key: cord-0808440-ezv6xp16
authors: Bhadra, Sanchita; Riedel, Timothy E.; Lakhotia, Simren; Tran, Nicholas D.; Ellington, Andrew D.
title: High-surety isothermal amplification and detection of SARS-CoV-2, including with crude enzymes
date: 2020-07-07
journal: bioRxiv
DOI: 10.1101/2020.04.13.039941
sha: 6548f56d0d109e8dec19397c787196d3a2c145c5
doc_id: 808440
cord_uid: ezv6xp16

Isothermal nucleic acid amplification tests (iNAT), such as loop-mediated isothermal amplification (LAMP), are good alternatives to polymerase chain reaction (PCR)-based amplification assays, especially for point-of-care and low resource use, in part because they can be carried out with relatively simple instrumentation. However, iNATs can generate spurious amplicons, especially in the absence of target sequences, resulting in false positive results. This is especially true if signals are based on non-sequence-specific probes, such as intercalating dyes or pH changes. In addition, pathogens often prove to be moving, evolving targets, and can accumulate mutations that will lead to inefficient primer binding and thus false negative results. Internally redundant assays targeting different regions of the target sequence can help to reduce such false negatives. Here we describe rapid conversion of three previously described SARS-CoV-2 LAMP assays that relied on non-sequence-specific readout into assays that can be visually read using sequence-specific fluorogenic oligonucleotide strand exchange (OSD) probes. We evaluate one-pot operation of both individual and multiplex LAMP-OSD assays and demonstrate detection of SARS-CoV-2 virions in crude human saliva.

Loop-mediated isothermal amplification (LAMP) uses the strand-displacing Bst DNA polymerase and 4 primers (FIP, BIP, F3, and B3) that bind to 6 target regions (B3, B2, B1, F1c, F2c and F3c) to generate 10 9 to 10 10 copies of DNA or RNA targets, typically within 1 to 2 h (Figure 1 ). 1 In greater detail, F2 in FIP (F1c-F2) and B2 in BIP (B1c-B2) initiate amplification. F1c and B1c self-prime subsequent amplification. F3 and B3-initiated DNA synthesis displaces FIP and BIP-initiated strands. 3′-ends of the resulting single-stranded, dumbbell-shaped amplicons are extended to hairpins by Bst polymerase. FIP and BIP hybridize to the single-stranded loops and initiate DNA synthesis that opens the hairpin to form concatameric amplicons containing self-priming 3′-end hairpins. The ensuing continuous amplification generates double-stranded concatameric amplicons with self-priming hairpins and single-stranded loops. 1 LAMP can rival PCR for sensitivity without thermocycling, 2 and additional stem and loop primers can accelerate amplification, with some LAMP assays being complete within 10 min. 3, 4 However, since LAMP is commonly read using non-specific methods (such as, Mg 2+ precipitation, intercalating dyes or labeled primers) that cannot distinguish spurious amplicons that frequently arise from continuous amplification, its utility can be limited. We have previously overcome these drawbacks using oligonucleotide strand exchange (OSD) probes, based in part on advances in strand exchange DNA computation (Figure 1) . Strand exchange occurs when two partially or fully complementary strands hybridize to each other by displacing pre-hybridized strand(s) ( Figure  1B) . Strand exchange usually initiates by basepairing at single-stranded 'toeholds' and progresses to form additional basepairs via branch migration, allowing the rational design of complex algorithms and programmable nanostructures [5] [6] [7] [8] [9] . The hemiduplex oligonucleotide probes contain a so-called 'toehold' that allows sequence-specific interaction with a target molecule, and have opposed fluor and quencher moieties. In the presence of a complementary target, the OSD probes can undergo strand exchange and separation, leading to an easily read fluorescent signal. 10 In essence the OSD probes are functional equivalents of TaqMan probes and have been shown to accurately report single or multiplex LAMP amplicons from few tens of targets without interference from non-specific amplicons or inhibitors. 10, 11 Of equal import, the programmability of OSD probes allows their adaptation to many different assay formats, including to off-the-shelf devices such as glucometers and pregnancy test strips. [12] [13] [14] [15] [16] Figure 1. LAMP-OSD schematic. 'c' denotes complementary sequences. F and Q on the OSD denote fluorophore and quencher, respectively. OSD and subsequent strand exchange intermediates are denoted by numbered domains, which represent short (usually <12 nt) sequences in an otherwise continuous oligonucleotide. Complementary domains are indicated by asterisk.

