Sensitive detection and typing of porcine reproductive and respiratory syndrome virus by RT-PCR amplification of whole viral genes

被引:131
作者
Oleksiewicz, MB
Botner, A
Madsen, KG
Storgaard, T [1 ]
机构
[1] Danish Vet Inst Virus Res, Kalvehave 4771, Denmark
[2] Danish Inst Plant & Soil Sci, Res Ctr, Slagelse 4200, Denmark
关键词
RT-PCR; PRRSV; RFLP; vaccinations; pig-viruses; diagnosis-viruses;
D O I
10.1016/S0378-1135(98)00254-5
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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. (C) 1998 Elsevier Science B.V. All rights reserved.
引用
收藏
页码:7 / 22
页数:16
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