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

Serologic studies have revealed strain variability between American and Eur opean isolates and among American isolates of porcine reproductive and resp iratory syndrome virus (PRRSV). The objective of this study was to develop an assay for the routine diagnosis of PRRSV in field specimens using revers e transcription-polymerase chain reaction (RT-PCR) amplification of conserv ed genomic regions. Twenty-four field isolates of PRRSV from different regi ons of the USA were analyzed in the study. Six primer pairs from open readi ng frames (ORFs) 4, 6, and 7 of the American strain (ATCC VR-2332) and from ORF Ib 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 nucl eocapsid 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 t he amplified products of ORF 7 from 7 field isolates and 2 reference strain s was confirmed by chemiluminescent hybridization using an internal digoxig enin-labeled DNA probe. Sequence analysis of this region indicated variatio n 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 variab ility should be considered when nucleic acid hybridization is used to confi rm the specificity of PCR amplification for diagnostic purposes.