[基质辅助激光解吸电离飞行时间质谱法与直接PCR测序法检测HBV-P基因突变的比较研究]

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

作者信息

Liu Fei, Xiao Ting, Wang Ling, Xie Jian-Ping, Li Guo-Hong, Liang Qiao-Ling, Luo Chun-Hui

机构信息

Department of Infectious Diseases, Xiangya Hospital, Central South University, Changsha 410008, China.

出版信息

Zhonghua Gan Zang Bing Za Zhi. 2011 Jun;19(6):436-9.

PMID:22053374

Abstract

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 x 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, TYPE4.0 software and Sequence Navigator software were used to analyze the results separately.

RESULTS

(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 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 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.

摘要

目的

比较基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）与直接PCR测序法对乙型肝炎病毒基因突变检测的灵敏度。

方法

收集100例慢性乙型肝炎患者的血清样本，其中包括90例（研究组）接受核苷类似物（NA）治疗1年以上且HBV DNA载量仍高于500拷贝/ml的慢性乙型肝炎患者的血清样本，以及10例（空白组）从未接受过抗病毒治疗且HBV-DNA载量高于1×10⁵拷贝/ml的慢性乙型肝炎患者的血清样本。同时采用MALDI-TOF MS和直接PCR测序法检测这些样本中9个与HBV P基因相关的已知突变，分别使用TYPE4.0软件和Sequence Navigator软件分析结果。

结果

（1）研究组中，MALDI-TOF MS检测到53个样本发生突变，总突变位点为86个，阳性检出率为58.89%；而直接PCR测序仅发现19个样本发生突变，共检测到28个突变位点，阳性检出率为21.11%。MALDI-TOF MS的阳性检出率高于直接PCR测序，差异有统计学意义（P<0.05）。空白组中，两种方法均未检测到突变。（2）研究组中，当HBV DNA载量分别为500-1000拷贝/ml、10³-10⁴拷贝/ml和10⁴-10⁵拷贝/ml时，MALDI-TOF MS的阳性突变检出率分别为50%、52.08%和77.27%，均高于直接PCR测序的0%、8.33%和45.45%。差异仍有统计学意义（P<0.05）。