Handyfuge-LAMP: low-cost and electricity-free centrifugation for isothermal SARS-CoV-2 detection in saliva

Abstract

<jats:p>Point of care diagnostics for COVID-19 detection are vital to assess infection quickly and at the source so appropriate measures can be taken. The loop-mediated isothermal amplification (LAMP) assay has proven to be a reliable and simple protocol that can detect small amounts of viral RNA in patient samples (&lt;10 genomes per μL) (Nagamine, Hase, and Notomi 2002) Recently, Rabe and Cepko at Harvard published a sensitive and simple protocol for COVID-19 RNA detection in saliva using an optimized LAMP assay (Rabe and Cepko, 2020). This LAMP protocol has the benefits of being simple, requiring no specialized equipment; rapid, requiring less than an hour from sample collection to readout; and cheap, costing around $1 per reaction using commercial reagents. The pH based colorimetric readout also leaves little ambiguity and is intuitive. However, a shortfall in many nucleic acid-based methods for detection in saliva samples has been the variability in output due to the presence of inhibitory substances in saliva. Centrifugation to separate the reaction inhibitors from inactivated sample was shown to be an effective way to ensure reliable LAMP amplification. However, a centrifuge capable of safely achieving the necessary speeds of 2000 RPM for several minutes often costs hundreds of dollars and requires a power supply. We present here an open hardware solution- Handyfuge - that can be assembled with readily available components for the cost of &lt;5 dollars a unit and could be used together with the LAMP assay for point of care detection of COVID-19 RNA from saliva. The device is then validated using the LAMP protocol from Rabe and Cepko. With the use of insulated coolers for reagent supply chain and delivery, the assay presented can be completed without the need for electricity or any laboratory scale infrastructure.</jats:p>

A materials-science perspective on tackling COVID-19

“…The investigation of new viral strains requires virus isolation and purification, size, structure and component determination, and analysis of infection, replication and spread. To prepare for new viral outbreaks and to accelerate and coordinate our response, portable and inexpensive devices for viral isolation and purification from samples such as blood or saliva are needed; for example, a hand-powered ultra-low-cost centrifuge 194,195 . To investigate virus structure and composition, electron microscopy, cryo-electron microscopy, confocal microscopy and sequencing technologies need to be applied.…”

Isothermal SARS-CoV-2 Diagnostics: Tools for Enabling Distributed Pandemic Testing as a Means of Supporting Safe Reopenings

“…Handyfuge-LAMP was recently developed as an optimized LAMP assay that is especially suited for POC diagnostics due to its low-cost and electricity-free centrifugation methods. 35 The group established a hardware system called “Handyfuge” that is cheap (<$5 per unit) and easily assembled for achieving the high speed centrifugation needed for separation of inhibitory components from inactivated saliva samples. The reagents also include a chaotropic salt binding solution and a silica binding solution that enables viral RNA capture.…”

Mobilefuge: A low-cost, portable, open source, 3D-printed centrifuge that can be used for purification of saliva samples for SARS-CoV2 detection

“…To demonstrate that Mobilefuge can be useful for the purification process during the LAMP assays, we separate the silica particles from a suspension consisting of water and "glassmilk" using the Mobilefuge. The "glass milk" suspension along with Chaotropic salts acts as a binding mix in the low-cost LAMP assays to capture the viral RNA from the cleared saliva solution containing the viral particles [11], [12]. This "glass milk" solution suspension was the predecessor to the commercial silica-based column purification kits.…”

“…Out of these four techniques, LAMP has high potential to be scaled up for point-of-care, near-patient-care and point-of-entry applications as it is highly reliable, sensitive, faster, cost-effective and doesn't require complex equipment [5], [6], [7]. Due to these benefits, a number of LAMP assays have been fast tracked by FDA for first at-home tests for COVID-19 [4,8] [9], [10] and have the potential to bring down the cost of each test to less than a dollar [11][12][13][14].…”