新型HIV-1核酸定量内标构建及验证

Construction and verification of a novel HIV-1 nucleic acid quantitative internal standard

目的构建一种稳定的以病毒颗粒为基础的HIV核酸定量检测内标。方法在实验室原有新型表型耐药载体pcDNA6.2-LTR gagpol的基础上,利用PCR扩增法在不改变p24保守区基础上突变核苷酸,经Gateway重组得到含突变碱基的pNL43-△ENV-LUC-ATTR表达载体,该载体与水泡性口膜炎病毒(VSV)质粒共转染293细胞48 h后收集上清中HIV-1内标假病毒颗粒,检测p24抗原含量,定量假病毒滴度,以倍比稀释内标质粒、内标质粒与阳性标本混合品、内标假病毒颗粒与阳性标本混合品为模板进行实时定量PCR法,观察内标质粒、内标假病毒颗粒内的核酸与内标引物探针的匹配情况,以及是否受样本探针引物的干扰。结果定点突变表型耐药载体pcDNA6.2-LTR gagpol内的p24保守区片段;获得仅具有1次感染性的HIV-1假病毒颗粒(3.61×10~9IU/ml);p24抗原检测含量为283.2 pg/ml;实时定量PCR法确定内标质粒、内标假病毒颗粒内的核酸与内标引物探针匹配良好,内标引物探针对阳性标本探针无干扰。结论成功建立了一种以假病毒颗粒为基础的HIV-1核酸定量检测内标。

Objective To construct a stable HIV-1 nucleic acid quantitative detection of internal standard based on virus particles.Methods p24 region was mutated without changing p24 conservative district and inserted into phenotype resistant vector pcDNA6.2-LTR gagpol by PCR amplification method,following by the construction of pNL43-AENV-LUC expression vector containing the specific mutation base by Gateway recombinant technology.This vector and VSV plasmid were cotransfected into 293 T cell lines and HIV-1 pseudotyped virus was collected from the supernatant 48 hours later.The loads of HIV-1 were quantified by detection of p24 antigen titers.The multiproportion diluted liquids of the internal standard plasmid,the mixture of the internal standard plasmid and positive samples and the mixture of the internal standard pseudotyped viral particles and positive samples were used as the template for the real-time quantitative PCR to verify whether the nucleic acids levels matched the probes of internal standard plasmid,and whether they could be interfered by the sample probe and primers.Results The fragment of specific site mutation in p24 conservative region was inserted into the phenotype resistant vector pcDNA6.2-LTR gagpol.The detective p24 antigen was 283.2 pg/ml,while HIV-1 pseudotyped virus was 3.61 × 10~9IU/ml.Internal standard plasmid and internal standard pseudotyped viral particles matched the probes of internal standard plasmid,which were confirmed using real-time quantitative PCR and not interfered by the sample probe and primers.Conclusion The novel internal standard construced with pseudotyped virus could be used to detect plasma HIV-1 level.