Molecular Techniques For Detection Of Pathogens Of Major Viral Diseases Of Swine

Under typical conditions of intensive swine production, it is common for swine to be simultaneously infected with two or more viral pathogens. A definitive diagnosis of multiple infections is often difficult in that clinical signs can be variable and thus fail to provide a clear indication of the most appropriate means of testing. As a result, a number of costly individual, virus-specific tests are sometimes performed initially to expedite the testing procedure. Real-time PCR and multiplex PCR are two relatively new testing procedures, and have been used for rapid identification of multiple viruses on the basis of their specific genome sequences.In the study reported here, a Real-time PCR of porcine reproductive and respiratory syndrome virus (PRRSV), and a triplex PCR test of three common DNA virus, namely, porcine circovirus type II (PCV-2), pseudorabies virus (PRV), and porcine parvovirus (PPV), were developed and subsequently evaluated for their efficiency as means to detect multiple viral infections of swine.The results of TaqMan based real-time PCR assay showed that there are no cross-reactions with other common porcine viruses. The sensitivity of this assay is 1.2 X 101 copy/ml of plasmid coding partial PRRSV N gene, which is 100 times lower than conventional PCR detection. It takes no more than 3 hours from viral RNA extraction to complete the real-time PCR, and the assay is simple and has good repeats.The lower limit of sensitivity of triplex PCR is approximately 4×10^-4ng/μl for double-strained DNA temple (for PRV) and 2×10⁵ng/μl for single-strained DNA temple (for PCV-2 and PPV), when a composite of all three viruses was tested as a single sample. It is also effective for detecting one or two of three viruses in various combinations in tissue specimens collected from diseased pigs and aborted fetuses. The results of relative efficiency (compared to performing separate assays for each virus) and apparent sensitivity of triplex PCR suggest its potential for application as routine molecular diagnostic purposes.A total of 190 clinical specimens including lymph nodes, lungs, spleens, and tonsils of sick pigs were subjected to the established PCR detection systems. The PRRSV infection was demonstrated in 38.9% of tissue samples (74/190), PCV-2 infection was 30.53% (58/190), PRV infection was 3.7% (11/190), and PPV infection was 3.7% (7/190). The rate of co-infection was 15.26% (29/190), and the PRRSV+ PCV-2 mixed infection was 75.86% (22/29).