Submitted to: American Association of Veterinary Laboratory Diagnosticians
Publication Type: Abstract Only
Publication Acceptance Date: 10/9/2003
Publication Date: 10/9/2003
Citation: RICHT, J., LAGER, K.M., SUAREZ, D.L., YOON, K.J. RAPID DETECTION OF SWINE VIRUSES USING REAL-TIME PCR ASSAYS. AMERICAN ASSOCIATION OF VETERINARY LABORATORY DIAGNOSTICIANS. 2003. ABSTRACT P. 150.
Technical Abstract: The choice of a diagnostic assay is based on a variety of selection criteria, e.g., sensitivity, specificity, cost, speed, and technical skills required to perform the assay. The choice to switch from a current diagnostic assay to a better assay is usually based on few criteria, e.g., the need to process a higher volume of samples or the need for a more rapid assay. Real-time PCR is a technology that can be utilized for rapid diagnostic assays. It can be performed in less than 2 hours, can be run in a single tube, thus reducing the potential for cross contamination, can be used on-site with portable equipment and can be automated (96/384-well format). This paper describes the development and evaluation of real-time PCR assays designed to detect porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), and porcine circovirus type 2 (PCV-2). The target gene for the PRRSV reverse transcription (RT)-PCR assay is open reading frame 7 (ORF 7). The ORF 7-based RT-PCR assay is able to detect U.S. PRRSV isolates with a sensitivity of ~25 RNA copies of in vitro generated RNA molecules and ~25 TCID50 per reaction. The target gene for the SIV RT-PCR assay is the matrix (M) gene. The M-gene based RT-PCR assay is able to detect H1- and H3-subtypes of SIVs with a sensitivity of ~5 RNA copies of in vitro generated M-specific RNA molecules and ~2 TCID*50 per reaction. The target gene for the PCV-2 PCR assay is the intergenic region between ORF 1 and ORF 2. The PCV-2-specific PCR assay is able to detect PCV-2 with a sensitivity of ~20 copies of in vitro generated DNA molecules per reaction. All three real-time PCR assays are currently validated using clinical samples (nasal swabs and lung lavage) submitted to the Veterinary Diagnostic Laboratory at the Iowa State University. These real-time PCR assays will allow rapid on-site pathogen detection and will have application for PRSSV, SIV and PCV-2 surveillance and disease management.