人类免疫缺陷病毒1型感染基因诊断方法的建立  

Development of HIV-1 laboratory diagnostic assay based on the nucleic acid detection

作　　者：魏民[1] 关琪[2] 梁浩[3] 陈健平[1] 陈钊[1] 冯毅[1] 洪坤学[1] 邢辉[1] 邵一鸣 

机构地区：[1]中国疾病预防控制中心性病艾滋病预防与控制中心病毒免疫室,北京100050 [2]沈阳医学院附属中心医院检验科 [3]华中科技大学同济医学院公共卫生学院

出　　处：《中华检验医学杂志》2003年第12期772-776,共5页Chinese Journal of Laboratory Medicine

基　　金：1999年度国家杰出青年科学基金资助项目(3992 50 30 );国家重点基础研究发展规则项目(G1 9990 541 0 7)

摘　　要：目的　建立人类免疫缺陷病毒 1型 (HIV 1)基因诊断方法。方法　随机选取 80份全国送检的已确认的HIV阳性样品 ,从健康献血员中选取 40份HIV阴性样品。分别提取人DNA和病毒RNA ,再分别扩增HIV 1的gp41、pol和gag各基因区的 3套反应系统 ,进行巢氏聚合酶链反应(nPCR)和逆转录聚合酶链反应 (RT PCR)。将结果与血清学方法比较 ,观察结果是否一致。同时检测 3套反应系统的敏感性、重复性和对HIV 1各亚型参考病毒株的扩增情况。结果　用 3套反应系统对 80份阳性样本进行nPCR检测的阳性率为 :gp41反应系统 88 8% ;pol反应系统 83 8% ;gag反应系统 81 3 % ;3套系统联合应用检出率 90 0 %。用RT PCR检测阳性率为 :gp41反应系统96 3 % ;pol反应系统 91 3 % ;gag反应系统 95 0 % ;3套反应系统联合应用检出率可达 98 8%。阴性样品扩增均阴性 ,特异性为 10 0 %。 3套反应系统不但可扩增HIV 1型M组的A、B、C、D、CRF0 1 AE、F、G、H亚型 ,还可扩增其N和O组毒株 ,且与HIV 2无交叉反应。nPCR最低检测限为 1拷贝 /PCR。gag和pol反应系统RT PCR最低检测限为 750拷贝 /ml;gp41反应系统最低检测限为 150拷贝 /ml。nPCR和RT PCR扩增gp41、pol和 gag基因区的重复性分别为 :93 3 %、90 0 %、 86 7%和10 0 %、96 7%、10 0 %?Objective To develop a HIV-1 laboratory diagnostic assay based on the nucleic acid detection. Methods Proviral DNA from whole blood and viral RNA from plasma were extracted from 80 HIV-positive samples and 40 HIV-negative samples. Three primer sets, amplifying HIV-1 gp41, pol and gag genes respectively, were used in nested polymerase chain reaction (nPCR) and reverse transcription (RT)-PCR. To cheap the cost, domestically produced Taq enzyme and reverse transcriptase were applied. Compare the nested PCR and RT-PCR results with serodiagnosis. Moreover, it was detected to the sensitivity, reproducibility and detection of different HIV-1 subtype reference strains based on the above three pairs of nested PCR and RT-PCR systems. Results For the 80 HIV positive samples, nested PCR systems detected samples as follows: gp41 primer sets, 71 (88 8%); pol primer sets, 67 (83 8%); gag primer sets, 65 (81 3%); and 72 samples (90 0%) in combination. RT-PCR systems detected samples as follows: gp41 primer sets, 77 (96 3%); pol primer sets, 73 (91 3%); gag primer sets, 76 (95 0%). In combination of three RT-PCR systems, only one sample was negative (98 8%, 79/80) For the 40 HIV negative samples, the results were all negativ e for three nested PCR and RT-PCR systems. Furthermore, the three reacti ve systems not only detected HIV-1 M group subtype A, B, C, D, CRF01-AE, F, G, H reference strains, but also amplify N and O group strains successfully. No cross-reaction to HIV-2 strains was found. The detection limit of nPCR was up to 1 copy/PCR, and the detection limit of RT-PCR was up to 750 copies/ml fo r gag and pol reactive systems, 150 copies/ml for gp41 system. The reproducibili ty of nested PCR was 93 3%, 90 0%, and 86 7% for gp41, pol, and gag systems, and the reproducibility of RT-PCR was 100%, 96 7%, and 100% for gp41, pol, and gag systems. Conclusions We have developed HIV-1 laboratory diagnostic assay based on the nucleic acid det ection with very low cost. The sensitivity, specificity and reproducibilit

关 键 词：人类免疫缺陷病毒1型感染 基因诊断 艾滋病 聚合酶链反应 血清学诊断 

分 类 号：R446.5[医药卫生—诊断学]