA titer still higher than 500 copies/ml and 10 serum samples （blank group） from 10 chronic hepatitis B patients never treated with antiviral therapy and HBV-DNA titer higher than 1×10^5 copies/ml. 9 known mutations associated with HBV P gene in these samples were detected by MALDI-TOF MS and direct PCR sequencing at the same time, TYPEA.0 software and Sequence Navigator software were used to analyze the results separately. Resuits （1） In study group, mutations were detected in 53 samples and the total mutation sites were 86 byMALDI-TOF MS with a positive detection rate of 58.89%, whereas only 19 samples were found with muta- tions and totally 28 mutation sites were detected by direct PCR sequencing, the positive detection rate was 21.11%. The positive detection rate by MALDI-TOF MS was higher than that by direct PCR sequencing and the difference was statistically significant （P 〈 0.05）. In blank group, no mutations were detected by any method. （2） In study group, when the HBV DNA titers were at 500-1000 copies/ml, 10^3-10^4 copies/ml and 10^4-10^5 copies/ml, the positive mutation detection rates by MALDI-TOF MS were 50%, 52.08% and 77.27% respectively, higher than that by direct PCR sequencing, which were only 0%, 8.33% and 45.45%. The differ- ence was still statistically significant （P 〈 0.05）. Conclusions MALDI-TOF MS had higher detection sensi- tivity for known mutation sites as compared to direct PCR sequencing method.

关 键 词：肝炎病毒 乙型 变异 光谱法 质量 基质辅助激光解析电离 聚合酶链反应 测序 

分 类 号：R4[医药卫生—临床医学] R51