RT Journal Article
T1 Development and Validation of a SYBR Green Real Time PCR Protocol for Detection and Quantification of Nervous Necrosis Virus (NNV) Using Different Standards
A1 Olveira Hermida, José Gabriel
A1 Souto Pereira, Sandra
A1 Bandín Matos, Isabel
A1 Pereira Dopazo, Carlos
K1 Diagnosis
K1 RT-qPCR
K1 Quantification standards
K1 Fish virus
AB The nervous necrosis virus (NNV) is a threat to fish aquaculture worldwide, especially in Mediterranean countries. Fast and accurate diagnosis is essential to control it, and viral quantification is required to predict the level of risk of new viral detections in field samples. For both, reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) is used by diagnostic laboratories. In the present study, we developed an RT-qPCR procedure for the diagnosis and simultaneous quantification of NNV isolates from any of the four genotypes. The method proved to be highly sensitive in terms of crude virus titer: 5.56–9.88 TCID50/mL (tissue culture infectious dose per mL), depending on the viral strain, and averaging 8.8 TCID50/mL or 0.08 TCID50/reaction. Other standards also yielded very low detection limits: 16.3 genome copies (cps) of purified virus per mL, 2.36 plasmid cps/mL, 7.86 in vitro synthetized RNA cps/mL, and 3.16 TCID50/mL of virus from infected tissues. The diagnostic parameters evaluated in fish samples were much higher in comparison to cell culture isolation and nested PCR. In addition, the high repeatability and reproducibility of the procedure, as well as the high coefficient of determination (R2) of all the calibration curves with any type of standard tested, ensure the high reliability of the quantification of NNV using this RT-qPCR procedure, regardless of the viral type detected and from the type of standard chosen
PB MDPI
YR 2021
FD 2021
LK http://hdl.handle.net/10347/25907
UL http://hdl.handle.net/10347/25907
LA eng
NO Animals 2021, 11(4), 1100; https://doi.org/10.3390/ani11041100
NO This research was partially funded by the Interreg VA Spain-Portugal cooperation program (POCTEP) 2014-2020, 0474_BLUEBIOLAB project, co-funded by FEDER
DS Minerva
RD 24 abr 2026