van der Vliet G M, Hermans C J, Klatser P R
N. H. Swellengrebel Laboratory of Tropical Hygiene, Royal Tropical Institute, Amsterdam, The Netherlands.
J Clin Microbiol. 1993 Mar;31(3):665-70. doi: 10.1128/jcm.31.3.665-670.1993.
The detection of amplified products resulting from polymerase chain reactions (PCRs) remains a complicated process. To simplify the detection procedures, we developed a colorimetric microtiter plate hybridization assay for the specific detection of 5'-biotinylated PCR fragments of Mycobacterium leprae DNA. For this assay, an M. leprae DNA capture probe was made and immobilized on the wells of a microtiter plate. Hybridization of the biotin-labeled PCR fragments was detected through enzymatic color development. The resulting optical densities showed a logarithm-linear relationship with the amount of template DNA and corresponded to the intensity of the bands obtained through gel analysis and Southern blotting of the PCR products. The sensitivity of the assay was found to be 125 fg of genomic M. leprae DNA, or 20 lysed bacilli, revealing a detection limit similar to that of agarose gel analysis. The efficient coamplification of human DNA was used as a positive control for the presence of inhibitory substances in clinical material. For detection of human PCR products, a human DNA capture probe was also constructed for the colorimetric assay. This dual setup for hybridization, which thus detected both M. leprae and human DNA PCR products, was useful for ascertaining the presence of inhibiting substances in clinical specimens. All biopsy specimens (n = 10) from untreated patients with leprosy were positive. Apparently, this assay is more sensitive than microscopy, because biopsy specimens from half of the patients were negative upon histopathological examination. Biopsy specimens from three treated patients were negative, as were those from the three patients who did not have leprosy. We conclude that this colorimetric assay can replace agarose gel analysis and Southern hybridization, because it is as sensitive as those methods. Its advantages over conventional gel analysis and Southern hybridization are that it is less cumbersome and more rapid.
聚合酶链反应（PCR）扩增产物的检测仍是一个复杂的过程。为简化检测程序，我们开发了一种比色微量滴定板杂交分析法，用于特异性检测麻风分枝杆菌DNA的5'-生物素化PCR片段。为此分析方法，制备了一种麻风分枝杆菌DNA捕获探针并将其固定在微量滴定板的孔中。通过酶促显色检测生物素标记的PCR片段的杂交情况。所得光密度与模板DNA量呈对数线性关系，且与通过PCR产物的凝胶分析和Southern印迹获得的条带强度相对应。该分析方法的灵敏度为125 fg麻风分枝杆菌基因组DNA，或20个裂解的杆菌，其检测限与琼脂糖凝胶分析相似。人类DNA的有效共扩增用作临床材料中是否存在抑制物质的阳性对照。为检测人类PCR产物，还构建了一种用于比色分析的人类DNA捕获探针。这种双重杂交设置可同时检测麻风分枝杆菌和人类DNA的PCR产物，有助于确定临床标本中是否存在抑制物质。所有未治疗的麻风病患者的活检标本（n = 10）均为阳性。显然，该分析方法比显微镜检查更灵敏，因为一半患者的活检标本经组织病理学检查为阴性。三名接受治疗患者的活检标本为阴性，三名非麻风病患者的活检标本也为阴性。我们得出结论，这种比色分析方法可替代琼脂糖凝胶分析和Southern杂交，因为它与那些方法一样灵敏。它相对于传统凝胶分析和Southern杂交的优势在于操作更简便、速度更快。
