Quick QUASR Test Lights up disease-carrying mosquitoes

Red means a disease is present to Sandia National Laboratories’ researchers Cameron Ball and Robert Meagher as they test their QUASR, for quenching of unincorporated amplification signal reporters, technique to detect the presence of malaria and viruses like West Nile. Simple enough for field labs and handheld devices, QUASR’s positive signal is 10 times brighter than a negative signal. (Photo by Dino Vournas)

Sandia National Laboratories: News Releases : Lighting up disease-carrying mosquitoes
Robert Meagher, a chemical engineer at Sandia National Laboratories, has developed a simple technique for simultaneously detecting RNA from West Nile and chikungunya virus in samples from mosquitoes. He is now working to add the ability to screen for Zika virus. “Our ultimate goal is to develop an autonomous device to passively monitor for mosquito-borne diseases,” Meagher explained. “But first you need an assay that is more robust than the gold standard in a laboratory and that has a very low false-positive rate.”

The technique is described in a paper published online in Analytical Chemistry,
Quenching of unincorporated amplification signal reporters (QUASR) in RT-LAMP enables bright, single-step, closed-tube, and multiplexed detection of RNA viruses

Cameron S. Ball, Yooli K. Light, Chung-Yan Koh, Sarah S. Wheeler, Lark L. Coffey, and Robert J. Meagher

Anal. Chem., Just Accepted Manuscript

DOI: 10.1021/acs.analchem.5b04054

Publication Date (Web): March 16, 2016

Copyright © 2016 American Chemical Society

Abstract

Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) has frequently been proposed as an enabling technology for simplified diagnostic tests for RNA viruses. However, common detection techniques used for LAMP and RT-LAMP have drawbacks, including: poor discrimination capability, inability to multiplex targets, high rates of false positives, and (in some cases) the requirement of opening reaction tubes post-amplification. Here, we present a simple technique that allows closed-tube, target-specific detection, based on inclusion of a dye-labeled primer that is incorporated into a target-specific amplicon if the target is present. A short, complementary quencher hybridizes to unincorporated primer upon cooling down at the end of the reaction, thereby quenching fluorescence of any unincorporated primer. Our technique, which we term QUASR (for Quenching of Unincorporated Amplification Signal Reporters, read “quasar”), does not significantly reduce the amplification efficiency or sensitivity of RT-LAMP. Equipped with a simple LED excitation source and a colored plastic gel filter, the naked eye or a camera can easily discriminate between positive and negative QUASR reactions, which produce a difference in signal of approximately 10:1 without background subtraction. We demonstrate that QUASR detection is compatible with complex sample matrices such as human blood, using a novel LAMP primer set for bacteriophage MS2 (a model RNA virus particle). Furthermore, we demonstrate single-tube duplex detection of West Nile virus (WNV) and chikungunya virus (CHIKV) RNA.

Publications Authored by Robert J Meagher