鸭源多杀性巴氏杆菌RPA-LFD检测方法的建立与应用  

Establishment and application of RPA-LFD detection method for Pasteurella multocida from duck

作　　者：龙宥茨 顾庆林 鲜思美[1,2] 郑维豪 吴琴 余梦怡 李京 吴帅斌 LONG Youci;GU Qinglin;XIAN Simei;ZHENG Weihao;WU Qin;YU Mengyi;LI Jing;WU Shuaibin(College of Animal Science,Guizhou University,Guiyang 550025,China;Institute of Animal Disease Research of Guizhou,Guiyang 550025,China)

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025 [2]贵州省动物疫病研究所,贵州贵阳550025

出　　处：《中国兽医学报》2025年第3期466-472,共7页Chinese Journal of Veterinary Science

基　　金：贵州省农业攻关资助项目(黔科合支撑[2016]2506号)。

摘　　要：根据GenBank上已公布的鸭源多杀性巴氏杆菌(Pasteurella multocida,Pm)kmt1基因序列,设计PCR扩增引物,将扩增所得kmt1基因克隆至pMD19-T载体中,构建重组质粒标准品pMD19-T-kmt1,并经PCR和测序鉴定。以pMD19-T-kmt1质粒为模板,kmt1基因为靶基因,设计并合成重组酶聚合酶扩增(recombinase polymerase amplification,RPA)引物;根据横向流动试纸条(lateral flow dipstick,LFD)要求设计1条探针(Pm-P),经反应条件优化,建立了检测Pm的RPA-LFD方法,并进行特异性、灵敏性试验,用所建方法对64份临床样品进行检测。结果显示:建立的Pm RPA-LFD方法在37℃15 min即可完成扩增反应;提取鸭源大肠杆菌(Escherichia coli,E.coli)、鸭源沙门菌(Salmonella enteriditis,SE)、鸭疫里默杆菌(Riemerella anatipestifer,RA)、葡萄球菌(Staphylococcus)、鹅细小病毒(goose parvovirus,GPV)、鸭瘟病毒(duck plague virus,DPV)、番鸭细小病毒(Muscovy duck parvovirus,MDPV)的DNA作为模板,以质粒标准品pMD19-T-kmt1为阳性对照进行RPA-LFD,显示除阳性对照外其他均为阴性;将质粒标准品pMD19-T-kmt1进行10倍倍比稀释,以浓度为10^(7)~10^(0)拷贝/μL的质粒标准品为模板,检测出敏感度为1.50×10^(1)拷贝/μL,比PCR方法高100倍。利用PCR、RPA和LAMP-LFD检测64份疑似RA临床样本,3者检出符合率为100%。结果表明,本试验建立的Pm RPA-LFD方法具有特异性强、检测速度快、敏感度高等特点,可应用于Pm的临床样本检测。This study aims to establish a convenient,new and visual detection method for the field diagnosis of Pasteurella multocida(Pm).With reference to the Pm kmt 1 gene conserved sequence published in GenBank,PCR amplification primers were designed,the amplified kmt1 gene was cloned into pMD19-T vector,and the recombinant plasmid standard pMD19-T-kmt1 was established and identified by PCR and sequencing.Using pMD19-T-kmt1 plasmid as template and kmt 1gene as target gene,basic primers were designed and synthesized.According to the requirements of LFD,a probe(Pm-P)was designed,and the RPA-LFD method for Pm detection was established by optimizing the reaction conditions.Specificity and sensitivity tests were carried out,and 64 clinical samples were tested by the method.The results showed that the established Pm RPA-LFD method could be amplified at 37℃for 15 min.Escherichia coli(E.coli),Salmonella enteriditis(SE),Riemerella anatipestifer Staphylococcus,goose parvovirus(GPV),duck plague virus(DPV),Muscovy duck parvovirus(MDPV)DNA was extracted as the template,and plasmid standard pMD19-T-kmt1 was used as the positive control.All the positive controls were negative,indicating that the method had good specificity.The plasmid standard pMD19-T-kmt1 was diluted with a 10-fold ratio,and the plasmid standard with a concentration of 10^(7)-10^(0)copies/μL was used as the template.The sensitivity was 1.50×10^(1)copies/μL,which was 100 times higher than that of PCR.A total of 64 clinical samples with suspected RA were subjected to testing using PCR,RPA and LAMP-LFD,with a 100%compliance rate for all three detection tests.The results show that the established RPA-LFD method has the characteristics of strong specificity,high sensitivity,fast speed and visualization,and can be applied to the field detection of Pm.

关 键 词：多杀性巴氏杆菌 kmt1基因 重组酶聚合酶扩增 横向流动试纸条 

分 类 号：S852.61[农业科学—基础兽医学]