摘要
作者用计算机辅助分析和比较１６种ＨＩＶ－１和ＨＩＶ－２序列并设计了一对用于检测ＨＩＶ－ｌ和ＨＩＶ－２的通用ＰＣＲ引物ＰＧ０３和ＰＧ０４．它们在ＡＲＢ－２病毒序列上分别位于４５７～４７８和７６６～７９８位核苷酸。两引物在ＡＲＶ－２基因上指导合成一个长３４２ｂｐ的ＤＮＡ片段。用ｐＡＲＶ－２／７Ａ和含有ｇａｇ基因的亚克隆质粒ｐＪＧ４２３作模板，均可扩增出特异性靶基因序列，而用不含ｇａｇ基因的亚克隆质粒ｐＪＥ３３２，ｐＪＰ１６３和ｐＪＰ０３２作模板则无扩增产物。将ｐＪＧ４２３进行稀释，结果该ＰＣＲ法可检出少至数个拷贝的质粒ＤＮＡ，表明此法敏感性极高。作者用ＰＣＲ法还直接从ＨＩＶ阳性血清中检测出ＨＩＶ基因序列，而正常对照血清却不能扩增出ＰＣＲ产物。
sixteen sequences of human immunodeficiency virus(HIV)type 1 and type 2 were compared withthe assistance of computer analyses.Two short sequences that is highly conserved in both HIV-1 and HIV-2were selected as the polymerase chain reaction(PCR)primers PG03 and PG04 for the detection of HIV infec-tions.The primers were designed degenerative sequences so that they may cover most HIV-4 and HIV-2strains.Primer PG03 and PG04 are located 457～ 478 and 766～ 798 nucleotide of ARV-2 and fland a DNAftagment of 342 bp long.ARV-2 plasmid and the subclone plasmid pJG423 which contains the gag gene frag-ment were used to test the primers,and both gave positive results. While the gag sequence- free ARV-2 sub-clone plasnid pJE332,pJP163 and pJP032 were all negative by PCR assays. The sensitivity of PCR was ex-tremely high that few copies of plasmid pJG423 could also be detected by fourty rounds of PCR amplifica-tions. This technique was further tested by direct detection of an HIV-positive serum.The result showedthat HIV proviral sequences were directly amplified from the HIV positive serum but not from normal humanserum.
出处
《第四军医大学学报》
1994年第1期1-4,共4页
Journal of the Fourth Military Medical University
关键词
聚合酶链反应
艾滋病毒
基因检测
polymerase chain reaction
human immunodeficiency virus
gene detection
serum
plasmid
