Name: reverse transcription-loop-mediated isothermal amplification (rt-lamp) assay for zika virus and housekeeping genes in urine, serum, and mosquito samples
Uploaded: 2018-09-14T15:00:00
Description: Watch this Scientific Journal Video about Reverse Transcription-Loop-mediated Isothermal Amplification RT-LAMP Assay for Zika Virus and Housekeeping Genes in Urine, Serum, and Mosquito Samples at JoVE

This work was supported by the Maureen and Ronald Hirsch family philanthropic contribution and Field Neurosciences Institute, St. Mary&#39;s of Michigan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We also thank Dr. Bernadette Zwaans and Elijah Ward for their critical review of the manuscript.

All methods described here have been approved by the Institutional Review Board (IRB) of Beaumont Health. All experiments were performed in accordance with relevant guidelines and regulations.
CAUTION: All potentially infectious materials should be handled according to Biosafety Level 2 standards including the use of personal protective equipment. Any procedures which may produce aerosol should be performed in a biosafety cabinet. Additionally, work with live mosquitoes should be performed in ...

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The ZIKV RT-LAMP assay described in this paper works using both human and mosquito samples6. The limit of detection was approximately 1 genome equivalent6, which should be sufficient since the typical viral load of a symptomatic ZIKV infected patient is 103 to&#160;106 PFU/mL7. Additionally, this method can detect ZIKV in samples without first isolating RNA and without virus amplification in cell culture. This signifi...

In 2015, Zika virus (ZIKV) gained prominent global attention as an infectious disease of concern because infection during pregnancy was linked to miscarriage, stillbirth, severe neurological birth defects including microcephaly, as well as other congenital birth defects1. In rare cases, ZIKV has been associated with Guillain-Barr&#233; syndrome. ZIKV is primarily transmitted by Aedes mosquitoes; however, it can also be spread through sexual contact1. Given that the infection with ZIKV is asymptomatic in most people or presents with mild flu-like symptoms that overlap with the symptoms of infection of other arborviruses1, there was a need for improved methods for rapid and cost-effective detection of ZIKV to screen both people as well as local mosquito populations.
Quantitative reverse transcription PCR (qRT-PCR) is a reliable assay for ZIKV detection; however, this technique requires expensive specialized equipment, trained personnel, and RNA isolation from the sample of interest. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a one-step nucleic acid amplification method based on PCR technology that only requires one incubation temperature due to the use of a thermophilic DNA polymerase with strand displacement properties. This circumvents the need for a thermocycler and decreases the length of time needed to complete the assay. Additional advantages of RT-LAMP include its high specificity and sensitivity, robustness at a range of pH levels and temperatures, and resistance to many PCR inhibitors2. It has a relatively low cost and reagents are stable at room temperature. Given these characteristics, RT-LAMP can be deployed in a laboratory or in the field. As such, LAMP reactions have been developed to detect a range of pathogens and other types of infection3,4,5. The goal of the RT-LAMP protocol described in this paper is to detect ZIKV without RNA isolation in human serum and urine samples as well as in a single infected mosquito within 30 min through RT-LAMP. This method can be used to replace qRT-PCR as it is a sensitive, rapid diagnostic tool that works quickly and in settings outside of the laboratory.

