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

目的 比较基质辅助激光解析电离飞行时间质谱法(MALDI-TOF MS)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、HBV DNA>500拷贝/ml,10例肝功能正常、HBV DNA>1×105拷贝/ml,未进行抗病毒治疗.对所有血清均采用MALDI-TOF MS法及PCR产物直接测序法(简称直接测序法)检测HBV P基因已知9个变异位点的变异情况,通过软件分析得出变异位点序列.根据样本量用相应的Pearson x2检验与Yates x2检验.结果 在服用LAM等核苷(酸)类似物的90份患者血清中,MALDI-TOF MS法测得53份阳性,检出率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%,仅发现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,HBV DNA水平在500～1000拷贝/ml、102～104拷贝/ml、104～105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HBV DNA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-TOF MS法检测已知耐药位点的敏感性高于PCR直接测序法.

Abstract：

Objective To compare the sensitivities of MALDI-TOF MS and direct PCR sequencing on gene mutations demction 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 nuclcoside 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 1×105 copies/m1.9 known mutations associated with HBV P gene in these samples were detected bv MALDI-TOF MS and direct PCR sequencing at the same time,TYPE4.0 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 by MALDI-TOF MS with a positive detection rate of 58.89%,whereas only 19 samples were found with mutations 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 RBV DNA titers were at 500-1000 copies/ml,103-104 copies/ml and 104-105 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 difference was still statistically significant (P < 0.05). Conclusions MALDI-TOF MS had higher detection sensitivity for known mutation sites as compared to direct PCR sequencing method.

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

Objective To compare the sensitivities of MALDI-TOF MS and direct PCR sequencing on gene mutations demction 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 nuclcoside 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 1×105 copies/m1.9 known mutations associated with HBV P gene in these samples were detected bv MALDI-TOF MS and direct PCR sequencing at the same time,TYPE4.0 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 by MALDI-TOF MS with a positive detection rate of 58.89%,whereas only 19 samples were found with mutations 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 RBV DNA titers were at 500-1000 copies/ml,103-104 copies/ml and 104-105 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 difference was still statistically significant (P < 0.05). Conclusions MALDI-TOF MS had higher detection sensitivity for known mutation sites as compared to direct PCR sequencing method.