Chirnside E D, Spaan W J
Institute of Virology, Faculty of Veterinary Sciences, University of Utrecht, The Netherlands.
J Virol Methods. 1990 Nov;30(2):133-40. doi: 10.1016/0166-0934(90)90014-7.
A technique is described for the amplification and specific identification of equine arteritis virus (EAV) nucleotide sequences. The polymerase chain reaction (PCR) was evaluated initially by amplification of cloned virus specific cDNA sequences prior to amplification of single-stranded (ss) cDNA produced by reverse transcription (RT) of viral genomic RNA. Three separate primer pairs were used for RT/PCR of EAV genomic RNA, each pair producing only one band in agarose gels of the predicted size from the genomic nucleotide sequence. The viral origin of cDNA products was confirmed by hybridisation analysis with EAV-specific probes. RT/PCR analysis of clinical material indicates the methodology is sensitive enough to detect 600 pfu/ml EAV in seminal plasma.
本文描述了一种用于扩增和特异性鉴定马动脉炎病毒（EAV）核苷酸序列的技术。最初通过对克隆的病毒特异性cDNA序列进行扩增来评估聚合酶链反应（PCR），然后再对病毒基因组RNA经逆转录（RT）产生的单链（ss）cDNA进行扩增。使用了三对不同的引物对EAV基因组RNA进行RT/PCR，每对引物在琼脂糖凝胶中仅产生一条预测大小的条带，该大小基于基因组核苷酸序列。通过与EAV特异性探针进行杂交分析，证实了cDNA产物的病毒来源。对临床材料的RT/PCR分析表明，该方法灵敏度足以检测精浆中600 pfu/ml的EAV。
