摘要
旨在建立一种能够同时鉴别H5、H7和H9亚型禽流感病毒、基因A型鸭甲肝病毒、鸭坦布苏病毒、鸭瘟病毒、减蛋综合征病毒、新城疫病毒、鸭圆环病毒、番鸭呼肠孤病毒和番鸭细小病毒9种常见鸭病毒病的GeXP多重PCR检测方法。根据这些病原体在GenBank上公布的保守基因序列,设计合成了12对特异性GeXP引物。用单一病毒或混合病毒样品的cDNA/DNA模板优化反应条件,同时以其他常见鸭病病原体及鸭组织器官核酸为对照,验证所建立的GeXP方法的特异性和准确性。以106至101拷贝·μL-1梯度稀释的克隆质粒或体外转录RNA来检测GeXP方法的敏感性。随机选取不同浓度的模板(103和107拷贝·μL-1)进行干扰性试验,并与单一病原体为模板的GeXP多重PCR的检测结果比较。最后用该方法检测150份临床样品,与普通PCR方法的结果作比较,所有阳性结果样品均进行测序,进一步验证所建立的GeXP检测方法的可靠性。结果显示,单重或多重模板的GeXP检测均能特异性扩增出相应片段,不能扩增其他常见鸭病病原体,并且可在103拷贝·μL-1水平上同时特异地检测出9种鸭病原体。GeXP干扰性试验结果显示,当3种不同浓度的模板组合时,本试验所建立的方法依然可同时检测出所有病原体,与单重检测比较,未影响其检出。比较GeXP多重PCR方法和普通PCR方法对150份临床样品的检测结果,结果显示GeXP方法更为敏感与准确。笔者建立的同时鉴别9种鸭病毒病的GeXP多重PCR检测方法,具有高通量、特异性强和灵敏度高的特点,为混合感染的鸭病毒病临床样品提供了快速分子诊断新方法。
This experiment was developed to simultaneously detect these nine viral pathogens that cause infections in ducks including avian influenza virus（AIV）subtypes H5,H7,and H9;duck hepatitis A virus（DHAV-A）;duck Tembusu virus（DTMUV）;duck enteritis virus（DEV）;egg drop syndrome virus（EDSV）;Newcastle disease virus（NDV）;duck circovirus（DuCV）;muscovy duck reovirus（MDRV）;and muscovy duck parvovirus（MDPV）.Twelve pairs of specific primers were designed according to the conserved sequences of the genes from each pathogen available in the GenBank database.Single,mixed pathogen cDNA/DNA templates,other common duck pathogens and duck organs nucleic acids were usedto evaluate the specificity of the GeXP-multiplex assay.Serial dilution from 106 to 101 copies·μL-1 of in vitro transcript RNA of target genes and plasmids were used to test the sensitivity of GeXP multiplex PCR assay.Different amounts of the templates（103and 107 copies·μL-1）were selected at random,mixed and tested in the GeXP multiplex PCR assay.The results were compared with those of the single template GeXP multiplex PCR assay.The GeXP assay was evaluated using 150 clinical specimens and compared with the single PCR.All positive specimens in the GeXP multiplex PCR and conventional PCR were identified via sequencing to verify the accuracy and reliability of the GeXP assay.These results showed that the corresponding specific fragments of genes were amplified by the single and multiplex GeXP PCR assay.Other pathogens did not result in amplification products. The detection limit of GeXP was 103copies·μL-1 when all nine types of duck virus were present.The results of the interference assay showed that three specific amplification peaks can still be observed in the case of combination of three different concentration templates.The results of these experiments showed that mixed infections can be detected by GeXP multiplex PCR with minimal interference.Compared with the results of conventional PCR,the GeXP multiplex PCR method was
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2016年第12期2457-2468,共12页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
广西科技攻关项目(10100014-5
14123001-8
AD16380009
AB16380054)
广西水产畜牧兽医局科技计划项目(桂渔牧科201452012)
国家"万人计划"领军人才专项(2016-37-88)
广西重大专项资金(桂科重14121003-4-2)
关键词
鸭病毒病
GeXP检测
多重PCR
特异性嵌合引物
duck virus disease
Genome Lab Gene Expression Profiler（GeXP）
multiplex PCR
specific chimeric primer
