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 tilapinevirus³. Genetic characterization of TiLV showed that the virus is a novel enveloped, negative-sense, single-stranded RNA virus that has 10 segments encoding 10 proteins^1,2,4. Various species of tilapia in the genus Sarotherodon, Oreochromis, and Tilapine and other warm water fish (e.g., giant gourami (Osphronemus goramy)) have been shown to be susceptible to TiLV^2,5. Currently, this virus continues to spread globally, possibly through the movement of infected live fish^6,7, while the risk of viral transmission via frozen tilapia or its product is limited⁸. Substantial mortality due to TiLV infection has the potential to have a significantly detrimental economic impact on the tilapia industry. For example, the economic impact of summer mortality syndrome in Egypt associated with TiLV infection was calculated to be US$100 million⁹. Accordingly, it is important to develop a rapid and proper diagnostic method to facilitate the control of this disease in fish farms.

Until now, the diagnosis of TiLVD has been based on molecular assays, viral isolation, and histopathology. Different PCR protocols and primers have been developed for TiLV diagnosis^10,11. For instance, a SYBR green-based reverse transcription quantitative PCR (RT-qPCR) method with the sensitivity to detect as few as two copies/µL of the virus has been developed and validated for TiLV detection¹⁰. Other PCR methods for TiLV detection include TaqMan quantitative PCR¹¹, RT-PCR², nested RT-PCR¹², and semi-nested RT-PCR¹³. However, these methods require sophisticated laboratory equipment and relatively extended periods of time to yield results due to the complexity of the reactions, which makes them unsuitable for field application.

The loop-mediated isothermal amplification (LAMP) assay is a rapid, simple, and practical for-field application^14,15. The technique employs the principle of a strand displacement reaction, while the amplification reaction runs under isothermal conditions without a sophisticated and expensive thermal cycler^14,15. Consequently, amplified LAMP products or RT-LAMP products are analyzed in ladder-like bands using agarose gel electrophoresis with a fluorescent stain for either the safe visualization of DNA or RNA¹⁴ or observation with the naked eye for the presence of turbidity or a white precipitate^16,17,18. For these reasons, this technique has been used for the on-site detection of different fish pathogens^{17,18,19,20,21,22,23,24,25,26,27}. The purpose of this study was to establish a rapid, sensitive, and accurate RT-LAMP assay for TiLV detection. The RT-LAMP assay offers screening for TiLV in fish samples within 30 min. The technique may be applied for the diagnosis and surveillance of TiLVD.

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 longitudinally (from anterior to posterior) into a 1.5 mL microcentrifuge tube.
3. Proceed to the RNA extraction step immediately or keep the collected sample at −80 °C until the experiment. As intact RNA is more sensitive than DNA, use RNase-free materials and reagents during the RNA extraction.

2. RNA extraction

1. To extract the RNA from the liver tissue, add 30–50 mg of the tilapia tissue to a 1.5 mL microcentrifuge tube containing 600–1,000 µL of guanidinium-acid-phenol extraction reagent (Table of Materials), and pulverize the sample until homogenous using a hand-held tissue homogenizer. The tissue samples require approximately 10% of the guanidinium-acid-phenol extraction reagent. For the mucus samples, use only 300 µL of the guanidinium-acid-phenol extraction reagent (3:1).
   CAUTION: The guanidinium-acid-phenol extraction reagent is toxic; hence, handling must be undertaken with care. Protective equipment, such as safety glasses, a laboratory gown, and safety gloves, must be worn.
2. Centrifuge the homogenized sample at 10,000 x g for 30 s at room temperature, and transfer the supernatant into a new sterile 1.5 mL microcentrifuge tube.
3. Add an equal volume of 95% ethanol to the tube and mix well.
4. Transfer the solution into a spin column (Table of Materials) placed in a collection tube and centrifuge at 10,000 x g for 30 s at room temperature. Discard the flow-through, and transfer the spin column to a new collection tube.
5. Add 400 µL of RNA Pre-Wash reagent (Table of Materials) to the column and centrifuge at 10,000 x g for 30 s at room temperature before discarding the flow-through. Repeat this step one more time.
   NOTE: To prepare the RNA Pre-Wash, add 10 mL of 95% ethanol to 40 mL of RNA Pre-Wash concentrate.
6. Add 700 µL of RNA Wash Buffer (Table of Materials) to the column and centrifuge at 10,000 x g for 2 min at room temperature. Transfer the column to a sterile 1.5 mL microcentrifuge tube.
   NOTE: To prepare RNA Wash Buffer, add 52 mL of 95% ethanol to 12 mL of RNA Wash Buffer concentrate.
7. Elute the RNA sample captured in the spin column matrix with 100 µL of nuclease-free water and centrifuge at 10,000 x g for 30 s at room temperature.
8. Measure the concentration of the extracted RNA using a microvolume spectrophotometer and dilute the RNA to a desired concentration using nuclease-free water.
   NOTE: The qualified absorbance value of OD260/OD280 ranges from 1.6 to 1.9, indicating the acceptable RNA purity for the RT-LAMP assay.
9. Proceed to the next step immediately, or keep the sample at −80 °C until use.

