多重PCR对禽分枝杆菌亚种鉴定的初步应用

Use of Multiplex PCR Assay for the Identification of Mycobacterium avium Sub-species

关键词:  禽分枝杆菌  亚型鉴定  多重PCR

Key words:  Mycobacterium avium  Subtype identification  multiplex PCR

摘要:

为了建立禽分枝杆菌多重PCR方法，给禽分枝杆菌亚种鉴定及禽结核病的诊断提供参考。本实验根据国外报道的诊断方法设计4对特异性引物，选用我国禽结核参考菌株（CVCC 68201）基因组DNA为模板，优化了退火温度，测定了DNA最小检测量，并进行了特异性检验。采用优化好的多重PCR体系，对国家兽医微生物菌种保藏中心保存的禽分枝杆菌标准菌株进行检测和分类。实验结果表明，禽结核参考菌株（CVCC 68201）能很好的扩增出577 bp（IS901基因片段）、385 bp（IS1245基因片段）和140 bp（dnaJ基因片段）三条目的条带，基因组DNA的最小检测量为0.38 ng/μL，目的条带在退火温度50 ℃～62 ℃之间均能得到有效扩增。对23株禽分枝杆菌菌株检测，并设立牛分枝杆菌作为阴性对照，最终将禽分枝杆菌菌株分为三大类：禽亚种/森林土壤亚种、副结核亚种、人猪亚种，与国家兽医微生物菌种保藏中心的分类结果相比不仅一致且更加详尽而有临床意义。因此，基于分子生物学方法建立的多重PCR方法可以对禽分枝杆菌亚型进行快速鉴定并对禽结核病的诊断提供参考。

Abstract:

In order to establish a multiplex PCR method for Mycobacterium avium(M. avium), to provide reference for the identification of Mycobacterium avium sub-species and the diagnosis of avium tuberculosis. In this study, four specific primers were designed according to the diagnostic methods reported in foreign countries. The genomic DNA of Bovine tuberculosis reference strain (CVCC 68201) was selected as the template, the minimum detection amount of DNA was determined, the annealing temperature was optimized and the specificity was tested. The Mycobacterium avium standard strains preserved by the China Veterinary Culture Collection Center (CVCC) were tested and classified by the optimized multiplex PCR system. The results showed that the reference strain (CVCC 68201) could amplify bands of 577 bp (IS901 gene fragment), 385 bp (IS1245 gene fragment) and 140 bp (dnaJ gene fragment), and the minimum detectable amount of genomic DNA is 0.38 ng/μL, the purpose bands can be effectively amplified between the annealing temperature of 50 ℃~62 ℃. Twenty-three strains of Mycobacterium avium were detected and the mycobacterium bovis was used as negative control. The Mycobacterium avium isolates were divided into three categories: M. avium subsp. Avium/ M. avium subsp. Silvaticum, M. avium subsp. Hominissuis, M. avium subsp. Paratuberculosis. Compared with the classification results of China Veterinary Culture Collection Center, our result are not only consistent but also more detailed and of clinical significance. Therefore, the multiplex PCR method based on molecular biology method can rapidly identify the subtype of Mycobacterium avium and provide reference for the diagnosis of avium tuberculosis.