Journal article
Sensitive detection and typing of porcine reproductive and respiratory syndrome virus by RT-PCR amplification of whole viral genes
Sektion for Eksotiske Virussygdomme, Division of Virology, National Veterinary Institute, Technical University of Denmark1
Division of Virology, National Veterinary Institute, Technical University of Denmark2
National Veterinary Institute, Technical University of Denmark3
Stald/vægterservice, Division of Virology, National Veterinary Institute, Technical University of Denmark4
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.
Language: | English |
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Publisher: | Elsevier Science B.V. |
Year: | 1998 |
Pages: | 7-22 |
ISSN: | 18732542 and 03781135 |
Types: | Journal article |
DOI: | 10.1016/S0378-1135(98)00254-5 |
PRRSV RFLP RT-PCR SDG 3 - Good Health and Well-being diagnosis-viruses pig-viruses vaccinations
Animals Antibodies, Monoclonal DNA Primers Denmark Diagnosis-viruses Electrophoresis, Agar Gel European Union Female Macrophages, Alveolar Male Open Reading Frames Pig-viruses Porcine Reproductive and Respiratory Syndrome Porcine respiratory and reproductive syndrome virus RNA, Viral Reverse Transcriptase Polymerase Chain Reaction Semen Sensitivity and Specificity Sequence Analysis, DNA Specific Pathogen-Free Organisms Swine United States Vaccinations