用逆转录-聚合酶链反应(RT-PCR)检测HIV-1感染

Using RT- PCR method to detect HIV- 1 infection

为了解决窗口期和发病期的 HIV- 1感染者中，抗体产生不足和 (或 )由于病毒大量复制中和了血液中的抗体，用检测抗体的常规方法难以确认，这一个困扰临床和防疫工作者的难题，我们建立了用逆转录-聚合酶链反应检测 HIV- 1感染者血清中病原体的方法。其优点为： (1)敏感性强，检测结果无假阴性反应； (2)扩增 HIV- 1的 gag,pol和 env三个基因区的保守基因片段，且每个片段都做巢式扩增，并规定扩增出两个片段才能确认，特异性为 100％； (3)所用的引物不仅能检测出中国的 HIV- 1流行株，也能检出非洲的 HIV- 1流行株； (4)使用了 RNA提取试剂盒，可直接利用筛选试验剩余的血清作标本，标本用量少 (200μ L)，方法相对较简便。

In window period and at AIDS stage, it is difficult to detect a low level of HIV antibodies by routine conventional method due to either no sufficient antibodies generated or antibodies neutralized by rapid active replication of HIV virus.This has been remained as a difficult problem in clinical and preventive medicine. Recently, we have developed a reverse transcriptase- PCR(RT- PCR) method to detect the pathogen in the serum of HIV carriers and AIDS patients. The advantages of our methods were discussed. First, RT- PCR had high sensitivity with no false negative reaction. Also, the specificity of this method was 100％ . Three sets of primers from the conserved regions of HIV genome were selected and used to amplify specifically the fragment of gag, pol, or env gene, respectively. Each fragment was amplified by nest method and was confirmed by the presence of the corresponding fragments. The primers used in this study can be used to detect not only HIV strains from Chinese population but also from African and European population. In addition, the detection procedure using RNA capture kit was relatively simple and only required a small amount of sample (200μ l serum).