3. Primer design

1. Use Primer Explorer version 4 to design the specific primers for the RT-LAMP. Go to the website, https://primerexplorer.jp/e/, and click on the PrimerExplorer V4 button.
2. Select the file containing the sequences of segment 3 of TiLV in FASTA format and then click the Primer Design button.
   NOTE: Retrieve the sequences in FASTA format from GenBank accession number KX631923, which represents tilapia lake virus TV1 segment 3¹.
3. To design the primers, input the following basic parameters:
   -   A GC content of 40%–60%
   -   An amplicon of ≥280 base pairs (bp)
   -   A similar melting temperature (Tm) among primers with a maximum difference of 5 °C
   NOTE: The primers must not complement each other
   1. Avoid sequence regions prone to forming secondary structures.
   2. Select the dimer primer analysis with a minimum of −3.5 for an optimal design for the largest ΔG, and select the ends of the primers with a maximum of −4 for an optimal design for the smaller ΔG.
4. Click the Generate button.
   NOTE: After the software finishes processing the input data, the primer results will be shown (Figure 1).

4. RT-LAMP assay

1. Prepare an RT-LAMP master mix containing 2.5 µL of 1x SD II reaction buffer, 3 µL of 6 mM MgSO₄, 1.4 µL of 1.4 mM dNTP set, 4 µL of 0.8 M betaine, 1.3 µL of 0.052 mM calcein mixture, 1 µL of 1.6 µM TiLV-F3 primer, 1 µL of 1.6 µM TiLV-B3 primer, 1 µL of 0.2 µM TiLV-BIP primer, 1 µL of 0.2 µM TiLV-FIP primer, 1 µL of 0.3 U Bst DNA polymerase, 1 µL of 0.1 U AMV reverse transcriptase, and 3.8 µL of nuclease-free water per reaction.
   NOTE: Prepare excess master mix comprising at least 10% of the total reaction volume.
2. Dispense 22 µL of the RT-LAMP master mix into a sterile 1.5 microcentrifuge tube.
3. Add 3 µL of the extracted RNA to the reaction tube, and mix the sample by vortexing. For the negative control, use distilled water instead of RNA materials.
4. Incubate the reaction at 65 °C for 60 min, followed by 80 °C for 10 min to terminate the reaction.
5. After incubation, visually observe the colorimetric changes with the naked eye. A positive result will appear as a fluorescent green color.

5. Agarose gel electrophoresis

1. Prepare a 1.5% w/v agarose gel by suspending 0.6 g of agarose powder in 40 mL of 1x TAE buffer. Melt the mixture by heating it in a microwave for 3–5 min until the agarose is completely dissolved, and swirl to mix.
2. Allow the agarose to cool down until the temperature reaches 65 °C. Pour 40 mL of the agarose solution onto a gel tray and add a comb to the gel mold.
3. Allow the agarose gel to set at room temperature for 20–30 min until it has completely solidified. Then remove the comb and place the gel in the gel tank.
4. Add a running buffer to cover the surface of the gel in the gel tank.
5. Add 10 µL of the RT-LAMP sample and 2 µL of 6x gel loading buffer to each well. Add 5 µL of a 1 kb DNA ladder to the reference lane.
6. Plug in the lid attached to the cathode and the anode connected to a power supply. Turn on the power supply, set it to a constant 100 V, and run for 40 min.
7. After completing the gel separation, remove the gel from the gel tray. Then stain the drained gel using ethidium bromide (EtBr) at a concentration of 10 mg/mL for 7 min and restain it in distilled water for 5 min.
   CAUTION: EtBr is toxic and considered a carcinogen; therefore, be careful when using this agent.
8. Expose the gel to UV light using a gel documentation system where bands appear, and take a picture of the gel.

6. Complementary DNA (cDNA) synthesis

1. Prepare a cDNA synthesis master mix containing 2 µL of 5x RT buffer, 0.5 µL of primer mix, 0.5 µL of RT enzyme, and 2 µL of nuclease-free water per reaction.
   NOTE: Prepare excess master mix comprising 1x the reaction due to potential loss during pipetting.
2. Dispense 5 µL of the master mix into a sterile 1.5 mL microcentrifuge tube.
3. Add 100 ng of the diluted extracted RNA (obtained from step 2.8) to the reaction tube, mix and spin down to move all the mixture to the bottom of the vessel.
4. Incubate the reactions at 42 °C for 60 min, followed by 98 °C for 5 min in a PCR machine. Store the cDNA at −20 °C until use.

7. RT-qPCR

1. Prepare a TiLV qPCR master mix containing 0.3 µL of 10 µM reverse primer, 0.3 µL of 10 µM forward primer, 5 µL of 2x SYBR Green DNA polymerase, and 0.4 µL of nuclease-free water per reaction. Use the following primers and controls:
   Forward primer (TiLV-112F):  5'-CTGAGCTAAAGAGGCAATATGGATT-3'
   Reverse primer (TiLV-112R):  5'-CGTGCGTACTCGTTCAGTATAAGTTCT-3'
2. Dispense 6 µL of the TiLV qPCR master mix into each well of the qPCR strip compatible with the real-time thermal cycler used.
3. Add 4 µL of the cDNA template, negative control (nuclease-free water), positive control (10 pg/µL), and pTiLV (plasmid)¹⁰ or serially diluted TiLV plasmid to the well, and mix each sample by flicking. Conduct each sample in triplicate.
4. Place the qPCR reactions into the programmed real-time thermal cycler. Set the qPCR program to perform the incubation at 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s before the melting curve step in which the temperature needs to increase from 65 °C to 95 °C at 0.5 °C/5 s increments.
5. Conduct the data analysis by evaluating the amplification and melting curves, and then compute the number of TiLV copies using the standard curve obtained from the data using the serially diluted plasmids.

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 met...

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

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