The project proposal is based upon ‘loop-mediated isothermal amplification’(1,2), or LAMP, a molecular detection strategy that allows identification of specific nucleic acids in samples, with the possibility for colorimetric or fluorescent visualization of results. The reaction uses 4-6 primers specific for short target regions (usually less than 300nt) and is run at a single temperature (about 65 degrees Celsius), meaning no special thermal cycler or even incubator is required. The reaction also completes in a short time (15-45min, depending on the target region)(3-6). Furthermore a special quenched fluorescence system (QUASR-rtLAMP) allows binary, "yes/no" detection of specific nucleic acid sequences and even multiplexed reactions (7,8). In summary, such loop-mediated isothermal amplification (LAMP) assays offer several advantages over qRT-PCR, the current standard for SARS CoV-2 laboratory diagnostics, as it is relatively inexpensive, does not necessarily require nucleic acid purification prior to assay, and provides a simple visual or fluorescent detection of positive samples. The proposed solution would consist ultimately of 8-tube strip ‘test kits’ containing lyophilised components (primers and enzyme) and including controls, so that they could be sent anywhere by post for validated detection of SARS CoV-2.
Parallel experiments by international partner laboratories from 6 countries (FR, ES, CH, USA, CM, CL) will ensure reliable development of the proposed #CoronaDetective kit.
Objective 1: Develop a functional Isothermal Amplification system, robust enough to detect SARS CoV-2 in samples.
Loop-mediated isothermal amplification can amplify a few copies of a nucleic acid target to 10^9 copies in less than one hour, even when large amounts of non-target DNA are present or other biological substances. RT-LAMP has been shown to be sensitive and specific enough to detect RNA viruses from biological samples such as blood, serum, urine, cerebrospinal fluid, tissue, and nasal swabs, without needing purification of RNA (12). We will test several openly available primer sets against SARS CoV-2 gene sequences and a control human gene to obtain optimal components for the #CoronaDetective kit. Most lab tests in the context of this proposal will evaluate reactions only with positive control DNA, but one partner’s access to the CDC lab in Atlanta means we can also validate our most efficient component mix with real viral samples.
Objective 2: Develop a kit that can be sent without depending on cold chain maintenance.
Lyophilised components driving LAMP reactions are surprisingly stable, and one partner has access to high-quality robotic tools and freeze-drying equipment, allowing large-scale production of the kit, based around 8-tube strips. These would be mailed off to partners around the world for tests, documentation and comparison of results.
Methodology (technical details)
(Day0) Reception of reagents (primers, enzymes, buffers, positive control DNA/RNA)
Primers would be ordered and obtained as lyophilised powder. Commercial enzymes and buffers and positive control plasmid (2019-nCoV_N_Positive Control plasmid from Integrated DNA Technologies) will be used, although there are hopes that open alternatives may become available. Two primer sets from the N gene region are initially proposed for this project, one from Zhang et al (9) and the other from the ‘Mammoth protocol.’ Additionally, a promising set of LAMP primers for the SARS CoV-2 Orf1ab gene (5), will be tested. Finally, a control primer set for the mRNA of the human housekeeping gene is proposed. These primers would be specific for mRNA, and can even be useful as an internal human RNA control for multiplex LAMP applications.
More details for how to design primers for the Quasr detection can be found here and here (in particular for these targets in the SARS CoV-2 N gene). Here is the open notebook (Benchling) page pulling details together.
(Days 1-14) First tests and primer set optimisation
Complete details for running basic isothermal amplification protocols can be found here.
Optimisation is performed by varying ratios of primer pairs in each set, and seeing which condition gives the fastest amplification time. Optimised primer concentrations are then titrated with varying amounts of MgSO4 to determine the best concentration for each primer set. Use of qPCR machines will facilitate the empirical detection of these optima. (CH, ES)
In parallel, viral RNAs can be tested at the CDC facilities with BSL3 conditions, to make sure the conditions are also suitable for such samples. (USA)
(By day 28) Validation of sensitivity and robust detection capacity
Dilution series with the positive control plasmid sequence (or maybe synthetic RNAs and hopefully viral samples at the CDC BSL3 lab) will allow determination of the limit of detection for the reactions.
If the colorimetric detection is sensitive and reliable enough, as being tested in the partner project #neb-lamp-test, the next section and other phrases in italics (below) can be omitted.
(From day 15) Labelled primers and quenchers arrive for the best N set of primers
For the labelling of one set of N primers, it looks like a fluorescein molecule at the 5’ end of the forward internal primer (FAM-FIP) with a Quencher at the 3’ end of a very short (10 bases) complementary sequence looks very promising, but awaits the empirical tests above.
To validate the best conditions for these labeled sets, a SYTO dye to detect in a different channel from fluorescein (i.e. ROX) will allow test reactions to be assessed in a qPCR system. Partners can also score the fluorescence of the results with the ‘GMO detective’ fluorescence detector tube holder.
If all looks well, tests of multiplexing will also be possible (with all 14 primers put together), which would require another (i.e. TRITC) label on the control gene set. Multiplexing would have the advantage that each set of 8 tubes could be used to test for 4 or 5 samples, rather than only 2 samples, as in the GMO Detective. With multiplexing, both positive results in one tube give yellow fluorescence. If one more control is desired for the combination, 4 samples could be tested from one strip (i.e tube 1, negative control; tube 2, mRNA + control, red; tube 3 N + control, green, tube 4 both + controls, then tubes 5-8 for samples). Feedback of end-users will be important to decide this detail.
(Day 30) Large-scale orders of best sets for lyophilisation (to be put together in FR) into the 8 tube strip format
Will need to get special approval to go into the lab to use the equipment there (FR), if France is still under lockdown.
Validation of the lyophilised ‘kits’ for detection
Mail tube strips to all partners (CH, ES, FR, USA, CL and CM) for tests, scoring fluorescence with the GMO detective rig and collecting all results on an open platform.
(Before day 60) Conclusions and broad dissemination of the open results
We will perform the final documentation of the project and its dissemination through open-source platforms, so anyone could benefit from these results and replicate the system.