Evaluation of real-time RT-PCR for the quantification of FCoV shedding in the faeces of domestic cats.
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 (C(T)) 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.
Dye
Charlotte
C
Small Animal Hospital, Department of Clinical Veterinary Science, University of Bristol, Langford House, Langford, Bristol BS40 5DU, UK. c.dye@bristol.ac.uk <c.dye@bristol.ac.uk>
Helps
Christopher R
CR
Siddell
Stuart G
SG
D000818:Animals; D002371:Cat Diseases / Q000175:diagnosis / Q000821:virology*; D002415:Cats; D018352:Coronavirus Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D016765:Coronavirus, Feline / Q000302:isolation & purification*; D005243:Feces / Q000821:virology*; D005260:Female; D008297:Male; D012367:RNA, Viral / Q000032:analysis*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards / Q000662:veterinary*; D012680:Sensitivity and Specificity; D017201:Virus Shedding
D012367:RNA, Viral