Sensitive and cost-effective citrate-based RNA extraction procedure for isolation of RNA from Tilapia Lake Virus-infected fish

S.R. Saranya; R. Sudhakaran

doi:10.7324/JABB.2024.196791

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Abstract

Aquaculture plays a crucial role in improving global nutrition and food security, with tilapia being a widely farmed fish species. However, the emergence of Tilapia Lake Virus Disease (TiLVD) poses a significant threat to the aquaculture industry and food security. This study aims to develop a novel and cost-effective RNA extraction method for the accurate diagnosis of TiLVD. It utilizes a Citrate buffer-based approach, which eliminates the need for harsh organic solvents and DNase treatment, simplifying the extraction process, reducing costs, and promoting environmental sustainability. The conventional Trizol method was used as a control for comparison. The results of the study demonstrate the efficiency and effectiveness of the Citrate buffer-based RNA extraction method in comparison to the Trizol technique. This novel method has the potential to enhance disease surveillance, facilitate early detection, and enable effective management strategies for TiLVD. Furthermore, high-quality RNA samples obtained through this method may contribute to a better understanding of TiLVD pathogenesis, viral load dynamics, and host–virus interactions. Overall, the development of a robust and affordable RNA extraction method specifically tailored for TiLVD diagnosis holds immense potential for the aquaculture industry. It addresses the limitations of existing techniques and contributes to the sustainable management of tilapia aquaculture.

Keyword: Tilapia TiLVD RNA extraction Citrate buffer method Inexpensive materials Environmental sustainability

Citation:

Saranya SR, Sudhakaran R. and Sensitive and cost-effective citrate-based RNA extraction procedure for isolation of RNA from Tilapia Lake Virus-infected fish. J App Biol Biotech. 2024. Online First. http://doi.org/10.7324/JABB.2024.196791

Copyright: Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license.

