Varrasso A, Dynon K, Ficorilli N, Hartley C A, Studdert M J, Drummer H E
Centre for Equine Virology, School of Veterinary Science, The University of Melbourne, Parkville, Victoria.
Aust Vet J. 2001 Aug;79(8):563-9. doi: 10.1111/j.1751-0813.2001.tb10751.x.
To develop and validate specific, sensitive and rapid (< 8 hour) diagnostic tests using polymerase chain reaction (PCR) for the diagnosis of abortion and respiratory disease caused by equine herpesvirus 1 (EHV1; equine abortion virus) and EHV4 (equine rhinopneumonitis virus).
Primer sets based on nucleotide sequences encoding glycoprotein H (gH) of EHV1 and gB of EHV4 were designed and used in single round and second round (seminested) PCRs, and in a multiplex PCR for the diagnosis of EHV1 and EHV4 infections.
Oligonucleotide primers were designed for each virus, PCR conditions were defined and the specificity and sensitivity of the assays were determined. The tests were applied to tissue samples from aborted equine foetuses and to nasopharyngeal swabs from horses with acute febrile respiratory disease.
Individual single round and a second round (seminested) EHV1 and EHV4 PCRs were specific in that EHV1 primers amplified all (n = 30) EHV1 isolates and did not amplify EHV4. Similarly EHV4 primers amplified all (n = 6) EHV4 isolates and did not amplify EHV1. Both PCRs were sensitive in that the first round EHV1 PCR detected 1220 molecules of EHV1 plasmid DNA and the first round EHV4 PCR detected 7280 molecules of EHV4 plasmid DNA. The EHV1 second round PCR was 100 times more sensitive in that it detected 12 molecules of EHV1 DNA and the EHV4 second round PCR was 1000 times more sensitive in that it detected 8 molecules of EHV4 DNA. There was a high correlation between detection of EHV1 by virus isolation and PCR when tissue samples from 71 aborted foetuses were examined; all samples positive by virus isolation were positive by PCR. Similarly the EHV4 PCR was at least as sensitive as virus isolation when applied to nasaopharyngeal swabs from horses with respiratory disease in that all samples positive by virus isolation were also positive by PCR.
Individual single round and second round (seminested) PCRs and a seminested multiplex PCR were developed that enabled reliable, rapid detection of EHV1 and EHV4 in aborted foetal tissues and nasopharyngeal swab samples.
开发并验证使用聚合酶链反应（PCR）的特异性、灵敏性且快速（<8小时）的诊断测试，用于诊断由马疱疹病毒1型（EHV1；马流产病毒）和EHV4（马鼻肺炎病毒）引起的流产和呼吸道疾病。
基于编码EHV1糖蛋白H（gH）和EHV4糖蛋白B（gB）的核苷酸序列设计引物组，并用于单轮和第二轮（半巢式）PCR以及用于诊断EHV1和EHV4感染的多重PCR。
为每种病毒设计寡核苷酸引物，确定PCR条件，并测定检测方法的特异性和灵敏性。将这些测试应用于流产马胎儿的组织样本以及患有急性发热性呼吸道疾病马匹的鼻咽拭子。
单独的单轮和第二轮（半巢式）EHV1和EHV4 PCR具有特异性，即EHV1引物扩增了所有（n = 30）EHV1分离株，而未扩增EHV4。同样，EHV4引物扩增了所有（n = 6）EHV4分离株，而未扩增EHV1。两种PCR均具有灵敏性，即第一轮EHV1 PCR检测到1220个EHV1质粒DNA分子，第一轮EHV4 PCR检测到7280个EHV4质粒DNA分子。EHV1第二轮PCR的灵敏性高100倍，因为它检测到12个EHV1 DNA分子，EHV4第二轮PCR的灵敏性高1000倍，因为它检测到8个EHV4 DNA分子。当检查71例流产胎儿的组织样本时，病毒分离法检测EHV1与PCR检测之间存在高度相关性；所有通过病毒分离呈阳性的样本通过PCR检测也呈阳性。同样，当将EHV4 PCR应用于患有呼吸道疾病马匹的鼻咽拭子时，其灵敏性至少与病毒分离法相同，因为所有通过病毒分离呈阳性的样本通过PCR检测也呈阳性。
开发了单独的单轮和第二轮（半巢式）PCR以及半巢式多重PCR，能够在流产胎儿组织和鼻咽拭子样本中可靠、快速地检测EHV1和EHV4。
