Detection of porcine reproductive and respiratory syndrome virus by reverse transcription-polymerase chain reaction using different regions of the viral genome

Department of Veterinary Diagnostic Medicine, College of Veterinary Medicine, University of Minnesota, St. Paul 55108, USA.

Serologic studies have revealed strain variability between American and European isolates and among American isolates of porcine reproductive and respiratory syndrome virus (PRRSV). The objective of this study was to develop an assay for the routine diagnosis of PRRSV in field specimens using reverse transcription-polymerase chain reaction (RT-PCR) amplification of conserved genomic regions. Twenty-four field isolates of PRRSV from different regions of the USA were analyzed in the study. Six primer pairs from open reading frames (ORFs) 4, 6, and 7 of the American strain (ATCC VR-2332) and from ORF 1b of the Lelystad strain were used for the amplification of the viral genome by PCR. Amplification products of the expected sizes were obtained from all isolates by PCR amplification of ORF 7, the gene encoding the nucleocapsid protein. Oligonucleotide primers designed to amplify ORFs 4 and 6 detected 92% and 96% of the isolates, respectively, whereas primers for the amplification of ORF 1b detected 88% of all isolates. The specificity of the amplified products of ORF 7 from 7 field isolates and 2 reference strains was confirmed by chemiluminescent hybridization using an internal digoxigenin-labeled DNA probe. Sequence analysis of this region indicated variation in the nucleotide sequence of 2 isolates that did not hybridize with the internal probe. These results indicate that ORF 7 may serve as a potential target for the detection of PRRSV strains by RT-PCR and that genomic variability should be considered when nucleic acid hybridization is used to confirm the specificity of PCR amplification for diagnostic purposes.