摘要
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.
出处
《西南师范大学学报(自然科学版)》
CAS
CSCD
北大核心
2012年第3期113-119,共7页
Journal of Southwest China Normal University(Natural Science Edition)
基金
"863"课题(Grant No.2008AA02Z424)
重庆市教委课题(Grant No KJ100309)