摘要
番鸭白肝病是2014年以来我国番鸭流行的一种新疫病,其病原为一种与鸭腺病毒B型法国GR株同源性较高的新型腺病毒,暂命名为鸭腺病毒B血清2型(DAdV-B2),将法国GR株命名为鸭腺病毒B血清1型(DAdV-B1)。为建立同时检测DAdV-B1和DAdV-B2的方法,本研究根据GenBank中DAdV-B1 fiber基因和DAdV-B2 fiber1基因序列特征,分别设计了2对特异性引物,通过优化反应条件,建立了可以同时检测这两种病毒的双重PCR方法。结果显示:优化后的PCR方法最佳退火温度为60.1℃,最适引物浓度为0.16μmol/L;利用该方法对DAdV-B1、DAdV-B2、鸭腺病毒A型(DAdV-A)、禽腺病毒血清4型(FAdV-4)、番鸭呼肠孤病毒(MDRV)、新型呼肠孤病毒(NDRV)、番鸭源鹅细小病毒(MDGPV)、番鸭细小病毒(MDPV)和禽坦布苏病毒(ATUMV)等检测,结果显示,该方法仅对DAdV-B1和DAdV-B2有特异性扩增,对其他鸭临床常见病原均无扩增,特异性较强。将DAdV-B1 fiber和DAdV-B2 fiber1质粒标准品等体积混合并作10倍倍比稀释后作为模板,利用该双重PCR方法检测,结果显示,该方法对DAdV-B1 fiber和DAdV-B2 fiber1质粒标准品的最低检测限分别为175拷贝/μL和163拷贝/μL,对DAdV-B2的最低检出滴度为1×10^(1) TCID_(50),敏感性较高。批内和批间的重复性试验检测结果均一致,重复性好。利用该双重PCR与单一PCR方法同时对64份临床样品检测,结果显示二者的符合率为100%,其中DAdV-B2的阳性率为79.7%(51/64),DAdV-B1的阳性率为4.7%(3/64)。以上结果表明本研究建立的双重PCR方法特异性较强、敏感性较高和稳定性较好,为DAdV-B1和DAdV-B2的临床鉴别检测及其分子流行病学调查提供了技术手段。
Muscovy duck Pale liver disease is a new epidemic disease in Muscovy ducks in China since 2014.Its pathogen is a new adenovirus homologous to duck adenovirus B French GR strain,temporarily named duck adenovirus B serotype 2(DAdV-B2),and French GR strain named duck adenovirus B serotype 1(DAdV-B1).In order to establish a method for simultaneous detection of DAdV-B1 and DAdV-B2,two pairs of specific primers were designed according to the sequence characteristics of DAdV-B1 fiber gene and DAdV-B2 fiber1 gene in GenBank.By optimizing the reaction conditions,a dual PCR method for simultaneous detection of these two viruses was established.The results showed that the optimum annealing temperature and primer concentration of the optimized PCR method are 60.1℃and 0.16μmol/L,respectively.This method was used to detect DAdV-B1,DAdV-B2,DAdV-A(duck adenovirus type A),fowl adenovirus serotype 4(FAdV-4),muscovy duck reovirus(MDRV),novel duck reovirus(NDRV),muscovy duck-origin parvovirus(MDGPV),muscovy duck parvovirus(MDPV)and avian Tambusu virus(ATUMV).The results showed that only DAdV-B1 and DAdV-B2 were specifically amplified,no other common clinical pathogens in ducks were detected,indicating the specificity of this method is strong.DAdV-B1 fiber and DAdV-B2 fiber1 plasmid standards were mixed in equal volume and diluted 10 times as templates.The duplex PCR method was used to detect.The results showed that the lowest detection limits of this method for DAdV-B1 fiber and DAdV-B2 fiber1 plasmid standards were 175copies/μL and 163copies/μL,respectively,and the lowest detection titer for DAdV-B2 was 1×10^(1)TCID_(50).The results of intra-batch and inter-batch repeatability tests were consistent and reproducible.The duplex PCR test and the single PCR test were used to detect 64 clinical samples,and the results showed that the coincidence rate was 100%,of which the positive rate of DAdV-B2 was 79.7%and the positive rate of DAdVB1 was 4.7%.The above results show that the duplex PCR method established in this study has strong
作者
江丹丹
林昶
黄志坚
肖世峰
王劭
林锋强
程晓霞
朱小丽
陈秀琴
董慧
陈少莺
陈仕龙
JIANG Dan-dan;LIN Chang;HUANG Zhi-jian;XIAO Shi-feng;WANG Shao;LIN Feng-qiang;CHENG Xiao-xia;ZHU Xiao-li;CHEN Xiu-qin;DONG Hui;CHEN Shao-ying;CHEN Shi-long(Institute of Animal Husbandry and Veterinary Medicine,Fujian Academy of Agriculture Sciences,Fuzhou 350013,China;Fujian Animal Diseases Control Technology Development Center,Fuzhou 350013,China;College of Animal Science,Fujian Agriculture and Forestry University(College of Bee Science),Fuzhou 350002,China;Sanming Green Food Development Center,Sanming 365000,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2022年第8期855-860,共6页
Chinese Journal of Preventive Veterinary Medicine
基金
福建省公益类科研院所专项(2019R1026-11)
福建省农业科学院创新团队项目(CXTD2021034)
福建省农业科学院“5511”协同创新工程(XTCXGC2021018、XTCXGC2021012)。
