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首页> 外文会议>International Colloquium on Paratuberculosis >Detection of Mycobacterium avium subsp. paratuberculosis IS900 in lesions of bovine paratuberculosis by a supersensitive in situ hybridization technique
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Detection of Mycobacterium avium subsp. paratuberculosis IS900 in lesions of bovine paratuberculosis by a supersensitive in situ hybridization technique

机译：检测分枝杆菌亚空分枝杆菌。 Paratuberculosis is900在原位杂交技术中超敏的牛解霉菌病变

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In process of intercellar survival of Mycobacterium avium subsp. parutuberculosis (MAP) during asymptomatic phase of the infection, it becomes harder to demonstrate MAP in tissue sections by established histological and immunohistochemical techniques by reason of transformation into cell wall deficient forms of the mycobacteria. In order to address this issue, a supersensitive in situ hybridization (ISH) technique was employed. Formalin-fixed and paraffin-embedded mirror sections of mesenteric lymph nodes were obtained from 5 cows clinically infected with MAP. A FITC-labeled double strand PCR probe targeted for MAP IS900 was synthesized using the DNA extracted from ATCC19698 strain by PCR technique with a pair of reported primers. Slides were deparaffinized and treated with 10 mug/ml of proteinase K at 37degC for 20 min, following incubation with hybridization solution containing 1 mug/ml of the purified probe for 16 hr at 50 degC. After wash with 5x, 2x and 0.2x SSC, immunological detection and visualization of hybridized probe were performed by a tyramide signal amplification method using a commercial kit (GenPoint~(TM) Fluorescein, DakoCytomation, Tokyo). Specific signals for MAP IS900 were detected in macrophages, epithelioid cells, multinucleate giant cells and Langhans-type giant cells located in the cortex of mesenteric lymph nodes.
机译：在分枝杆菌的跨跨亚阶段的过程中。在感染的无症状期间，在无症状期间，通过在细胞壁缺陷形式的细胞壁缺陷形式的转化中通过建立的组织学和免疫组织化学技术，在组织切片中展示地图变得更难。为了解决这个问题，采用了原位杂交（ISH）技术的超敏反应。乳液固定的和石蜡嵌入镜面部分的肠系膜淋巴结从临床感染映射的5个牛获得。使用PCR技术通过PCR技术与一对报道的引物通过PCR技术从ATCC19698菌株中提取的DNA合成靶向MAP IS900的FITC标记的双链PCR探针。在37degc下用10麦克风/ mL蛋白酶K在37degc下用10麦克/ mug / mL蛋白酶K处理，然后在50 deGC下培养16小时的杂交探针进行杂交溶液。后，用5倍，2倍和0.2×SSC，免疫学检测和杂交的探针可视化洗涤，通过使用商业试剂盒（GenPoint〜（TM）荧光素，DakoCytomation公司，东京）酪胺信号扩增方法进行。在巨噬细胞，上皮细胞，多核巨细胞和位于肠系膜淋巴结的皮质中的巨噬细胞，上皮细胞，多核巨细胞和羊膜型巨细胞中检测到MAP的特定信号。

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