Classical swine fever virus: a ring test to evaluate RT-PCR detection methods

Six laboratories participated in an exercise to compare the sensitivity and specificity of RT-PCR tests for the detection of classical swine fever vir us (CSFV). Two sets of coded samples were prepared by serial dilution of po sitive samples and then distributed to each of the laboratories. One set co mprised 34 samples of random primed cDNA. These had been synthesised from v iral RNA representative of seven different genetic subtypes of CSFV. The ot her set comprised 40 clinical samples containing tonsil, spleen, whole bloo d or serum from a pig that had been experimentally infected with CSFV. Each laboratory tested the samples using one or more PCR/RT-PCR tests that they were accustomed to using. The methods and results of the laboratories were compared with one another. The RT-PCR results obtained from testing the cl inical samples were also compared with those obtained by virus isolation an d antigen ELISA. The cDNA from three CSFV isolates was detected poorly or not at all by some of the PCR tests. For clinical samples, the order of sensitivity was RT-ne sted PCR>RT-PCR>virus isolation>ELISA. Both RT-PCR and RT-nested PCR appear ed to give some false positive results. Several of the PCR tests appear sui table in terms of specificity and sensitivity. Further trials are necessary to compare results when the same test is performed by different laboratori es, and to show that improved control procedures can eliminate problems due to false positive reactions. A limited comparison of extraction and reverse transcription procedures sho wed similar results in each of three participating laboratories, even thoug h the methods were not standardised. (C) 2000 Elsevier Science B.V. All rig hts reserved.