新型HIV-1核酸定量内标构建及验证
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摘要
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.
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.
引文
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[20]徐树莹,乔录新,史宣玲,等.HIV-1假病毒包装方法的探讨[J/CD].中华实验和临床感染痫杂志(电子版),2014,8(1):10-13.
[2]张岭,蒋岩,潘品良.HIV-I病毒载量检测常用技术及研究进展[J].传染病信息,2015,28(6):352-356.
[3]刘敏,陈秀英,梅少林,等.不同核酸提取方法检测HIV病毒载量和耐药基因的研究[J].中国现代医生,2016,54(20):111-114.
[4]John B,Lupiwa T,Tollman P,et al.Validation of the Roche AMPLICOR HIV DNA test version 1.5 for early infant diagnosis of HIV in Papua New Guinea[J].PNG Med J,2012,55(l-4):16-23.
[5]付龙亭,顾士民.HIV-1核酸扩增(PCR).荧光定量检测试剂盒的临床应用评价[C].第二届全国现代免疫诊断技术学术研讨会论文汇编,2005:551-554.
[6]朱碧姝.HIV抗原抗体诊断试剂在献血者筛查中的应用[J].中国输血杂志,2006,19(4):304-305.
[7]Mayer MP.A new set of useful cloning and expression vectors derived from pBlueScript[J].Gene,1995,163(1):41-46.
[8]李华琴,林陈水,张文倩,等.不依赖连接反应的高通量克隆方法[J].氨基酸和生物资源,2013,35(4):43-46.
[9]张阳璞,杨淑慎.几种新型植物基因表达载体的构建方法[j].生物工程学报,2015,31(3):311-327.
[10]罗官生.HIV病毒分子生物学检测技术最新进展[J].中国保健营养(中旬刊),2013,24(12):688-689.
[11]郭宏雄,还锡萍,周莹,等.环介导等温扩增技术检测HIV-1DNA[J].江苏大学学报(医学版),2015,25(6):528-531.
[12]宋震,周常勇,刘科宏,等.柑橘碎叶病毒巢式RT-PCR检测方法建立及应用[J].果树学报,2011,28(3):458-462.
[13]孔丰,袁远华,肖成谋,等.新型鸭呼肠孤病毒RT-PCR检测方法建立与初步应用[J].广东畜牧兽医科技,2014,39(1):24-27.
[14]Piras-Straub K,Khairzada K,Kocabayoglu P,et al.A-1573T>C SNP within the human TRAIL promoter determines TRAIL expression and HCC tumor progression[J].Cancer Med,2016,5(10):2942-2952.
[15]Di Domenico M,Di Giuseppe M,Rodriguez JD,et al.Validation of a fast real-time PCR method to detect fraud and mislabeling in milk and dairy products[J].J Dairy Sci,2017,100(1):106-112
[16]Muller J,Eis-Hubinger AM,Daumer M,et al.A novel internally controlled real-time reverse transcription-PCR assay for H1V-1 RNA targeting the pol integrase genomic region[J].J Virol Methods,2007,142(1-2):127-135.
[17]Dreier J,Stormer M,Kleesiek K.Use of bacteriophage MS2 as an internal control in viral reverse transcription-PCR assays[J].J Clin Microbiol,2005,43(9):4551-4557.
[18]Polonis VR,Brown BK,Rosa Borges A,et al.Recent advances in the characterization of HIV-1 neutralization assays for standardized evaluation of the antibody response to infection and vaccination[J].Virology,2008,375(2):315-320.
[19]DePolo NJ,Reed JD,Sheridan PL,et al.VSV-G pseudotyped lentiviral vector particles produced in human cells are inactivated by human serum[J].Mol Ther,2000,2(3):218-222.
[20]徐树莹,乔录新,史宣玲,等.HIV-1假病毒包装方法的探讨[J/CD].中华实验和临床感染痫杂志(电子版),2014,8(1):10-13.