通过聚合酶链反应（PCR）和比色微量滴定板杂交测定法（CMHA）对贝氏柯克斯体进行定量分析。

Quantification of Coxiella burnetii by polymerase chain reaction (PCR) and a colorimetric microtiter plate hybridization assay (CMHA).

作者信息

Fritz E, Thiele D, Willems H, Wittenbrink M M

机构信息

Institut für Hygiene und Infektionskrankheiten der Tiere, Justus-Liebĩg-Universität, Giessen, Germany.

出版信息

Eur J Epidemiol. 1995 Oct;11(5):549-57. doi: 10.1007/BF01719307.

DOI:10.1007/BF01719307

PMID:8549729

Abstract

A colorimetric microtiter plate hybridization assay (CMHA) for the quantitative determination of Coxiella burnetii DNA after amplification by externally controlled polymerase chain reaction (PCR) is described. The quantification assay is based on an enzyme linked immunosorbent assay (ELISA) format. Cloned DNA, representing a sequence complementary to an internal part of the diagnostic amplicon, was noncovalently attached to the wells of a microtiter plate. Biotinylated PCR product was hybridized to the immobilized capture probe. Bound product was detected via streptavidin horse-radish peroxidase. The devised nonisotopic technique allows specific, rapid, and convenient quantification of C. burnetii DNA. Additionally, it is compatible with standard laboratory ELISA equipment, making this assay amenable to automation and permitting processing of large sample numbers.

摘要

本文描述了一种比色微量滴定板杂交测定法（CMHA），用于在通过外部控制的聚合酶链反应（PCR）扩增后定量测定伯氏考克斯体DNA。该定量测定基于酶联免疫吸附测定（ELISA）形式。代表与诊断扩增子内部部分互补序列的克隆DNA非共价连接到微量滴定板的孔中。生物素化的PCR产物与固定的捕获探针杂交。通过链霉亲和素辣根过氧化物酶检测结合的产物。所设计的非同位素技术能够对伯氏考克斯体DNA进行特异性、快速且便捷的定量。此外，它与标准实验室ELISA设备兼容，使得该测定法适合自动化操作并允许处理大量样品。