摘要
本试验旨在构建一种以不同分支杆菌特异片段为目的基因的多重PCR检测方法。采用GenBank公布的结核分支杆菌RD10序列、牛分支杆菌moaB3序列、鸟分支杆菌16-23SrDNA序列设计并合成特异性引物,扩增产物条带分别为954bp、297bp、119bp。结果显示,三段目的基因都有很高的特异性,对比菌株均无扩增产物出现。对倍比稀释的模板质粒进行检测,该方法的最低检出量为103 copies/μL的DNA模板,该方法特异、敏感、重复性好。首次应用的moaB3基因所得扩增效果良好,成功区分出牛分支杆菌。此多重PCR方法可用于结核分支杆菌、牛分支杆菌、鸟分支杆菌的鉴别和牛结核病的快速临床检测。
We established a multiplex PCR detection method with three specific gene fragments to de tect M. tuberculosis, M. bovis and M. avium. The primers were designed and synthesized based on the spe cific genes of Mycobacterium tuberculosis＇s RD10 gene, M. bovis＇s moaB3 gene and M. avium＇s 16-23S （in ternal transcribed spacer, ITS） gene sequences published in GenBank, the corresponding products were 954 bp, 297 bp, 119 bp. Experiment results showed that the fragments we chose were highly specific, the reference strains showed positive results in gene amplification. It was found that the sensitivity of this method was 103 copies of template DNA with good reproducibility. The moaB3 gene was the first time to be used for detecting M. boris, we got satisfactory result and it showed high efficiency amplification. The muhiplexPCR assay can be used for distinguishing M. tuberculosis, M. bovis and M. avium, also can be used for clinical diagnosis of Tuberculosis.
出处
《中国兽医杂志》
CAS
北大核心
2012年第9期16-18,共3页
Chinese Journal of Veterinary Medicine
基金
广州市科技计划项目(2009Z1-E731)
广东省科技计划项目(2008A020100012)
广东省科技基础条件建设项目(2010B060200040)