LAMP-OSD is designed consciously to be easy to use and interpret, which makes it a reliable choice for either screening or validation of disease states. Base-pairing to the toehold region is extremely sensitive to mismatches, ensuring specificity, and the programmability of both primers and probes makes possible rapid adaptation to new diseases or new disease variants. We have shown that higher order molecular information processing is also possible, such as integration of signals from multiple amplicons 17 . Overall, the use of sequence-specific probes allow construction of strand exchange computation circuits that act as 'matter computers', 5-8 something that is not generally possible within the context of a PCR reaction (which would of necessity melt the computational devices).

We have taken pains to make LAMP-OSD robust for resource-poor settings. Lyophilized master mixes are stable without cold chain for extended durations and can be operated simply upon rehydration and addition of crude sample. 18 The one-pot operation, direct analysis of crude specimens, and easy yes/no visual readout make LAMP-OSD ideal for field operation with minimal training and resources.

OSD probes can be readily designed for integration into existing LAMP assays without significant disruption to standard assay practice. To that end, here we demonstrate the conversion of three recently pre-published LAMP primer sets described for detection of SARS-CoV-2, but that used non-specific readout methods ( Table 1) . The LAMP-OSD versions of these assays maintain the simplicity of visual yes/no readout, while endowing the assays with the inherent accuracy of probe-based signal transduction. We also perform multiplex execution of some of these LAMP-OSD assays and demonstrate the feasibility of sample-to-answer operation by directly analyzing human saliva spiked with SARS-CoV-2 virions. 

All chemicals were of analytical grade and were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise indicated. All enzymes and related buffers were purchased from New England Biolabs (NEB, Ipswich, MA, USA) unless otherwise indicated. All oligonucleotides and gene blocks ( Table 1) were obtained from Integrated DNA Technologies (IDT, Coralville, IA, USA). SARS-CoV-2 N gene synthetic transcript was a gift from the Schoggins lab at UT Southwestern Medical Center, Dallas, TX. SARS-CoV-2 genomic RNA and heat-inactivated virions were obtained from American Type Culture Collection.

We chose to design OSD probes for three of these pre-published LAMP primer sets, from here on referred to as the Tholoth, Lamb, and NB primers. The three primer sets target different regions in the ORF1AB and N genes of the SARS-CoV-2 genome. Fluorogenic OSD probes were designed for each of these primer sets using previously described principles and the Nucleic acid circuit design software NUPACK available freely at http://www.nupack.org/. 10, 19 Briefly, the target derived loop regions between the F1c and F2 primer binding sites were chosen as OSD binding regions for each of the three LAMP primer sets. The long OSD strand was designed to be complementary to this loop region. Single stranded 10-12 nucleotides long toehold regions were designated on one end of this long strand while a complementary short OSD strand was designed to hybridize to the remaining portion of the long strand. The long strand was labeled with a fluorescein moiety at the terminus not acting as the toehold. The short strand was labeled with a quencher and all free 3'-OH ends were blocked with inverted dT to prevent extension by DNA polymerase. Amplicon accumulation was measured by adding OSD probes. First, Tholoth, Lamb, and NB OSD probes were prepared by annealing 1 µM of the fluorophore-labeled OSD strand with 2 µM, 3 µM, and 5 µM, respectively of the quencher-labeled strand in 1X Isothermal buffer. Annealing was performed by denaturing the oligonucleotide mix at 95 °C for 1 min followed by slow cooling at the rate of 0.1 °C/s to 25 °C. Excess annealed probes were stored at -20 °C. Annealed Tholoth, Lamb, and NB OSD probes were added to their respective LAMP reactions at a final concentration of 100 nM, 100 nM, and 120 nM, respectively of the fluorophore-bearing strand. Multiplex LAMP-OSD assays comprising both Tholoth and NB primers and probes were set up using the same conditions as above except, the total LAMP primer amounts were made up of equimolar amounts of Tholoth and NB primers.

