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 de-tection of DAdV-3 and FAdV-4
周珈羽 1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 2陈红梅 2傅秋玲 2刘荣昌 2黄小红 1黄瑜2
作者信息
- 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
- 折叠
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.
Abstract
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.
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Key words
duck adenovirus type 3/fowl adenovirus serotype 4/real-time quantitative PCR/fiber2 gene/clinical detection引用本文复制引用
基金项目
福建省属公益类科研院所基本科研专项资助项目(2022R1026005)
福建省属公益类科研院所基本科研专项资助项目(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)
出版年
2024
NETL
NSTL
万方数据