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		The Real-Time TaqMan PCR and Applications in Veterinary Medicine



		· Introduction · From PacMan to TaqMan - a computer game revisited · The advantages of real-time TaqMan PCR over conventional quantitative PCR · Applications in Veterinary Medicine · Allelic discrimination · Discussion · The veterinarian and his relationship with the next-generation PCR technology · Acknowledgements · References
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Discussion
PCR has proved to be a useful tool in research and diagnosis. However, its use has also brought new challenges to research. The sensitivity found in PCR technology and the availability of quantitative results will bring new problems to the interpretation of these results. As illustrated with feline infectious peritonitis, a fatal disease caused by mutant feline coronavirus (FCoV) strains [66], its diagnosis is difficult, especially when patients lack typical clinical signs of the disease. The causative agent, FIP virus (FIPV), is a macrophage-trophic mutant of the ubiquitous feline enteric coronavirus (FECV) [49, 66] and can be detected in blood and faecal samples from healthy cats [27]. Interpretation of positive PCR results in these cats is difficult, especially when the cat appears to be healthy. In this situation, the positive and negative predictive values are the basis for correct interpretation. By testing large numbers of blood samples from healthy cats, cats with non-FIP disease and cats with FIP-related disease, diagnostic values can be calculated; the diagnostic specificity gives the percentage of negative FCoV-PCR tests in healthy cats and the diagnostic sensitivity is the percentage of positive FCoV-PCR tests in sick cats. If the diagnostic specificity is 100%, then no FCoV has been found in a healthy cat and therefore any positive results must be associated with disease, either with non-FIP or with FIP. If this were the case, the FCoV PCR would be easy to apply. But this is rarely the case and, therefore, intense investigations are necessary to get these values. So far, the positive predictive values for FIP RT-PCR have not been determined carefully, which makes interpretation difficult. Every new PCR test to be used for the purpose of diagnosis should be evaluated carefully according to this procedure. The same information is lacking for FeLV, FHV and many other infections. A great deal of work is needed to generate a basis of knowledge for correct interpretation of these tests. In veterinary medicine, PCR-based diagnostics are just becoming widely used and because of the increased cost-effectiveness of the newer assays, knowledge for their interpretation will soon become available.