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1. Naylor RL, Goldburg RJ, Primavera JH, Kautsky N, Beveridge MCM, Clay J, et al. Effect of aquaculture on world fish supplies. Nature. 2000; 405(6790):1017-24. https://doi.org/10.1038/35016500
2. Coad BW, Murray AM. Fishes of the World. Joseph S. Nelson. Can Field Nat. 2006;120(1):116. https://doi.org/10.22621/cfn.v120i1.262
3. Eyngor M, Zamostiano R, Tsofack JEK, Berkowitz A, Bercovier H, Tinman S, et al. Identification of a novel RNA virus lethal to tilapia. J Clin Microbiol. 2014;52(12):4137-46. https://doi.org/10.1128/JCM.00827-14
4. Tsofack JEK, Zamostiano R, Watted S, Berkowitz A, Rosenbluth E, Mishra N, et al. Detection of tilapia lake virus in clinical samples by culturing and nested reverse transcription-PCR. J Clin Microbiol. 2017;55(3):759-67. https://doi.org/10.1128/JCM.01808-16
5. Surachetpong, W, Dvm. Enhancing capacity/risk reduction of emerging Tilapia Lake Virus (TiLV) to African tilapia aquaculture: Intensive Training Course on TiLV Kenya in cooperation with Kenya Marine Fisheries Research Institute (KMFRI) and Kenya Fisheries Service (KeFS) 2. FAO/ASTF Project: GCP/RAF/510/MUL. Session: Diagnostic PCR method for Tilapia Lake Virus (TiLV); 2018.
6. Ferguson HW, Kabuusu R, Beltran S, Reyes E, Lince JA, del Pozo J. Syncytial hepatitis of farmed tilapia, Oreochromis niloticus (L.): a case report. J Fish Dis. 2014;37(6):583-9. https://doi.org/10.1111/jfd.12142
7. del-Pozo J, Mishra N, Kabuusu R, Cheetham S, Eldar A, Bacharach E, et al. Syncytial Hepatitis of Tilapia (Oreochromis niloticus L.) is associated with orthomyxovirus-like virions in hepatocytes. Vet Pathol. 2017;54(1):164-70. https://doi.org/10.1177/0300985816658100
8. Bacharach E, Mishra N, Briese T, Zody MC, Kembou Tsofack JE, Zamostiano R, et al. Characterization of a novel orthomyxo-like virus causing mass die-offs of tilapia. mBio. 2016;7(2):e00431-16. https://doi.org/10.1128/mBio.00431-16
9. Waiyamitra P, Piewbang C, Techangamsuwan S, Liew WC, Surachetpong W. Infection of Tilapia tilapinevirus in Mozambique Tilapia (Oreochromis mossambicus), a globally vulnerable fish species. Viruses. 2021;13(6). https://doi.org/10.3390/v13061104
10. Tavares L, Alves PM, Ferreira RB, Santos CN. Comparison of different methods for DNA-free RNA isolation from SK-N-MC neuroblastoma. BMC Res Notes. 2011;4(1):3. https://doi.org/10.1186/1756-0500-4-3
11. Mészáros E. Comprehensive introduction to DNA extraction. Mol Biol Blog. 2021. Available from: https://www.integra-biosciences.com/china/en
12. Saranya SR, Sudhakaran R. Report on prevalence of tilapia lake virus infection in tilapia fishes (Oreochromis niloticus). Biocatal Agric Biotechnol. 2020;27:101665. https://doi.org/10.1016/j.bcab.2020.101665
13. Jaemwimol P, Sirikanchana K, Tattiyapong P, Mongkolsuk S, Surachetpong W. Virucidal effects of common disinfectants against tilapia lake virus. J Fish Dis. 2019;42(10):1383-9. https://doi.org/10.1111/jfd.13060
14. Hasson K, Fan Y, Reisinger T, Venuti J, Varner P. White-spot syndrome virus (WSSV) introduction into the Gulf of Mexico and Texas freshwater systems through imported, frozen bait-shrimp. Dis Aquat Organ. 2006;71:91-100. https://doi.org/10.3354/dao071091
15. Mônica Ghislaine Oliveira A, Pérez-Sayáns M, Padín-Iruegas ME, Reboiras-López MD, Suarez-Peñaranda JM, López-López R, et al. Comparison of RNA extraction methods for molecular analysis of oral cytology. Acta Stomatol Croat. 2016;50(2):108-15. https://doi.org/10.15644/asc50/2/2
16. Xiang X, Qiu D, Hegele RD, Tan WC. Comparison of different methods of total RNA extraction for viral detection in sputum. J Virol Methods. 2001;94(1-2):129-35. https://doi.org/10.1016/S0166-0934(01)00284-1
17. Heath . Low pH RNA isolation reagents, method, and kit. United States; 5,973,137. 1999.
18. Oñate?Sánchez L, Verdonk JC. Citrate?citric acid RNA isolation (CiAR) for fast, low?cost, and reliable RNA extraction from multiple plant species and tissues. Curr Protoc. 2021;1(12):e298. https://doi.org/10.1002/cpz1.298
19. Wozniak A, Cerda A, Ibarra-Henríquez C, Sebastian V, Armijo G, Lamig L, et al. A simple RNA preparation method for SARS-CoV-2 detection by RT-qPCR. Sci Rep. 2020;10(1):16608. https://doi.org/10.1038/s41598-020-73616-w
20. Fanson BG, Osmack P, Di Bisceglie AM. A comparison between the phenol-chloroform method of RNA extraction and the QIAamp viral RNA kit in the extraction of hepatitis C and GB virus-C/hepatitis G viral RNA from serum. J Virol Methods. 2000;89(1-2):23-7. https://doi.org/10.1016/S0166-0934(00)00192-0
21. Kim TK. T test as a parametric statistic. Korean J Anesthesiol. 2015;68(6):540. https://doi.org/10.4097/kjae.2015.68.6.540
22. Zhang B, Chen K, Ni E. Comparison of three methods for extraction of HCV RNA in sera collected from individuals with hyperlipidemia, hyperbilirubinemia and hyperglobulinemia. J Virol Methods. 2015;212:44-6. https://doi.org/10.1016/j.jviromet.2014.11.004
23. Roy D, Tomo S, Modi A, Purohit P, Sharma P. Optimising total RNA quality and quantity by phenol-chloroform extraction method from human visceral adipose tissue: a standardisation study. MethodsX. 2020;7:101113. https://doi.org/10.1016/j.mex.2020.101113
24. Deng MY, Wang H, Ward GB, Beckham TR, McKenna TS. Comparison of six RNA extraction methods for the detection of classical swine fever virus by real-time and conventional reverse transcription-PCR. J Vet Diagn Invest. 2005;17(6):574-8. https://doi.org/10.1177/104063870501700609
25. Possebon FS, Ullmann LS, Malossi CD, Miodutzki GT, da Silva EC, Machado EF, et al. A fast and cheap in-house magnetic bead RNA extraction method for COVID-19 diagnosis. J Virol Methods. 2022;300:114414. https://doi.org/10.1016/j.jviromet.2021.114414
26. Deng MY, Wang H, Ward GB, Beckham TR, McKenna TS. Comparison of six RNA extraction methods for the detection of classical swine fever virus by real-time and conventional reverse transcription-PCR. J Vet Diagn Invest. 2005;17(6):574-8. https://doi.org/10.1177/104063870501700609
27. Wilfinger WW, Mackey K, Chomczynski P. Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. Biotechniques. 1997;22(3):474-81. https://doi.org/10.2144/97223st01
28. Tellez-Bañuelos MC, Santerre A, Casas-Solis J, Zaitseva G. Endosulfan increases seric interleukin-2 like (IL-2L) factor and immunoglobulin M (IgM) of Nile tilapia (Oreochromis niloticus) challenged with Aeromona hydrophila. Fish Shellfish Immunol. 2010;28(2):401-5. https://doi.org/10.1016/j.fsi.2009.11.017

