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用寡色谱试纸条检测血吸虫聚合酶链反应扩增的DNA

Detection of schistosomes polymerase chain reaction amplified DNA by oligochromatographic dipstick.

作者信息

Akinwale O P, Laurent T, Mertens P, Leclipteux T, Rollinson D, Kane R, Emery A, Ajayi M B, Akande D O, Fesobi T W

机构信息

Zoology Department, Wolfson Wellcome Biomedical Laboratories, Natural History Museum, London, UK.

出版信息

Mol Biochem Parasitol. 2008 Aug;160(2):167-70. doi: 10.1016/j.molbiopara.2008.04.003. Epub 2008 Apr 12.

DOI:10.1016/j.molbiopara.2008.04.003
PMID:18501978
Abstract

The applications of highly specific and sensitive molecular techniques based on polymerase chain reaction (PCR) have constituted a valuable tool for the diagnosis of schistosomiasis and also for the detection of schistosome infections in the snail intermediate hosts. The common method of detecting PCR amplicons is gel electrophoresis in the presence of ethidium bromide, a carcinogen, which is followed by UV transillumination. Other methods, which are available for detecting PCR products, are real-time PCR, PCR-enzyme-linked immunosorbent assay (PCR-ELIZA) and mass spectrometry but they are cumbersome while they are sometimes complex and expensive. Therefore, a simple method of PCR product detection would be a welcome idea and a most valuable tool particularly in disease endemic countries with limited research facilities and resources. In this study, we applied a simple and rapid method for the detection of Schistosoma haematobium and Schistosoma mansoni PCR amplified DNA products using oligochromatographic (OC) dipstick. The amplicons are visualized by hybridization with a gold conjugated probe, while a control for the chromatographic migration is incorporated in the assay. The lower detection limit observed was 10fg of genomic DNA from each of the two species, while the dipstick was also specific for each of the species used in this study.

摘要

基于聚合酶链反应(PCR)的高特异性和高灵敏度分子技术的应用,已成为诊断血吸虫病以及检测钉螺中间宿主中血吸虫感染的宝贵工具。检测PCR扩增产物的常用方法是在存在致癌物溴化乙锭的情况下进行凝胶电泳,随后进行紫外透射照明。其他可用于检测PCR产物的方法有实时PCR、PCR-酶联免疫吸附测定(PCR-ELISA)和质谱分析,但这些方法操作繁琐,有时还复杂且昂贵。因此,一种简单的PCR产物检测方法将是一个受欢迎的想法,也是一种非常有价值的工具,特别是在研究设施和资源有限的疾病流行国家。在本研究中,我们应用了一种简单快速的方法,使用寡色谱(OC)试纸条检测埃及血吸虫和曼氏血吸虫的PCR扩增DNA产物。扩增产物通过与金标记探针杂交进行可视化,同时在检测中加入了色谱迁移对照。观察到的最低检测限为这两个物种各自基因组DNA的10fg,并且试纸条对本研究中使用的每个物种也具有特异性。

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