The project is financially funded by Thailand Research Fund (TRF) grant number RDG6050078 and the Center for Advanced Studies for Agriculture and Food, Institute for Advanced Studies, Kasetsart University, Bangkok, Thailand under the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, Ministry of Education, Thailand. The research is supported in part by the Graduate Program Scholarship from the Graduate School, Kasetsart University. The authors would like to thank Dr. Kwanrawee Sirikanchana for the narrative speaking of the video and Piyawatchara Sikarin for editing the video.

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
<ol>
	<li>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.</li>
	<li>Use sterile mayo scissors and forceps to cut open the abdomen of the postmortem tilapia and excise approximately 30&#8211;50 mg of liver tissue, or using a microscope cover glass, collect 100&#160;&#181;L of mucus by scraping the fish skin layer longitudinally (from anterior to posterior) into a 1.5 mL microcentrifuge tube.</li>
	<li>Proceed to the RNA extraction step immediately or keep the collected sample at &#8722;80 &#176;C until the experiment. As intact RNA is more sensitive than DNA, use RNase-free materials and reagents during the RNA extraction.</li>
</ol>
2. RNA extraction
<ol>
	<li>To extract the RNA from the liver tissue, add 30&#8211;50 mg of the tilapia tissue to a 1.5 mL microcentrifuge tube containing 600&#8211;1,000 &#181;L 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 &#181;L 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.</li>
	<li>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.</li>
	<li>Add an equal volume of 95% ethanol to the tube and mix well.</li>
	<li>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.</li>
	<li>Add 400 &#181;L 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.</li>
	<li>Add 700 &#181;L 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.</li>
	<li>Elute the RNA sample captured in the spin column matrix with 100 &#181;L of nuclease-free water and centrifuge at 10,000 x g for 30 s at room temperature.</li>
	<li>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.</li>
	<li>Proceed to the next step immediately, or keep the sample at &#8722;80 &#176;C until use.</li>
</ol>
3. Primer design
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>To design the primers, input the following basic parameters: 
	-&#160; &#160;A GC content of 40%&#8211;60% 
	-&#160; &#160;An amplicon of &#8805;280 base pairs (bp) 
	-&#160; &#160;A similar melting temperature (Tm) among primers with a maximum difference of 5 &#176;C 
	NOTE: The primers must not complement each other
	<ol>
		<li>Avoid sequence regions prone to forming secondary structures.</li>
		<li>Select the dimer primer analysis with a minimum of &#8722;3.5 for an optimal design for the largest &#916;G, and select the ends of the primers with a maximum of &#8722;4 for an optimal design for the smaller &#916;G.</li>
	</ol>
	</li>
	<li>Click the Generate button. 
	NOTE: After the software finishes processing the input data, the primer results will be shown (Figure 1).</li>
</ol>
4. RT-LAMP assay
<ol>
	<li>Prepare an RT-LAMP master mix containing 2.5 &#181;L of 1x SD II reaction buffer, 3 &#181;L of 6&#160;mM MgSO4, 1.4 &#181;L of 1.4 mM dNTP set, 4 &#181;L of 0.8 M betaine, 1.3 &#181;L of 0.052 mM calcein mixture, 1 &#181;L of 1.6 &#181;M TiLV-F3 primer, 1 &#181;L of 1.6 &#181;M TiLV-B3 primer, 1 &#181;L of 0.2 &#181;M TiLV-BIP primer, 1 &#181;L of 0.2 &#181;M TiLV-FIP primer, 1 &#181;L of 0.3 U Bst DNA polymerase, 1 &#181;L of 0.1 U AMV reverse transcriptase, and 3.8 &#181;L of nuclease-free water per reaction. 
