反向线性探针杂交技术检测慢性乙型肝炎病毒拉米夫定耐药研究

Reverse Line Probe Hybridization Assay for Detection of Lamivudine-Resistant Chronic Hepatitis B Virus

目的:建立起简单、快速、灵敏、准确的慢性乙型肝炎病毒拉米夫定耐药位点的反向线性探针(reverse lineprobe,RLP)检测方法.方法:根据HBV野生及耐药基因序列设计通用探针、3’、5’对称加有poly-C的特异性探针和5’标记生物素的扩增引物.将探针线性固定在硝酸纤维膜上,使HBV PCR扩增产物与探针进行杂交.通过优化杂交条件,建立RLP检测方法.利用该方法对重庆地区86个慢性乙肝病人进行检测,同时与直接测序结果比较.结果:半巢式PCR可对103拷贝/ml的血清样本进行有效特异扩增,新建的RLP检测方法可对PCR扩增产物在1ng/ml以上,或血清样本中突变型DNA占野生型DNA比例为5%以上的均可有效检测,86例临床的检测灵敏度为100%,野生型和耐药型的检测准确性分别为98.09%(103/105)、100%(43/43),与直接测序法比,RLP检测野生与耐药混合型准确性更好.结论:反向线性探针杂交检测方法检测HBV拉米夫定耐药位点方便、灵敏、准确,是HBV拉米夫定治疗有效的监控工具,该方法适合临床应用.

Objective： To establish a simple,rapid,sensitive and accurate reserve line probe（RLP） method for the detection of chronic HBV（hepatitis B virus） lamivudine-resistant locus.Methods： Based on the gene sequence of HBV wild-type and lamivudine-resistant drug,universal probe,specific probe of 3＇,5＇ added poly-C and primers of 5＇ labeled biotin were designed.The specific probes were fixed on the nitrocellulose membrane linear;so as to make HBV PCR amplification products hybridize with the probes.By optimizing the hybridization conditions,a reverse line probe hybridization test method was established,which was then used to detect 86 chronic hepatitis B patients in Chongqing,and the results were compared with those of the direct sequencing method.Results： On the condition that HBV DNA in the serum had more than 103 copies/ml,it could be amplified effectively by semi-nested PCR.At the same time,when cDNA was more than 1ng/ml or the mutant proportion was more than 5% in wild-type of serum samples,lamivudine-resistant to chronic hepatitis B virus could be effectively detected by the new RLP hybridization method.The sensitivity in 86 clinic cases was 100%（86/86）.The detection accuracy of the wild type and the drug resistant type was 98.09%（103/105）,100%（43/43）,respectively.Conclusions： The reverse linear probe hybridization method to HBV lamivudine resistance is convenient,sensitive and accurate.It is an effective monitoring tool for HBV lamivudine therapy and suitable for clinical application.