摘要
灵敏、准确、多样的检测方法对有效防控猪瘟疫情极其重要,为了丰富现有的猪瘟病毒(CSFV)检测技术,本研究拟建立基于RAA-Cas13a的检测方法。基于全序列比对的基础上分别设计1对引物和两条cr RNA,对不同浓度的阳性质粒、阳性样品和7种其他猪病病毒样品进行RAA扩增,扩增产物以Cas13a酶切体系进行荧光检测;结果显示,Cas13a酶切体系可以放大普通RAA的检测信号,RAA-Cas13a可以将检测的灵敏度提高至102copies/μL,不能检出其他7种猪病病毒,检测变异系数小于5%,临床样本检测结果与荧光RT-PCR一致性高。结果表明,本研究建立CSFV的RAA-Cas13a灵敏度较高、特异性和重复性好,具有推广应用的潜力。
Sensitive,accurate and diverse detection methods are essential to effective prevention and control classic swine fever epidemic.In order to enrich the existing detection technology of classic swine fever virus,we intended to develop a detection method based on RAA-Cas13a.In this study,one set of primers and two crRNAs were designed based on the whole sequence alignment.The primers were applied to RAA in different concentrations of positive plasmids,positive samples and 7 other swine disease viruses.The coefficient of variation was less than 5%.The amplicons were detected by Cas13a enzyme digestion system.The results showed that the high amplification efficiency of RAA primers was conducive to thefluorescence peak of Cas13a enzyme digestion system.RAA-Cas13a improved the sensitivity of detection to 102 copies per microliter but did not detect other 7 viruses.The detection results of clinical samples were highly in accordance with real-time RT-PCR.In conclusion,the RAA-Cas13a developed in this study had high sensitivity,repeatability and specificity and might be widely used for prevention and control of classical swine fever.
作者
薛俊欣
韩伟
朱忠武
熊炜
黄忠荣
李春阳
赵新宇
孔祥翔
李健
蒋原
XUE Junxin;HAN Wei;ZHU Zhongwu;XIONG Wei;HUANG Zhongrong;LI Chunyang;ZHAO Xinyu;KONG Xiangxiang;LI Jian;JIANG Yuan(Shanghai Customs District P.R.China,Shanghai 200135,China;Changsha Customs District P.R.China,Changsha 410004,China;Shanghai University,Shanghai 200444,China)
出处
《中国动物传染病学报》
CAS
北大核心
2023年第6期78-84,共7页
Chinese Journal of Animal Infectious Diseases
基金
上海市自然科学基金(18ZR1413400)
浦东新区科技发展基金(PKJ2019-N09)。
