A sensitive nonisotopic hybridization assay for HIV-1 DNA.
A sensitive nonisotopic hybridization assay for HIV-1 DNA.
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HIV-1 DNA 的灵敏非同位素杂交测定。
DOI：
10.1016/0003-2697(89)90007-9
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发表时间：
1989
影响因子：
2.9
通讯作者：
Manak,MM
中科院分区：
文献类型：
--
作者：
Keller,GH;Huang,DP;Manak,MM
我们开发了一种基于微滴度的夹心杂交检测方法，用于检测低拷贝数HIV-1序列。该分析采用捕获DNA序列共价偶联到微滴孔通过连接臂。检测探针是生物素标记的DNA片段，来源于与捕获序列相邻的序列。在样品核酸存在下杂交后，只有当样品包含跨越捕获探针和检测探针之间连接处的互补序列时，检测探针才保持结合。用过氧化物酶-链亲和素缀合物和比色过氧化物酶底物孵育，定量检测探针结合的量。该试验已与酶靶扩增相结合，以实现患者样品中HIV-1的敏感检测。使用聚合酶链反应技术扩增HIV-1 DNA后，产生190 bp的产物。该产品易于使用夹心杂交法进行定量分析。由此产生的测试可以在105个细胞或大约30个HIV-1 DNA分子中检测到一个HIV-1感染的细胞。
We have developed a microtiter-based sandwich hybridization assay for the detection of low copy number HIV-1 sequences. The assay employs a capture DNA sequence covalently coupled to microtiter wells through linker arms. The detection probe is a biotin-labeled DNA fragment derived from sequences adjacent to the capture sequence. After hybridization in the presence of sample nucleic acid, the detection probe remains bound only if the sample contained complementary sequences spanning the junction between capture and detection probes. The amount of detection probe bound is quantified by incubation with a peroxidase-streptavidin conjugate and a colorimetric peroxidase substrate. This assay has been combined with enzymatic target amplification to achieve sensitive detection of HIV-1 in patient samples. Following amplification of HIV-1 DNA using the polymerase chain reaction technique, a 190-bp product is produced. This product is easily and specifically quantified using the sandwich hybridization assay. The resulting test can detect one HIV-1-infected cell in 105cells or about 30 molecules of HIV-1 DNA.