	NOTE: Prepare excess master mix comprising at least 10% of the total reaction volume.</li>
	<li>Dispense 22 &#181;L of the RT-LAMP master mix into a sterile 1.5 microcentrifuge tube.</li>
	<li>Add 3 &#181;L 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.</li>
	<li>Incubate the reaction at 65 &#176;C for 60 min, followed by 80 &#176;C for 10 min to terminate the reaction.</li>
	<li>After incubation, visually observe the colorimetric changes with the naked eye. A positive result will appear as a fluorescent green color.</li>
</ol>
5. Agarose gel electrophoresis
<ol>
	<li>Prepare a 1.5% w/v agarose gel by suspending&#160;0.6 g of agarose powder in 40 mL of 1x TAE buffer. Melt the mixture by heating it in a microwave for 3&#8211;5 min until the agarose is completely dissolved, and swirl to mix.</li>
	<li>Allow the agarose to cool down until the temperature reaches 65 &#176;C. Pour 40 mL of the agarose solution onto a gel tray and add a comb to the gel mold.</li>
	<li>Allow the agarose gel to set at room temperature for 20&#8211;30 min until it has completely solidified. Then remove the comb and place the gel in the gel tank.</li>
	<li>Add a running buffer to cover the surface of the gel in the gel tank.</li>
	<li>Add 10 &#181;L of the RT-LAMP sample and 2 &#181;L of 6x gel loading buffer to each well. Add 5 &#181;L of a 1 kb DNA ladder to the reference lane.</li>
	<li>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.</li>
	<li>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&#160;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.</li>
	<li>Expose the gel to UV light using a gel documentation system where bands appear, and take a picture of the gel.</li>
</ol>
6. Complementary DNA (cDNA) synthesis
<ol>
	<li>Prepare a cDNA synthesis master mix containing 2 &#181;L of 5x RT buffer, 0.5 &#181;L of primer mix, 0.5 &#181;L of RT enzyme, and 2 &#181;L of nuclease-free water per reaction. 
	NOTE: Prepare excess master mix comprising 1x the reaction due to potential loss during pipetting.</li>
	<li>Dispense 5 &#181;L of the master mix into a sterile 1.5 mL microcentrifuge tube.</li>
	<li>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.</li>
	<li>Incubate the reactions at 42 &#176;C for 60 min, followed by 98 &#176;C for 5 min in a PCR machine. Store the cDNA at &#8722;20 &#176;C until use.</li>
</ol>
7. RT-qPCR
<ol>
	<li>Prepare a TiLV qPCR master mix containing 0.3 &#181;L of 10 &#181;M reverse primer, 0.3 &#181;L of 10 &#181;M&#160;forward primer, 5 &#181;L of 2x SYBR Green DNA polymerase, and 0.4 &#181;L of nuclease-free water per reaction. Use the following primers and controls: 
	Forward primer (TiLV-112F):&#160; 5&#39;-CTGAGCTAAAGAGGCAATATGGATT-3&#39; 
	Reverse primer (TiLV-112R):&#160; 5&#39;-CGTGCGTACTCGTTCAGTATAAGTTCT-3&#39;</li>
	<li>Dispense 6 &#181;L of the TiLV qPCR master mix into each well of the qPCR strip compatible with the real-time thermal cycler used.</li>
	<li>Add 4 &#181;L of the cDNA template, negative control (nuclease-free water), positive control (10 pg/&#181;L), and pTiLV (plasmid)10 or serially diluted TiLV plasmid to the well, and mix each sample by flicking. Conduct each sample in triplicate.</li>
	<li>Place the qPCR reactions into the programmed real-time thermal cycler. Set the qPCR program to perform the incubation at 95 &#176;C for 3 min, followed by 40 cycles of 95 &#176;C for 10 s and 60 &#176;C for 30 s before the melting curve step in which the temperature needs to increase from 65 &#176;C to 95 &#176;C at 0.5 &#176;C/5 s increments.</li>
	<li>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.</li>
</ol>