<table border="0" cellpadding="0" cellspacing="0" style="width:901px;" width="902">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Bst 2.0 WarmStart DNA polymerase</td>
			<td style="width:223px;">New England Biolabs</td>
			<td style="width:223px;">M0537S</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isothermal Amplification Buffer&#160;</td>
			<td>New England Biolabs</td>
			<td style="width:223px;">B0537S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">WarmStart RTx Reverse Transcriptase</td>
			<td>New England Biolabs</td>
			<td style="width:223px;">M0380S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antarctic Thermolabile UDG</td>
			<td>New England Biolabs</td>
			<td style="width:223px;">M0372S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4</td>
			<td>New England Biolabs</td>
			<td style="width:223px;">B1003S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100mM dATP Solution</td>
			<td>New England Biolabs</td>
			<td style="width:223px;">N0440S</td>
			<td>Deoxynucleotide Set N0446S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100mM dCTP Solution</td>
			<td>New England Biolabs</td>
			<td style="width:223px;">N0441S</td>
			<td>Deoxynucleotide Set N0446S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100mM dTTP Solution</td>
			<td>New England Biolabs</td>
			<td style="width:223px;">N0443S</td>
			<td>Deoxynucleotide Set N0446S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100mM dGTP Solution</td>
			<td>New England Biolabs</td>
			<td style="width:223px;">N0442S</td>
			<td>Deoxynucleotide Set N0446S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100mM dUTP Solution</td>
			<td style="width:223px;">Thermo Fisher Scientific</td>
			<td style="width:223px;">R0133</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SYBR Green I Nucleic Acid Gel Stain</td>
			<td style="width:223px;">Invitrogen</td>
			<td style="width:223px;">S7563</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nancy-520</td>
			<td style="width:223px;">Sigma Aldrich</td>
			<td style="width:223px;">01494</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Low DNA Mass Ladder</td>
			<td style="width:223px;">Invitrogen</td>
			<td style="width:223px;">10-068-013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Zika Postive Control</td>
			<td style="width:223px;">Robert Koch Institute, Germany</td>
			<td style="width:223px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Agarose</td>
			<td style="width:223px;">Thermo Fisher Scientific</td>
			<td style="width:223px;">BP1600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">BlueJuice Gel Loading Buffer</td>
			<td style="width:223px;">Invitrogen</td>
			<td style="width:223px;">10816015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Primers</td>
			<td style="width:223px;">Integrated DNA Technologies</td>
			<td style="width:223px;">Custom Oligo</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Nuclease-Free Water</td>
			<td style="width:223px;">Ambion</td>
			<td style="width:223px;">AM9938</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Urine Preservative</td>
			<td style="width:223px;">Norgen Biotek</td>
			<td style="width:223px;">18126</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">ZIKV PCR Standard</td>
			<td>Robert Koch Institute</td>
			<td style="width:223px;">N/A</td>
			<td>Vero E6 cell supernatants</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">DENV2 New Guinea C Control</td>
			<td style="width:223px;">Connecticut Agricultural Experiment Station</td>
			<td style="width:223px;">N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

reverse transcription-loop-mediated isothermal amplification (rt-lamp) assay for zika virus and housekeeping genes in urine, serum, and mosquito samples

Infection with Zika virus (ZIKV) can be asymptomatic in adults, however, infection during pregnancy can lead to miscarriage and severe neurological birth defects. The goal of this protocol is to quickly detect ZIKV in both human and mosquito samples. The current gold standard for ZIKV detection is quantitative reverse transcription PCR (qRT-PCR); reverse transcription loop-mediated isothermal amplification (RT-LAMP) may allow for a more efficient and low-cost testing without the need for expensive equipment. In this study, RT-LAMP is used for ZIKV detection in various biological samples within 30 min, without first isolating the RNA from the sample. This technique is demonstrated using ZIKV infected patient urine and serum, and infected mosquito samples. 18S ribosomal ribonucleic acid and actin are used as controls in human and mosquito samples, respectively.

RT-LAMP reactions can be analyzed using three different methods. First, with the addition of a fluorescent nucleic acid dye, positive reactions will be yellow/green in color where negative reactions will appear orange in color to the naked eye. Second, the addition of the fluorescent nucleic acid dye to RT-LAMP reaction results in a fluorescent signal when the samples are excited by UV light. Negative reactions will not have a detectable fluorescent signal over any background fluorescence...

Watch this Scientific Journal Video about Reverse Transcription-Loop-mediated Isothermal Amplification RT-LAMP Assay for Zika Virus and Housekeeping Genes in Urine, Serum, and Mosquito Samples at JoVE

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

This protocol provides an efficient and low-cost method to detect Zika virus or control targets in human urine and serum samples or in mosquitoes by reverse transcription loop-mediated isothermal amplification (RT-LAMP). This method does not require RNA isolation and can be done within 30 min.

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

Infection with Zika virus (ZIKV) can be asymptomatic in adults, however, infection during pregnancy can lead to miscarriage and severe neurological birth ...

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.

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