Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Specific and Rapid Detection of Tilapia Lake Virus

Summary

We present an RT-LAMP assay for the detection of TiLV in tilapia fish using simple instruments over a relatively short period of time compared to conventional RT-PCR techniques. This protocol may help control the epidemic spread of TiLVD, especially in developing countries.

Abstract

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the aquaculture industry that has resulted in the mass morbidity and mortality of tilapia in many parts of the world. An effective, rapid, and accurate diagnostic assay for TiLV infection is therefore necessary to detect the initial infection and to prevent the spread of the disease in aquaculture farming. In this study, a highly sensitive and practical reverse transcription loop-mediated isothermal amplification (RT-LAMP) method is presented to detect tilapia lake virus in fish tissue. A comparison of the RT-qPCR and RT-LAMP assays of infected samples revealed positive results in 63 (100%) and 51 (80.95%) samples, respectively. Moreover, an analysis of uninfected samples showed that all 63 uninfected tissues yielded negative results for both the RT-qPCR and RT-LAMP assays. The cross-reactivity with five pathogens in tilapia was evaluated using RT-LAMP, and all the tests showed negative results. Both the liver and mucus samples obtained from infected fish showed comparable results using the RT-LAMP method, suggesting that mucus can be used in RT-LAMP as a nonlethal assay to avoid killing fish. In conclusion, the results demonstrated that the presented RT-LAMP assay provides an effective method for TiLV detection in tilapia tissue within 1 h. The method is therefore recommended as a screening tool on farms for the rapid diagnosis of TiLV.

Introduction

Tilapia lake virus disease (TiLVD) is a viral disease in tilapia (Oreochromis spp.) that reportedly causes tilapia deaths in many regions of the world, including Asia^1,2, Africa, and America. The disease was first recognized during the mass mortality of tilapia in 2009 in Israel, where the number of wild tilapia in Lake Kinneret plummeted dramatically from 257 to 8 tons per year². The disease is caused by the tilapia lake virus (TiLV), which has been assigned to the family Amnoonviridae as a new genus Tilapinevirus and a new species Tilapia tilapinevirus

Protocol

This experiment, which involved the use of animal tissue, was approved by the Institutional Animal Care and Use Committee of Kasetsart University, Bangkok, Thailand (protocol number ACKU61-VET-009).

1. Tissue collection

1. Euthanize tilapia fish using an overdose of clove oil (i.e., more than 3 mL/L). Tricaine methanesulfonate can be used as an alternative to clove oil.
2. Use sterile mayo scissors and forceps to cut open the abdomen of the postmortem tilapia and excise approximately 30–50 mg of liver tissue, or using a microscope cover glass, collect 100 µL of mucus by scraping the fish skin layer longitudin....

Results

In this study, an RT-LAMP assay was developed to detect TiLV infection in tilapia. The tested samples were collected from 14 farms located in different parts of Thailand between 2015 and 2016. The infected and uninfected fish were primarily grouped based on physical diagnoses and the appearances of symptomatic TiLVD. TiLV infection was subsequently confirmed using RT-PCR after the collection process. Agarose gel electrophoresis and the detection of a luminescent green color were selected as the evaluation methods of the LA.......

Discussion

The aquaculture industry is continuously threatened by viral infections that cause substantial economic losses^9,23,28. For instance, the emerging TiLV poses a major threat to tilapia-producing countries in many parts of the world^1,6,9. Until now, there have been no specific therapeutics available to prevent TiLVD. While the development .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tissue collection:
Clove oil	Better Pharma	N/A
Tricaine methanesulfonate	Sigma-Aldrich	E10521	An alternative option to clove oil
RNA extraction:
Acid guanidinium-phenol based reagent (TRIzol reagent)	ThermoFisher Scientific Corp.	15596026
Acid guanidinium-phenol based reagent (GENEzol reagent)	Geneaid	GZR100
Direct-zol RNA Kit:	Zymo Research	R2071
- Direct-zol RNA PreWash
- RNA Wash Buffer
- DNase/RNase-free water
- Zymo-spin IIICG columns
- Collection Tubes
RT-LAMP:
1x SD II reaction buffer	Biotechrabbit	BR1101301
Magnesium sulfate (MgSO4)	Sigma-Aldrich	7487-88-9
dNTP set	Bioline	BIO-39053
Betaine	Sigma-Aldrich	B2629
Calcein mixture	Merck	1461-15-0
Bst DNA polymerase	Biotechrabbit	BR1101301
AMV reverse transcriptase	Promega	M510A
Nuclease-free water	Invitrogen	10320995
Elite dry bath incubator, single unit	Major Science	EL-01-220
Gel electrophoresis:
Agarose	Vivantis Technologies	PC0701-500G
Tris-borate-EDTA (TBE) buffer	Sigma-Aldrich	SRE0062
Tris-acetic-EDTA (TAE) buffer:
- Tris	Vivantis Technologies	PR0612-1KG
- Acetic acid (glacial), EMSURE	Merck Millipore	1000632500
- Disodium Ethylenediaminetetraacetate dihydrate (EDTA), Vetec	Sigma-Aldrich	V800170-500G
Neogreen	NeoScience Co., Ltd.	GR107
DNA gel loading dye (6X)	ThermoFisher Scientific Corp.	R0611
DNA ladder and markers	Vivantis Technologies	PC701-100G
Mini Ready Sub-Cell GT (Horizontal electrophoresis system)	Bio-Rad	1704487
PowerPac HC power supply	Bio-Rad	1645052
Gel Doc EZ System	Bio-Rad	1708270
UV sample tray	Bio-Rad	1708271
NαBI imager	Neogene Science
cDNA synthesis:
ReverTra Ace qPCR RT Kit	Toyobo	FSQ-101
Viva cDNA Synthesis Kit	Vivantis Technologies	cDSK01	An alternative option for cDNA synthesis
NanoDrop2000 (microvolume spectrophotometer)	ThermoFisher Scientific Corp.	ND-2000
T100 Thermal Cycler	Bio-Rad	1861096
RT-qPCR:
iTaq Universal SYBR Green Supermix	Bio-Rad	1725120
Nuclease-free water, sterile water	MultiCell	809-115-CL
8-tube PCR strips, white	Bio-Rad	TLS0851
Flat PCR tube 8-cap strips, optical	Bio-Rad	TCS0803
CFX96 Touch Thermal Cycler	Bio-Rad	1855196
General equipment and materials:
Mayo scissors		N/A
Forceps		N/A
Vortex Genie 2 (vortex mixer)	Scientific Industries
Microcentrifuge LM-60	LioFuge	CM610
Corning LSE mini microcentrifuge	Corning	6765
Pipettes	Rainin	Pipete-Lite XLS
QSP filtered pipette tips	Quality Scientific Plastics	TF series
Corning Isotip filtered tips	Merck	CLS series
Nuclease-free 1.5 mL microcentrifuge tubes, NEST	Wuxi NEST Biotechnology	615601