Fletcher H A, Barton R C, Verweij P E, Evans E G
Department of Microbiology, University of Leeds, UK.
J Clin Pathol. 1998 Aug;51(8):617-20. doi: 10.1136/jcp.51.8.617.
To develop a DNA based plate hybridisation assay for the detection of polymerase chain reaction (PCR) products amplified from Aspergillus fumigatus DNA; and to determine the sensitivity of this technique and compare it with Southern blotting.
A half-log dilution series of DNA extracted from A fumigatus was amplified with specific primers, one of which was 5' end labelled with biotin. PCR products were subsequently detected by agarose gel electrophoresis, Southern blotting, and binding of the products to a streptavidin coated microtitre well, followed by non-radioactive colorimetric detection. Amplification was carried out 10 times for each DNA dilution and a plot of initial DNA concentration against signal intensity was made.
A DNA concentration of 1.5 pg could be detected by agarose gel electrophoresis and Southern blotting with a non-radioactively labelled aspergillus specific probe; 1.5 pg was detectable by streptavidin binding of the PCR products to a microtitre plate. The signal from the microtitre plate detection was proportional to the amount of DNA in the PCR reaction on a log-log scale between 100 and 1 pg of DNA.
A DNA based plate hybridisation assay for the detection of A fumigatus PCR products is as sensitive as Southern blotting. However, results are obtained in three hours rather than the three days required for agarose gel electrophoresis, blotting, hybridisation, and detection.
开发一种基于DNA的平板杂交检测方法，用于检测从烟曲霉DNA扩增的聚合酶链反应（PCR）产物；并确定该技术的灵敏度，并与Southern印迹法进行比较。
用特异性引物扩增从烟曲霉中提取的DNA的半对数稀释系列，其中一个引物的5'端用生物素标记。随后通过琼脂糖凝胶电泳、Southern印迹法检测PCR产物，并将产物与链霉亲和素包被的微量滴定孔结合，然后进行非放射性比色检测。对每个DNA稀释度进行10次扩增，并绘制初始DNA浓度与信号强度的关系图。
通过琼脂糖凝胶电泳和用非放射性标记的曲霉特异性探针进行Southern印迹法可检测到1.5 pg的DNA浓度；通过将PCR产物与微量滴定板进行链霉亲和素结合可检测到1.5 pg。在100至1 pg DNA之间的对数-对数尺度上，微量滴定板检测的信号与PCR反应中的DNA量成正比。
用于检测烟曲霉PCR产物的基于DNA的平板杂交检测方法与Southern印迹法一样灵敏。然而，三小时即可获得结果，而不是琼脂糖凝胶电泳、印迹、杂交和检测所需的三天时间。
