Zammatteo N, Moris P, Alexandre I, Vaira D, Piette J, Remacle J
Laboratoire de Biochimie Cellulaire, Facultés Notre-Dame de la Paix, Namur, Belgium.
J Virol Methods. 1995 Oct;55(2):185-97. doi: 10.1016/0166-0934(95)00050-5.
A new bioluminescent detection system combined with a sandwich DNA hybridisation reaction in microwells has been developed for the detection of human immunodeficiency virus type 1 (HIV-1) provirus DNA amplified by the polymerase chain reaction (PCR). First, a fragment of the HIV-1 gag gene was amplified. The amplified DNA fragments were denatured and hybridised to a capture probe immobilised in microwells and to a biotinylated detection probe. A streptavidin-pyruvate kinase conjugate could then react on the biotinylated probe and the kinase activity detected by means of the luciferin-luciferase system, with production of a bioluminescent signal. This sandwich assay followed by a bioluminescent reaction detected as little as 7 amol of target DNA. The bioluminescent assay detected 5 HIV copies generated after one round of PCR, even if no band was seen on an agarose gel. The assay was applied to the detection of HIV-proviral DNA in peripheral blood mononuclear cells after one round of PCR and allowed to clearly identify a positive sample as compared to nested PCR.
一种结合微孔夹心DNA杂交反应的新型生物发光检测系统已被开发出来，用于检测通过聚合酶链反应（PCR）扩增的人类免疫缺陷病毒1型（HIV-1）前病毒DNA。首先，扩增HIV-1 gag基因的一个片段。将扩增的DNA片段变性，并与固定在微孔中的捕获探针以及生物素化的检测探针杂交。然后，链霉亲和素-丙酮酸激酶偶联物可与生物素化探针反应，并通过荧光素-荧光素酶系统检测激酶活性，产生生物发光信号。这种夹心测定法随后进行生物发光反应，可检测低至7 amol的靶DNA。即使在琼脂糖凝胶上未看到条带，生物发光测定法也能检测到一轮PCR后产生的5个HIV拷贝。该测定法应用于一轮PCR后外周血单个核细胞中HIV前病毒DNA的检测，与巢式PCR相比，能够清晰地鉴定出阳性样本。
