通过聚合酶链反应扩增和微量滴定夹心杂交法检测血清中的乙型肝炎病毒DNA
Detection of hepatitis B virus DNA in serum by polymerase chain reaction amplification and microtiter sandwich hybridization.
作者信息
Keller G H, Huang D P, Shih J W, Manak M M
机构信息
Biotech Research Laboratories, Inc., Rockville, Maryland 20850.
出版信息
J Clin Microbiol. 1990 Jun;28(6):1411-6. doi: 10.1128/jcm.28.6.1411-1416.1990.
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.
我们开发了一种用于检测聚合酶链反应（PCR）扩增的乙型肝炎病毒（HBV）序列的微量滴定夹心杂交检测法。该检测法采用类似酶联免疫吸附测定的形式，其中包含与一条PCR产物链的一半互补序列的克隆DNA被固定在微量滴定孔中。与同一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。通过PCR程序对69份乙型肝炎表面抗原阳性血清样本和16份抗原阴性对照样本的DNA进行扩增，并通过Southern杂交和夹心杂交检测产物。两种检测方法都能够检测到低至5份HBV DNA。与Southern杂交相比，夹心杂交检测法在检测扩增的HBV序列时灵敏度为100%，特异性为95%。然而，与Southern杂交不同的是，夹心杂交检测法使用非放射性探针，并且便于大量样本的处理。在74%的抗原阳性样本中检测到了DNA。通过两种方法检测，所有抗原阴性样本（健康献血者）的HBV DNA均为阴性。
Zotero 插件