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. 2023 Dec;43(1):1-12.
doi: 10.1080/01652176.2023.2272188. Epub 2023 Nov 1.

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

Kannika Phongroop  1   2   3 , Jatuporn Rattanasrisomporn  4 , Sahatchai Tangtrongsup  3   5 , Anudep Rungsipipat  1 , Chutchai Piewbang  1   2 , Somporn Techangamsuwan  1   2
Affiliations

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

Kannika Phongroop et al. Vet Q. 2023 Dec.

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; cat; feline calicivirus; polymerase chain reaction.

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Conflict of interest statement

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

Figures

Figure 1.
Figure 1.
Feline calicivirus (FCV) strain differentiation by high-resolution melting (HRM) analysis. (A) HRM normalized the melt curve of eight unique patterns. (B) HRM difference graph of the eight FCV strains using the vaccine strain (nobivac® 1-HCPCh) as a reference strain. HRM normalized and difference graphs could distinguish eight FCV-strain typing patterns from 11 strain templates.
Figure 2.
Figure 2.
Linear regression analysis of the relationship between the percentage of the C:G content of the PCR product and Tm value.
Figure 3.
Figure 3.
Differentiation efficacy of qPCR-HRM between FCV-Vac, VS-FCV, and FCV-TH wild type (A) HRM normalized the melt curve of ten unique patterns. (B) HRM difference graph of the ten FCV strains using the vaccine strain (purevax® RCPCh) as a reference strain. HRM normalized and difference graphs could distinguish between FCV-Vac, VS-FCV, and FCV-TH wild type.
Figure 4.
Figure 4.
Analytical sensitivity of the high-resolution melting (HRM) assay. (A) The quantitative assessment in a tenfold dilution of feline calicivirus (FCV)-synthesis string DNA (6.18 × 101 and 6.18 × 108 copies/µl) was evaluated using qPCR and analyzed by HRM software. (B) The normalized graph of HRM analysis revealed a normalization region between 80 and 89 °C with a confidence threshold of 95%. The cutoff Ct value of the positive sample was set at Ct < 35. (C) The standard curve for indicating the efficiency of the qPCR detection assay by the x-axis refers to the tenfold dilution of FCV-synthesis string DNA (ng/µl). In contrast, the y-axis refers to the corresponding Ct values. The assay is linear in the range of 6.18 × 101 to 6.18 × 108 template copies/µl, with a determination coefficient (R2) of 0.998 and reaction efficiency of 96.13%.
Figure 5.
Figure 5.
Analytical specificity of the high-resolution melting (HRM) assay. The graph shows positive results from the positive control (PTC: string DNA fragment from the feline calicivirus [FCV] vaccine strain with concentration at 6.2 × 105 copies/µl) and FCV-positive clinical sample. The non-template control (NTC) and other feline virus templates, including FHV-1, FIV, FeLV, FCoV, and FeMV, showed amplification signals over positive cutoff values (Ct > 35).
Figure 6.
Figure 6.
Analytical efficacy of the high-resolution melting (HRM) assay for strain differentiation between the wild-type feline calicivirus-Thai (FCV-TH) strain and vaccine strain (FCV-Vac). The patterns of the HRM normalized graph (A) and HRM difference graph (B) demonstrate detectability patterns at each various concentration ratio. Melt curve analysis from the HRM assay (C) indicated the derivative melt peak patterns that seem likely to correspond to the divergence ratio of the strain mixture.
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