Oleksiewicz M B, Bøtner A, Madsen K G, Storgaard T
Danish Veterinary Institute for Virus Research, Lindholm, Kalvehave.
Vet Microbiol. 1998 Nov;64(1):7-22. doi: 10.1016/s0378-1135(98)00254-5.
Following the recent use of a live vaccine against porcine reproductive and respiratory syndrome virus (PRRSV) in Denmark, both American (vaccine) and European-type PRRSV now coexist in Danish herds. This situation highlighted a requirement for supplementary tests for precise virus-typing. As a result, we developed a RT-PCR assay able to detect as well as type PRRSV. To provide maximal sequence information, complete viral open reading frames (ORFs 5 and 7) were targeted for amplification. The RT-PCR test was able to amplify complete PRRSV ORFs from complex materials such as boar semen containing as little as 1 TCID50 ml(-1) of PRRSV. Typing of viruses was accomplished by any one of three strategies: (i) use of type-specific PCR primers, (ii) size determination of ORF 7 amplicons, (iii) DNA sequencing. All three typing strategies showed complete concordance with the currently used method of typing with monoclonal antibodies (MAbs) when used on a panel of PRRSV field isolates covering the period 1992-1997. The ORF 7-based test had particularly desirable characteristics, namely, highly sensitive detection of PRRSV without apparent type bias, typing of the detected virus, discrimination between pure and mixed virus populations, and semi-quantitative assessment of type ratios in mixed populations, all in a single PCR reaction. In addition, the obtained sequence data were used to predict two simple and rapid strategies (single-enzyme restriction length polymorphy analysis and oligonucleotide hybridization) for confirmation of the specificity of ORF 7 RT-PCR reactions. As such, the RT-PCR assay provides a new, powerful diagnostic tool to study the population dynamics between present and emerging PRRSV-types.
近期丹麦使用了一种针对猪繁殖与呼吸综合征病毒（PRRSV）的活疫苗后，美国型（疫苗）和欧洲型PRRSV目前在丹麦猪群中共存。这种情况凸显了进行精确病毒分型补充检测的必要性。因此，我们开发了一种能够检测并分型PRRSV的RT-PCR检测方法。为了提供最大程度的序列信息，针对病毒完整开放阅读框（ORF 5和7）进行扩增。RT-PCR检测能够从复杂材料（如每毫升含低至1个半数组织培养感染剂量（TCID50）的PRRSV的公猪精液）中扩增出完整的PRRSV ORF。病毒分型可通过三种策略中的任何一种完成：（i）使用型特异性PCR引物，（ii）确定ORF 7扩增子的大小，（iii）DNA测序。当对1992 - 1997年期间的一组PRRSV田间分离株进行检测时，所有这三种分型策略与目前使用单克隆抗体（MAb）进行分型的方法完全一致。基于ORF 7的检测具有特别理想的特性，即在一次PCR反应中，能高度灵敏地检测PRRSV且无明显型别偏向、对检测到的病毒进行分型、区分纯病毒群体和混合病毒群体以及对混合群体中的型别比例进行半定量评估。此外，所获得的序列数据被用于预测两种简单快速的策略（单酶限制性片段长度多态性分析和寡核苷酸杂交）以确认ORF 7 RT-PCR反应的特异性。因此，RT-PCR检测为研究当前和新出现的PRRSV型别之间的群体动态提供了一种新的、强大的诊断工具。