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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tilapia+lake+virus:+a+threat+to+the+global+tilapia+industry.">Tilapia lake virus: a threat to the global tilapia industry.</a> Reviews in Aquaculture. 11 (3), 725-739 (2019).</li><li>Yin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+simple+and+rapid+reverse+transcription-loopmediated+isothermal+amplification+(RT-LAMP)+assay+for+sensitive+detection+of+tilapia+lake+virus.">Development of a simple and rapid reverse transcription-loopmediated isothermal amplification (RT-LAMP) assay for sensitive detection of tilapia lake virus.</a> Journal of Fish Diseases. 42 (6), 817-824 (2019).</li><li>Savan, R., Kono, T., Itami, T., Sakai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification:+an+emerging+technology+for+detection+of+fish+and+shellfish+pathogens.">Loop-mediated isothermal amplification: an emerging technology for detection of fish and shellfish pathogens.</a> Journal of Fish Diseases. 28 (10), 573-581 (2005).</li><li>Phusantisampan, T., Tattiyapong, P., Mutrakulcharoen, P., Sriariyanun, M., Surachetpong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+of+tilapia+lake+virus+using+a+one-step+reverse+transcription+loop-mediated+isothermal+amplification+assay.">Rapid detection of tilapia lake virus using a one-step reverse transcription loop-mediated isothermal amplification assay.</a> Aquaculture. 507, 35-39 (2019).</li><li>Liamnimitr, P., Thammatorn, W., U-thoomporn, S., Tattiyapong, P., Surachetpong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-lethal+sampling+for+Tilapia+Lake+Virus+detection+by+RT-qPCR+and+cell+culture.">Non-lethal sampling for Tilapia Lake Virus detection by RT-qPCR and cell culture.</a> Aquaculture. 486, 75-80 (2018).</li><li>Yin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+simple+and+rapid+reverse+transcription-loopmediated+isothermal+amplification+(RT-LAMP)+assay+for+sensitive+detection+of+tilapia+lake+virus.">Development of a simple and rapid reverse transcription-loopmediated isothermal amplification (RT-LAMP) assay for sensitive detection of tilapia lake virus.</a> Journal of Fish Diseases. 42 (6), 817-824 (2019).</li><li>Khan, R. S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+of+infectious+bursal+disease+by+loop-mediated+isothermal+amplification+for+field+analysis.">Rapid detection of infectious bursal disease by loop-mediated isothermal amplification for field analysis.</a> Iranian Journal of Veterinary Research. 19 (2), 101-107 (2018).</li><li>Nagamine, K., Hase, T., Notomi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+reaction+by+loop-mediated+isothermal+amplification+using+loop+primers.">Accelerated reaction by loop-mediated isothermal amplification using loop primers.</a> Molecular and Cellular Probes. 16 (3), 223-229 (2002).</li><li>Debode, F., Marien, A., Janssen, E., Bragard, C., Berben, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+amplicon+length+on+real-time+PCR+results.">The influence of amplicon length on real-time PCR results.</a> Biotechnology, Agronomy, Society and Environment. 21 (1), 3-11 (2017).</li></ol>

The authors have nothing to disclose.

The aquaculture industry is continuously threatened by viral infections that cause substantial economic losses9,23,28. For instance, the emerging TiLV poses a major threat to tilapia-producing countries in many parts of the world1,6,9. Until now, there have been no specific therapeutics available to prevent TiLVD. While the development ...

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 Asia1,2, 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 proteins1,2,4. 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 TiLV2,5. Currently, this virus continues to spread globally, possibly through the movement of infected live fish6,7, 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 diagnosis10,11. For instance, a SYBR green-based reverse transcription quantitative PCR (RT-qPCR) method with the sensitivity to detect as few as two copies/&#181;L 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 application14,15. The technique employs the principle of a strand displacement reaction, while the amplification reaction runs under isothermal conditions without a sophisticated and expensive thermal cycler14,15. 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 precipitate16,17,18. For these reasons, this technique has been used for the on-site detection of different fish pathogens17,18,19,20,21,22,23,24,25,26,27. 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.

