High-resolution melting analysis for simultaneous detection and discrimination between wild-type and vaccine strains of feline calicivirus

Abstract

High-resolution melting (HRM) analysis, a post-polymerase chain reaction (PCR) application in a single closed tube, is the straightforward method for simultaneous detection, genotyping, and mutation scanning, enabling more significant dynamic detection and sequencing-free turnaround time. This study aimed to establish a combined reverse-transcription quantitative PCR and HRM (RT-qPCR-HRM) assay for diagnosing and genotyping feline calicivirus (FCV). This developed method was validated with constructed FCV plasmids, clinical swab samples from living cats, fresh-frozen lung tissues from necropsied cats, and four available FCV vaccines. We performed RT-qPCR to amplify a 99-base pair sequence, targeting a segment between open reading frame (ORF) 1 and ORF2. Subsequently, the HRM assay was promptly applied using Rotor-Gene Q® Software. The results significantly revealed simultaneous detection and genetic discrimination between commercially available FCV vaccine strains, wild-type Thai FCV strains, and VS-FCV strains within a single PCR reaction. There was no cross-reactivity with other feline common viruses, including feline herpesvirus-1, feline coronavirus, feline leukemia virus, feline immunodeficiency virus, and feline morbillivirus. The detection limit of the assay was 6.18 × 10¹ copies/µl. This study, therefore, is the first demonstration of the uses and benefits of the RT-qPCR-HRM assay for FCV detection and strain differentiation in naturally infected cats.

Keywords:

• High-resolution melting analysis
• feline calicivirus
• cat
• polymerase chain reaction

Acknowledgements

K.P. received grant funding from the Ph.D. program at Chiang Mai University. C.P. received a grant from the Ratchadapisek Somphot Fund for Postdoctoral Fellowship at Chulalongkorn University.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Ethical approval

All procedures in this study were approved by the Chulalongkorn University Animal Care and Use Committee (No. 1631002). The cats’ owners were requested to sign a consent form before sampling.

Data availability statement

The authors guarantee that the data supporting the findings in this study are available within this article and its supplementary material. The raw data that encourage the finding of this study are available from the corresponding author, upon reasonable request. Nine sequences of the FCV-TH strains (FCV-TH/KP80, FCV-TH/KP82, FCV-TH/KP100, FCV-TH/KP103, FCV-TH/KP105, FCV-TH/KP181, FCV-TH/KP276, FCV-TH/KP313, and FCV-TH/KP361) obtained from this study have been deposited in NCBI GenBank under accession numbers OM982622, OM982623, OM982625, OM982626, OM982627, OM982629, OM982630, OM982631, and MZ542330, respectively.

Additional information

Funding

This study was supported by the 90th Anniversary of the Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund). ST was partly supported by the National Research Council of Thailand (NRCT) through R. Thanawongnuwech NRCT Senior Scholar 2022 #N42A650553.