Information on new diagnostic QPCR assays currently being investigated
Feline calicivirus QRT-PCR
Feline calicivirus (FCV) is a small single-stranded RNA virus that is widespread in the feline population. Clinical signs of FCV infection include pyrexia, oral ulceration and upper respiratory tract disease. Diagnosis of FCV infection has traditionally been by virus isolation from oropharyngeal swabs, since the virus grows very well in cultures of feline cells. Replication of this RNA virus leads to the evolution of many different strains, which all vary in their genetic sequence. This genetic diversity means that the development of a sensitive PCR assay is extremely difficult since FCV isolates have different sequences and some may not be detected by the assay.
The Molecular Diagnostic Unit is currently working on the development of a sensitive and specific quantitative reverse transcription PCR (QRT-PCR) assay to detect a wide range of FCV strains. Reverse transcription is the process by which the RNA of the virus is converted to DNA so that we can detect it in the PCR. Development of an assay for FCV is being done by aligning whole FCV genome sequences from 10-12 different strains and searching for regions of genetic identity (similarity), which can subsequently be used for QRT-PCR assay design. Due to the high genetic diversity between different FCV strains there are relatively few target sites to which QRT-PCR primers can be designed. Of the likely primer sites that we have found, the genetic identity is not always perfect for all of the strains used, meaning that sequence differences must be incorporated in to the primers (i.e. they are made degenerate). Development is therefore ongoing and any FCV QRT-PCR assays that are developed will be tested for their ability to detect a wide range of FCV strains from our diagnostic sample archive to minimise the chance of false-negative results. We hope that this test will be available in the near future so we appreciate your patience. All tests that the Molecular Diagnostic Unit run are rigorously tested before release to ensure that we are providing high quality reliable assays that you trust.
Feline coronavirus QRT-PCR and FIP
Feline coronavirus (FCoV) is a large single-stranded RNA virus which is very stable and can survive for many weeks in the environment. Infection with FCoV is very common in cats, particularly in multi-cat households, but only a small proportion of FCoV infected cats will develop feline infectious peritonitis (FIP), which is usually fatal. Many cats infected with FCoV show no clinical signs of infection, whilst others develop transient diarrhoea and/or vomiting due to FCoV primarily remaining within the enteric system; a few cats go on to develop FIP due to extensive systemic spread of the virus.
Assays to detect FCoV infection include serology (to detect antibodies against the virus that are present in the blood) and reverse transcription PCR (RT-PCR), which amplifies FCoV in the blood. However, these methods will only indicate that a cat has been, or is infected with FCoV, they WILL NOT tell you if a cat has FIP because neither method can distinguish cats with FIP from those infected with FCoV. It is important to appreciate this when interpreting test results. Although some laboratories are offering RT-PCR for the detection of FCoV, more research is required to validate how useful this test is in the diagnosis of FIP. The design of the RT-PCR assay is very important as some published FCoV assays have been found to also amplify cat genomic DNA, which obviously produces unreliable results. The Molecular Diagnostic Unit is therefore currently investigating the use of a newly designed quantitative RT-PCR (QRT-PCR) assay in cats with and without FIP to determine its usefulness before offering it commercially. It is hoped that this assay will be useful for the diagnosis of FIP when run on tissues or effusion samples, and that detection of FCoV in faeces may be helpful for multi-cat households to identify cats shedding the virus.
For more information about the QPCR diagnostic assays offered please see our list of publications