摘要
目的应用巢式PCR和Sanger测序技术,建立一种人类免疫缺陷病毒(HIV)-1 pol基因蛋白酶(PR)区及逆转录酶(RT)区耐药突变检测的方法。方法根据HIV-1 pol基因保守区域设计特异性的2对扩增引物和6条测序引物,使用巢式PCR特异性扩增HIV-1 PR区以及RT区耐药突变基因序列,进而对靶标基因区域进行双向Sanger测序检测,实现了HIV-1基因PR区和RT区基因耐药突变检测的高灵敏性、特异性、准确性。结果本研究所建立的针对HIV-1 pol基因PR区和RT区耐药突变的检测方法,巢式PCR结果经琼脂糖凝胶电泳鉴定有清晰、单一条带,符合扩增产物大小,其Sanger测序检测的HIV-1 PR区以及RT区耐药突变基因序列分析结果与HIV-1耐药性分析国家参考品一致。灵敏度:1×10^(3)copies/mL浓度的标准品重复测20次,检出率为100%;突变率检测:20%突变率的样本20个重复检测均100%为突变型;特异性验证:检测HBV、HCV、EBV血清样本,均为阴性。结论本研究建立的以巢式PCR和Sanger测序相结合的检测方法,具有灵敏度高、准确性高、结果可靠等优点,适用于HIV-1pol基因PR区以及RT区耐药突变的体外检测。
Objective To establish a method for the detection of human immunodeficiency virus(HIV)-1 drug resistance mutations in the protease(PR) and reverse transcriptase(RT) regions of pol gene by the integration of Sanger sequencing and nested RT-PCR techniques.Methods A detection method was established in which HIV-1 drug resistance mutations in the PR and RT regions was specifically amplified using the nested RT-PCR,and the target gene was tested with the method of Bidirectional Sanger sequencing.For this method,specific primers including two pairs of amplification primers and six pairs of sequencing primers targeting the conserved region of HIV-1 pol gene were designed,and consequently,contributing to the high sensitivity,specificity and accuracy for the HIV-1 drug resistance mutations test in the PR and RT regions.Results In this research,a detection method of HIV-1 drug resistance mutations in the PR and RT regions of pol gene had been established.The result of nested PCR in this method was identified by agarose gel electrophoresis,which has a clear and single band and was in line with the size of the amplified product.The Sanger sequence analysis results were in line with the result of HIY-1 drug resistance analysis national reference.The limit of detection:the positive rate of 20 repeated tests of the standard substance with a concentration of 1×10^(3) copies/mL was 100%.The lowest mutation detection rate:the result of the sample with 20% mutation rate were 100% mutant in 20 repeated tests.The specificity:there was no cross-reaction observed from HBV,HCV and EBV positive serum samples.Conclusions This laboratory-developed method had demonstrated to be sensitive,rapid,accurate and reliable in the detection of HIV-1 drug resistant mutations in the PR and RT regions.Our results indicate a potential of this method as a useful in vitro diagnostic procedure in the HIV-related clinical field.
作者
刘悦
董子维
陈悦宁
谢龙
李瑞玲
蒋析文
LIU Yue;DONG Zi-wei;CHEN Yue-ning;XIE Long;LI Rui-ling;JIANG Xi-wen(National and Regional Joint Engineering Laboratory for Clinical Medical Molecular Diagnostics,Guangdong Province Nucleic Acid Molecular Diagnostics Engineering Technology Research Center,Guangdong Provincial Clinical Medical Molecular Diagnostics Engineering Technology Center,Daan Gene Co.,Ltd.,Guangzhou,Guangdong 510665,China)
出处
《热带医学杂志》
CAS
2021年第9期1115-1118,1142,共5页
Journal of Tropical Medicine
基金
国家科技重大专项(2018ZX10732401,2018ZX10306414)
广州市产业技术重大攻关计划联盟产学研重大专项(201802030001)。
