Vet World Vol.17 May-2024 Article - 2
Research Article
Veterinary World, 17(5): 946-955
https://doi.org/10.14202/vetworld.2024.946-955
One-step triplex TaqMan quantitative reverse transcription polymerase chain reaction for the detection of feline coronavirus, feline panleukopenia virus, and feline leukemia virus
2. Guangxi Center for Animal Disease Control and Prevention, Nanning 530001, China.
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.
Keywords: detection method, feline coronavirus, feline leukemia virus, feline panleukopenia virus, multiplex reverse transcription-quantitative polymerase chain reaction.
How to cite this article: He M, Feng S, Shi K, Shi Y, Long F, Yin Y, and Li Z (2024) One-step triplex TaqMan quantitative reverse transcription polymerase chain reaction for the detection of feline coronavirus, feline panleukopenia virus, and feline leukemia virus, Veterinary World, 17(5): 946–955.
Received: 2024-01-23 Accepted: 2024-04-10 Published online: 2024-05-04
Corresponding author: E-mail:
DOI: 10.14202/vetworld.2024.946-955
Copyright: He, et al. This article is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/ by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
