SIMPLE, SENSITIVE, AND SPECIFIC DETECTION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 IN CLINICAL SPECIMENS BY POLYMERASE CHAIN-REACTION WITH NESTED PRIMERS
SIMPLE, SENSITIVE, AND SPECIFIC DETECTION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 IN CLINICAL SPECIMENS BY POLYMERASE CHAIN-REACTION WITH NESTED PRIMERS
复制标题
DOI：
10.1128/jcm.28.7.1560-1564.1990
复制
发表时间：
1990-07-01
影响因子：
9.4
通讯作者：
FENYO, EM
中科院分区：
文献类型：
--
作者：
ALBERT, J;FENYO, EM
描述了一种简单、灵敏和特异的检测人类免疫缺陷病毒1型(HIV-1)的聚合酶链式反应(PCR)方法。我们改进了所有三个PCR步骤：样品准备、DNA扩增和扩增产物的检测。之前已经描述了一些改进，但它们从未被合并到一个完整的PCR协议中。外周血单个核细胞直接在含有十二烷基硫酸钠、Triton X-100和蛋白酶K的缓冲液中裂解。粗细胞裂解产物在两步聚合酶链式反应中扩增，首先用外引物扩增，然后用嵌套在第一引物内的内引物扩增。用琼脂糖凝胶电泳法和溴化乙锭染色对扩增产物进行显色。因此，我们避免了传统的DNA提取以及杂交检测聚合酶链式反应产物。用四套套式引物(JA4至JA7，JA9至JA12，JA13至JA16，JA17至JA20)分别扩增HIV-1 Gag、env gp120、env gp41和pol序列。我们能够从90名HIV-1血清阳性患者的所有样本中扩增出HIV-1基因序列，这些患者大多症状较轻。在这些人中，24人的HIV-1分离呈阴性，9人被选中，因为他们感染了非洲和海地的HIV-1毒株。85个(94%)的个体至少有三组四对引物呈阳性。从26名健康献血员以及体外感染人类免疫缺陷病毒2型和人类T细胞白血病病毒I型的细胞中提取的样本均为阴性，表明该扩增具有特异性。
A simple, sensitive, and specific polymerase chain reaction (PCR) protocol for the detection of human immunodeficiency virus type 1 (HIV-1) is described. We have improved all three PCR steps: sample preparation, DNA amplification, and detection of the amplified product. Some of the improvements have been described previously, but they have never been combined into a complete PCR protocol. Peripheral blood mononuclear cells were lysed directly in a buffer containing sodium dodecyl sulfate, Triton X-100, and proteinase K. This crude cell lysate was amplified in a two-step PCR, first with outer primers and then with inner primers nested within the first primers. The PCR product was visualized by agarose gel electrophoresis and ethidium bromide staining. Thus, we avoided conventional DNA extraction as well as hybridization for the detection of the PCR product. The sampling was analyzed with four sets of nested primers (JA4 through JA7, JA9 through JA12, JA13 through JA16, and JA17 through JA20) designed to amplify HIV-1 gag, env gp120, env gp41, and pol sequences, respectively. We were able to amplify HIV-1 sequences in all samples from 90 HIV-1-seropositive individuals with mostly mild symptoms. Of these individuals, 24 were negative in HIV-1 isolation and 9 were selected because they were infected by African and Haitian HIV-1 strains. Eighty-five (94%) individuals were positive with at least three of four primer sets. Samples from 26 healthy blood donors, as well as cells infected in vitro with human immunodeficiency virus type 2 and human T-cell leukemia virus type I, were negative in PCR, thus demonstrating the specificity of the amplification.