Detection of hepatitis B virus DNA in serum by polymerase chain reaction amplification and microtiter sandwich hybridization
Detection of hepatitis B virus DNA in serum by polymerase chain reaction amplification and microtiter sandwich hybridization
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聚合酶链式反应扩增和微量滴定夹心杂交检测血清中乙型肝炎病毒DNA
DOI：
10.1128/jcm.28.6.1411-1416.1990
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发表时间：
1990
影响因子：
9.4
通讯作者：
M. Manak
中科院分区：
文献类型：
--
作者：
G H Keller;D. Huang;J W Shih;M. Manak
我们已经开发了一种用于检测聚合酶链反应（PCR）扩增的B型肝炎病毒（HBV）序列的微量滴定夹心杂交法。该测定采用酶联免疫吸附测定样形式，其中将含有与一条PCR产物链的一半互补的序列的克隆DNA固定在微量滴定威尔斯孔中。将与相同PCR产物链的另一部分互补的生物素标记的DNA序列用作探针。对69份B肝炎表面抗原阳性血清和16份抗原阴性对照血清进行PCR扩增，并进行Southern杂交和夹心杂交检测。两种检测方法都能够检测到少至5个拷贝的HBV DNA。与Southern杂交法相比，夹心杂交法检测HBV扩增序列的敏感性为100%，特异性为95%。然而，与Southern杂交不同的是，夹心杂交试验采用非放射性探针，并允许容易地处理大量样品。在74%的抗原阳性样本中检测到DNA。所有抗原阴性样本（健康献血员）经两种方法检测均为HBV DNA阴性。
We have developed a microtiter sandwich hybridization assay for the detection of polymerase chain reaction (PCR)-amplified hepatitis B virus (HBV) sequences. This assay utilizes an enzyme-linked immunosorbent assay-like format in which cloned DNA containing a sequence complementary to half of one PCR product strand is immobilized in microtiter wells. A biotin-labeled DNA sequence complementary to the other portion of the same PCR product strand is used as the probe. The DNAs from 69 hepatitis B surface antigen-positive serum samples and 16 antigen-negative control samples were amplified by the PCR procedure, and the product was detected by Southern and sandwich hybridization. Both detection procedures were capable of detecting as few as five copies of HBV DNA. Compared with Southern hybridization, the sandwich hybridization assay exhibited a sensitivity of 100% and a specificity of 95% for the detection of amplified HBV sequences. Unlike Southern hybridization, however, the sandwich hybridization assay employs a nonradioactive probe and allows easy handling of large numbers of samples. DNA was detected in 74% of the antigen-positive samples. All of the antigen-negative samples (healthy blood donors) were negative for HBV DNA by both procedures.