Hashimoto A, Koga H, Kohno S, Miyazaki Y, Ohno H, Ogawa K, Higashiyama Y, Tomono K, Kaku M, Hara K
Second Department of Internal Medicine, Nagasaki University School of Medicine, Japan.
Kekkaku. 1994 Dec;69(12):767-78.
We developed the rapid detection and identification method of mycobacteria, involving amplification of mycobacterial 16S rRNA gene by nested PCR and identification of M. tuberculosis complex or M. avium-intracellulare complex (MAC) by hybridization protection assay (HPA) using the acridinium-ester (AE) labeled DNA probe. The specificity of the nested PCR combined with DNA probe test was excellent in terms of detection of mycobacterial organisms and identification of M. tuberculosis or MAC. The detection limits of the present method were 10 fg DNA for M. tuberculosis, and 100 fg DNA for MAC, respectively. We further investigated on the optimum temperature for hybridization in HPA with AE labeled DNA probe because there was the difference in the mode of DNA-RNA hybridization from that of DNA-DNA hybridization. In our method, the optimum temperature of hybridization was estimated as 55 +/- 1 degrees C. In preliminary experiments on two clinical cases, we practically detected and identified M. tuberculosis and MAC in clinical specimens, such as sputa, by using this newly devised method. We concluded that this method is useful for rapid detection and identification of M. tuberculosis and MAC in clinical specimens.
我们开发了分枝杆菌的快速检测和鉴定方法，该方法包括通过巢式PCR扩增分枝杆菌16S rRNA基因，并使用吖啶酯（AE）标记的DNA探针通过杂交保护分析（HPA）鉴定结核分枝杆菌复合群或鸟分枝杆菌-胞内分枝杆菌复合群（MAC）。就分枝杆菌的检测以及结核分枝杆菌或MAC的鉴定而言，巢式PCR与DNA探针检测相结合的特异性极佳。本方法对结核分枝杆菌的检测限为10 fg DNA，对MAC的检测限为100 fg DNA。由于DNA-RNA杂交模式与DNA-DNA杂交模式存在差异，我们进一步研究了使用AE标记DNA探针的HPA中的最佳杂交温度。在我们的方法中，杂交的最佳温度估计为55±1℃。在对两个临床病例的初步实验中，我们使用这种新设计的方法实际检测并鉴定了临床标本（如痰液）中的结核分枝杆菌和MAC。我们得出结论，该方法对于临床标本中结核分枝杆菌和MAC的快速检测和鉴定很有用。
