Tilapia lake virus is an emerging virus and contagious pathogens, that cause high mortality in tilapia and its hybrid species. As tilapia is a main protein source for many developing countries, the new virus infection has a great impact to tilapia aquaculture, and also threatens the world food security. The development of a fast, accurate, and sensitive method to detect the virus in fish tissue, will help the disease control, and reduce the impact of the virus.
In this video, we will show the reverse transcription loop-mediated isothermal amplification, or RT-LAMP assay, for rapid detection of tilapia lake virus in fish samples. Part two, RNA extraction. Firstly, add 3250 milligrams of liver tissue into a sterile 1.5-milliliters microcentrifuge tube, containing 600 to 1000 microliters acid guanidinium-phenol base reagent, and mince until homogeneous.
Centrifuge at 10, 000 RCF for 30 seconds, and transfer the supernatant into a new tube. Add an equal volume of 95%ethanol to the tube, and mix thoroughly. Transfer the solution into a spin column, placed in a collection tube, then centrifuge at 10, 000 RCF for 30 seconds.
Discard the flow-through, and transfer the column into a new collection tube. Add 400 microliters of RNA PreWash into the column, and centrifuge at 10, 000 RCF for 30 seconds. Discard the flow-through, and repeat this step.
Add 700-microliter RNA Wash Buffer into the column, and centrifuge at 10, 000 RCF for two minutes. Afterwards, transfer the column into a sterile 1.5-milliliter microcentrifuge tube. Finally, elute RNA into the column matrix with a 100-microliter, nuclease-free water, and centrifuge at 10, 000 RCF for 30 seconds.
Part three, primer design. The PrimerExplorer Version 4 is the most common bioinformatics tool for designing efficient LAMP, or RT-LAMP primers, in which good manuals for the software are available on their website. Here, four specific primers, composed of two internal and two external primers, were designed to recognize six distinct regions in the segment 3 of TiLV, originated from Thailand.
After entering the website, where the link is shown in the video, click on PrimerExplorer V4.Choose the file containing the sequence of the segment 3 of TiLV, in FASTA format, retrieved from GenBank-accessed number, KX631923, or tilapia lake virus TV1 segment 3, and then click Primer Design button. Once clicking on Generate button, the software will process, and the results will show up. After optimizing the sequences for LAMP amplification, in total, one pair of external primers, and two pairs of internal primers, were selected for this study, to detect the TiLV infection.
This set of the primers are shown here. Part four, demonstration of RT-LAMP. The master mix for RT-LAMP specific for tilapia lake virus, comprises of the following chemicals.
Prepare a master mix using the chemicals shown in the table. Dispense 22 microliters of master mix into a sterile 1.5-milliliter microcentrifuge tube. Then, add three microliters of RNA template into the reaction tube.
For the negative control, use distilled water instead of RNA materials, and mix well. Incubate the reactions at 65 degrees Celsius for 60 minutes, followed by 80 degrees Celsius for 10 minutes. After the incubation for 30 minutes, the calorimetric changes are visually observed by naked eyes.
Where appropriate, we can confirm RT-LAMP assay results, by performing gel electrophoresis on 1.5%agarose gel, running with a 100-Volt constant, for 40 minutes, whereby one-kilobase-pair molecular-size DNA Ladder is loaded along as a marker to determine fragment size. Part five, representative results and interpretation. After the incubation for 30 minutes, the grain fluorescence reaction suggesting the positive amplification of TiLV in fish tissues, were obviously observed in infected samples under UV light at a wavelength of 365 nanometers, while those of negative control had a clear and slightly colorless solution.
Correspondingly, the amplification of RT-LAMP products was confirmed on the agarose gel electrophoresis, showing various sizes of the multiple amplifications of inverted DNA repeats, forming stem-loop structures under UV lamp in TiLV-infected samples, whereas no bands were displayed in the wells of the negative control, where the results were in accordance with those of the RT-LAMP assay. So, to reduce the spread of tilapia lake virus, an immediate screening of these infected fish, using an effectively diagnostic method, is one of the important keys. So, the newly developed RT-LAMP assay should offer a rapid and sensitive diagnostic tool to be applied in the process of biosecurity measure.
The assay could be used either in the laboratory, or on-site facilities. We hope that the method will be implement to screen infected-fish population that could lead to the reduction of the spread of tilapia lake virus.
