利用PCR法检测HIV—1 DNA

Detection of HIV-1 DNA by PCR

通过比较多个HIV(人免疫缺陷病毒)分离株的核苷酸序列,我们选择膜基因上7373—7514位的一段保守区为目的片段合成了引物1(5′—AGCAGCAGGAAGCACTATGGGC—3′)和引物2(5′—CCAGACTGTGAGTTGCAACA—3′),并分别以质粒PⅢexE7和MT4-HIV-1 DNA,为模板进行了PCR反应及敏感性试验。结果表明,用PCR法可检测出1～10个质粒分子及1×10~6个细胞中一个感染细胞。因此我们推论,本法可应用于AIDS临床标本的检测。

A single pair of oligonucleotide primers within the envelope gene of HIV1(primer 1: 5'-AGCAGCAGGAAGCACTATGGGC-3'; primer 2: 5'-CCAGACTGAGTTGCAACA-3'), 142bp apart, were synthesized and used to amplify a specific fragment of the HIV-1 DNA in infected cell cultures(MT4 HIV 1), and in plasmid pⅢexE7 carrying HIV-1 envelope gene by means of polymerase chain reaction(PCR). Amplified product was identified as 142bp fragment by agrose electrophoresis and cut into a 50bp and a 92bp fragment with restriction endonuclease HaeⅢ. Hybridization was carried out with α-^(32)P labeled gene fragment corresponding to the relevent region of the HIV-1 genome. All the experimental results sbowed that the amplified product was the faithful copy of the envelope gene sequence. 10^(-5)pg of plasmid DNA (about 1—10 plasmid molecules)and one infected cell in 10~6 cells could be detected by this method. From this data, we conclude that PCR is a specific, sensitive and quantitative assay.