Abstract
The veterinary surgeon took a sample of the cat's blood and popped it into the PCR machine, saying to the client “We'll have the result in a couple of hours.”
Science fiction, perhaps … or a very real possibility in the next few years? Ten years ago polymerase chain reaction (PCR) tests were almost unheard of in the veterinary surgery, they were something esoteric that researchers and forensic scientists did. Now PCR tests appear on the menu and price lists of many commercial veterinary laboratories, so it is timely that this edition of JFMS contains an excellent review of PCR by Hans Lutz and his colleagues (pp. 89–100).
In their review, the authors explain what PCR is and how it has been used in feline medicine so far. In essence, PCR amplifies a vanishingly small amount of a specific piece of DNA sufficiently for it to be visualised as a band on a gel following electrophoresis (as shown in figure 1 of Professor Lutz's paper). Detection of RNA is more complex since RNA cannot directly be used as a test sample in a PCR reaction. To overcome this problem a DNA copy of the RNA is made by the enzyme reverse transcriptase, hence the term reverse-transcriptase PCR, or RT-PCR. The nucleotide sequence of the target DNA to be detected must be known. The specificity of the PCR is ensured by using as primers for the reaction small pieces of DNA matching the nucleotide sequence on either side of the DNA fragment being sought. The length of the amplified DNA fragment can be predicted so that the band which is found on the gel should be the right size. In most PCRs these are the only measures taken to ensure specificity but more are available. For example, the PCR product can be treated with restriction enzymes which cut it into several bands with a characteristic pattern for each microbe. Another method is to show that a labelled probe specific for the target DNA actually binds to the product. In the final analysis, the nucleotide sequence of the whole length of the product can be determined to ensure that it matches that of the target.
Many controls are needed in a PCR because of the potential for contamination. Should even a single molecule of DNA from a positive sample contaminate a negative sample, a false positive result will occur, such is the power of the reaction. In PCR of RNA samples (eg feline coronavirus) another type of contamination can occur: the presence of ribonuclease, a ubiquitous enzyme which destroys RNA, can lead to false negative results.
PCR, like every other test in veterinary practice, has its pitfalls and veterinary surgeons need to understand what those are before they can rely on the technique with confidence. As Professor Lutz points out, just because the PCR test is specific for the virus or bacterium being sought, does not mean that the test is specific for diagnosis. For example, PCRs claiming to be diagnostic of feline infectious peritonitis (FIP) are misleading. What they are actually detecting is feline coronavirus (FCoV), and while some cats with FIP have FCoV in their blood, so do many healthy cats and cats suffering from other diseases. Those PCRs which claim to have detected the mutation which differentiates virulent FCoVs from avirulent FCoVs have never been validated by the scientific community. Professor Lutz rightly points out that the presence of a viral genome does not necessarily indicate that the virus is causing disease in the cat being tested, whether it be FCoV, feline leukaemia virus (FeLV) or feline herpesvirus, and that the predictive positive value of PCR or RT-PCRs needs to be carefully evaluated before the test is put into widespread use. A useful assurance for the veterinary surgeon is to ask whether the PCR used by a particular laboratory has featured in a scientific paper, since to do so it would have been refereed by others in the field who would know whether or not its results were valid.
The new kid on the PCR block is the technology such as Taqman to quantify DNA or RNA. The capacity to measure the amount of viral or bacterial nucleic acid may become very useful in the future, not only in diagnosis but also in the prognosis of chronic infections such as FCoV, FeLV or feline immunodeficiency virus as it is already used in humans with HIV.
I was recently asked whether our laboratory was able to identify the genes involved in polycystic kidney disease in a cat. So far, PCR has not been used to screen cats for genetic diseases, but it no doubt will be in the near future. Another use of PCR is in the identification of individual animals and their relatives. Perhaps the most sensational use of PCR in this respect was when the DNA from the hairs of a cat were found on a bloodstained jacket and were used to implicate the owner of the jacket as the murderer! At a more mundane level, these techniques are now being used to study the population dynamics of cats and the impact of viruses on these populations.
Obviously a review of this sort cannot include every PCR test ever used on a feline problem, but the authors provide a comprehensive list of references for those who want to follow up particular examples.