Vilcek S
Univerzita veterinárskeho lekárstva, Kosice.
Vet Med (Praha). 1994;39(11):687-700.
The development of PCR assays for detection of BHV-1, BRSV, BVDV and another pestiviruses is summarized. A polymerase chain reaction assay based on primers selected from the viral gI glycoprotein gene detected 3 fg pure BHV-1 DNA, 0.1-1.0 TCID50 or a single infected cell. No amplification was observed with DNA from BHV-2, BHV-3, BHV-4, OHV-1 or OHV-2. However, a fragment of the correct size (468 bp) was amplified using DNA from herpesviruses isolated from reindeer, red deer and goat. The PCR assay was able to detect virus in nasal swabs 1-14 days after experimental infection of cattle and there was a good correlation when PCR was compared to virus isolation for the detection of BHV-1 in clinical field samples. Detection of BHV-1 in fetal bovine serum and semen samples was also successful. PCR detecting a broad range of BVDV, BDV and HCV was developed. Of six sets of primers selected from different parts of the pestivirus genome the best results were provided by a pair 324/326 from the highly conserved 5'-non-coding region which gave an amplification with all 129 isolates tested. This panel consisted of 79 isolates from cattle, 33 from pigs and 17 from sheep. Differentiation between viruses was achieved by cleavage of the PCR-amplified products (288 bp) with the restriction endonucleases AvaI and BglI. The BVDV products were cleaved by AvaI, HCV by BglI and AvaI. Both enzymes, AvaI and BglI, did not cut the BDV products. A nested polymerase chain reaction assay was developed for the detection of bovine respiratory syncytial virus (BRSV). Primers were selected from the gene encoding the F fusion protein. The sensitivity of PCR assay was 0.1 TCID50. No cross reaction was observed with nine heterologous respiratory viruses. PCR products of bovine and human RSV strains were discriminated using endonuclease ScaI, which specifically cleaved products of BRSV. PCR assay detected BRSV in nasal swabs collected from cattle in the acute stage of respiratory disease. In vitro amplification detected 31 positive samples of 35 while immunofluorescence only 23 samples.
总结了用于检测牛疱疹病毒1型（BHV - 1）、牛呼吸道合胞病毒（BRSV）、牛病毒性腹泻病毒（BVDV）及其他瘟病毒的聚合酶链反应（PCR）检测方法的进展。基于从病毒gI糖蛋白基因中选择的引物建立的聚合酶链反应检测方法，能检测到3 fg的纯BHV - 1 DNA、0.1 - 1.0半数组织培养感染剂量（TCID50）或单个感染细胞。用BHV - 2、BHV - 3、BHV - 4、奥卡病毒1型（OHV - 1）或奥卡病毒2型（OHV - 2）的DNA未观察到扩增。然而，使用从驯鹿、马鹿和山羊分离的疱疹病毒的DNA扩增出了正确大小（468 bp）的片段。该PCR检测方法能够在牛实验感染后1 - 14天从鼻拭子中检测到病毒，并且在临床现场样本中检测BHV - 1时，将PCR与病毒分离法进行比较，两者具有良好的相关性。在胎牛血清和精液样本中检测BHV - 1也取得了成功。开发了能检测多种BVDV、牛痘病毒（BDV）和丙型肝炎病毒（HCV）的PCR检测方法。从瘟病毒基因组不同部位选择的六组引物中，来自高度保守的5' - 非编码区的一对引物324/326效果最佳，对所有129株测试分离株均能进行扩增。该组分离株包括79株来自牛的、33株来自猪的和17株来自羊的。通过用限制性内切酶AvaI和BglI切割PCR扩增产物（288 bp）实现病毒间的鉴别。BVDV产物能被AvaI切割，HCV能被BglI和AvaI切割。AvaI和BglI这两种酶都不能切割BDV产物。开发了一种巢式聚合酶链反应检测方法用于检测牛呼吸道合胞病毒（BRSV）。引物是从编码F融合蛋白的基因中选择的。PCR检测方法的灵敏度为0.1 TCID50。与九种异源呼吸道病毒未观察到交叉反应。使用特异性切割BRSV产物的核酸内切酶ScaI区分牛和人呼吸道合胞病毒株的PCR产物。PCR检测方法在呼吸道疾病急性期从牛采集的鼻拭子中检测到了BRSV。体外扩增检测出35个样本中有31个阳性，而免疫荧光仅检测出23个样本。
