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| 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) |
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
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
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