Suzuki H, Fukushima K, Matsuo M, Hashimoto N, Yoshihara N
AIDS Research Center, National Institute of Health, Tokyo.
Rinsho Byori. 1996 Dec;44(12):1169-74.
We have developed a microplate hybridization(MH) technique, which utilizes the non-isotopic method of enzyme-linked assay for detection of HIV in the amplified product after PCR. HIV RNA extracted from plasma was amplified by RT-Nested-PCR using biotinylated-inner primers of gag, pol and env regions, respectively. The PCR product was visualized by 5% polyacrylamide gel electrophoresis and ethidium bromide staining. The heat-denatured PCR product was hybridized with HIVcDNA of each region which was immobilized to a microplate. The hybridized microplate was reacted with streptavidin-conjugate peroxidase and then the optical density(O.D.) was read at 490nm. The cut off value was determined at O.D. 0.25. The results of the electrophoresis of gag, pol and env regions were all positive in 53 HIV-1 seropositive samples from Japan and the USA, and all negative in 55 HIV-1 seronegative samples. Using the MH technique, USA samples showed a higher O.D. than the Japan samples, particularly in the pol region. The results of MH technique in gag and pol regions coincided with that of electrophoresis. But, one of 27 Japanese HIV-1 seropositive samples showed O.D. of less than 0.2 in only env region. This particular sample was classified by V3 peptide-based enzyme immunoassay as subtype E, which differs from the typical subtype B of Japan and USA samples. This suggests the presence of several genotypes in HIV-1 seropositive individuals in Japan. Based on this data, the MH technique using gag, pol and env region is a simple, sensitive, safe and specific assay for detection of HIV-1 RNA in plasma, and would be useful in clinical testing.
我们开发了一种微孔板杂交（MH）技术，该技术利用酶联检测的非同位素方法来检测PCR扩增产物中的HIV。从血浆中提取的HIV RNA分别使用gag、pol和env区域的生物素化内引物通过RT-巢式PCR进行扩增。PCR产物通过5%聚丙烯酰胺凝胶电泳和溴化乙锭染色进行可视化。热变性的PCR产物与固定在微孔板上的每个区域的HIV cDNA进行杂交。杂交后的微孔板与链霉亲和素偶联的过氧化物酶反应，然后在490nm处读取光密度（O.D.）。临界值确定为O.D. 0.25。来自日本和美国的53份HIV-1血清阳性样本中，gag、pol和env区域的电泳结果均为阳性，而55份HIV-1血清阴性样本的电泳结果均为阴性。使用MH技术，美国样本的O.D.值高于日本样本，特别是在pol区域。gag和pol区域的MH技术结果与电泳结果一致。但是，27份日本HIV-1血清阳性样本中有1份仅在env区域显示O.D.值小于0.2。该特殊样本通过基于V3肽的酶免疫测定被分类为E亚型，这与日本和美国样本的典型B亚型不同。这表明日本HIV-1血清阳性个体中存在几种基因型。基于这些数据，使用gag、pol和env区域的MH技术是一种用于检测血浆中HIV-1 RNA的简单、灵敏、安全且特异的检测方法，在临床检测中将会很有用。
