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

Summary

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.

Abstract

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 "approved" 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.

Introduction

Mosquito-borne virus infections, including dengue, chikungunya, and Zika viruses are on the rise and demand immediate management strategies. Dengue and chikungunya viruses are already endemic in many of the tropical regions where Zika is now spreading in the Western Hemisphere¹. Zika virus, like dengue, is a member of the Flaviviridae family and is native to Africa with one Asian and two African genetic lineages². Even though the identification of the Zika virus dates back to 1947, Zika infection in humans remained sporadic for a half century before emerging in the Pacific and the Americas. The first reported ou....

Protocol

NOTE: Mosquitoes were the only animals directly used in this study. The procedures to manage chickens, whose blood was used to feed the infected mosquitoes, were approved as IACUC Protocol #201507682 by the University of Florida Institutional Animal Care and Use Committee.Virus propagation and mosquito infection studies were performed at the BSL-3 facility of the Florida Medical Entomology Laboratory in Vero Beach, FL. RT-LAMP experiments were performed in the BSL-2 laboratory shared by FfAME and Firebird Biomolecular Sciences LLC in Alachua, FL.

1. Design of Primers and Strand Displacing Probes

1. Extract viral sequences for Dengu....

Results

Initially, the performances of each RT-LAMP primer (Table 1) with its corresponding viral RNA substrate as well as negative controls were assessed by gel electrophoresis. RT-LAMP primers were designed to target NS5 region (RNA-dependent RNA polymerase) for Zika and Dengue 1, and nsP2 region (non-structural protein P2) for Chikungunya. Templates were total RNA extracted from viral stocks cultured in African green monkey kidney (Vero) cells. In one case, total nucleic acid .......

Discussion

Mosquito-borne viruses including Zika, chikungunya, and dengue threaten the public health and recent Zika outbreaks highlight the need for low-cost point-of-care detection alternatives for patient diagnostics, as well as for mosquito surveillance. Isothermal amplification methods were developed as affordable alternatives to PCR-based systems. Particularly, RT-LAMP-based platforms have been applied to detect a wide range of pathogens. However, the use of isothermal platforms has been mainly limited to single target detect.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
SafeBlue Illuminator/ Electrophoresis System, MBE-150-PLUS	Major Science	MBE-150	Gel electrophoresis
G:BOX F3	Syngene	G:BOX F3	Gel imaging
LightCycler 480 Instrument II, 96-well	Roche Applied Science	05 015 278 001	Real-time PCR
Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-10 membrane	Millipore Sigma	UFC501096	ultrafiltraton membrane for dialysis
Eppendorf 5417C Centrifuge	Marshall Scientific	EP-5417C	centrifuge
Myblock Mini Drybath	Benchmark Scientific	BSH200	drybath
FreeZone Plus 6 Liter Cascade Console Freeze Dry System	Labconco	7934020	lyophilizer
all priers and probes	IDT	custom	RT-LAMP primers and probes
dNTP set	Bioline	BIO-39049
Deoxyuridine Triphosphate (dUTP)	Promega	U1191
Bst 2.0 WarmStart DNA Polymerase	New England Biolabs	M0538L	enzyme
WarmStart RTx Reverse Transcriptase	New England Biolabs	M0380L	enzyme
RNase Inhibitor, Murine	New England Biolabs	M0314L	enzyme
Antarctic Thermolabile UDG	New England Biolabs	M0372L	enzyme
50bp DNA Step Ladder	Promega	G4521	marker
LightCycler 480 Multiwell Plate 96, white	Roche Applied Science	4729692001	real-time RT-LAMP analysis