Abstract
Current multiplexed diagnostics for Zika, dengue, and chikungunya viruses are situated outside the intersection of affordability, high performance, and suitability for use at the point-of-care in resource-limited settings. Consequently, insufficient diagnostic capabilities are a key limitation facing current Zika outbreak management strategies. Here we demonstrate highly sensitive and specific detection of Zika, chikungunya, and dengue viruses by coupling reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with our recently developed quenching of unincorporated amplification signal reporters (QUASR) technique. We conduct reactions in a simple, inexpensive and portable “LAMP box” supplemented with a consumer class smartphone. The entire assembly can be powered by a 5 V USB source such as a USB power bank or solar panel. Our smartphone employs a novel algorithm utilizing chromaticity to analyze fluorescence signals, which improves the discrimination of positive/negative signals by 5-fold when compared to detection with traditional RGB intensity sensors or the naked eye. The ability to detect ZIKV directly from crude human sample matrices (blood, urine, and saliva) demonstrates our device’s utility for widespread clinical deployment. Together, these advances enable our system to host the key components necessary to expand the use of nucleic acid amplification-based detection assays towards point-of-care settings where they are needed most.
Highlights
IntroductionPeripheral components including expensive and power-hungry thermal cyclers (for rapidly heating and cooling samples) with built-in fluorimeter units (for performing real-time fluorescence detection)
Peripheral components including expensive and power-hungry thermal cyclers with built-in fluorimeter units
We aimed to develop a rapid and reliable quenching of unincorporated amplification signal reporters (QUASR) Reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay for Zika virus (ZIKV), while relying on previously reported RT-LAMP assays for chikungunya virus (CHIKV) and DENV17,19,22
Summary
Peripheral components including expensive and power-hungry thermal cyclers (for rapidly heating and cooling samples) with built-in fluorimeter units (for performing real-time fluorescence detection). Post-reaction analysis that requires opening the tube is undesirable, both for reasons of complexity and the risk of amplicon contamination, whereas non-specific indicators such as turbidity or HNB may be prone to detection of false-positive amplification events (a common, if under-reported problem with isothermal amplifications such as LAMP). We address these issues by implementing RT-LAMP for Zika, dengue, and chikungunya in a closed-tube, multiplexable, target-specific format. The app acquires images from the phone camera and processes them through a novel color and luminance-based detection algorithm capable of detecting multiplexed QUASR assay signals with greater accuracy than conventional image intensity analysis
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