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1. Naylor RL, Goldburg RJ, Primavera JH, Kautsky N, Beveridge MCM, Clay J, et al. Effect of aquaculture on world fish supplies. Nature. 2000; 405(6790):1017-24. https://doi.org/10.1038/35016500

2. Coad BW, Murray AM. Fishes of the World. Joseph S. Nelson. Can Field Nat. 2006;120(1):116. https://doi.org/10.22621/cfn.v120i1.262

3. Eyngor M, Zamostiano R, Tsofack JEK, Berkowitz A, Bercovier H, Tinman S, et al. Identification of a novel RNA virus lethal to tilapia. J Clin Microbiol. 2014;52(12):4137-46. https://doi.org/10.1128/JCM.00827-14

4. Tsofack JEK, Zamostiano R, Watted S, Berkowitz A, Rosenbluth E, Mishra N, et al. Detection of tilapia lake virus in clinical samples by culturing and nested reverse transcription-PCR. J Clin Microbiol. 2017;55(3):759-67. https://doi.org/10.1128/JCM.01808-16

5. Surachetpong, W, Dvm. Enhancing capacity/risk reduction of emerging Tilapia Lake Virus (TiLV) to African tilapia aquaculture: Intensive Training Course on TiLV Kenya in cooperation with Kenya Marine Fisheries Research Institute (KMFRI) and Kenya Fisheries Service (KeFS) 2. FAO/ASTF Project: GCP/RAF/510/MUL. Session: Diagnostic PCR method for Tilapia Lake Virus (TiLV); 2018.

6. Ferguson HW, Kabuusu R, Beltran S, Reyes E, Lince JA, del Pozo J. Syncytial hepatitis of farmed tilapia, Oreochromis niloticus (L.): a case report. J Fish Dis. 2014;37(6):583-9. https://doi.org/10.1111/jfd.12142

7. del-Pozo J, Mishra N, Kabuusu R, Cheetham S, Eldar A, Bacharach E, et al. Syncytial Hepatitis of Tilapia (Oreochromis niloticus L.) is associated with orthomyxovirus-like virions in hepatocytes. Vet Pathol. 2017;54(1):164-70. https://doi.org/10.1177/0300985816658100

8. Bacharach E, Mishra N, Briese T, Zody MC, Kembou Tsofack JE, Zamostiano R, et al. Characterization of a novel orthomyxo-like virus causing mass die-offs of tilapia. mBio. 2016;7(2):e00431-16. https://doi.org/10.1128/mBio.00431-16

9. Waiyamitra P, Piewbang C, Techangamsuwan S, Liew WC, Surachetpong W. Infection of Tilapia tilapinevirus in Mozambique Tilapia (Oreochromis mossambicus), a globally vulnerable fish species. Viruses. 2021;13(6). https://doi.org/10.3390/v13061104

10. Tavares L, Alves PM, Ferreira RB, Santos CN. Comparison of different methods for DNA-free RNA isolation from SK-N-MC neuroblastoma. BMC Res Notes. 2011;4(1):3. https://doi.org/10.1186/1756-0500-4-3

11. Mészáros E. Comprehensive introduction to DNA extraction. Mol Biol Blog. 2021. Available from: https://www.integra-biosciences.com/china/en

12. Saranya SR, Sudhakaran R. Report on prevalence of tilapia lake virus infection in tilapia fishes (Oreochromis niloticus). Biocatal Agric Biotechnol. 2020;27:101665. https://doi.org/10.1016/j.bcab.2020.101665