In some experiments, LAMP-OSD reactions were assembled using Bst-LF instead of Bst 2.0 and RTX. This was done by replacing Bst 2.0 and RTX in the LAMP-OSD reactions with ~2 x 10 7 CFU of rehydrated Bst-LF cellular reagents. 20 These cellular reagents were prepared in an E. coli BL21(DE3) derivative ∆endA ∆recA strain using previously described protocols. 20 Briefly, chemically competent bacteria were transformed with Bst-LF expression plasmid. Overnight 3 ml cultures of these transformed bacteria were grown in 2X YT broth containing 100 µg/mL ampicillin. Subsequently, sub-cultures were initiated at 1:200 dilution using 50 mL Superior Broth TM (Athena Environmental Sciences, Inc., Baltimore, MD, USA) containing 100 µg/ml ampicillin. These cultures were incubated at 37 °C and constant 225 rpm agitation till they reached the log phase of growth (A600 = 0.4 to 0.7). Protein production was initiated by adding 20ng/ml anhydrotetracycline (aTC) followed by 3 h incubation at 37 °C with 225 rpm agitation.

After induction, bacteria were collected by centrifugation followed by washing twice in cold 1X PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.47 mM KH2PO4, pH 7.4). The bacterial pellets were resuspended in cold 1X PBS at a density of A600 = 3.5 to 6.5. Some 2x10 8 induced bacteria (estimated from the A600 value using the relation 0.5 optical density = 5x10 8 bacteria/ml) were aliquoted into individual 0.2 ml PCR tubes and frozen at -80 °C overnight prior to lyophilization for 3 h at 197 mTorr and -108 °C using a VirTis Benchtop Pro lyophilizer (SP Scientific, Warminster, PA, USA). Immediately before use, each tube of cellular reagents was hydrated in 30 µL water and 3 µL of this suspension was added to each LAMP reaction.

Templates were serially diluted in TE buffer ( 

Integration of OSD probes into pre-published SARS-CoV-2 LAMP primer sets A series of 11 published primer sets for SARS-CoV-2 were screened using New England Biolab's WarmStart® Colorimetric LAMP 2X Master Mix (NEB, Ipswich, MA, USA) according to the manufacturer's protocol ( Table 2) . We found that 9 of the 11 sets showed significant no-template amplification in over 10% of the replicates in less than an hour of incubation at 65 °C (data not shown). These results are consistent with other published results that rely on colorimetric LAMP reactions, rather than on probe-based detection. 21 In fact, for many published assays, color changes must be read within a narrow window of time in order to minimize spurious conclusions, a consideration that does not scale well for diagnostic screening, especially at point-of-care or as an early part of a clinical diagnostics pipeline. To suppress potential false positive readout, we chose to develop OSD probes for three of the LAMP primer sets, termed herein as NB, Lamb, and Tholoth ( Table 1) . These primer sets target three different regions of the viral genome, the N gene, the NSP3 coding region of ORF1AB, and the RNA-dependent RNA polymerase coding region of ORF1AB. Of the three primer sets, the NB assay had the lowest propensity for spurious signal when analyzed by non-specific colorimetric readout (data not shown). To create LAMP-OSD versions of these assays, we designed OSD probes that were complementary to one of the loop sequences in each of the three LAMP amplicons. Subsequently, Tholoth, Lamb, and NB LAMP-OSD assays were setup individually by mixing separate reaction components as indicated in the Methods section. Each individual assay contained its specific OSD probes along with both inner primers FIP and BIP and both outer primers F3 and B3. In addition, each assay also received the backward loop (LB) primer that bound to the amplicon loop between B1c and B2 sites that was not recognized by the respective OSD probe. The forward loop (LF) primer that overlapped the Tholoth and NB OSD binding region was excluded. The LF primer was also initially excluded in Lamb LAMP-OSD assay even though the amplicon loop that bound this loop primer was long enough to accommodate a nonoverlapping OSD reporter; this was done to fairly compare the amplification kinetics of all three assays in a 5-primer format. In later versions of the assay with the Lamb primers, all 6 primers were included (designated as "6 primer Lamb" in Figures 3, 6, and 7) . Figure 2 , in response to target templates, all three LAMP-OSD assays generated strong OSD signal that could be measured both in real-time and observed visually at endpoint without interference from noise. We then tested the three LAMP-OSD assays using SARS-CoV-2 genomic RNA as templates.

While the NB and Tholoth LAMP-OSD assays were performed using 5 primers (FIP, BIP, F3, B3, and LB), the Lamb LAMP-OSD assay was tested using either 5 primers (FIP, BIP, F3, B3, and LB) or 6 primers (FIP, BIP, F3, B3, LB, and LF). Following 90 min of amplification at 65 °C, presence or absence of OSD fluorescence at endpoint was visually observed. As shown in Figure  3 , presence of SARS-CoV-2 genomic RNA resulted in bright, easily detected fluorescence in all three LAMP-OSD assays. The 6-primer version of Lamb LAMP-OSD could detect fewer genomic RNA copies compared to the 5-primer version of the assay. In contrast, all assays showed no signal in the presence of only human genomic DNA. 

