熒光原位雜交技術可快速鑒定病原菌，較傳統(tǒng)病原分離鑒定方法具有明顯優(yōu)勢。針對我國動物布魯氏菌病和牛結核病這兩個重要的人獸共患病，目前還沒有標準的熒光原位雜交檢測方法可供參考。為建立布魯氏菌和牛結核分枝桿菌熒光原位雜交檢測方法，快速診斷動物布魯氏菌病和牛結核病，利用布魯氏菌探針Bru-996和結核分枝桿菌探針MTB770，通過優(yōu)化雜交溫度、雜交時間和樣品處理等關鍵條件，確定最佳檢測程序；根據(jù)已知背景的菌株和臨床樣品，對Bru-996和MTB770探針的特異性和敏感性進行評價，最終建立了熒光復位雜交診斷方法。結果顯示：反應條件優(yōu)化后，該方法可在4 h內(nèi)完成布魯氏菌檢測；熒光標記Bru-996探針與布魯氏菌待檢菌株的雜交結果均為陽性，而與結核分枝桿菌、禽結核分枝桿菌和大腸桿菌雜交結果均為陰性，并從5個已知背景的組織病料中成功檢出布魯氏菌。牛結核分枝桿菌檢測則需要6~8 h，雜交前必須對樣品用二甲苯和溶菌酶進行處理；MTB770探針可特異性識別并能從牛肺部結節(jié)中檢出牛結核分枝桿菌。結果表明，熒光原位雜交方法快速、簡便，而且Bru-996和MTB770探針分別在布魯氏菌和牛結核分枝桿菌檢測上具有較高的特異性，可替代傳統(tǒng)的病原分離鑒定，作為動物布魯氏菌病和牛結核病的實驗室確診方法。
Application of Fluorescence in Situ Hybridization in Detection
of Brucella and Mycobacterium bovis
Compared with traditional methods of pathogen isolation and identification，fluorescence in situ hybridization（FISH）technology could be used to rapidly identify pathogenic bacteria due to its obvious advantages. However，there has been no any formal FISH technology for two kinds of major zoonoses（animal brucellosis and bovine tuberculosis）in China. In order to develop the FISH technology for rapidly diagnosing the two diseases，the probes of Brucella（Bru-996）and Mycobacterium tuberculosis（MTB770）were used to determine the optimal detection procedure by optimizing the temperature and time of hybridization，dealing 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 positive，and that between Bru-996 probe and Mycobacterium bovis，Mycobacterium avium and Escherichia coli were all negative；and Brucella was successfully detected from five tissue materials with known background. It would take 6 to 8 hours when Mycobacterium bovis was detected，and 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 convenience，and the high specificity of Bru-996 and MTB770 probes in detection of Brucella and Mycobacterium bovis，it was believed that the developed FISH technology could replace relevant traditional methods and be used to diagnose animal brucellosis and bovine tuberculosis in laboratories.
全文下載鏈接：http://kns.cnki.net/KCMS/detail/37.1246.S.20190903.1708.032.html
當前位置：