1种检测基因点突变的新型MLP-RT-PCR技术构建

A new MLP-RT-PCR method for the detecion of point mutations

目的构建1种检测基因点突变的新型MLP-RT-PCR技术。方法将多重连接探针扩增(multiplex ligation-dependent probe amplification,MLPA)技术与实时PCR技术相结合,优化杂交和连接反应条件,建立MLP-RT-PCR反应步骤。设计检测HBV耐药相关基因5个突变位点的4类MLPA探针,根据反应特异性筛选出高特异性探针。同时对5个突变位点进行检测,考察该方法的特异性。检测不同浓度突变株的样本考察该方法的灵敏度。结果统一优化了各个位点的探针反应条件,提高了检测通量;选择单个错配碱基的MLPA探针,可提高检测特异性,5个突变位点检测之间无交叉反应;该方法的扩增效率可达99%～105%,可检测最低0.1%突变率的样本。结论成功构建了检测基因点突变的新型MLP-RT-PCR技术,该方法可在1个反应管中同时检测HBV耐药相关基因的5种突变形式,1次检测时间为4～5 h,为临床提供1种能够快速检测HBV耐药相关基因点突变的方法。

This study aimed to develop a multiplex ligation-dependent probe real-time PCR（MLP-RTPCR） method for simultaneously detecting five antiviral drug-resistant mutations of HBV. The method was developed by combining the high-throughout of multiplex ligation-dependent probe amplification（MLPA） with the rapid and sensitive detection of real time PCR. The MLPA procedures were optimized by changing the conditions of hybridization and ligation. Four kinds of MLPA probes were designed to evaluate the specificity of MLP-RT-PCR.The probes contained a mismatched base were finally chosen to detect clinical samples. Data showed that there was no cross-reaction among the five mutations and the five pairs of MLPA probes; the amplification efficiency of realtime PCR for each mutation was very high（99% to 105%）. To better evaluate the MLP-RT-PCR method for detecting low-frequency mutations, target sequences with mutations were mixed with wild sequences in different ratios copies. We found that the sequence with the mutations whose frequencies were 0.1% were detected by MLPRT-PCR. In conclusion, we develop a new method to rapidly detect the usual drug-resistant mutations of HBV,which can simultaneously detect five mutations in one tube. The entire assay can be completed in 4 to 5 h.