首页|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

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

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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.
Establishment and application of a duplex fluorescence quantitative PCR for de-tection of DAdV-3 and FAdV-4
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 eco-nomic losses to the poultry industry.Till date,there is no duplex fluorescence real-time quantita-tive PCR assay that can rapid and simultaneously detect these two pathogens.Based on the homol-ogous 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 quantita-tive PCR assay for simultaneous detection of DAdV-3 and FAdV-4 was established.The phyloge-netic 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-3 and FAdV-4 detection.The sensitivity of this duplex qPCR assay was 45 copies/μL and 17 copies/μL for DAdV-3 and FAdV-4,respectively.A total of 34 clinical suspected samples with hepatitises hydropericardium syndrome were collected and tested in 2022,and the positive rates of DAdV-3 and FAdV-4 were 17.65％and 2.94％,respectively.This study will provide a foundation for further research on the molecular epidemiology and pathogenic mechanism of DAdV-3 and FAdV-4 co-in-fections in poultry.

duck adenovirus type 3fowl adenovirus serotype 4real-time quantitative PCRfiber2 geneclinical detection

周珈羽、朱春华、陈珍、程龙飞、陈翠腾、傅光华、万春和、施少华、梁齐章、陈红梅、傅秋玲、刘荣昌、黄小红、黄瑜

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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

福建省农业科学院畜牧兽医研究所,福建福州 350013

鸭腺病毒3型 禽腺病毒4型 Fiber2基因 实时定量PCR 临床检测

福建省属公益类科研院所基本科研专项资助项目福建省属公益类科研院所基本科研专项资助项目福建省自然科学基金面上资助项目国家现代农业产业技术体系资助项目

2022R10260052021R102600122022J01466CARS-42

2024

10.16303/j.cnki.1005-4545.2024.01.10

中国兽医学报
吉林大学

中国兽医学报

CSTPCD北大核心
影响因子：0.702
ISSN：1005-4545
年,卷(期)：2024.44(1)
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