Multiplex assays designed to detect multiple sequences from an organism are often employed to improve the accuracy of identification. 22, 23 CDC recommended diagnostic protocol for SARS-CoV-2 includes RT-qPCR amplification of at least two different regions of the viral genome. In fact, a recent pre-publication demonstrated a multiplex PCR approach to enhance efficiency of detecting SARS-CoV-2 at low copy numbers. 24 Having determined that the individual LAMP-OSD assays with NB, Tholoth, and Lamb primers could signal the presence of SARS-CoV-2 RNA, we sought to execute these assays in a multiplexed format to create an internally redundant assay for SARS-CoV-2. We chose to multiplex the NB and the Tholoth assays because they target different viral genes: the N gene and the ORF1AB gene, respectively. We first tested the ability of both primer sets to amplify their respective synthetic targets (in vitro RNA transcripts of N gene and ORF1AB gBlock DNA templates) in a multiplex assay format by assembling LAMP-OSD reactions containing equimolar amounts of both Tholoth and NB LAMP primer sets with either only one or both OSD probes.

When these multiplex assays were seeded with both types of target templates, both Tholoth and NB primer sets led to an increase in OSD fluorescence, measured both in real-time and visually observed at endpoint (Figure 4) . Multiplex assays containing both OSD probes demonstrated an additive effect, with OSD signal being brighter than assays containing only one type of OSD. Having confirmed that both the NB and Tholoth primer sets are able to amplify their respective targets in one-pot individual and multiplex reactions containing N gene and ORF1AB sequences (synthetic or viral, Figures 3, 4) , we tested the multiplex assay containing both Tholoth and NB primers and OSD probes using full length SARS-CoV-2 viral genomic RNA (Figure 5) . Visual observation of endpoint fluorescence revealed a bright signal in multiplex assays containing as few as 10 copies of genomic RNA while reactions containing non-specific human DNA remained dark ( Figure 5 ). Compared to individual LAMP-OSD assays of SARS-CoV-2 viral genomic RNA (Figure 3) , it seems that multiplex LAMP-OSD assay is detecting fewer genomic RNA copies ( Figure 5 ). For instance, while NB and Tholoth LAMP-OSD assays could routinely detect 100 and 1000 genomic RNA copies, respectively, the multiplex LAMP-OSD assay could signal the presence of as few as 10 genomic RNA copies. 

Given the low limits of detection we have observed, it is possible that LAMP-OSD might be used as part of diagnostics pipelines, or in direct patient screening. However, for this the reactions would need to operate under conditions commensurate with sample collection, especially in resource poor settings. Collection of nasopharyngeal and oropharyngeal swab specimens causes considerable discomfort to patients and requires supplies in the form of sterile swabs and transport media. Moreover, these samples are relatively difficult to self-collect. In contrast, saliva can be non-invasively collected simply by spitting in a sterile collection vessel and it can be done just as easily in a clinic as well as at home. Moreover, studies have shown that SARS-CoV-2 can be consistently detected in patient saliva with median viral loads of 3.3 × 10 6 copies/mL. 25 We tested the direct sample analysis ability of individual and multiplex LAMP-OSD assays by seeding them with 3 µL of human saliva spiked with SARS-CoV-2 virions. As control, duplicate LAMP-OSD reactions were seeded with virions suspended in 3 µL of TE buffer. Following 90 min incubation at 65 °C, endpoint observation of presence or absence of OSD fluorescence revealed that all assays seeded with SARS-CoV-2 virions, whether in human saliva or TE buffer, were brightly fluorescent (Figure 6 ). In contrast, assays lacking specific templates remained dark. These results suggest that LAMP-OSD assays might be used for direct analysis of human saliva samples in order to amplify and detect genetic signatures from SARS-CoV-2 virions. Considering potential supply chain issues, especially for resource poor settings, we sought to determine whether Bst-LF (an open source enzyme that maybe produced on site, see also attached protocol for production, Supplementary information) as a cellular reagent could be used for LAMP-OSD assays with SARS-CoV-2 targets. The previously described NB, Tholoth, and 6 primer Lamb LAMP-OSD assays were carried out with SARS-CoV-2 viral genomic RNA and Bst-LF enzyme as a cellular reagent 20 in an E. coli BL21(DE3) derivative ∆endA ∆recA strain (see also attached protocol for production, Methods).

