A new CRISPR-based diagnostic test to detect tilapia lake virus

Dr. Subramaniam
Dr. Kuttichantran Subramaniam

By DeLene Beeland

Tilapia is a popular firm, flaky-white meat for people who enjoy eating freshwater fish. It is native to lakes in Africa and is grown in aquaculture farms throughout the world. Tilapia were first found in US lakes in the mid-1970s. According to the Florida Fish and Wildlife Conservation Commission, tilapia are now widespread in Florida lakes south of Lake Okeechobee.

Tilapia can be infected by Tilapia tilapinevirus, commonly called tilapia lake virus, which damages tissues in the fish’s brain, spleen, liver and kidney. Tilapia lake virus is usually fatal and can devastate 90% of infected tilapia fish in aquaculture or wild environments. Given the $14 billion global value of farmed tilapia and the role of wild tilapia in balancing algae and mosquito larvae in wild waterways, the tilapia lake virus poses a looming threat.

“The U.S. has no surveillance network in place to monitor for tilapia lake virus,” said Kuttichantran Subramaniam, Ph.D., a research associate professor in the UF College of Veterinary Medicine. “And part of the reason for this is that we don’t have a validated diagnostic that can be done in the field [pondside] or laboratories with limited resources.”

Subramaniam, along with Ph.D. student Dorothea Megarani, plan to develop a rapid diagnostic to help fish professionals detect tilapia lake virus in the field or in laboratories with limited resources. Subramaniam directs the Wildlife and Aquatic Veterinary Disease Laboratory within the Aquatic Pathobiology Laboratory and is the principal investigator on the diagnostic development project. This project is supported by the intramural research program of the U.S. Department of Agriculture National Institute of Food and Agriculture.

Diagram of diagnostic assay that uses CRISPR-based technology
A diagram of the diagnostic assay process that uses cutting-edge CRISPR-based technology. (Graphic created by Dorothea Megarani)

The diagnostic assay uses cutting-edge CRISPR-based technology in a single tube, or “one-pot,” that eliminates the need for a central lab and specialized equipment. The one-pot test should have results ready in an hour, Subramaniam said. Work performed for this project in the Aquatic Pathobiology Laboratory helped validate the CRISPR technology for use in aquatic diseases, Subramaniam said.

Internal tissue from a fish is needed to test for the virus, and Megarani and Subramaniam are pioneering a new sampling method. Standard testing is lethal and relies on killing the fish to detect the virus, he said. But he and Megarani are testing the feasibility of using a bit of gill tissue instead. This would flip the sampling to a humane, nonlethal method.

Tissue from the gill will then be used to extract RNA, Subramaniam explained. Then it will be mixed with a reagent mixture consisting of Cas (CRISPR-associated) enzyme, primers and fluorescent tags. The reaction mixture will then go into a chamber of the testing device where a CRISPR-based reaction takes place at 62°C for an hour. The Cas enzyme and single-guide RNA (sgRNA) will probe for an amplified target region of the tilapia lake virus genome. If they find their target, the Cas enzyme will get activated, and fluorescent tags will be cleaved and light up. The fluorescence will then be read by an optic visualizer, producing a positive result. If no amplified target region of the tilapia lake virus genome is found, the device will show a negative result.

“There are other CRISPR applications developed for tilapia lake virus elsewhere in the world, but we do not yet have this in the U.S.” Subramaniam said. Subramaniam and Megarani also acknowledge Dr. Piyush Jain and his graduate student, Lilia Yang, at the UF Herbert Wertheim College of Engineering, Department of Chemical Engineering, for their technical assistance in this project. The Aquatic Pathology Laboratory, operated by the Emerging Pathogens Institute at UF, offers investigators lab space to research infectious diseases affecting fresh- and saltwater organisms.

Editor’s note: This story was originally written by DeLene Beeland to highlight pathogens research at the Emerging Pathogens Institute.


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