<table><tbody><tr><td>&lt;strong&gt;Tissue collection:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Clove oil</td><td>Better Pharma</td><td>N/A</td><td></td></tr><tr><td>Tricaine methanesulfonate</td><td>Sigma-Aldrich</td><td>E10521</td><td>An alternative option to clove oil</td></tr><tr><td>RNA extraction:</td><td></td><td></td><td></td></tr><tr><td>Acid guanidinium-phenol based reagent (TRIzol reagent)</td><td>ThermoFisher Scientific Corp.</td><td>15596026</td><td></td></tr><tr><td>Acid guanidinium-phenol based reagent (GENEzol reagent)</td><td>Geneaid</td><td>GZR100</td><td></td></tr><tr><td>Direct-zol RNA Kit:</td><td>Zymo Research</td><td>R2071</td><td></td></tr><tr><td>- Direct-zol RNA PreWash</td><td></td><td></td><td></td></tr><tr><td>- RNA Wash Buffer</td><td></td><td></td><td></td></tr><tr><td>- DNase/RNase-free water</td><td></td><td></td><td></td></tr><tr><td>- Zymo-spin IIICG columns</td><td></td><td></td><td></td></tr><tr><td>- Collection Tubes</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;RT-LAMP:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1x SD II reaction buffer</td><td>Biotechrabbit</td><td>BR1101301</td><td></td></tr><tr><td>Magnesium sulfate (MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>7487-88-9</td><td></td></tr><tr><td>dNTP set</td><td>Bioline</td><td>BIO-39053</td><td></td></tr><tr><td>Betaine</td><td>Sigma-Aldrich</td><td>B2629</td><td></td></tr><tr><td>Calcein mixture</td><td>Merck</td><td>1461-15-0</td><td></td></tr><tr><td>Bst DNA polymerase</td><td>Biotechrabbit</td><td>BR1101301</td><td></td></tr><tr><td>AMV reverse transcriptase</td><td>Promega</td><td>M510A</td><td></td></tr><tr><td>Nuclease-free water</td><td>Invitrogen</td><td>10320995</td><td></td></tr><tr><td>Elite dry bath incubator, single unit</td><td>Major Science</td><td>EL-01-220</td><td></td></tr><tr><td>&lt;strong&gt;Gel electrophoresis:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Agarose</td><td>Vivantis Technologies</td><td>PC0701-500G</td><td></td></tr><tr><td>Tris-borate-EDTA (TBE) buffer</td><td>Sigma-Aldrich</td><td>SRE0062</td><td></td></tr><tr><td>Tris-acetic-EDTA (TAE) buffer:</td><td></td><td></td><td></td></tr><tr><td>- Tris</td><td>Vivantis Technologies</td><td>PR0612-1KG</td><td></td></tr><tr><td>- Acetic acid (glacial), EMSURE</td><td>Merck Millipore</td><td>1000632500</td><td></td></tr><tr><td>- Disodium Ethylenediaminetetraacetate dihydrate (EDTA), Vetec</td><td>Sigma-Aldrich</td><td>V800170-500G</td><td></td></tr><tr><td>Neogreen</td><td>NeoScience Co., Ltd.</td><td>GR107</td><td></td></tr><tr><td>DNA gel loading dye (6X)</td><td>ThermoFisher Scientific Corp.</td><td>R0611</td><td></td></tr><tr><td>DNA ladder and markers</td><td>Vivantis Technologies</td><td>PC701-100G</td><td></td></tr><tr><td>Mini Ready Sub-Cell GT (Horizontal electrophoresis system)</td><td>Bio-Rad</td><td>1704487</td><td></td></tr><tr><td>PowerPac HC power supply</td><td>Bio-Rad</td><td>1645052</td><td></td></tr><tr><td>Gel Doc EZ System</td><td>Bio-Rad</td><td>1708270</td><td></td></tr><tr><td>UV sample tray</td><td>Bio-Rad</td><td>1708271</td><td></td></tr><tr><td>N&amp;alpha;BI imager</td><td>Neogene Science</td><td></td><td></td></tr><tr><td>&lt;strong&gt;cDNA synthesis:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ReverTra Ace qPCR RT Kit</td><td>Toyobo</td><td>FSQ-101</td><td></td></tr><tr><td>Viva cDNA Synthesis Kit</td><td>Vivantis Technologies</td><td>cDSK01</td><td>An alternative option for cDNA synthesis</td></tr><tr><td>NanoDrop2000 (microvolume spectrophotometer)</td><td>ThermoFisher Scientific Corp.</td><td>ND-2000</td><td></td></tr><tr><td>T100 Thermal Cycler</td><td>Bio-Rad</td><td>1861096</td><td></td></tr><tr><td>&lt;strong&gt;RT-qPCR:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>iTaq Universal SYBR Green Supermix</td><td>Bio-Rad</td><td>1725120</td><td></td></tr><tr><td>Nuclease-free water, sterile water</td><td>MultiCell</td><td>809-115-CL</td><td></td></tr><tr><td>8-tube PCR strips, white</td><td>Bio-Rad</td><td>TLS0851</td><td></td></tr><tr><td>Flat PCR tube 8-cap strips, optical</td><td>Bio-Rad</td><td>TCS0803</td><td></td></tr><tr><td>CFX96 Touch Thermal Cycler</td><td>Bio-Rad</td><td>1855196</td><td></td></tr><tr><td>&lt;strong&gt;General equipment and materials:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Mayo scissors</td><td></td><td>N/A</td><td></td></tr><tr><td>Forceps</td><td></td><td>N/A</td><td></td></tr><tr><td>Vortex Genie 2 (vortex mixer)</td><td>Scientific Industries</td><td></td><td></td></tr><tr><td>Microcentrifuge LM-60</td><td>LioFuge</td><td>CM610</td><td></td></tr><tr><td>Corning LSE mini microcentrifuge</td><td>Corning</td><td>6765</td><td></td></tr><tr><td>Pipettes</td><td>Rainin</td><td>Pipete-Lite XLS</td><td></td></tr><tr><td>QSP filtered pipette tips</td><td>Quality Scientific Plastics</td><td>TF series</td><td></td></tr><tr><td>Corning Isotip filtered tips</td><td>Merck</td><td>CLS series</td><td></td></tr><tr><td>Nuclease-free 1.5 mL microcentrifuge tubes, NEST</td><td>Wuxi NEST Biotechnology</td><td>615601</td><td></td></tr></tbody></table>

