牛冠状病毒交叉引物扩增快速检测方法的建立

Establishment of a rapid detection method for BCoV by cross-primers amplification

牛冠状病毒(BCoV)引起犊牛腹泻,对养牛业造成极大危害。本研究采用交叉引物扩增(CPA)方法和核酸检测装置相结合,建立了BCoV的可视化快速检测方法。结果表明,CPA体系Mg2+最佳物质浓度为2.0 mmol/L,甜菜碱最佳物质浓度为0.4 mmol/L,d NTPs最佳物质浓度为0.6 mmol/L,Bst DNA聚合酶最佳浓度为1.5 U/μL,60℃为最佳反应温度,60 min为最佳反应时间。采用CPA BCoV-N反应体系同时检测沙门菌、大肠埃希菌、弯曲杆菌、牛冠状病毒、牛病毒性腹泻病毒、牛轮状病毒等犊牛腹泻相关病原,层析试纸条检测显示只有牛冠状病毒呈现检测线,未出现交叉反应。结果显示,CPA BCoV-N具有较高的特异性,CPA检测BCoV不需要昂贵的PCR仪器,简单的水浴或培养箱即可完成DNA扩增,封闭式核酸检测装置避免了气溶胶污染,对结果的判断更加直观客观,是一种简便、快速、灵敏的BCoV检测方法。

Bovine coronavirus(BCoV)causes calf diarrhea,resulting in great harm to cattle production.The crosspriming amplification(CPA)approach and nucleic acid detection device were used to establish a visual rapid detection method for BCoV.The results showed when the amplification reaction was carried out in the amplification system at optimal concentrations of 2.0 mmol/L for Mg2+,0.4 mmol/L for betaine,0.6 mmol/L for dNTPs,and 1.5 U/μL for Bst DNA polymerase at 60℃for just 60 min,CPA had the highest detection efficiency.Salmonella,Escherichia coli,Campylobacter,Bovine coronavirus,Bovine viral diarrhea virus and Bovine rotavirus were added into the CPA BCoV-N reaction system,and only Bovine coronavirus was detected by test paper with test line without cross-reaction,indicating that CPA BCoV-N had high specificity.CPA detection of BCoV does not require an expensive PCR instrument;a simple water bath or incubator is sufficient for complete DNA amplification,and the closed nucleic acid detection device avoids aerosol pollution,making judgment of results more intuitive and objective.The CPA assay would be a promising simple,rapid and sensitive method for identification of BCoV.