Affiliation:
1. Instituto de Acuicultura, Department of Microbiology, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain
Abstract
The viral nervous necrosis virus (VNNV) is the causative agent of an important disease affecting fish species cultured worldwide. Early and accurate diagnosis is, at present, the most effective control and prevention tool, and molecular techniques have been strongly introduced and accepted by official organizations. Among those, real-time quantitative polymerase chain reaction (rt-qPCR) is nowadays displacing other molecular techniques. However, another PCR-based technology, droplet digital PCR (ddPCR), is on the increase. It has many advantages over qPCR, such as higher sensitivity and more reliability of the quantification. Therefore, we decided to design and validate a protocol for the diagnosis and quantification of SJ and RG type VNNV using reverse transcription-ddPCR (RT-ddPCR). We obtained an extremely low limit of detection, 10- to 100-fold lower than with RT-qPCR. Quantification by RT-ddPCR, with a dynamic range of 6.8–6.8 × 104 (SJ type) or 1.04 × 101–1.04 × 105 (RG type) cps/rctn, was more reliable than with RT-qPCR. The procedure was tested and validated in field samples, providing high clinical sensitivity and negative predictive values. In conclusion, we propose this method to substitute RT-qPCR protocols because it exceeds the expectations of qPCR in the diagnosis and quantification of VNNV.
Funder
Ministerio de Ciencia e Innovación
European Union NextGenerationEU
European Maritime and Fisheries Fund
MiCIN with ERDF
Subject
Infectious Diseases,Microbiology (medical),General Immunology and Microbiology,Molecular Biology,Immunology and Allergy
Reference27 articles.
1. Bandín, I., and Souto, S. (2020). Betanodavirus and VER Disease: A 30-year Research Review. Pathogens, 9.
2. World Organization of Animal Health (WOAH) (2019). Manual of Diagnostic Tests for Aquatic Animals, World Organization of Animal Health. Available online: https://www.woah.org/fileadmin/Home/eng/Health_standards/aahm/current/chapitre_viral_encephalopathy_retinopathy.pdf.
3. Development and validation of a real-time TaqMan PCR assay for the detection of Betanodavirus in clinical specimens;Panzarin;Arch. Virol.,2010
4. Olveira, J.G., Souto, S., Bandín, I., and Dopazo, C.P. (2021). Development and validation of a SYBR Green real time PCR protocol for detection and quantification of nervous necrosis virus (NNV) using different standards. Animals, 11.
5. Gezer, U., Bronkhorst, A.J., and Holdenrieder, S. (2022). The Clinical Utility of Droplet Digital PCR for Profiling Circulating Tumor DNA in Breast Cancer Patients. (Special Issue Cell-Free Nucleic Acids—New Insights into Physico-Chemical Properties, Analytical Considerations, and Clinical Applications). Diagnostics, 12.