Osterrieder N, Hübert P H, Brandmüller C, Kaaden O R
Institute for Medical Microbiology, Ludwig-Maximilians-University Munich, Germany.
J Virol Methods. 1994 Dec;50(1-3):129-36. doi: 10.1016/0166-0934(94)90169-4.
More than 50 reference strains and field isolates of equine herpesvirus type 1 (EHV-1) were examined by a touchdown PCR. Primers for specific amplification of EHV-1 DNA were chosen from the terminal and internal repeat regions of the EHV-1 genome where the high-passaged live vaccine strain RacH displays symmetric 850 bp deletions. The positive strand and one negative strand primer were designed to encompass the deletions present in RacH, and the second negative strand primer was designed to hybridize within these deletions. Discrimination between field isolates and the vaccine strain was achieved by the generation of amplification products of different size: In all EHV-1 reference strains and field isolates, a 495 bp DNA fragment was amplified specifically, whereas a 310 bp fragment was amplified when DNA of the vaccine strain RacH was used as a template. PCR amplification was only obtained in the presence of 8-10% dimethylsulfoxide and when the primer annealing temperatures were decreased stepwise from 72 degrees C to 60 degrees C. Under these conditions as little as 100 fg template DNA, corresponding to about 100 genome equivalents, could be detected. The PCR assay allows fast and sensitive discrimination of the modified live vaccine strain RacH from field strains of EHV-1 since it is applicable to viral DNA extracted from organ samples and paraffin-embedded tissues. It may thus be helpful for examining the potential involvement of the RacH live vaccine strain in abortions of vaccinated mares.
采用降落PCR对50多株1型马疱疹病毒（EHV-1）参考毒株和野外分离株进行了检测。用于特异性扩增EHV-1 DNA的引物是从EHV-1基因组的末端和内部重复区域中选取的，其中高代次活疫苗株RacH在这些区域存在对称的850 bp缺失。正向引物和一条反向引物的设计涵盖了RacH中存在的缺失，第二条反向引物则设计为在这些缺失区域内杂交。通过产生不同大小的扩增产物实现了野外分离株与疫苗株的区分：在所有EHV-1参考毒株和野外分离株中，特异性扩增出一条495 bp的DNA片段，而以疫苗株RacH的DNA为模板时，则扩增出一条310 bp的片段。只有在存在8-10%二甲基亚砜且引物退火温度从72℃逐步降至60℃时才能获得PCR扩增。在这些条件下，低至100 fg的模板DNA（约相当于100个基因组当量）都能被检测到。该PCR检测方法能够快速、灵敏地区分改良活疫苗株RacH与EHV-1的野外毒株，因为它适用于从器官样本和石蜡包埋组织中提取的病毒DNA。因此，它可能有助于检测RacH活疫苗株在接种疫苗母马流产中可能的作用。
