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

Summary

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.

Abstract

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.

Introduction

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-Barré 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 arbor....

Protocol

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 the appropriate Arthropod Containment Level 1-3 facility. Given the association of ZIKV infection with congenital abnormalities, women who are pregnan....

Results

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.......

Discussion

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 10⁶ PFU/mL⁷. Additionally, this method can detect ZIKV in samples without first isolating RNA and without virus amplification in cell culture. This signifi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bst 2.0 WarmStart DNA polymerase	New England Biolabs	M0537S
Isothermal Amplification Buffer	New England Biolabs	B0537S
WarmStart RTx Reverse Transcriptase	New England Biolabs	M0380S
Antarctic Thermolabile UDG	New England Biolabs	M0372S
MgSO4	New England Biolabs	B1003S
100 mM dATP Solution	New England Biolabs	N0440S	Deoxynucleotide Set N0446S
100 mM dCTP Solution	New England Biolabs	N0441S	Deoxynucleotide Set N0446S
100 mM dTTP Solution	New England Biolabs	N0443S	Deoxynucleotide Set N0446S
100 mM dGTP Solution	New England Biolabs	N0442S	Deoxynucleotide Set N0446S
100 mM dUTP Solution	Thermo Fisher Scientific	R0133
SYBR Green I Nucleic Acid Gel Stain	Invitrogen	S7563
Nancy-520	Sigma Aldrich	01494
Low DNA Mass Ladder	Invitrogen	10-068-013
Zika Postive Control	Robert Koch Institute, Germany	N/A
Agarose	Thermo Fisher Scientific	BP1600
BlueJuice Gel Loading Buffer	Invitrogen	10816015
Primers	Integrated DNA Technologies	Custom Oligo
Nuclease-Free Water	Ambion	AM9938
Urine Preservative	Norgen Biotek	18126
ZIKV PCR Standard	Robert Koch Institute	N/A	Vero E6 cell supernatants
DENV2 New Guinea C Control	Connecticut Agricultural Experiment Station	N/A