reverse transcription loop-mediated isothermal amplification (rt-lamp) assay for the specific and rapid detection of tilapia lake virus

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.

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 &#64257;sh were primarily grouped based on physical diagnoses and the appearances of symptomatic TiLVD. TiLV infection was subsequently con&#64257;rmed using RT-PCR after the collection process. Agarose gel electrophoresis and the detection of a luminescent green color were selected as the evaluation met...

Watch this Scientific Journal Video about Reverse Transcription Loop-Mediated Isothermal Amplification RT-LAMP Assay for the Specific and Rapid Detection of Tilapia Lake Virus at JoVE.com

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

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.

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

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the ...

reverse-transcription-loop-mediated-isothermal-amplification-rt-lamp

Research

JoVE Journal

Immunology and Infection

12.7K Views.  King Mongkut's University of Technology North Bangkok (KMUTNB). Tilapia lake virus is een opkomende virus en besmettelijke ziekteverwekkers, die hoge sterfte veroorzaken in tilapia en zijn hybride soorten. Aangezien tilapia een belangrijke eiwitbron is voor veel ontwikkelingslanden, heeft de nieuwe virusinfectie een grote impact op de tilapia-aquacultuur en bedreigt ook de voedselzekerheid in de wereld. De ontwikkeling van een snelle, nauwkeurige en gevoelige methode om het virus in visweefsel op te sporen, zal de ziektebestrijding helpen en de impact van...

