Detection of Clostridium piliforme by enzymatic assay of amplified cDNA segment in microtitration plates.
Detection of Clostridium piliforme by enzymatic assay of amplified cDNA segment in microtitration plates.
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通过酶法检测微量滴定板中扩增的 cDNA 片段来检测毛状梭菌。
DOI：
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发表时间：
1996
期刊：
影响因子：
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通讯作者：
T. Itoh
中科院分区：
文献类型：
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作者：
K. Goto;T. Itoh
我们开发了一个简单的酶检测系统，使用逆转录聚合酶链反应产物在微量滴定板检测组织标本中的梭状芽孢杆菌。逆转录-聚合酶链反应是用生物素标记的引物进行的，该引物针对C. piliforme 16 S rRNA基因。将扩增的cDNA与微管中的碱性磷酸酶标记的DNA探针杂交，并将混合物施加到用链霉亲和素预涂覆的微量滴定孔中。然后通过与碱性磷酸酶底物反应观察微量滴定板中的杂交信号。当在405 nm处光密度> 0.200被认为是阳性结果时，我们发现该系统可以检测到来自少至10个生物体的RNA。该系统还能够检测实验感染小鼠肝脏、肠系膜淋巴结和回肠中的微生物。回肠中C.感染后1 ~ 21 d检测到piliforme基因。这一结果表明，肠上皮是持续感染的部位之一。结论：该逆转录-聚合酶链反应产物酶法检测方法简便、快速、准确，可用于临床念珠菌的检测。毛状的
We developed a simple enzymatic assay system using reverse transcription-polymerase chain reaction products in a microtitration plate to detect Clostridium piliforme in tissue specimens. The reverse transcription-polymerase chain reaction was performed with a biotin-labeled primer directed in a highly conserved area of the C. piliforme 16S rRNA gene. The amplified cDNA was hybridized to an alkaline phosphatase-labeled DNA probe in a microtube, and the mixture was applied to a microtitration well precoated with streptavidin. Then hybridization signals in the microtitration plate were visualized by reaction with substrate for the alkaline phosphatase. When an optical density > 0.200 at 405 nm was considered a positive result, we found that the system could detect the RNA from as few as 10 organisms. The system was also capable of detecting organisms from the liver, mesenteric lymph nodes, and ileum of experimentally infected mice. In the ileum the C. piliforme gene was detected from 1 to 21 days after infection. This result suggests that the intestinal epithelium is one of the sites of persistent infection. We conclude that this enzymatic assay system of reverse transcription-polymerase chain reaction products is simple, rapid, and convenient for the detection of C. piliforme.