Detection of Feline Coronavirus Membrane Gene Based on Conventional Revere Transcription-Polymerase Chain Reaction, Nested Reverse Transcription-Polymerase Chain Reaction, and Reverse Transcription-Quantitative Polymerase Chain Reaction: A Comparative Study

Abstract

Feline coronavirus (FCoV) is a major pathogen causing feline infectious peritonitis (FIP), a lethal disease in cats, necessitating accurate diagnostic methods. This study developed and compared novel primers targeting the FCoV membrane (M) gene for enhanced detection. Specific primers were designed for the M gene and their performance evaluated using reverse transcription-PCR (RT-PCR), nested RT-PCR, and reverse transcription-quantitative PCR (RT-qPCR) on 80 clinical effusion samples from cats suspected of FIP. Specificity of assays was tested against other feline viruses, with sensitivity being assessed via serial dilutions of FCoV RNA. RT-qPCR had the highest sensitivity, detecting 9.14 × 10¹ copies/µL, identifying 93.75% of positive samples, followed by nested RT-PCR (87.50%, 9.14 × 10⁴ copies/µL) and RT-PCR (61.25%, 9.14 × 10⁶ copies/µL). All assays had 100% specificity, with no cross-reactivity to other viruses. The nested RT-PCR and RT-qPCR outperformed RT-PCR significantly, with comparable diagnostic accuracy. The novel primers targeting the FCoV M gene, coupled with RT-qPCR, delivered unparalleled sensitivity and robust reliability for detecting FCoV in clinical settings. Nested RT-PCR was equally precise and amplified diagnostic confidence with its high performance. These cutting-edge assays should revolutionize FCoV detection, offering trusted tools that seamlessly integrate into veterinary practice, empowering clinicians to manage feline infectious peritonitis with unprecedented accuracy and speed.

Keywords: FIP; RT-PCR; RT-qPCR; feline coronavirus; nested RT-PCR.

Figure 1

Temperature optimization. (A) RT-PCR; (B) nested RT-PCR (outer primers); (C) nested RT-PCR (inner primers). Agarose-gel electrophoresis (1.5%, 100 V, 30 min) shows amplicons generated at the indicated annealing-temperature gradient. Lane M, 100 bp DNA ladder (Marker III, Yeastern Biotech, New Taipei City, Taiwan); lane NTC, negative control (RNase-free water). (D) Evaluation of optimal annealing temperature using RT-qPCR.

Figure 2

(A) RT-PCR, (B) nested RT-PCR assay. Specificity was evaluated by agarose gel electrophoresis using various feline and related viruses. Amplicons were detected only in the FCoV clinical sample and the vaccine-derived positive control. No amplification was observed in other viral targets, internal controls, or the negative control (NTC). Lane M: 100 bp DNA ladder (Yeastern Biotech, New Taipei City, Taiwan); lanes 1–10: FIV, FeLV, FPV, FHV, FCV, FCoV clinical sample, CCoV, TGEV, CRFK cells, and whole blood cells, respectively. (C) Evaluation of specificity using RT-qPCR assay.

Figure 3

Similarity in BLAST analysis of FCoV clinical sample No. KU80 sequence.

Figure 4

Comparison of RT-PCR, nested RT-PCR, and RT-qPCR sensitivity for FCoV detection. (A) RT-PCR products resolved on a 1.5% agarose gel. Lane M, 100 bp ladder; lanes 1–9, 10-fold serial dilutions from 9.14 × 10⁹ to 9.14 × 10¹ copies µL; lane NTC, negative control. (B) Nested RT-PCR products analysed under identical gel conditions. (C) RT-qPCR standard curve derived from the same dilution series (9.14 × 10⁹ to 9.14 × 10⁰ copies µL), showing the lower detection limit of each assay.

Figure 5

Schematic representation of the FCoV genome, comprising the 5′ untranslated region (UTR), ORF1a/1b, spike (S), ORF3a, ORF3b, ORF3c, envelope (E), membrane (M), nucleocapsid (N), ORF7a, ORF7b, and the 3′ UTR. Locations of the RT-PCR outer primers (MF, MR) and nested RT-PCR/RT-qPCR inner primers (N-Inner MF, N-Inner MR) within the M gene are indicated.