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

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different downstream patient-to-patient transmission potentials, and require very different patient treatments. Thus, recent Zika outbreaks make it urgent to develop tools that rapidly discriminate these viruses in patients and trapped mosquitoes, to select the correct patient treatment, and to understand and manage their epidemiology in real time.
Unfortunately, current diagnostic tests, including those receiving 2016 emergency use authorizations and fast-track status, detect viral RNA by reverse transcription polymerase chain reaction (RT-PCR), which requires instrumentation, trained users, and considerable sample preparation. Thus, they must be sent to &#34;approved&#34; reference laboratories, requiring time. Indeed, in August 2016, the Center for Disease Control (CDC) was asking pregnant women who had been bitten by a mosquito and developed a Zika-indicating rash to wait an unacceptable 2 to 4 weeks before learning whether they were infected. We very much need tests that can be done on site, with few resources, and by trained but not necessarily licensed personnel.
This video demonstrates an assay that meets these specifications, working with urine or serum (for patients) or crushed mosquito carcasses (for environmental surveillance), all without much sample preparation. Mosquito carcasses are captured on paper carrying quaternary ammonium groups (Q-paper) followed by ammonia treatment to manage biohazards. These are then directly, without RNA isolation, put into assay tubes containing freeze-dried reagents that need no chain of refrigeration. A modified form of reverse transcription loop-mediated isothermal amplification with target-specific fluorescently tagged displaceable probes produces readout, in 30 min, as a three-color fluorescence signal. This is visualized with a handheld, battery-powered device with an orange filter. Forward contamination is prevented with sealed tubes, and the use of thermolabile uracil DNA glycosylase (UDG) in the presence of dUTP in the amplification mixture.

Current multiplexed diagnostics to detect Zika, chikungunya, and dengue viruses require complex sample preparation and expensive instrumentation, and are difficult to use in low resource environments. We show a diagnostic that uses isothermal amplification with target-specific strand displaceable probes to detect and differentiate these viruses with high sensitivity and specificity.

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different ...

Biochemistry

Evaluation of a Universal Nested Reverse Transcription Polymerase Chain Reaction for the Detection of Lyssaviruses

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription polymerase chain reaction (nested RT-PCR) was developed to detect the conserved region of the nucleoprotein (N) gene of lyssaviruses. The method applies reverse transcription (RT) using viral RNA as template and oligo (dT)15 and random hexamers as primers to synthesize the viral complementary DNA (cDNA). Then, the viral cDNA is used as a template to amplify an 845 bp N gene fragment in first-round PCR using outer primers, followed by second-round nested PCR to amplify the final 371 bp fragment using inner primers. This method can detect different genetic clades of rabies viruses (RABV). The validation, using 9,624 brain specimens from eight domestic animal species in 10 years of clinical rabies diagnoses and surveillance in China, showed that the method has 100% sensitivity and 99.97% specificity in comparison with the direct fluorescent antibody test (FAT), the gold standard method recommended by the World Health Organization (WHO) and the World Organization for Animal Health (OIE). In addition, the method could also specifically amplify the targeted N gene fragment of 15 other approved and two novel lyssavirus species in the 10th Report of the International Committee on Taxonomy of Viruses (ICTV) as evaluated by a mimic detection of synthesized N gene plasmids of all lyssaviruses. The method provides a convenient alternative to FAT for rabies diagnosis and has been approved as a National Standard (GB/T36789-2018) of China.

A pan-lyssavirus nested reverse transcription polymerase chain reaction has been developed to detect specifically all known lyssaviruses. Validation using rabies brain samples of different animal species showed that this method has a sensitivity and specificity equivalent to the gold standard fluorescent antibody test and could be used for routine rabies diagnosis.

