牛化脓性隐秘杆菌病PCR诊断方法的建立

Establishment of PCR Method for Diagnosing Bovine Arcanobacterium pyogenes

摘要:

近几年犊牛化脓性肺炎发病率逐年升高, 经检测该病原以化脓性隐秘杆菌为主, 研究建立快速检测化脓性隐秘杆菌的PCR诊断方法。根据化脓性隐秘杆菌16S rRNA基因设计并合成一对特异性引物, 对PCR条件进行优化后通过特异性试验和敏感性试验检测其特异性和敏感性。扩增出927 bp目的基因, 最佳引物浓度为0.2 μmol/L、退火温度58°C、Mg2+浓度1.5 mmol/L, 特异性试验结果表明化脓性隐秘杆菌参考菌株能扩增出927 bp目的基因, 而大肠杆菌、金黄色葡萄球菌、绿脓杆菌、化脓链球菌、蜡样芽孢杆菌、沙门氏菌、肺炎克雷伯氏菌和变形杆菌等的扩增结果均为阴性。敏感性试验结果表明, PCR的最低检出量为42个化脓性隐秘杆菌。建立的牛化脓性隐秘杆菌的PCR诊断方法, 具有较高的特异性和敏感性, 为化脓性隐秘杆菌引起的牛化脓性肺炎的快速诊断及流行病学调查提供了新的手段。

Abstract:

Calf pneumonia apostematosa has increased in recent years and major pathogen was Arcanobacterium pyogenes by detecting. The purpose of this study is to develop a PCR method for Arcanobacterium pyogenes diagnosis. A pair of specific primer was designed and synthesized according to 16S rRNA of Arcanobacterium pyogenes. After PCR method was optimized, the specificity and sensitivity was assayed. The specific DNA fragment (927 bp) was successfully amplified with the following condition: 0.2 μmol/L optimal concentration of primer, 58°C anneal temperature and 1.5 mmol/L Mg2+ concentration. The specific test results showed that Arcanobacterium pyogenes could be amplified to obtain 927 bp, however the amplified results of Escherichia coli, Staphylococcus aureus, Bacillus pyocyaneus, Streptococcus pyogenes, Bacillus cereus, Salmonella, Klebsiella pneumoniae and Bacillus proteus were negative. The results of sensitivity test indicated that the minimum detection limits of PCR were 42 Arcanobacterium pyogenes. The PCR diagnosis method of bovine Arcanobacterium pyo-genes was successfully established and this method has high specificity and sensitivity. This research provides new means for the rapid diagnosis of the bovine purulent pneumonia and epidemiological in-vestigations.