禽腺病毒DAdV-3和FAdV-4双重荧光定量PCR检测方法的建立及应用  

Establishment and application of a duplex fluorescence quantitative PCR for detection of DAdV-3 and FAdV-4

作　　者：周珈羽 朱春华[2] 陈珍[2] 程龙飞[2] 陈翠腾[2] 傅光华[2] 万春和[2] 施少华[2] 梁齐章 陈红梅[2] 傅秋玲[2] 刘荣昌[2] 黄小红 黄瑜[2] ZHOU Jiayu;ZHU Chunhua;CHEN Zhen;CHENG Longfei;CHEN Cuiteng;FU Guanghua;WAN Chunhe;SHI Shaohua;LIANG Qizhang;CHEN Hongmei;FU Qiuling;LIU Rongchang;HUANG Xiaohong;HUANG Yu(Fujian Key Laboratory of Traditional Chinese Veterinary Medicine and Animal Health,College of Animal Science,Fujian Agriculture and Forestry University,Fuzhou 350002,China;Institute of Animal Husbandry and Veterinary Medicine,Fujian Academy of Agricultural Sciences,Fuzhou 350013,China)

机构地区：[1]福建农林大学动物科学学院福建省兽医中药与动物保健重点实验室,福建福州350002 [2]福建省农业科学院畜牧兽医研究所,福建福州350013

出　　处：《中国兽医学报》2024年第1期66-73,共8页Chinese Journal of Veterinary Science

基　　金：福建省属公益类科研院所基本科研专项资助项目(2022R1026005,2021R10260012);福建省自然科学基金面上资助项目(2022J01466);国家现代农业产业技术体系资助项目(CARS-42)。

摘　　要：禽腺病毒属成员鸭腺病毒3型(duck adenovirus type 3,DAdV-3)和禽腺病毒4型(fowl adenovirus serotype-4,FAdV-4)是养殖场主要流行的病原,对养禽业造成巨大的经济损失。目前尚未见可同时快速检测这2种病原的双重荧光定量PCR检测方法,本研究基于DAdV-3 GDMM10毒株和FAdV-4 GDMZ毒株Fiber2基因序列比对及遗传进化分析,分别在Fiber2基因保守区设计特异性引物,建立了能同时鉴别临床上常见的DAdV-3和FAdV-4双重荧光定量PCR检测方法。结果表明,DAdV-3 GDMM10和FAdV-4 GDMZ Fiber2基因处于两个不同进化分支上,同源性为46.10%。建立的检测DAdV-3和FAdV-4双重荧光定量PCR方法特异性强、灵敏度高,最低可检出45拷贝/μL的DAdV-3样品和17拷贝/μL的FAdV-4样品;且检测结果可直接通过Tm值差异进行判定,简化操作时间。利用该双重荧光定量PCR方法对2022年度临床上采集的34份肝炎-心包积液疑似样品进行检测,DAdV-3和FAdV-4检出率分别为17.65%和2.94%。为进一步开展禽源DAdV-3和FAdV-4的分子流行病学及共感染致病机制奠定了基础。Duck adenovirus type 3(DAdV-3)and fowl adenovirus serotype 4(FAdV-4),members of the genus Aviadenovirus,are the main prevalent pathogens in poultry farms,causing huge economic losses to the poultry industry.Till date,there is no duplex fluorescence real-time quantitative PCR assay that can rapid and simultaneously detect these two pathogens.Based on the homologous sequences alignment and genetic evolution analysis of DAdV-3 and FAdV-4 fiber2 genes,two pairs of specific primers were designed in the conservative regions of fiber2 genes from DAdV-3 GDMM10 and FAdV-4 GDMZ strains,respectively,and a duplex fluorescence real-time quantitative PCR assay for simultaneous detection of DAdV-3 and FAdV-4 was established.The phylogenetic analysis showed that the fiber2 genes of DAdV-3 GDMM10 and FAdV-4 GDMZ strains were in two different evolutionary branches,and the homology was 46.10%.The established dual fluorescence quantitative PCR method exhibited high specificity and sensitivity for both DAdV-3and FAdV-4detection.The sensitivity of this duplex qPCR assay was 45copies/μL and 17copies/μL for DAdV-3and FAdV-4,respectively.A total of 34clinical suspected samples with hepatitises hydropericardium syndrome were collected and tested in 2022,and the positive rates of DAdV-3and FAdV-4were 17.65%and 2.94%,respectively.This study will provide a foundation for further research on the molecular epidemiology and pathogenic mechanism of DAdV-3and FAdV-4co-infections in poultry.

关 键 词：鸭腺病毒3型 禽腺病毒4型 Fiber2基因 实时定量PCR 临床检测 

分 类 号：S852.65[农业科学—基础兽医学]