Identification of duck plague virus by polymerase chain reaction
Identification of duck plague virus by polymerase chain reaction
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DOI：
10.2307/1592768
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发表时间：
1999-01-01
期刊：
影响因子：
1.4
通讯作者：
Knudson, DL
中科院分区：
文献类型：
--
作者：
Hansen, WR;Brown, SE;Knudson, DL
建立了检测鸭瘟病毒的聚合酶链反应（PCR）方法。从鸭瘟疫苗（DP-VAC）病毒基因组中克隆了一个765 bp的EcoR 1片段，并对其进行了序列测定。经GenBank比对发现，该片段序列与其它疱疹病毒的开放阅读框3'端和DNA聚合酶蛋白基因相似。4对引物中有3对对引物对DP-VAC病毒特异，对鹤类病毒包涵体病的DNA扩增率为100%（7/7）。用来自其他禽类疱疹病毒的基因组模板测试一个引物组的特异性，所述其他禽类疱疹病毒包括来自金雕、秃鹰、大角猫头鹰、雪鸮、游隼、草原隼、鸽子、鹦鹉和鸡（传染性喉气管炎）的基因组模板，但也未产生扩增子。因此，该PCR检测方法对鸭瘟病毒DNA具有高度特异性。两套引物能够检测到来自鸭瘟疫苗株的1 fg DNA，相当于5个基因组拷贝。此外，组织培养感染剂量与来自感染的鸭胚细胞的鸭瘟疫苗病毒基因组拷贝数的比例被确定为1：100，使得PCR检测鸭瘟病毒的灵敏度比组织培养高20倍。该方法的快速性、敏感性和特异性为鸭瘟的诊断和研究提供了一种新的手段。
A polymerase chain reaction (PCR) assay was developed for detecting duck plague virus. A 765-bp EcoR1 fragment cloned from the genome of the duck plague vaccine (DP-VAC) virus was sequenced for PCR primer development. The fragment sequence was found by GenBank alignment searches to be similar to the 3' ends of an undefined open reading frame and the gene for DNA polymerase protein in other herpesviruses, Three of four primer sets were found to be specific for the DP-VAC virus and 100% (7/7) of field isolates but did not amplify DNA from inclusion body disease of cranes virus. The specificity of one primer set was tested with genome templates from other avian herpesviruses, including those from a golden eagle, bald eagle, great horned owl, snowy owl, peregrine falcon, prairie falcon, pigeon, psittacine, and chicken (infectious laryngotracheitis), but amplicons were nor produced. Hence, this PCR test is highly specific for duck plague virus DNA. Two primer sets were able to detect 1 fg of DNA from the duck plague vaccine strain, equivalent to five genome copies. In addition, the ratio of tissue culture infectious doses to genome copies of duck plague vaccine virus from infected duck embryo cells was determined to be 1:100, making the PCR assay 20 times more sensitive than tissue culture for detecting duck plague virus. The speed, sensitivity and specificity of this PCR provide a greatly improved diagnostic and research tool for studying the epizootiology of duck plague.