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荧光原位杂交在布鲁氏菌和牛结核分枝杆菌检测中的应用

点击数:4530  时间:2019-09-17 00:00:00  来源: 中国动物检疫      作者: 孙明军,王 萍,张喜悦,等

荧光原位杂交技术可快速鉴定病原菌,较传统病原分离鉴定方法具有明显优势。针对我国动物布鲁氏菌病和牛结核病这两个重要的人兽共患病,目前还没有标准的荧光原位杂交检测方法可供参考。为建立布鲁氏菌和牛结核分枝杆菌荧光原位杂交检测方法,快速诊断动物布鲁氏菌病和牛结核病,利用布鲁氏菌探针Bru-996和结核分枝杆菌探针MTB770,通过优化杂交温度、杂交时间和样品处理等关键条件,确定最佳检测程序;根据已知背景的菌株和临床样品,对Bru-996MTB770探针的特异性和敏感性进行评价,最终建立了荧光复位杂交诊断方法。结果显示:反应条件优化后,该方法可在4 h内完成布鲁氏菌检测;荧光标记Bru-996探针与布鲁氏菌待检菌株的杂交结果均为阳性,而与结核分枝杆菌、禽结核分枝杆菌和大肠杆菌杂交结果均为阴性,并从5个已知背景的组织病料中成功检出布鲁氏菌。牛结核分枝杆菌检测则需要6~8 h,杂交前必须对样品用二甲苯和溶菌酶进行处理;MTB770探针可特异性识别并能从牛肺部结节中检出牛结核分枝杆菌。结果表明,荧光原位杂交方法快速、简便,而且Bru-996MTB770探针分别在布鲁氏菌和牛结核分枝杆菌检测上具有较高的特异性,可替代传统的病原分离鉴定,作为动物布鲁氏菌病和牛结核病的实验室确诊方法。

Application of Fluorescence in Situ Hybridization in Detection

of Brucella and Mycobacterium bovis

Compared with traditional methods of pathogen isolation and identificationfluorescence in situ hybridizationFISHtechnology could be used to rapidly identify pathogenic bacteria due to its obvious advantages. Howeverthere has been no any formal FISH technology for two kinds of major zoonosesanimal brucellosis and bovine tuberculosisin China. In order to develop the FISH technology for rapidly diagnosing the two diseasesthe probes of BrucellaBru-996and Mycobacterium tuberculosisMTB770were used to determine the optimal detection procedure by optimizing the temperature and time of hybridizationdealing with samples and optimizing other important conditions. Then the specificity and sensitivity of the two probes were evaluated based on strains with known background and clinical samples. The results showed that Brucella could be detected within 4 hours after reaction conditions were optimized. The hybridizations between Bru-996 probe and Brucella strains were positiveand that between Bru-996 probe and Mycobacterium bovisMycobacterium avium and Escherichia coli were all negativeand Brucella was successfully detected from five tissue materials with known background. It would take 6 to 8 hours when Mycobacterium bovis was detectedand samples must be treated with Xylene and Lysozyme prior to hybridization. Mycobacterium bovis could be identified and detected from bovine pulmonary nodules by MTB770 probe. In view of the advantages of FISH technology of rapidity and convenienceand the high specificity of Bru-996 and MTB770 probes in detection of Brucella and Mycobacterium bovisit was believed that the developed FISH technology could replace relevant traditional methods and be used to diagnose animal brucellosis and bovine tuberculosis in laboratories.

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