Souto, S.; Olveira, J.G.; López-Vázquez, C.; Bandín, I.; Dopazo, C.P. Designing and Validation of a Droplet Digital PCR Procedure for Diagnosis and Accurate Quantification of Nervous Necrosis Virus in the Mediterranean Area. Pathogens2023, 12, 1155.
Souto, S.; Olveira, J.G.; López-Vázquez, C.; Bandín, I.; Dopazo, C.P. Designing and Validation of a Droplet Digital PCR Procedure for Diagnosis and Accurate Quantification of Nervous Necrosis Virus in the Mediterranean Area. Pathogens 2023, 12, 1155.
Souto, S.; Olveira, J.G.; López-Vázquez, C.; Bandín, I.; Dopazo, C.P. Designing and Validation of a Droplet Digital PCR Procedure for Diagnosis and Accurate Quantification of Nervous Necrosis Virus in the Mediterranean Area. Pathogens2023, 12, 1155.
Souto, S.; Olveira, J.G.; López-Vázquez, C.; Bandín, I.; Dopazo, C.P. Designing and Validation of a Droplet Digital PCR Procedure for Diagnosis and Accurate Quantification of Nervous Necrosis Virus in the Mediterranean Area. Pathogens 2023, 12, 1155.
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 the 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, the 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 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-folds lower than with RT-qPCR. Quantification by RT-ddPCR, with a dynamic range of 6.8 – 6.8 x 104 (SJ type) or 1.04 x 101 – 1.04 x 105 (RG) cps/rctn, was more reliable than with RT-qPCR. The procedure was tested and validated in field samples, providing the 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.
Keywords
VNNV; Diagnosis; Validation; dPCR; droplet digital PCR.
Subject
Biology and Life Sciences, Virology
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
