结核分枝杆菌和牛分枝杆菌的二重PCR快速检测

Development of a rapid and sensitive method of duplex PCR to detect Mycobacterium tuberculosis complex and Mycobacterium bovis in dairy cow

目的建立一种鉴别结核分枝杆菌和牛分枝杆菌的二重PCR快速检测方法。方法根据已发表的结核分枝杆菌23SrRNA和牛分枝杆菌特殊基因序列,设计并合成了两对可扩增结核分枝杆菌和牛分枝杆菌特异性基因片段的引物,建立二重PCR快速检测结核分枝杆菌和牛分枝杆菌的方法,测定其特异性和敏感性,并对238份牛临床样品的DNA分别进行了检测。结果该方法对牛分枝杆菌能扩增出838bp和631bp的特异性基因片段,结核分枝杆菌只扩增出838bp基因片段而扩增不出631bp的特异性基因片段,对参试的其它牛菌株的DNA扩增结果均为阴性。敏感度检测可达到50pg的DNA含量。临床样品检测结核分枝杆菌阳性有21份,牛分枝杆菌阳性有1份,其它临床样品扩增结果全部为阴性。结论本方法可作为牛分枝杆菌的快速检测和流行病学调查的工具。

A rapid and sensitive method of duplex PCR was developed for the detection of Mycobacterium tuberculosis complex and Mycobacterium boris in dairy cow, in which two pairs of primers were designed and synthesized according to the specific gene of M. boris and the 23S rRNA of Mycobacterium tuberculosis complex, and thereby a duplex PCR amplification and detection platform to diagnose M, boris and M, tuberculosis complex infection simultaneously in cow milk and nasal secre- tions was developed. With this method of duplex PCR, specific gene fragment of M. tuberculosis complex in length of 838 bps and that of M. boris in length of 631 bps could be amplified respectively. The DNAs extracted from 238 clinical samples of milk and reference strains showed positive results in DNA amplification, but other bacterial strains from cow milk showed negative results. The detection limit of this assay was 50 pg DNA extracted in 238 clinical samples examined, 21 samples were positive for M. tuberculosis complex and one specimen positive for M. boris. It is evident that this method of assay can be used as a rapid method of detection for M. boris infection as well as employed as a means for epidemiological investigation.