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

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-LAM...

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Bibliographic Details
Published in:Animals (Basel) 2022-08, Vol.12 (16), p.2075
Main Authors: Rapichai, Witsanu, Saejung, Wichayet, Khumtong, Kotchaporn, Boonkaewwan, Chaiwat, Tuanthap, Supansa, Lieberzeit, Peter A., Choowongkomon, Kiattawee, Rattanasrisomporn, Jatuporn
Format: Article
Language:English
Subjects:
Assaying
Cats
Cell culture
Colorimetry
Coronaviruses
Cross-reactivity
Design
Domestic animals
Effusion
Feline infectious peritonitis
Gene amplification
Genes
Genomes
Infectious diseases
Inspection
molecular diagnosis
Mutation
Nucleotides
Peritonitis
Polymerase chain reaction
Proteins
Reverse transcription
reverse transcription loop-mediated isothermal amplification
Virulence
Viruses
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Summary: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 °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/µL. 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.
ISSN:2076-2615
2076-2615
DOI:10.3390/ani12162075