Efficiency of Tilapia Lake Virus Detection in Nile Tilapia by Nested RT-PCR and Semi- Nested RT-PCR

Authors

  • Umaporn Khimmakthong Faculty of Veterinary Science, Rajamangala University of Technology Srivijaya, Nakhon Si Thammarat 80240, Thailand, Animal Infectious Diseases in South of Thailand Research Unit, Faculty Veterinary Science, Rajamangala University of Technology Srivijaya, Nakorn Si Thammarat 80240, Thailand
  • Sulaiman Madyod Faculty of Veterinary Science, Rajamangala University of Technology Srivijaya, Nakhon Si Thammarat 80240, Thailand
  • Supaporn Noochu Faculty of Veterinary Science, Rajamangala University of Technology Srivijaya, Nakhon Si Thammarat 80240, Thailand

Keywords:

Detection, Nested RT-PCR, Semi-nested RT-PCR, Tilapia, TiLV

Abstract

There are several methods for detecting Tilapia Lake virus (TiLV). The most popular methods for detecting RNA viruses are nested RT-PCR and semi-nested RT-PCR. The primers currently used are varied and are often designed to be specific for viruses found in each area. This study aimed to develop a detection method for TiLV in tilapia by designing two pairs of TiLVspecific primers and then detecting TiLV by nested RT-PCR and semi-nested RT-PCR, which examined suspected tilapia samples from fish cages in the Tapi River, Nakhon Si Thammarat Province. The results showed that nested RT-PCR (PCR product size of 307 bp) and semi-nested RT-PCR 2 (PCR product size of 496 bp) gave more accurate results than semi-nested RT-PCR 1 (PCR product size of 600 bp) while nested RT-PCR was more sensitive than semi-nested RT-PCR 2. The sensitivity and specificity of the nested RT-PCR method were each 100%.

References

Ferguson HW, Kabuusu R, Beltran S, Reyes E, Lince JA and del Pozo J. Syncytial hepatitis of farmed tilapia, Oreochromis niloticus ( L. ) : a case report. J Fish Dis. 2014;37:583-89.

Eyngor M, Zamostiano R, Tsofack JEK, Berkowitz A, Bercovier H, Tinman S, Lev M, Hurvitz A, Galeotti M, Bacharach E and Eldar A. Identification of novel RNA virus lethal to tilapia. J Clin Microbiol. 2014;52:4137-46.

Dong HT, Siriroob S, Meemetta W, Santimana-wong W, Gangnonngiw W, Pirarat N, Khunrae P, Rattanarojpong T, Vanichviri-yakit R and Senapin S. Emergence of tilapia lake virus in Thailand and an alternative semi-Nested RT-PCR for detection. Aquaculture. 2017;476:111-8.

Surachetpong W, Janetanakit T, Nonthabenjawan N, Tattiyapong P, Sirikanchana K and Amonsin A. Outbreaks of tilapia lake virus infection, Thailand, 2015- 2016. Emerg Infect Dis. 2017;23(6):1031-3.

Tsofack JEK, Zamostiano R, Watted S, Berkowitz A, Rosenbluth E, Mishra N, Briese T, Lipkin WI, Kabuusu RM, Ferguson H, del Pozo J, Eldar A, and Bacharach E. Detection of tilapia lake virus in clinical samples by culturing and nested reverse transcription-PCR. J Clin Microbiol. 2017;55(3):759-67.

Al-Hussinee L, Subramaniam K, Surachetpong W, Popov V, Hartman K, Starzel K, Yanong R, Watson C, Ferguson H, Frasca Jr S and Waltzek T. Tilapia lake virus (TiLV): a globally emerging threat to tilapia aquaculture. UF/IFAS. 2019;2019(2):1-7.

Wang E, Wang K, Chen D, Wang J, He Y, Long B, Yang L, Yang Q, Geng Y, Huang X, Ouyang P and Lai W. Evaluation and selection of appropriate reference genes for real- time quantitative PCR analysis of gene expression in nile tilapia (Oreochromisniloticus) during vaccination and infection. Int J Mol Sci. 2015;16(5):9998-10015.

Jacobson RH. Principles of validation of diagnostic assays for infectious disease. In: Manual of diagnostic tests and vaccines for terrestrial animals. Paris, France: World Organisation for Animal Health (OIE); 2006. Chapter 1.1.4.

Waiyamitra P, Tattiyapong P, Sirikanchana K, Mongkolsuk S, Nicholson P and Surachetpong W. A TaqMan RT-qPCR assay for tilapia lake virus (TiLV) detection in tilapia. Aquaculture. 2018;497:184-8.

Tattiyapong P, Dachavichitlead W and Surachetpong W. Experimental infection of tilapia lake virus (TiLV) in Nile tilapia (Oreochromis niloticus) and red tilapia (Oreochromis spp.). Vet Microbiol. 2017;207:170-7.

Tattiyapong P, Sirikanchana K and Surachetpong W. Development and validation of a reverse transcription quantitative polymerase chain reaction for tilapia lake virus detection in clinical samples and experimentally challenged fish. J Fish Dis. 2018;41(2):255-61.

Liamnimitr P, Thammatorn W, Uthoomporn S, Tattiyapong P and Surachetpong W. Non-lethal sampling for Tilapia Lake Virus detection by RT-qPCR and cell culture. Aquaculture. 2018;486:75-80.

Carr J, Williams DG and Hayden RT. Molecular detection of multiple respiratory viruses. Mol Diagn. 2010;289-300.

Levin RE, Ekezie FGC. and Sun DW. DNAbased technique: polymerase chain reaction (PCR). In: Sun DW, editors. Modern techniques for food authentication. 2nd ed. London, UK: Academic Press; 2018. p. 527-616.

Downloads

Published

2023-09-26

How to Cite

Umaporn Khimmakthong, Sulaiman Madyod, & Supaporn Noochu. (2023). Efficiency of Tilapia Lake Virus Detection in Nile Tilapia by Nested RT-PCR and Semi- Nested RT-PCR. Science & Technology Asia, 28(3), 239–246. Retrieved from https://ph02.tci-thaijo.org/index.php/SciTechAsia/article/view/248798

Issue

Section

Biological sciences