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

M.B. Oleksiewicz, Anette Bøtner, K.G. Madsen, Torben Storgaard

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    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.
    Original languageEnglish
    JournalVeterinary Microbiology
    Volume64
    Issue number1
    Pages (from-to)7-22
    ISSN0378-1135
    DOIs
    Publication statusPublished - 1998

    Keywords

    • pig-viruses
    • PRRSV
    • RT-PCR
    • diagnosis-viruses
    • RFLP
    • vaccinations

    Fingerprint Dive into the research topics of 'Sensitive detection and typing of porcine reproductive and respiratory syndrome virus by RT-PCR amplification of whole viral genes'. Together they form a unique fingerprint.

    Cite this