One-Pot RT-LAMP CRISPR/Cas12b Platform for Rapid Detection of Tilapia Lake Virus
Abstract
Tilapia lake virus disease (TiLVD) is a viral disease that has been associated with high morbidity and mortality in cultured and wild tilapia worldwide.1-4 Although several diagnostic tools have been developed for TiLV detection,1,5-8 they require advanced equipment and time-consuming procedures, making them impractical for laboratories with limited resources or pondside use. Addressing this challenge, we have developed and partially validated an innovative, rapid, and cost-effective one-pot diagnostic assay that combines thermostable Cas12b enzyme with RT-LAMP amplification, targeting a conserved region within segment 4 of the genome. Notably, this assay can be conveniently used as the incubation process is done at 62°C for 75 minutes, and the results can be observed using an inexpensive portable fluorescence viewer. The TiLV one-pot assay is sensitive and specific, with a limit of detection of 50 RNA viral copies, and no cross-reaction was detected with other fish DNA and RNA viruses. The analysis of 43 positive and 40 negative samples previously determined by TaqMan qPCR assay8 resulted in both diagnostic sensitivity and specificity of 100%. Additional samples from different populations or field outbreaks will be utilized to validate the diagnostic performance of the assay further. The present study outlines a report on the development and partial validation of a diagnostic assay for TiLV in stages 1 and 2, following the guidelines from WOAH.9 Finally, this assay can serve as a valuable tool in global surveillance efforts that fish health professionals can easily employ to screen, monitor, and control TiLV disease in tilapia production.
Acknowledgments
The authors thank Dr. Piyush Jain from the Department of Chemical Engineering, University of Florida, for providing the Cas12b enzyme and technical support. The authors also thank Dr. Robert Ossiboff and Dr. Nicole Stacy from the College of Veterinary Medicine, University of Florida, and Dr. Roy Yanong from the Tropical Aquaculture Laboratory, School of Forest, Fisheries, and Geomatics Sciences, Institute of Food and Agricultural Sciences, University of Florida, for their valuable insights and for reviewing the abstract. This study is supported by the intramural research program of the U.S. Department of Agriculture, National Institute of Food and Agriculture.
*Presenting author
+Student presenter
Literature Cited
1. Eyngor M, Zamostiano R, Tsofack JEK, et al. Identification of a novel RNA virus lethal to tilapia. J Clin Microbiol. 2014;52:4137–4146.
2. Fathi M, Dickson C, Dickson M, et al. Identification of Tilapia lake virus in Egypt in Nile tilapia affected by ‘summer mortality’ syndrome. Aquaculture. 2017;473:430–432.
3. Surachetpong W, Janetanakit T, Nonthabenjawan N, Tattiyapong P, Sirikanchana K, Amonsin A. Outbreaks of Tilapia lake virus infection, Thailand, 2015–2016. Emerg Infect Dis. 2017;23:1031–1033.
4. Nicholson P, Fathi MA, Fischer A, et al. Detection of Tilapia lake virus in Egyptian fish farms experiencing high mortalities in 2015. J Fish Dis. 2017;40:1925–1928.
5. Dong HT, Siriroob S, Meemetta W, et al. Emergence of Tilapia lake virus in Thailand and an alternative semi-nested RT-PCR for detection. Aquaculture. 2017;476:111–118.
6. Tsofack JEK, Zamostiano R, Watted S, et al. Detection of Tilapia lake virus in clinical samples by culturing and nested. J Clin Microbiol. 2017;55:759–767.
7. Tattiyapong P, Sirikanchana K, 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;41:255–261.
8. Megarani DV, Al-Hussinee L, Subramaniam K, et al. Development of a TaqMan quantitative reverse transcription PCR assay to detect Tilapia lake virus. Dis Aquat Org. 2022;152:147–158.
9. World Organization for Animal Health. Manual of diagnostic tests for aquatic animals 2023. 10th ed [Internet]. Paris: Office International des épizooties; 2023. https://www.woah.org/fileadmin/Home/eng/Health_standards/aahm/current/1.1.02_VALI DATION.pdf. (VIN editor: Link was not accessible as of 4/25/24.)