多杀性巴氏杆菌荧光RAA快速检测方法的建立

Development of A Rapid Fluorescent RAA Detection Method for Pasteurella multocida

为建立一种敏感特异、简便快速的多杀性巴氏杆菌检测方法,根据多杀性巴氏杆菌kmt1基因保守区序列设计特异性引物和探针,通过对反应条件优化筛选,建立了重组酶介导的等温扩增(RAA)荧光检测方法。结果显示,建立的多杀性巴氏杆菌RAA荧光检测方法在39℃恒温条件下20 min内即可特异性的检出多杀性巴氏杆菌,与鸭疫里默氏杆菌、副猪嗜血杆菌、支原体、链球菌、大肠杆菌、附红细胞体、新孢子虫无交叉反应,方法的最低检出限为10 copies/μL,组内和组间重复性试验的变异系数均小于10%,用该方法对45份临床样本进行检测,结果阳性率为33.33%,与荧光定量PCR方法检测结果一致。本研究建立的多杀性巴氏杆菌荧光RAA检测方法操作简单、反应时间短、具有较高的特异性和敏感性,不依赖于复杂的仪器,适合临床上对多杀性巴氏杆菌的快速检测。

In order to develop a sensitive,specific,simple and rapid method to detect Pasteurella multocida,the specific primers and probes were designed based on the conserved region sequence of P.multocida KMT1 gene,and a fluorescence detection method of recombinase aided amplification(RAA)was developed through screening and optimization of reaction conditions.The results showed that the fl uorescent RAA method could be completed for P.multocida detection within 20 minutes under 39℃and the low detection limit reached to 10 copies per microlite.There was no cross reaction with Mycoplasma,Riemerella anatipestifer,Haemophilus parasuis,Streptococcus,Escherichia coli,Etoerythrocyte bodies and Neosporidium.The coefficient of variation in both intra-and inter-group repeatability tests was less than 10%.A total of 45 clinical samples were tested using this method and the positive rate was 33.33%,which was consistent with the fl uorescent quantitative PCR.The fl uorescence RAA detection method developed in this study had the advantages such as simplicity in operation,shortness in response time,high specificity and sensitivity and no need for complex instruments.Therefore,this method might be suitable for rapid detection of P.multocida in clinical sites.