13. Jaemwimol P, Sirikanchana K, Tattiyapong P, Mongkolsuk S, Surachetpong W. Virucidal effects of common disinfectants against tilapia lake virus. J Fish Dis. 2019;42(10):1383-9. https://doi.org/10.1111/jfd.13060

14. Hasson K, Fan Y, Reisinger T, Venuti J, Varner P. White-spot syndrome virus (WSSV) introduction into the Gulf of Mexico and Texas freshwater systems through imported, frozen bait-shrimp. Dis Aquat Organ. 2006;71:91-100. https://doi.org/10.3354/dao071091

15. Mônica Ghislaine Oliveira A, Pérez-Sayáns M, Padín-Iruegas ME, Reboiras-López MD, Suarez-Peñaranda JM, López-López R, et al. Comparison of RNA extraction methods for molecular analysis of oral cytology. Acta Stomatol Croat. 2016;50(2):108-15. https://doi.org/10.15644/asc50/2/2

16. Xiang X, Qiu D, Hegele RD, Tan WC. Comparison of different methods of total RNA extraction for viral detection in sputum. J Virol Methods. 2001;94(1-2):129-35. https://doi.org/10.1016/S0166-0934(01)00284-1

17. Heath . Low pH RNA isolation reagents, method, and kit. United States; 5,973,137. 1999.

18. Oñate?Sánchez L, Verdonk JC. Citrate?citric acid RNA isolation (CiAR) for fast, low?cost, and reliable RNA extraction from multiple plant species and tissues. Curr Protoc. 2021;1(12):e298. https://doi.org/10.1002/cpz1.298

19. Wozniak A, Cerda A, Ibarra-Henríquez C, Sebastian V, Armijo G, Lamig L, et al. A simple RNA preparation method for SARS-CoV-2 detection by RT-qPCR. Sci Rep. 2020;10(1):16608. https://doi.org/10.1038/s41598-020-73616-w

20. Fanson BG, Osmack P, Di Bisceglie AM. A comparison between the phenol-chloroform method of RNA extraction and the QIAamp viral RNA kit in the extraction of hepatitis C and GB virus-C/hepatitis G viral RNA from serum. J Virol Methods. 2000;89(1-2):23-7. https://doi.org/10.1016/S0166-0934(00)00192-0

21. Kim TK. T test as a parametric statistic. Korean J Anesthesiol. 2015;68(6):540. https://doi.org/10.4097/kjae.2015.68.6.540

22. Zhang B, Chen K, Ni E. Comparison of three methods for extraction of HCV RNA in sera collected from individuals with hyperlipidemia, hyperbilirubinemia and hyperglobulinemia. J Virol Methods. 2015;212:44-6. https://doi.org/10.1016/j.jviromet.2014.11.004

23. Roy D, Tomo S, Modi A, Purohit P, Sharma P. Optimising total RNA quality and quantity by phenol-chloroform extraction method from human visceral adipose tissue: a standardisation study. MethodsX. 2020;7:101113. https://doi.org/10.1016/j.mex.2020.101113

24. Deng MY, Wang H, Ward GB, Beckham TR, McKenna TS. Comparison of six RNA extraction methods for the detection of classical swine fever virus by real-time and conventional reverse transcription-PCR. J Vet Diagn Invest. 2005;17(6):574-8. https://doi.org/10.1177/104063870501700609

25. Possebon FS, Ullmann LS, Malossi CD, Miodutzki GT, da Silva EC, Machado EF, et al. A fast and cheap in-house magnetic bead RNA extraction method for COVID-19 diagnosis. J Virol Methods. 2022;300:114414. https://doi.org/10.1016/j.jviromet.2021.114414

26. Deng MY, Wang H, Ward GB, Beckham TR, McKenna TS. Comparison of six RNA extraction methods for the detection of classical swine fever virus by real-time and conventional reverse transcription-PCR. J Vet Diagn Invest. 2005;17(6):574-8. https://doi.org/10.1177/104063870501700609

27. Wilfinger WW, Mackey K, Chomczynski P. Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. Biotechniques. 1997;22(3):474-81. https://doi.org/10.2144/97223st01

28. Tellez-Bañuelos MC, Santerre A, Casas-Solis J, Zaitseva G. Endosulfan increases seric interleukin-2 like (IL-2L) factor and immunoglobulin M (IgM) of Nile tilapia (Oreochromis niloticus) challenged with Aeromona hydrophila. Fish Shellfish Immunol. 2010;28(2):401-5. https://doi.org/10.1016/j.fsi.2009.11.017

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