实验动物四种病原体多重PCR方法的建立与应用

发布时间：2018-04-22 23:41

本文选题：多杀巴氏杆菌 + 支气管鲍特杆菌　；参考：《中国比较医学杂志》2017年08期

【摘要】：目的建立快速检测多杀巴氏杆菌、支气管鲍特杆菌、支原体和肺炎克雷伯杆菌4种实验动物病原体的多重PCR方法。方法根据Gen Bank基因设计出特异性引物;经过多重PCR的优化,特异性和敏感性的检测,建立多重PCR体系。应用该PCR体系检测人工感染样本和实验动物的气管分泌物,并与传统方法做对比。结果多重PCR扩增出多杀巴氏杆菌(356 bp)、支气管鲍特杆菌(237 bp)、支原体(266 bp)和肺炎克雷伯杆菌(142 bp)的目的条带。肺炎克雷伯杆菌敏感性为10 pg,多杀巴氏杆菌、支气管鲍特杆菌、支原体敏感性为1 pg,特异性检测未从其他病原菌中检测出目的条带。应用建立的多重PCR体系检测人工感染样本的不同组合,45只实验动物气管检测出15只多杀巴氏杆菌阳性,9只肺支原体阳性,但传统培养方法和血清学方法未检测出阳性标本。结论本文建立的多重PCR方法操作简单、快速、特异性强、灵敏度高,能够实现对多杀巴氏杆菌、支气管鲍特杆菌、支原体和肺炎克雷伯杆菌4种实验动物病原体的快速检测。
[Abstract]:Objective to establish a multiplex PCR method for rapid detection of four pathogens of Pasteurella multocida, Bacillus bronchus, Mycoplasma pneumoniae and Klebsiella pneumoniae. Methods specific primers were designed according to the Gen Bank gene, and the multiplex PCR system was established by the optimization of multiple PCR and the detection of specificity and sensitivity. The PCR system was used to detect trachea secretions from artificially infected samples and experimental animals, and compared with traditional methods. Results the target bands of Pasteurella multocida, Pasteurella pneumoniae, Mycoplasma pneumoniae and Klebsiella pneumoniae were amplified by multiplex PCR. The sensitivity of Klebsiella pneumoniae was 10 PG, that of Pasteurella multocida, Bacillus bronchus and Mycoplasma was 1 PG. Using the multiplex PCR system to detect the different combinations of artificial infection samples of 45 experimental animals, 15 of them were positive for Pasteurella multocida in the trachea, 9 were positive for Mycoplasma multogenes, but no positive specimens were detected by traditional culture methods and serological methods. Conclusion the multiplex PCR method established in this paper is simple, rapid, specific and sensitive. It can be used to detect the pathogens of Pasteurella multocida, Pasteurella bronchus, Mycoplasma pneumoniae and Klebsiella pneumoniae.
【作者单位】：河北省实验动物重点实验室河北医科大学实验动物学部;柏乡县中心医院;河北医科大学第四医院;
【基金】：国家科技支撑计划(2015BAI07B02-05)
【分类号】：Q78;R-33

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