One-step Triplex TaqMan Quantitative Reverse Transcription Polymerase Chain Reaction for the Detection of Feline Coronavirus, Feline Panleukopenia Virus, and Feline Leukemia Virus

Overview

Journal Vet World

Specialty Veterinary Medicine

Date 2024 Jun 24

PMID 38911097

Authors

Mengyi He

Shuping Feng

Kaichuang Shi

Yandi Shi

Feng Long

Yanwen Yin

Zongqiang Li

Affiliations

Soon will be listed here.

Abstract

Background And Aim: Feline coronavirus (FCoV), feline panleukopenia virus (FPV), and feline leukemia virus (FeLV) are prevalent throughout China and significantly threaten cat health. These viruses cause similar manifestations and pathological damage. Rapid and accurate diagnosis depends on detection in the laboratory. This study aimed to establish a reliable and rapid method for accurate detection of FCoV, FPV, and FeLV so that a definite diagnosis can be made and effective measures can be taken to prevent and control viral infection.

Materials And Methods: We designed three pairs of specific primers and probes for the detection of FCoV 5' untranslated region, FPV viral protein 2, and FeLV pol genes. Recombinant plasmid constructs were generated for use as standard plasmid constructs. Optimal reaction conditions, including primer and probe concentrations, reaction cycles, and annealing temperatures, were obtained on the basis of optimization tests. One-step triplex real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was successfully established to simultaneously detect FCoV, FPV, and FeLV. The specificity, sensitivity, and repeatability of the assay were analyzed, and its applicability was validated by testing 1175 clinical samples.

Results: One-step triplex RT-qPCR had a high degree of specificity only for the detection of FCoV, FPV, and FeLV; it had high sensitivity with limits of detection of 139.904, 143.099, and 152.079 copies/reaction for p-FCoV, p-FPV, and p-FeLV standard plasmid constructs, respectively, and it had reliable repeatability with 0.06%-0.87% intra-assay coefficients of variations. A total of 1175 clinical samples were examined for FCoV, FPV, and FeLV using triplex RT-qPCR, and the FCoV, FPV, and FeLV positivity rates were 18.47%, 19.91%, and 47.57%, respectively. The clinical sensitivity and specificity of one-step triplex RT-qPCR were 93.07% and 97.99%, respectively.

Conclusion: We developed a rapid and reliable one-step triplex RT-qPCR method for the detection of FCoV, FPV, and FeLV, which could be used as a diagnostic tool for clinical monitoring and diagnosis.

Citing Articles

Genetic and Phylogenetic Analysis of Feline Coronavirus in Guangxi Province of China from 2021 to 2024.

Shi K, He M, Shi Y, Long F, Shi Y, Yin Y Vet Sci. 2024; 11(10).

PMID: 39453047 PMC: 11512343. DOI: 10.3390/vetsci11100455.

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