Development and Validation of a SYBR Green Real Time PCR Protocol for Detection and Quantification of Nervous Necrosis Virus (NNV) Using Different Standards
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 tra...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-04-01
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| Series: | Animals |
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| Online Access: | https://www.mdpi.com/2076-2615/11/4/1100 |
| _version_ | 1797537982614339584 |
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| author | José G. Olveira Sandra Souto Isabel Bandín Carlos P. Dopazo |
| author_facet | José G. Olveira Sandra Souto Isabel Bandín Carlos P. Dopazo |
| author_sort | José G. Olveira |
| collection | DOAJ |
| description | 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 TCID<sub>50</sub>/mL (tissue culture infectious dose per mL), depending on the viral strain, and averaging 8.8 TCID<sub>50</sub>/mL or 0.08 TCID<sub>50</sub>/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 TCID<sub>50</sub>/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 (R<sup>2</sup>) 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. |
| first_indexed | 2024-03-10T12:23:58Z |
| format | Article |
| id | doaj.art-4ca19700694b4563b5f51665e7b198ed |
| institution | Directory Open Access Journal |
| issn | 2076-2615 |
| language | English |
| last_indexed | 2024-03-10T12:23:58Z |
| publishDate | 2021-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Animals |
| spelling | doaj.art-4ca19700694b4563b5f51665e7b198ed2023-11-21T15:13:42ZengMDPI AGAnimals2076-26152021-04-01114110010.3390/ani11041100Development and Validation of a SYBR Green Real Time PCR Protocol for Detection and Quantification of Nervous Necrosis Virus (NNV) Using Different StandardsJosé G. Olveira0Sandra Souto1Isabel Bandín2Carlos P. Dopazo3Unidad de Ictiopatología, Instituto de Acuicultura y Departamento de Microbiología, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, SpainUnidad de Ictiopatología, Instituto de Acuicultura y Departamento de Microbiología, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, SpainUnidad de Ictiopatología, Instituto de Acuicultura y Departamento de Microbiología, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, SpainUnidad de Ictiopatología, Instituto de Acuicultura y Departamento de Microbiología, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, SpainThe 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 TCID<sub>50</sub>/mL (tissue culture infectious dose per mL), depending on the viral strain, and averaging 8.8 TCID<sub>50</sub>/mL or 0.08 TCID<sub>50</sub>/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 TCID<sub>50</sub>/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 (R<sup>2</sup>) 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.https://www.mdpi.com/2076-2615/11/4/1100diagnosisRT-qPCRquantification standardsfish virus |
| spellingShingle | José G. Olveira Sandra Souto Isabel Bandín Carlos P. Dopazo Development and Validation of a SYBR Green Real Time PCR Protocol for Detection and Quantification of Nervous Necrosis Virus (NNV) Using Different Standards Animals diagnosis RT-qPCR quantification standards fish virus |
| title | Development and Validation of a SYBR Green Real Time PCR Protocol for Detection and Quantification of Nervous Necrosis Virus (NNV) Using Different Standards |
| title_full | Development and Validation of a SYBR Green Real Time PCR Protocol for Detection and Quantification of Nervous Necrosis Virus (NNV) Using Different Standards |
| title_fullStr | Development and Validation of a SYBR Green Real Time PCR Protocol for Detection and Quantification of Nervous Necrosis Virus (NNV) Using Different Standards |
| title_full_unstemmed | Development and Validation of a SYBR Green Real Time PCR Protocol for Detection and Quantification of Nervous Necrosis Virus (NNV) Using Different Standards |
| title_short | Development and Validation of a SYBR Green Real Time PCR Protocol for Detection and Quantification of Nervous Necrosis Virus (NNV) Using Different Standards |
| title_sort | development and validation of a sybr green real time pcr protocol for detection and quantification of nervous necrosis virus nnv using different standards |
| topic | diagnosis RT-qPCR quantification standards fish virus |
| url | https://www.mdpi.com/2076-2615/11/4/1100 |
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