Molecular Detection of Feline Coronavirus Based on Recombinase Polymerase Amplification Assay.

Rea Maja Kobialka,Ahmed Abd El Wahed,Michelle Bergmann,Katrin Hartmann,Uwe Truyen,Arianna Ceruti

doi:10.3390/pathogens10101237

Rea Maja Kobialka, Ahmed Abd El Wahed + Show 4 more

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https://doi.org/10.3390/pathogens10101237

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Abstract

Feline coronavirus (FCoV) is endemic in cat populations worldwide. Persistently, subclinically infected cats play a significant role in spreading the infection. Testing fecal samples of cats may facilitate efforts to decrease the viral burden within a population. Real-time RT-PCR is highly sensitive and specific for the detection of FCoV but must be performed in a fully equipped laboratory. A simple and accurate assay is needed to identify FCoV at the point-of-need. The aim of this study was to develop a rapid FCoV detection assay based on isothermal amplification technology, i.e., reverse transcription-recombinase polymerase amplification (RT-RPA). Primers were designed to target the highly conserved 3′ untranslated region of the 7b gene. Running on a constant temperature of 42 °C, reverse transcription as well as DNA amplification and detection was achieved in a maximum of 15 min. A probit analysis revealed a detection limit of 58.5 RNA copies/reaction. For cross-detection, nucleic acids from 19 viruses were tested. Both RT-RPA and real-time RT-PCR showed cross-detection with canine coronavirus and transmissible gastroenteritis virus, but not with other pathogens. To evaluate clinical performance, RNA was extracted from 39 fecal samples from cats. All samples were tested simultaneously with real-time RT-PCR resulting in a RT-RPA sensitivity and specificity of 90.9% and 100%, respectively. RT-RPA can be considered a promising simple method for rapid detection of FCoV.

Highlights

Feline coronavirus (FCoV) is an enveloped single-stranded positive-sense RNA virus, belonging to the genus Alphacoronavirus, family Coronaviridae, and order Nidovirales [1]
Based on pathogenicity and gene mutations, FCoV can be divided into two biotypes: the common avirulent feline enteric coronavirus (FECV) and the highly virulent feline infectious peritonitis virus (FIPV) [2,3]
Forward Primer 1 (FP1) + Reverse Primer 3 (RP3) primers were aligned to various FCoV and FIPV sequences to assure the coverage of circulating variants (Figure S7) and were used for further validation steps

Summary

Introduction

Feline coronavirus (FCoV) is an enveloped single-stranded positive-sense RNA virus, belonging to the genus Alphacoronavirus, family Coronaviridae, and order Nidovirales [1]. Based on pathogenicity and gene mutations, FCoV can be divided into two biotypes: the common avirulent feline enteric coronavirus (FECV) and the highly virulent feline infectious peritonitis virus (FIPV) [2,3]. It is known that multi-cat environments show a higher prevalence of FCoV infections than single-cat environments and different factors are discussed to play an important role in the spread of the virus [12,13,14]. Most FCoV-infected cats remain without clinical signs or only develop mild enteric signs. Sudden changes in the virus tropism to macrophages and monocytes instead of enterocytes due to mutations in the spike gene [20] lead or contribute to the development of the fatal systemic disease, FIP, in 5–12% of infected cats in multi-cat environments [21]. New studies with antiviral therapy resulted in promising results, but the medication is only available for routine clinical use in few countries so far [23,24]

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