Evaluation of real-time RT-PCR for the quantification of FCoV shedding in the faeces of domestic cats

Abstract

Faecal samples were taken from cats living in multi-cat households with endemic feline coronavirus (FCoV) infection. Total RNA was extracted from faecal suspensions and FCoV RNA was quantified using a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay. The real-time RT-PCR threshold cycle (CT) values were consistently high suggesting that the samples contained very little viral RNA. However, experiments in which RNA extracted from FCoV-infected cell culture supernatants was combined with RNA extracted from faecal suspensions revealed the presence of faecal factors that significantly inhibited the reverse transcription reaction. Consequently, three methods of RNA extraction were investigated and RNA dilution was undertaken to investigate whether the effects of the faecal inhibitors could be reduced. Our results show that using the QIAgen RNA mini kit for RNA extraction and dilution of the RNA samples helps to reduce the inhibitory effects. However, because the extent of the inhibitory effects varied between faecal samples, accurate quantification proved difficult. We, therefore, conclude that although real-time RT-PCR provides an excellent method for detecting the presence of viral shedding, quantification of FCoV RNA in faecal material has to take into account the possible effects of RT-PCR inhibitors. It is, therefore, essential that all new assays, and the methods of sample preparation, are carefully evaluated before being used in a clinical setting.

A retrospective clinical and epidemiological study on feline coronavirus (FCoV) in cats in Istanbul, Turkey

“…FCoVs primarily infect enterocytes and spread from the intestine by monocyte-associated viremia (Gunn-Moore et al, 1998;Kipar et al, 2005). They have also been shown to replicate in monocytes/macrophages of healthy cats (Can-Sahna et al, 2007;Dye et al, 2008). Vertical transmission has not been demonstrated (Foley et al, 1997).…”

“…Vertical transmission has not been demonstrated (Foley et al, 1997). Persistently infected, asymptomatic carriers spread FCoV since most of these cats shed the virus for a period of months or years, either continuously or transiently (Foley et al, 1997;Cave et al, 2004;Dye et al, 2008;Kipar et al, 2010;Sabshin et al, 2012).…”

“…Worldwide the prevalence of FCoV infections may be up to 90% in multi-cat environments and 10-60% in household cats (Herrewegh et al, 1997;Pedersen et al, 2004;Bell et al, 2006;Addie et al, 2009;Sharif et al, 2009;Taharaguchi et al, 2012). Detection of FCoV antibodies in the early stage of infection can be useful to minimize the spread of FCoVs in a breeding cattery, multi-cat household and FCoV-free household (Cave et al, 2004;Dye et al, 2008;Drechsler et al, 2011). Therefore, it is important to monitor cats living in multi-cat environments in order to reduce and control FCoV infection.…”

A real-time TaqMan® RT-PCR assay with an internal amplification control for rapid detection of transmissible gastroenteritis virus in swine fecal samples

“…Nucleic acids extracted from fecal samples may contain unknown additional molecules, usually referred to as PCR inhibitors, which can interfere with the PCR amplification (Dye et al, 2008;Scipioni et al, 2008;Oikarinen et al, 2009). Presence of PCR inhibitors can lead to false negative results.…”

Detection of subgenomic mRNA of feline coronavirus by real-time polymerase chain reaction based on primer-probe energy transfer (P-sg-QPCR)

“…Another explanation for the surprisingly low copy number of the FCoV sg-mRNA in the gut may be explained with the balanced immune response of the host animals that keeps FCoV replication at low level, and as a consequence, the subgenomic RNA levels may reach much lower rates then their genomic counterparts. Whichever explanation is true, evaluation of quantitative PCR results has to take into consideration the inhibitory compounds of the faecal samples (Dye et al, 2008). The different melting point values of the faecal samples indicating variable number of nucleotide substitutions at the probe-binding site (Rasmussen et al, 2003;Hakhverdyan et al, 2006) revealed at least four different FCoV virus genetic variants in the targeted region of the M gene.…”