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription ...

Genetics

Detection of Tilapia Lake Virus Using Conventional RT-PCR and SYBR Green RT-qPCR

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be used as part of surveillance programs, biosecurity measures and in TiLV basic research laboratories. The gold standard of virus diagnostics typically involves virus isolation followed by complementary techniques such as reverse-transcription polymerase chain reaction (RT-PCR) for further verification. This can be cumbersome, time-consuming and typically requires tissue samples heavily infected with virus. The use of RT-quantitative (q)PCR in the detection of viruses is advantageous because of its quantitative nature, high sensitivity, specificity, scalability and its rapid time to result. Here, the entire method of PCR based approaches for TiLV detection is described, from tilapia organ sectioning, total ribonucleic acid (RNA) extraction using a guanidium thiocyanate-phenol-chloroform solution, RNA quantification, followed by a two-step PCR protocol entailing, complementary deoxyribonucleic acid (cDNA) synthesis and detection of TiLV by either conventional PCR or quantitative identification via qPCR using SYBR green I dye. Conventional PCR requires post-PCR steps and will simply inform about the presence of the virus. The latter approach will allow for absolute quantification of TiLV down to as little as 2 copies and thus is exceptionally useful for TiLV diagnosis in sub-clinical cases. A detailed description of the two PCR approaches, representative results from two laboratories and a thorough discussion of the critical parameters of both have been included to ensure that researchers and diagnosticians find their most suitable and applicable method of TiLV detection.

This protocol diagnoses Tilapia Lake Virus (TiLV) in tilapia tissues using RT-PCR methodologies. The entire method is described from tissue dissection to total RNA extraction, followed by cDNA synthesis and detection of TiLV by either conventional PCR or quantitative PCR using dsDNA binding a fluorescent binding dye.

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be ...

Environment

Field Postmortem Rabies Rapid Immunochromatographic Diagnostic Test for Resource-Limited Settings with Further Molecular Applications

Functional rabies surveillance systems are crucial to provide reliable data and increase the political commitment necessary for disease control. To date, animals suspected as rabies-positive must be submitted to a postmortem confirmation using classical or molecular laboratory methods. However, most endemic areas are in low- and middle-income countries where animal rabies diagnosis is restricted to central veterinary laboratories. Poor availability of surveillance infrastructure leads to serious disease underreporting from remote areas. Several diagnostic protocols requiring low technical expertise have been recently developed, providing opportunity to establish rabies diagnosis in decentralized laboratories. We present here a complete protocol for field postmortem diagnosis of animal rabies using a rapid immunochromatographic diagnostic test (RIDT), from brain biopsy sampling to the final interpretation. We complete the protocol by describing a further use of the device for molecular analysis and viral genotyping. RIDT easily detects rabies virus and other lyssaviruses in brain samples. The principle of such tests is simple: brain material is applied on a test strip where gold conjugated antibodies bind specifically to rabies antigens. The antigen-antibody complexes bind further to fixed antibodies on the test line, resulting in a clearly visible purple line. The virus is inactivated in the test strip, but viral RNA can be subsequently extracted. This allows the test strip, rather than the infectious brain sample, to be safely and easily sent to an equipped laboratory for confirmation and molecular typing. Based on a modification of the manufacturer&#39;s protocol, we found increased test sensitivity, reaching 98% compared to the gold standard reference method, the direct immunofluorescence antibody test. The advantages of the test are numerous: rapid, easy-to-use, low cost and no requirement for laboratory infrastructure, such as microscopy or cold-chain compliance. RIDTs represent a useful alternative for areas where reference diagnostic methods are not available.

We present a complete protocol for postmortem diagnosis of animal rabies under field conditions using a rapid immunochromatographic diagnostic test (RIDT), from brain biopsy sampling to final interpretation. We also describe further applications using the device for molecular analysis and viral genotyping.

