Mallet F, Hebrard C, Brand D, Chapuis E, Cros P, Allibert P, Besnier J M, Barin F, Mandrand B
Unité Mixte de Recherche 103, Centre National de la Recherche Scientifique-bioMérieux, Ecole Normale Supérieure de Lyon, France.
J Clin Microbiol. 1993 Jun;31(6):1444-9. doi: 10.1128/jcm.31.6.1444-1449.1993.
An enzyme-linked oligosorbent assay (ELOSA) was developed for the detection on microtiter plates of polymerase chain reaction (PCR)-amplified human immunodeficiency virus type 1 (HIV-1) DNA. The denatured PCR product was hybridized with a passively adsorbed oligonucleotide capture probe and a horseradish peroxidase-labeled oligonucleotide detection probe. The sensitivity and specificity of the PCR-ELOSA technique depended to some extent on the nucleotide sequences of the oligonucleotide primer and probe quartet used in the amplification and detection. We evaluated five oligonucleotide quartets located in the gag, pol, vpr, env, and nef regions of HIV-1. DNAs from 39 HIV-1-seropositive individuals and 27 healthy HIV-1-seronegative controls were amplified by the PCR procedure, and the products were detected by ELOSA. Ten copies of HIV-1 DNA against a background of 1 microgram of human DNA were specifically detected by PCR-ELOSA. Specificities and sensitivities were, respectively, 100 and 95% for the gag system, 100 and 97% for the pol system, 100 and 85% for the vpr system, 96 and 95% for the env system, and 100 and 95% for the nef system. The simplicity of ELOSA makes it suitable for automation and applicable to genetic testing and detection of viral and bacterial DNAs or RNAs in most routine laboratories.
开发了一种酶联寡吸附测定法（ELOSA），用于在微量滴定板上检测聚合酶链反应（PCR）扩增的1型人类免疫缺陷病毒（HIV-1）DNA。变性的PCR产物与被动吸附的寡核苷酸捕获探针和辣根过氧化物酶标记的寡核苷酸检测探针杂交。PCR-ELOSA技术的灵敏度和特异性在一定程度上取决于扩增和检测中使用的寡核苷酸引物和探针四重奏的核苷酸序列。我们评估了位于HIV-1的gag、pol、vpr、env和nef区域的五个寡核苷酸四重奏。通过PCR程序扩增39名HIV-1血清阳性个体和27名健康HIV-1血清阴性对照的DNA，并通过ELOSA检测产物。PCR-ELOSA可特异性检测出在1微克人类DNA背景下的10个HIV-1 DNA拷贝。gag系统的特异性和灵敏度分别为100%和95%，pol系统为100%和97%，vpr系统为100%和85%，env系统为96%和95%，nef系统为100%和95%。ELOSA的简便性使其适用于自动化，适用于大多数常规实验室的基因检测以及病毒和细菌DNA或RNA的检测。
