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

Abstract

Feline infectious peritonitis is one of the most severe devastating diseases of the Felidae. Upon the appearance of clinical signs, a cure for the infected animal is impossible. Therefore rapid and proper diagnosis for both the presence of the causative agent, feline coronavirus (FCoV) and the manifestation of feline infectious peritonitis is of paramount importance. In the present work, a novel real-time RT-PCR method is described which is able to detect FCoV and to determine simultaneously the quantity of the viral RNA. The new assay combines the M gene subgenomic messenger RNA (sg-mRNA) detection and the quantitation of the genome copies of FCoV. In order to detect the broadest spectrum of potential FCoV variants and to achieve the most accurate results in the detection ability the new assay is applying the primer-probe energy transfer (PriProET) principle. This technology was chosen since PriProET is very robust to tolerate the nucleotide substitutions in the target area. Therefore, this technology provides a very broad-range system, which is able to detect simultaneously many variants of the virus(es) even if the target genomic regions show large scale of variations. The detection specificity of the new assay was proven by positive amplification from a set of nine different FCoV strains and negative from the tested non-coronaviral targets. Examination of faecal samples of healthy young cats, organ samples of perished animals, which suffered from feline infectious peritonitis, and cat leukocytes from uncertain clinical cases were also subjected to the assay. The sensitivity of the P-sg-QPCR method was high, since as few as 10 genome copies of FCoV were detected. The quantitative sg-mRNA detection method revealed more than 10-50,000 times increase of the M gene sg-mRNA in organ materials of feline infectious peritonitis cases, compared to those of the enteric FCoV variants present in the faeces of normal, healthy cats. These results indicate the applicability of the new P-sg-QPCR test as a powerful novel tool for the better detection and quantitation of FCoV and for the improved diagnosis of feline infectious peritonitis, this important disease of the Felidae, causing serious losses in the cat populations at a global scale.

Discrepancies between feline coronavirus antibody and nucleic acid detection in effusions of cats with suspected feline infectious peritonitis

“…Molecular methods have been used for detection of FCoV RNA in the effusions of cats with suspected FIP (Gut et al, 1999;Simons et al, 2004;Hornyák et al, 2012;Soma et al, 2013;Doenges et al, 2017;Felten et al, 2017;Longstaff et al, 2017). However, these methods display similar issues related to the diagnostic performances (lack of sensitivity and specificity).…”

Detection of feline coronavirus mutations in paraffin-embedded tissues in cats with feline infectious peritonitis and controls

“…Previous studies have looked into the use of PCR in the detection of FCoV in various sample materials of cats with FIP, reporting conflicting results. 13,[15][16][17][18][19][20] So far, no other study has investigated paraffin-embedded tissues using the recently commercially available genotypediscriminating RT-qPCR.…”

An update on feline infectious peritonitis: Diagnostics and therapeutics

“…A remaining problem is the high degree of viral genome diversity, which affects the binding of primers used in RT-PCR tests. An attempt was made to address this by designing a RT-PCR that takes into account a degree of genomic variability in various feline coronaviruses (Hornyák et al, 2012). Most of these technical aspects, although daunting, can be overcome with attention to detail.…”