Development and Validation of a SYBR Green Real Time PCR Protocol for Detection and Quantification of Nervous Necrosis Virus (NNV) Using Different Standards

Abstract

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.

Keywords: RT-qPCR; diagnosis; fish virus; quantification standards.

Figure 1

Calibration curves using different standards: averaging data from the four strain types. All the data from replicas and repeats were averaged to obtain a general calibration curve for each standard: viral titer (A), RNA copies from crude virus (B) or from purified virus (C), pDNA copies (D), ivRNA (E), or for viral quantification from infected tissues (F).

Figure 2

Comparison of standard curves. The regression lines with each type of standard averaging the data from the 4 strain types assessed are compared.