mgLAMP Reagent Preparation
One key strategy in addressing the COVID-19 pandemic is the detection and quantification of SARS-CoV-2 in wastewater, as a means to both monitor the levels of infections and to act as an early warning system among a community population: a process known as wastewater-based epidemiology (WBE). While centralized WBE can be performed using standard polymerase chain reaction (PCR) based techniques, like reverse transcription quantitative PCR (RT-qPCR), these systems can be unfeasible for point-of-sampling applications in remote or developing systems, as they require expensive thermocycling equipment, trained laboratory personnel to prepare chemical reagent solutions and perform the tests, and require a constant chemical supply chain and reliable freezer storage for reagents. To address the technological gap for point-of-sampling or point-of-care SARS-CoV-2 detection and quantification, alternative techniques have been developed, including loop-mediated isothermal amplification (LAMP), as well as other LAMP-based derivatives, including those developed in the Hoffmann Lab at Caltech: in-gel LAMP (gLAMP) and membrane-based gLAMP (mgLAMP) [see figure below].
Our novel mgLAMP technology functions as a point-of-sampling technology, better suited for rapid (1 h) applications in small, remote, decentralized and/or developing communities; a common demographic which can’t currently benefit from SARS-CoV-2 WBE efforts. The mgLAMP system is an inexpensive and user-friendly molecular diagnostics technique that requires no specialized lab equipment, whereby sensitive and absolute quantification of SARS-CoV-2 in wastewater can be obtained within 1 hour, from time of sampling to automatic interpretation of post-amplification results. The mgLAMP system 1) integrates the enrichment of target SARS-CoV-2 particles through a simple sample filtration system, which retains the target particles (e.g., virus or bacteria) on a polycarbonate track etch (PCTE) membrane, while filtering out co-occurring water constituents including LAMP inhibitors, and 2) includes the in-assay viral lysis through including 0.5% Triton X-100 in the LAMP reaction system. End-point quantification is achieved through the automatic counting of amplicon dots after incubation, via smartphone camera and developed amplicon counting software, delivering an absolute pathogen concentration.
While LAMP-based techniques can eliminate the prohibitive costs associated with PCR techniques, one key aspect of these systems that may present itself as a bottleneck to their wide adoption for remote and developing community applications is with respect to both the storage and preparation of process reagents. Typically, SARS-CoV-2 LAMP detection and quantification, for example, can use up to seven reagents that require freeze-storage in between usage, with one reagent mix containing five primers that also need to be prepared into a single solution (i.e., for our SARS-CoV-2 mgLAMP process, a total of 11 components are needed to facilitate the amplification of SARS-CoV-2). If these LAMP amplification reagents are not stored in the freezer, their shelf lives have been observed to significantly decrease after a few days. A segment of the intended market for the point-of-sampling and point-of-care systems is in small, remote, and developing communities, where reliable freeze-storage is often not available. Moreover, the frequent and reliable chemical supply chain to provide LAMP reagents to these vulnerable communities is problematic. The second complication associated with the LAMP reagents is their preparation procedures, which involve the precise and repetitive pipetting and mixing of low volumes (1-150 μL) of up to seven reagents to make a reagent master mix.
One solution that addresses the complications associated with reagent storage and preparation is lyophilization (i.e., freeze-drying). Lyophilization of amplification reagents (both PCR and LAMP processes) significantly increases reagent shelf lives up to several months and even one year in ambient temperature (20°C) conditions [1,2,3]. In addition to non-freezer storage and an increased shelf life, a lyophilized master mix of all LAMP reagents would significantly reduce the complexity and time required for reagent preparation. Instead of pipetting and mixing up to seven reagents, which typically requires a trained microbiology lab technician to undertake, a single rehydration step would be required through addition of water or buffer to a premeasured lyophilized master mix powder. This effectively turns a seven-step process requiring multiple pipettes into a single step, allowing a local water operator or health official to conduct the diagnostic procedure, on-site, and on-demand. The following outlines the novel procedures to lyophilize the SARS-CoV-2 LAMP reagents master mix, including novel QUASR primers, as well as a resuspension/delivery system.
The addition of cryoprotectant chemicals to the reagent master mix prior to lyophilization is necessary, in order to preserve the LAMP reagents throughout the lyophilization process. Without the addition of trehalose, the LAMP reagents were damaged and observed to not facilitate SARS-CoV-2 amplification after resuspension [see figure below]. Moreover, glycerol-free reagents are needed for successful lyophilization to take place. Many reagents and enzymes are stored and shipped by chemical companies in glycerol solutions in order to increase stability in regular (-20°C) freezers, however glycerol interferes with the lyophilization process.
While the use of trehalose was found to be necessary for the fluorescent primer to not be damaged during lyophilization, the addition of three additional cryoprotectants were also investigated, namely mannitol, glycine and polyethylene glycol (PEG). They added effect on fluorescent intensity, signal to noise ratio and reagent storage lifetime were all important variables being monitored. Using trehalose, as well as a mixture of trehalose and the additional cryoprotectants were monitored over a period of six months [see figure below]. Over the six month tests, no significant decrease in LAMP reagent performance was observed when compared to control tests (i.e., freshly prepared LAMP reagents).
Some differences in amplicon shape were observed with the various added cryoprotectants, all solutions provided results that were easily countable and discernable with the developed software [see figure below].
In addition to the lyophilization process and cryoprotectants used to preserve the self-designed QUASR signal primers and other LAMP reagents, for extending shelf life and simplifying preparation procedures for field-use, a novel microfluidic delivery system can be used. For LAMP application (e.g., in-vial incubation and amplification), a microfluidic chamber of approximately 40 µL in volume can be used to store the lyophilized reagent master mix, as well as accommodate its resuspension and mixing. A channel of with lyophilized reagents accommodates fluid flow for reagent resuspension and convective mixing [see figure below]. The inlet will accommodate a pre-measured and packaged syringe of nuclease-free water, which is pushed through the microfluidic chip and collected at the outlet in another syringe.
