Ⅰ群禽腺病毒通用型二温式PCR和VPCR方法的建立及初步应用  

Establishment and Preliminary Application of Two-temperature PCR and VPCR for Rapid Detection ofⅠGroup of Fowl Aviadenovirus

作　　者：赵磊[1] 刘畅[1] 刘欣超 王旋[1] 黄佳佳 阮来田 张训海[1] ZHAO Lei;LIU Chang;LIU Xinchao;WANG Xuan;HUANG Jiajia;RUAN Laitian;ZHANG Xunhai(Anhui Key Laboratory of Poultry Infectious Disease Prevention and Control,Anhui Science and Technology University,Fengyang 233100,China;Guichi Animal Health Inspection Institution,Chizhou 247000,China)

机构地区：[1]安徽科技学院家禽疫病防控监测安徽省重点实验室,凤阳233100 [2]池州市贵池区动物卫生监督所,池州247000

出　　处：《中国动物传染病学报》2021年第3期47-55,共9页Chinese Journal of Animal Infectious Diseases

基　　金：安徽省教育厅重点项目(KJ2019A0800,KJ2017A504);安徽省家禽产业技术体系项目(AHCYJSTX-06);高校重点学科(AKZDXK2015B03)。

摘　　要：为建立快速检测Ⅰ群禽腺病毒(FAdV-I)血清型通用PCR方法,根据FAdV-Ⅰ12个血清型基因保守区设计引物,建立了二温式PCR和VPCR方法。结果显示:本研究建立的FAdV-Ⅰ二温式PCR方法,在90℃~94℃变性3 s、70℃~74℃退火兼延伸8 s,循环35~40次时,最快可在15 min内完成扩增;对分属5个种的5个血清型毒株检测都呈阳性,对EDSV、NDV、沙门菌等12种禽常见病原核酸检测呈阴性;最低检测质粒拷贝数为13.9 copies/μL,最低检测病毒滴度为100.3 EID50/0.1 mL;对源于FAdV-4感染致死SPF鸡的心脏、肝脏、脾脏等14种样品核酸提取物检测均为阳性。建立的VPCR,在92℃变性0 s、70℃退火延伸0 s,40次循环时,可23 min完成,其最低检测质粒拷贝数亦为13.9 copies/μL;对150份采集自安徽省部分地区样品及送检病料的检测与分析显示,所建立的二温式PCR和VPCR总阳性检出均率为30.7%,其符合率为100%。本研究表明,所建立的通用型二温式PCR和VPCR方法,均可用于Ⅰ群FAdV的快速检测与鉴定。To establish PCR methods for rapid and universal detection ofⅠgroup of Fowl aviadenovirus(FAdV-I),primers were designed based on the conserved regions of FAdV-I including twelve serotypes.Rapid two-temperature PCR and VPCR methods were established and optimized,then analyzed its characteristics.The results showed that the two-temperature PCR for rapid detection of FAdV-I was established,and its amplification could be completed within 15 min by denatured at 90℃-94℃for 3 s and annealing/extended at70℃-74℃for 8 s for 35-40 cycles.It was positive for 5 strains from different serotype and belonging to five species of FAdV-I A-E.It tested negative for twelve types of conventional avian virus including EDSV,NDV,Salmonella pullorum and so on.The lowest detectable concentration of FAdV-4 strains was 100.3 EID50/0.1mL,and the minimum detection limits to standard plasmid was 13.9 copies/μL.Nucleic acids extracted from heart,liver,spleen,lung,kidney,glandular stomach,small intestine,cecal tonsil,trachea,thymus,pancreas,bursa,brain and cloacal swab samples of SPF chickens infected with FAdV-4 could be detected positive.The optimized results of VPCR were completed within 23 min by denatured at 92℃for 0 s and annealing/extended at 70℃for 0 s for 40 cycles,and its minimum detection limits to standard plasmid was 13.9 copies/μL.Clinical tests showed that the established two-temperature PCR and VPCR methods had detected 150 samples collected from some areas in Anhui province,and its positive rate was 30.7%.In conclusion,the established two-temperature PCR and VPCR methods could be used for the rapid detection and identification of FAdV-I.

关 键 词：Ⅰ群禽腺病毒 通用型 二温式PCR VPCR 快速诊断 

分 类 号：S852.659.1[农业科学—基础兽医学]