应用MALDI-TOF-MS技术高通量检测乙肝病毒耐药基因变异

High-throughput Detection of Drug-resistance Gene mutations in HBV Using MALDI-TOF Mass Spectrometry

目的建立基于MALDI-TOF-MS技术的高通量检测乙型肝炎病毒耐药基因变异的临床检测方法。方法应用MassARRAY Assay Design软件设计iPLEX引物，按iPLEX反应的操作说明进行PCR扩增、SAP反应、引物延伸、脱盐、点样，MALDI-TOF-MS采集、分析iPLEX反应物的数据，判读基因变异位点。批量检测、分析138例HBV耐药包括拉米夫定、阿德福韦、恩替卡韦单药耐药及多重耐药的慢性乙型肝炎患者血清标本。并对MALDI-TOF-MS所检测的HBV基因变异位点所在区域进行DNA测序，与MALDI-TOF-MS检测结果对比、分析。结果建立了基于MALDI-TOF-MS技术的HBV基因突变检测平台，实现了临床血清标本的高通量检测。能一次获得138份标本的HBV基因突变临床检测结果。MALDI-TOF-MS技术和DNA测序同时检测的33份标本中，有10份检测结果不一致，其中有2份MALDI-TOF-MS技术未检测到，有1例出现2个检测位点不一致。结论MALDI-TOF-MS技术灵敏度高、准确度高，可高通量、快速检测HBV基因变异，适用于HBV临床诊治及监测。

Objective To develop a high-throughput clinical method on drug-resistance gene mutations of HBV using MALDI-TOF-MS. Method Using MassArray Assay Design software designed the iPLEX primers and followed the iPLEX instruction for amplification, SAP reaction, primer extenction, desalination, dispensing, MALDI-TOF-MS screening and data analysis of the gene mutation locus. 138 serum samples of chronic HBV patients with single drug-resistance or multiple drug-resistance on Lamivudin, adefovi, Entecavir were detected. Result The HBV gene mutation platform was successfully developed and applied on the high-throughput dectection of clinical serum samples. It was also a high throughput assay which could be used to detect for more than 138 samples once. The MALDI-TOF-MS technology and the DNA sequencing simultaneously examine 33 samples, in which result of 10 sample is inconsistent, the including 2 samples by MALDI-TOF-MS technology has not tested, 1 sample has 2 inconsistent mutations. Conclusion Detction of HBV gene mutations using MALDI- TOF-MS is highly-sensitive, highly-accuate, high-thoughput ,fast achieved and suitble to use in the diagnosis and monitoring of HBV.