Vet World Vol.17 October-2024 Article - 10
Research Article
Veterinary World, 17(10): 2286-2294
https://doi.org/10.14202/vetworld.2024.2286-2294
Molecular detection and quantification of canine parvovirus 2 using a fast and sensitive SYBR® green-based quantitative polymerase chain reaction assay in dogs affected with gastroenteritis
2. Facultad de Ingeniería y Ciencias Aplicadas, Carrera de Ingeniería en Biotecnología, Universidad de Las Américas (UDLA), Antigua Vía a Nayón S/N, Quito EC 170124, Ecuador.
3. Facultad de Ciencias de la Salud, Carrera de Medicina Veterinaria, Universidad de Las Américas (UDLA), Quito, Ecuador, Antigua Vía a Nayón S/N, Quito EC 170124, Ecuador.
4. Facultad de Industrias Agropecuarias y Ciencias Ambientales. Carrera agropecuaria. Universidad Po-litécnica Estatal del Carchi (UPEC). Antisana S/N y Av Universitaria, Tulcán EC 040102, Ecuador.
5. Facultad de Ciencias Veterinarias, Universidad Nacional de Rosario (UNR). Boulevard Ovidio Lagos y Ruta 33 Casilda – Santa Fe – Argentina.
6. Facultad de Medicina Veterinaria y Zootecnia, Universidad Central Del Ecuador, Quito, Ecuador, Gatto Sobral y Jerónimo Leiton, Quito EC 170521, Ecuador.
7. Clínica Veterinaria Docente, Universidad de Las Américas (UDLA), Calle Shuara N40-55y Av. De Los Granados, Quito, EC 170503, Ecuador.
8. One Health Research Group, Universidad de Las Américas (UDLA), Antigua Vía a Nayón S/N, Quito EC 170124, Ecuador.
Background and Aim: Viral gastroenteritis in canines is primarily caused by the canine parvovirus 2 (CPV-2). Infections by this virus can cause severe consequences in dogs, such as fever, vomiting, diarrhea, septicemia, systemic inflammation, and immunosuppression. Therefore, the mortality rate of persistent infections caused by this virus is significantly high. The capsid protein VP2 genome of canine parvovirus has undergone many changes, resulting in the emergence of different genotypes, including CPV-2a, CPV-2b, and CPV-2c. Diagnostic procedures often lack the necessary specificity for early infection diagnosis. Early detection of the infection enhances the likelihood of canine survival because the canine will receive prompt therapy. Hence, this study aimed to develop a quantitative polymerase chain reaction (qPCR)-based diagnostic technique using SYBR Green for the rapid and accurate detection and quantification of CPV-2.
Materials and Methods: The assay was specifically designed to identify a portion of the conserved NS gene using primers that amplify a 125-bp fragment. The qPCR method was executed in the fast mode to expedite the process using Power up SYBR Green Master Mix reagent. A standard curve was constructed using the amplified and purified PCR product of the NS gene.
Results: The limit of detection and quantification were determined in the one amplified-DNA copy. The standard curve showed an efficiency of 99.5% and inter- and intra-assay coefficients of variation of 0.387%–0.976% and 0.085%–0.430%, respectively. The assay was specific for the amplification of CPV-2, as no amplification was observed for other viral genomes (canine adenovirus II, canine distemper virus, canine coronavirus, and canine astrovirus) or from the negative controls. Inter- and intra-tests for repeatability showed low test variability around the run time. To validate the present assay, 200 samples of fezzes from canines with gastroenteritis and symptoms associated with enteric infection were tested using the qPCR protocol. From the analyzed samples, 136 were positive for CPV-2 by qPCR assay, of which 110 were before diagnostic positive for the virus by endpoint PCR, showing high sensitivity of the current assay. CPV-2 was detected in dogs over 2 weeks old up to dogs 9 years old, where the highest viral concentration found was 16429595 gene copies in dogs aged 2 weeks.
Conclusion: In the present study, a rapid, specific, repeatable, and sensitive assay was developed for the detection and quantification of CPV-2. Furthermore, it was demonstrated that in the population of domestic dogs in Ecuador affected with gastrointestinal disease, the virus is presented in dogs of different ages and not only in young dogs.
Keywords: canine parvovirus, gastroenteritis, quantitative polymerase chain reaction, SYBR green.
How to cite this article: Loor-Giler A, Castillo-Reyes S, Santander-Parra S, Campos M, Mena-Pérez R, Prado-Chiriboga S, and Nuñez L (2024) Molecular detection and quantification of canine parvovirus 2 using a fast and sensitive SYBR® green-based quantitative polymerase chain reaction assay in dogs affected with gastroenteritis, Veterinary World, 17(10): 2286-2294.
Received: 2024-05-08 Accepted: 2024-09-11 Published online: 2024-10-17
Corresponding author: E-mail: fabiann7@yahoo.es
DOI: 10.14202/vetworld.2024.2286-2294
Copyright: Loor-Giler, 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.
