Development of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Detecting Feline Coronavirus

Simple Summary Feline coronavirus infecting domestic cats can cause feline infectious peritonitis (FIP), a fatal infectious disease. Several relevant clinical diagnoses and molecular methods are complicated and often ambiguous for veterinarians. In this work developed a rapid, sensitive, specific, and easy-to-visualize colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay with a novel LAMP primer set that has high specificity was developed using neutral red as an indicator dye. This proposed procedure could reliably detect FCoV RNA from effusion fluids comparable to the conventional PCR method. Considering these advantages, the RT-LAMP developed here has great potential on FIP-associated FCoV surveillance. Together with other sophisticated molecular diagnostic tools, this method can further be exploited in clinical laboratories to inspect suspected cats with effusive FIP. Feline infectious peritonitis (FIP) is a worldwide fatal disease caused by a mutant feline coronavirus (FCoV). Simple and efficient molecular detection methods are needed. Here, sensitive, specific, rapid, and reliable colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed to detect the ORF1a/1b gene of FCoV from cats with suspected FIP using neutral red as an indicator. Novel LAMP primers were specifically designed based on the gene of interest. The isothermal assay could visually detect FCoV at 58 degrees C for 50 min. The RT-LAMP assay was highly specific and had no cross-reactivity with other related feline viruses. The detection limit of FCoV detection by RT-LAMP was 20 fg/mu L. A blind clinical test (n = 81) of the developed RT-LAMP procedure was in good agreement with the conventional PCR method. In the light of its performance specificity, sensitivity, and easy visualization, this neutral-red-based RT-LAMP approach would be a fruitful alternative molecular diagnostic tool for veterinary inspection of FCoV when combined with nucleotide sequencing or specific PCR to affirm the highly virulent FIP-associated FCoV.

Automated summary

A rapid, sensitive, specific, and easy-to-visualize colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay using neutral red as an indicator dye was developed to detect feline coronavirus (FCoV) associated with feline infectious peritonitis (FIP). It showed great potential for FIP surveillance and could be used as an alternative molecular diagnostic tool for the veterinary inspection of FCoV.