多重PCR对禽分枝杆菌亚种鉴定的初步应用

发布时间：2018-06-05 22:19

本文选题：禽分枝杆菌 + 亚型鉴定　；参考：《中国兽药杂志》2017年10期

【摘要】：为建立禽分枝杆菌多重PCR鉴定方法,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(dna J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 multiple PCR identification methods for Mycobacterium avian Mycobacterium, 4 pairs of specific primers were designed according to the diagnostic methods reported abroad, and the genomic DNA of China's avian tuberculosis reference strain (CVCC 68201) was selected as the template. The annealing temperature was optimized. The minimum detection amount of DNA was measured and the specificity test was tested. The optimized multiple PCR system was used for the national veterinarian. The experimental results showed that the reference strain of avian tuberculosis (CVCC 68201) could well amplify 577 BP (IS901 gene fragment), 385 BP (IS1245 gene fragment) and 140 BP (DNA J gene fragment), and the minimum detection amount of genomic DNA was 0.38 ng/. L, the target band can be effectively amplified between the annealing temperature of 50 C ~62 C and the detection of 23 strains of Mycobacterium avian Mycobacterium and the establishment of Mycobacterium bovis as negative control. Finally, the Mycobacterium avian Mycobacterium strain is divided into three categories: avian subspecies / forest soil subspecies, subspecies subspecies, human pig subspecies, and national veterinary microorganism Preservation Center The classification results are not only consistent and more detailed but also have clinical significance. Therefore, the multiple PCR method based on molecular biological methods can be used to quickly identify the subtypes of Mycobacterium avian Mycobacterium and provide reference for the diagnosis of avian tuberculosis.
【作者单位】：中国兽医药品监察所;山东农业大学动物科技学院;
【基金】：国家重点研发计划(2016YFD0500902)
【分类号】：S852.618

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