Functional rabies surveillance systems are crucial to provide reliable data and increase the political commitment necessary for disease control. To date, ...

Human Respiratory Bacterial Infection Diagnosis Using Isothermal Amplification

Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

Author Spotlight: Advancing Rapid Detection of Respiratory Pathogens Using Microfluidic Chip 

Various bacterial pathogens can cause respiratory tract infections and lead to serious health issues if not detected accurately and treated promptly. Rapid and accurate detection of these pathogens via&#160;loop-mediated isothermal amplification provides effective management and control of respiratory tract infections in clinical settings.

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will ...

Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation

Loop-mediated isothermal amplification (LAMP) has emerged as a powerful nucleic acid amplification test for the rapid detection of numerous bacterial, fungal, parasitic, and viral agents. Salmonella is a bacterial pathogen of worldwide food safety concern, including food for animals. Presented here is a multi-laboratory-validated Salmonella LAMP protocol that can be used to rapidly screen animal food for the presence of Salmonella contamination and can also be used to confirm presumptive Salmonella isolates recovered from all food categories. The LAMP assay specifically targets the Salmonella invasion gene (invA) and is rapid, sensitive, and highly specific. Template DNAs are prepared from enrichment broths of animal food or pure cultures of presumptive Salmonella isolates. The LAMP reagent mixture is prepared by combining an isothermal master mix, primers, DNA template, and water. The LAMP assay runs at a constant temperature of 65 &#176;C for 30 min. Positive results are monitored via real-time fluorescence and can be detected as early as 5 min. The LAMP assay exhibits high tolerance to inhibitors in animal food or culture medium, serving as a rapid, reliable, robust, cost-effective, and user-friendly method for screening and confirming Salmonella. The LAMP method has recently been incorporated into the U.S. Food and Drug Administration&#8217;s Bacteriological Analytical Manual (BAM) Chapter 5.

Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation

Loop-mediated isothermal amplification (LAMP) is an isothermal nucleic acid amplification test (iNAAT) that has attracted broad interest in the pathogen detection field. Here, we present a multi-laboratory-validated Salmonella LAMP protocol as a rapid, reliable, and robust method for screening Salmonella in animal food and confirming presumptive Salmonella from culture isolation.

Loop-mediated isothermal amplification (LAMP) has emerged as a powerful nucleic acid amplification test for the rapid detection of numerous bacterial, ...

Biology

Using Colorimetric and RT-LAMP Analysis to Detect SARS-CoV-2 

Detecting SARS-CoV-2 Virus by Reverse Transcription-Loop-Mediated Isothermal Amplification

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has dramatically impacted human health. It continues to be a threat to modern society because many people die as a result of infection. The disease is diagnosed using serologic and molecular tests, such as the gold standard real-time polymerase chain reaction (RT-PCR). The last has several disadvantages because it requires specialized infrastructure, costly equipment, and trained personnel. Here, we present a protocol outlining the steps required to detect the SARS-CoV-2 virus using reverse transcription-loop-mediated isothermal amplification (RT-LAMP) in human samples. The protocol includes instructions for designing primers in silico, preparing reagents, amplification, and visualization. Once standardized, this method can be easily implemented and adapted to any laboratory or point-of-care within 60 min at a low cost and using inexpensive equipment. It is adaptable to detecting different pathogens. Thus, it can potentially be used in the field and in health centers to carry out timely epidemiological surveillance.

Author Spotlight: Advancing Pathogen Diagnostics with Standardized LAMP

Here we provide a complete protocol to standardize and implement the method for detecting the SARS-CoV-2 virus in human samples by reverse transcription loop-mediated isothermal amplification (RT-LAMP). This method, done within 60 min, could be adapted to any laboratory or point-of-care at a low cost and using inexpensive equipment.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has dramatically impacted human health. It continues to be a threat to modern ...

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

Samenvatting

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