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

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.

Ultra-rapid detection of SARS-CoV-2 in public workspace environments

“…As an alternative to this saliva sampling method, saliva was absorbed on to Q-paper, which was placed after a brief time (5 min) at room temperature (to simulate how processing might occur in a workplace lobby) and directly added into the RT-LAMP mixture. Analogous to what is seen with mosquito carcasses[16], 100 copies of viral RNA could be detected by simply spotting SARS-CoV-2 RNA onto saliva coated Q-paper from which it was directly amplified by RT-LAMP. Visualization of the positive signals was obtained similarly using blue LED and orange filter combination ( Fig 4D ).…”

A Mosquito Workshop and Community Intervention: A Pilot Education Campaign to Identify Risk Factors Associated with Container Mosquitoes in San Pedro Sula, Honduras

“…Therefore, we only collected data from one community and trained one cohort of individuals. Future work could expand training to multiple groups in other areas of Honduras and collecting similar data from diverse communities, as well as the inclusion of a rapid and inexpensive multiplexed test for dengue, chikungunya, and Zika viruses in clinical and mosquito samples [54,55]. While the results of our study are specific to this community in San Pedro Sula, the methods can be generalized to target other communities and create highly precise information based on the community that is being targeted or the group that is being trained.…”

Ultra-rapid detection of SARS-CoV-2 in public workspace environments

“…RT-LAMP primers were designed as described previously [ 17 , 18 ]. The DP-RT-LAMP primers and strand displaceable probes were purchased from Integrated DNA Technologies (IDT, Coralville, IA).…”

“…Using various nucleic acid based architectural innovations, we have developed a variety of kits that meet these specs with untrained and unlicensed users outside of traditional laboratory settings, including in the field [ 17 – 19 ]. These have focused on detecting environmental pathogens, including detecting arboviral RNA in infected insects and ticks [ 20 ] since these require no regulatory supervision.…”