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首页> 外文期刊>Journal of Clinical Microbiology >Simple colorimetric microtiter plate hybridization assay for detection of amplified Mycobacterium leprae DNA.
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Simple colorimetric microtiter plate hybridization assay for detection of amplified Mycobacterium leprae DNA.

机译：简单的比色微量滴定板杂交检测扩增的麻风分枝杆菌DNA。

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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.
机译：检测聚合酶链反应（PCR）产生的扩增产物仍然是一个复杂的过程。为了简化检测程序，我们开发了比色微量滴定板杂交检测法，用于特异性检测麻风分枝杆菌DNA的5'-生物素化PCR片段。对于该测定，制备了麻风杆菌DNA捕获探针并将其固定在微量滴定板的孔上。通过酶促显色检测生物素标记的PCR片段的杂交。所得的光密度显示出与模板DNA的量的对数线性关系，并且对应于通过PCR产物的凝胶分析和Southern印迹获得的条带的强度。发现该测定方法的灵敏度为125 fg基因组麻风分枝杆菌DNA或20个裂解的杆菌，揭示了与琼脂糖凝胶分析相似的检测限。人DNA的有效共扩增被用作临床材料中抑制性物质存在的阳性对照。为了检测人类PCR产物，还构建了人类DNA捕获探针用于比色测定。这种双重杂交设置可同时检测麻风分枝杆菌和人DNA PCR产物，可用于确定临床标本中抑制物质的存在。未经治疗的麻风患者的所有活检标本（n = 10）均为阳性。显然，这种分析比显微镜更为灵敏，因为一半患者的活检标本在组织病理学检查中呈阴性。来自三名接受治疗的患者的活检标本为阴性，来自三位没有麻风病的患者的活检标本均为阴性。我们得出结论，这种比色测定法可以代替琼脂糖凝胶分析和Southern杂交，因为它与那些方法一样灵敏。与传统的凝胶分析和Southern杂交相比，它的优点是麻烦小且快速。

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