猪δ冠状病毒Basic RT-RPA和LF-RT-RPA方法的建立与应用  

Establishment and Application of Basic RT-RPA and LF-RT-RPA of Porcine Deltacoronavirus

作　　者：魏语泽 孟凡茹[1] 裴志花[1] 丁梓健 王雪磊 王淑洁 王开[1] 胡桂学[1] WEI Yuze;MENG Fanru;PEI Zhihua;DING Zijian;WANG Xuelei;WANG Shujie;WANG Kai;HU Guixue(College of Veterinary Medicine,Jilin Agricultural University,Changchun 130118,China;Jilin Xinfangyuan Animal Husbandry Technology Co.,Ltd.,Changchun 130031,China)

机构地区：[1]吉林农业大学动物医学院,长春130118 [2]吉林新方圆牧业科技有限公司,长春130031

出　　处：《经济动物学报》2024年第4期311-318,共8页Journal of Economic Animal

基　　金：国家自然科学基金项目(32072857),吉林省重点研发项目(20200402055NC)。

摘　　要：为建立快速检测猪δ冠状病毒(PDCo V)的重组酶聚合酶扩增(RPA)方法。针对PDCo V N基因设计了7对引物和1条探针,首先建立Basic RT-RPA方法,获得最佳引物;然后建立测流层析(LF)-RT-RPA反应的最佳体系、反应温度和时间,同时验证2种方法的特异性和敏感性。用建立的Basic RT-RPA方法结合LF-RT-PCR方法,对来自吉林省部分地区的162份临床腹泻仔猪病料进行检测。结果表明:Basic RT-RPA检测方法最佳反应条件为41.6℃、25 min,最低检测限度为3.34×10 copies/μL;LF-RT-RPA检测方法最佳反应条件为39℃、12 min,最低检测限度为3.34×10^(2) copies/μL,2种方法均具有良好的特异性和敏感性。腹泻仔猪病料PDCo V核酸阳性率为12.34%。建立的LF-RT-RPA检测方法可用于PDCo V感染的临床现场检测,以期对吉林省生猪该病的防控提供帮助。This study was purposed to establish a recombinase polymerase amplification(RPA)method for rapid detection of porcine deltacoronavirus(PDCo V).In total,seven pairs of primers and one probe were designed according to the PDCo V N gene.First of all,the Basic RT-RPA method was established to identify the best primers.Then,the optimal system,reaction temperature and time were determined for the LF-RT-RPA reaction,while the specificity and sensitivity of both methods were verified simultaneously.The established RPA method combined with the RT-PCR method was adopted to detect 162 clinical porcine diarrhea materials collected from some regions of Jilin province.According to the test results,the optimal reaction conditions of the Basic RT-RPA assay were 41.6℃for 25 min,and the detection limit was 3.34×10 copies/μL of viral nucleic acid.The optimal reaction conditions of the LF-RT-RPA assay were 39℃for 12 min,and the detection limit was 3.34×10^(2) copies/μL of viral nucleic acid.Both methods were demonstrated with high specificity and sensitivity,and the nucleic acid positive rate was 12.34%of PDCo V in diarrhea piglets from some regions of Jilin province.The LF-RT-RPA assay proposed in this study was applicable for on-site clinical detection for infection with PDCo V to prevent and control of the disease of pigs in Jilin province.

关 键 词：猪δ冠状病毒 重组酶聚合酶扩增技术 侧流层析技术 

分 类 号：S852[农业科学—基础兽医学]