摘要
为建立塞内卡病毒(SVA)的快速检测方法,本研究在SVA高度保守区域,分别设计了5对特异性LAMP引物,并通过反应条件优化、敏感性试验、特异性试验以及临床样本检测,建立了一种经济、特异、敏感的SVA检测方法。在62℃水浴55 min扩增出大于220 bp的特异性阶梯状条带,以质粒为模板进行敏感性试验结果表明该方法在5.1 copies/μL时仍可以检测出SVA,比普通PCR敏感1000倍。特异性试验结果显示该方法与猪肺炎支原体(MPH)、猪伪狂犬病病毒(PRV)、猪圆环病毒2型(PCV2)、猪圆环病毒3型(PCV3)、猪瘟病毒(CSFV)、猪繁殖与呼吸综合增病毒(PRRSV)均无交叉反应,应用该方法检测72例临床疑似病料,阳性率为33%(24例)。与普通PCR相比较,该方法操作简便、快速、特异、经济,本研究提供了一种更适于临床检测的SVA检测方法。
In order to develop a rapid detection method for Seneca virus A(SVA),five pairs of specific LAMP primers in the highly conserved region of SVA genome were designed in this study.A specific ladder-like band larger than 220 bp was amplified in 62℃water bath for 55 min.The resulting plasmid was used as the template for sensitivity test.The results showed that the method detected SVA at 5.1 copies/μL,which was 1000 times more sensitive than the conventional PCR.The results of specificity test showed that there was no cross-reactivity with Mycoplasma hyopneumoniae(MPH),porcine pseudorabies virus(PRV),porcine circovirus type 2(PCV2),porcine circovirus type 3(PCV3),swine fever virus(CSFV)a nd porcine reproductive and respiratory syndrome virus(PRRSV).In addition,72 cases suspected of SVA infection was tested using this method and the positive rate was 33%(24/72).Compared with the conventional PCR,the LAMP visual method developed here was simple,rapid,specific and economical,which provided a more suitable method for clinical detection of SVA.
作者
田瑶
李琛
杨莹
李佳昕
王硕
时建立
彭喆
徐绍建
吴晓燕
刘畅
韩红
韩先杰
李俊
TIAN Yao;LI Chen;YANG Ying;LI Jiaxin;WANG Shuo;SHI Jianli;PENG Zhe;XU Shaojian;WU Xiaoyan;LIU Chang;HAN Hong;HAN Xianjie;LI Jun(College of Veterinary Medicine,Qingdao Agricultural University,Qingdao 266109,China;Institute of Animal Science and Veterinary Medicine Shandong Academy of Agricultural Sciences,Jinan 250100,China;College of Life Sciences,Shandong Normal University,Jinan 250100,China)
出处
《中国动物传染病学报》
CAS
北大核心
2024年第3期88-94,共7页
Chinese Journal of Animal Infectious Diseases
基金
山东省重大科技创新工程(2023CXGC010705)
山东省现代农业产业技术体系(SDAIT-08-06)
山东省科技型中小企业创新能力提升工程(2023TSGC0734)
国家自然基金(32302911)
山东省自然基金(ZR2022MC011)
济南市“新高校20条”(202228112)
山东省农业科学院科技创新工程(CXGC2018E10)。
关键词
塞内卡病毒
环介导等温扩增
快速检测
Seneca virus A
loop-mediated isothermal amplification
rapid detection
