Reverse Transcription-Loop-mediated Isothermal Amplification (RT-LAMP) Assay for Zika Virus and Housekeeping Genes in Urine, Serum, and Mosquito Samples

This method can quickly determine if samples contain Zika virus. This is useful for pregnant women, couples trying to conceive, or surveillance of local mosquito populations that are at risk for Zika infection. The main advantage of this technique is that it’s fast, easy, does not require expensive equipment, and can be done without an RNA isolation step.

The implications of this technique extend towards diagnosis of Zika infection, because infection during pregnancy is linked to miscarriage, stillbirth, and other severe congenital, neurological birth defects. Though this method can provide insight into Zika, it can also be applied to other infectious diseases such as dengue, chikungunya, or West Nile virus by using virus-specific primers. To begin, reconstitute each RT-LAMP primer in molecular grade water to a final concentration of 100 micromolar.

Briefly vortex the primer solution to ensure the solution is homogeneous. Centrifuge for about five seconds at maximum speed to collect the primer solution. Then prepare a 10x RT-LAMP primer mix as outlined in table two of the tech’s protocol.

Vortex briefly to ensure homogeneity. And centrifuge briefly at maximum speed to avoid any loss. Store the primers at minus 20 degrees Celsius, between uses, avoiding any freeze-thaw cycles.

For human urine samples, use either fresh urine, frozen urine or urine in preservative. Thaw any frozen samples on ice before use. Human serum samples should be either fresh or frozen, while ZIKV infected cell lines should contain either conditioned media or cell lysates.

Finally, for Aedes aegypti mosquitoes, use whole carcasses, either fresh or frozen. Thaw any frozen mosquito carcasses on ice. Place an individual mosquito in 100 microliters of PBS.

Using a P10 pipette tip crush the mosquito 10 times, and homogenize it to create a crude mosquito lysate. Briefly centrifuge the crude lysate to pellet any debris and use the supernatant for downstream RT-LAMP analysis. To begin, prepare an RT-LAMP master mix reaction on ice for each primer set to be used, as outline by table three in the tech’s protocol.

Vortex each sample to ensure it is well mixed. Then briefly spin them down to prevent volume loss. For each reaction, pipette 23.0 microliters of the RT-LAMP master mix into a 200 microliter PCR tube.

Then, add two microliters of sample to bring the total volume to 25.0 microliters. Use molecular grade water for the negative control, and make sure to include a positive control. Optionally, include a specificity control reaction of a related arbovirus such as dengue virus, as outlined in tech’s protocol.

Using a heat block, water bath, or a thermocycler, heat the samples at 61 degrees Celsius for 30 minutes. After this, increase the heat to 80 degree Celsius for 10 minutes to heat deactivate the polymerase. Perform the RT-LAMP analysis in a separate enclosed space.

Dilute the fluorescent nucleic acid dye one to ten in TAE buffer. Add two micorliters of the fluorescent nucleic acid dye solution to 12 micorliters of the RT-LAMP reaction. Assess the RT-LAMP reactions visually by looking for the presence of a color change.

Optionally use a camera to take pictures of RT-LAMP reactions on a white background. Next, place the samples under a 302 nanometer UV light to confirm the presence of RT-LAMP products by fluorescence. Use a camera to take a picture of the result.

To begin performing electrophoresis pour 2%agarose gel in 1x TAE buffer with a nucleic acid stain for visualization. Add five microliters of a DNA ladder in the first lane to compare the molecular weights. For each RT-LAMP reaction, mix 13 microliters of RT-LAMP reaction mixture with two microliters of DNA loading dye.

Load this 15 microliter mixture. Run the gel at 90 volts for 90 minutes or until the bands are separated. An image with 302 nanometer UV light.

When finished, dispose of RT-LAMP reactions in double sealed bags. In this study, three different methods are utilized to analyze RT-LAMP reactions. When using fluorescent nucleic acid dye positive reactions become yellow-green in color, while negative reactions appear orange.

This dye also results in a fluorescent signal when samples are excited by UV light. Lastly, RT-LAMP reactions can be run out on an agarose gel. Positive RT-LAMP reactions will have a banding pattern, whereas negative reactions will have no DNA bands.

These methods are used to demonstrate the specificity of the ZIKV RT-LAMP reaction. Only the ZIKV molecular control has a positive RT-LAMP reaction. Furthermore, while ZIKV is detected in both the urine and serum of a patient with known ZIKV infection, it is not seen in the asymptomatic control patient without ZIKV infection.

The samples are then tested for human 18S rRNA. Only the samples taken from patients are seen to be positive for 18S rRNA. Mosquito samples can be similarly tested for ZIKV or the RT-LAMP quality control AEDAE.

Only the positive molecular control for ZIKV and the mosquito infected with ZIKV are seen to be positive for ZIKV. However all the mosquitoes are positive for AEDAE by RT-LAMP. Once mastered, this technique can be done in under 30 minutes if it is performed properly.

It’s important to remember that RT-LAMP reactions are prone to false positive reactions. Prevention steps include using a thermolabile uracil DNA glycosylase in reactions, using a lateral workflow, performing analysis in a separate space, and reducing time post amplification tubes are open. Following this procedure other methods like QPCR can be performed in order to answer additional questions, like how many copies the virus, or other targets, are present in the sample.

After watching this video, you should have a a good understanding of how to use RT-LAMP to detect Zika virus and target controls in human urine and serum, as well as mosquitos. Don’t forget that working with Zika virus and nucleic acid stain can be extremely hazardous, and precautions such as wearing gloves should always be taken while performing this procedure. Given the association of Zika virus infection with congenital abnormalities, women who are pregnant, trying to conceive, or partners of these women, should significantly minimize their laboratory exposure to Zika virus.