多引物嵌套式聚合酶链反应在艾滋病病毒感染诊断中的应用

Diagnosis of HIV Infection by Multiple Primers of Nested PCR

目的 评价多引物嵌套式聚合酶链反应(Nested-PCR)在艾滋病病毒(HIV)感染诊断中的应用。方法 采用柱离心式小量组织/细胞基因组脱氧核糖核酸(DNA)抽提试剂盒提取DNA。用3套分别来自于长末湍重复序列(LTR)-gag、pol、env不同区域的序列引物进行嵌套式PCR扩增。结果 用多引物嵌套式PCR对28例不同来源标本进行检测,其中13例为HIV感染者,4例为HIV可疑感染者,4例为人嗜T淋巴细胞病毒(HTLV)感染者且HIV抗体检测为阴性,7例为正常者。经过检测每一套引物均漏检了1份标本,即每一套引物敏感性均为92.3％。HTLV标本经检测均为HIV阴性。根据判断标准,该检测方法敏感性和特异性均达到100％,最低检测线达到103外周血单核细胞(PBMC)。结论 多引物嵌套式PCR具有高敏感性及特异性,在鉴定HTV感染方面有重要的意义.可作为蛋白印迹试验的补充和辅助诊断。

Objective To evaluate the application of multiple primes of Nested-PCR for the diagnosis of HIV infection. Methods We used Tissue/cell Genomic DNA isolation Kit as a method of DNA extraction. In our laboratory, a diagnosis Nested PCR was carried out on proviral HIV-1 DNA, using three HIV-1 primer sets. The three primer sets, amplifying a fragment in the LTR-gag gene, in the pol gene and in the env gene, were situated within conserved regions of the HIV-1 genome. Results A panel of 28 samples was tested by Nested PCR. The panel contained 13 seropositives, 4 suspected specimen, 4 specimen of HTLV infection but with HIV antibody negative and 7 healthy seronegatives, No false positives were found, but in each of the primers sets one sample was missed. For each of the primer sets, the sensitivity was 92.3% .According to the testing algorithm, the sensitivity and specificity of Nested PCR was 100%, respectively. We were able to detect HIV-1 DNA from 103 PBMC.Conclusion Multiple primers of Nested PCR is highly sensitive and specific and has important significance for the diagnosis of HIV infection, so it may be used as an supplementary test to Western Blot.