Following 90 min incubation at 65 °C, visual observation of endpoint OSD fluorescence revealed that the Tholoth LAMP-OSD assay containing 1000 copies of SARS-CoV-2 genomic RNA was brighter compared to assays containing fewer or no SARS-CoV-2 genomic RNA (Figure 7) . Similarly, 6 primer Lamb LAMP-OSD assays containing as few as 100 SARS-CoV-2 genomes were bright compared to controls containing no viral genomes. In contrast, all tubes of NB LAMP-OSD assay demonstrated background fluorescence with no virus-specific increase in OSD signal. These results are consistent with previous reports from NEB, the manufacturer of Bst 2.0 (https://www.neb.com/products/m0374-bst-3-0-dna-polymerase#Product%20Information), that while Bst-LF cannot support all reverse transcription LAMP reactions, for the Tholoth and Lamb primer sets it is suitable for the detection of SARS-CoV-2. 

In summary, we have demonstrated a facile way to rapidly configure LAMP assays for accurate probe-based readout of SARS-CoV-2 by integrating OSD probes into individual and multiplex assays. The SARS-CoV-2 LAMP-OSD assays can be executed in one-pot reactions assembled using individual reverse transcription LAMP reagents, including with trivially produced cellular reagents. Moreover, these assays can be used to directly analyze crude human samples, such as saliva, to detect SARS-CoV-2 without interference from spurious signal. A few hundreds to a few tens of virion genomic RNA can be identified using individual or multiplex LAMP-OSD assays, respectively. We suggest that while LAMP-OSD may not have the same sensitivity as 'gold standard' RT-qPCR assays, that the versatility of LAMP-OSD, especially for resource poor settings with limited infrastructure, might prove useful for screening for positives, which could then be followed up with more limited or difficult to execute RT-qPCR tests.

Bst-LF purification is described in Reference 27 and detailed as a protocol in Supplementary information. Expression plasmids will be available from Addgene shortly, or can be obtained via https://reclone.org/. The production of cellular reagents is described in Reference 20 and detailed in the Methods section.

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We acknowledge Dr. Andre Maranhao for kindly providing a protocol for expression and purification of Bst-LF enzyme. We also acknowledge the FRI DIY Spring 2020 Peer Mentor Cohort for their project initiation and screenings of the preprint COVID- 19 

Purification of Bst-LF or equivalent enzyme: For sites wishing to produce their own LAMP enzymes, the following protocol can be used. In brief, an E. coli protein expression strain such as BL21 was transformed with Bst-LF expression plasmid that contained an amino terminal six histidine tag for purification. An individual transformant colony was picked into desired media with appropriate antibiotics (e.g. ampicillin) and 1-2% glucose. This starter culture was grown overnight in shaking incubator at 37˚C.

On day three, cultures were diluted 1:200 or 250 mL to 1 L of fresh medium. Inoculated cultures were grown to OD600 between 0.5−1 before inducing protein expression by adding anhydrotetracycline (aTC) to a final concentration of 200 ng/mL aTC. Induced cultures were allowed to express 3−7 hr for expression. Following expression, cells were harvested by centrifuging cultures at 4˚C, 5000 xg for 20 min and then flash frozen in liquid nitrogen. Flashfrozen cell pellets may be stored at -80˚C or allowed to thaw on ice for same day purification.After thawing on ice, resuspend cell pellets in 30 mL cold Lysis Buffer. Transfer the resuspended cell pellet to a small 50 mL beaker with clean stir bar and place securely in an ice bath. With moderate stirring, sonicate the sample using 40% amplitude and 1 sec ON / 4 sec OFF for 4 min total sonication time.Following sonication, centrifuge the resulting lysate at 4˚C, 35,000 xg for 30 min. Carefully transfer the supernatant to a clean ultracentrifugation tube and incubate in a thermomixer at 400 rpm, 65˚C for 20 min. Then place the heat-treated lysate on ice for 10 min. Centrifuge the heat-treated lysate at 4˚C, 35,000 xg for 30 min. Carefully transfer the supernatant to a clean tube and filter the clarified lysate using a 0.2 μm filter.Add clarified, filtered lysate to 1 mL of pre-equilibrated HisPur Ni-NTA resin and incubate for 30 min at 4 °C with end-over-end mixing for batch binding. Apply lysate and resin slurries to gravity columns and allow to drain. Wash columns three times with 10 mL of Wash Buffer and then elute with four 1 mL aliquots of Elution Buffer. Finally, pool elutions and dialyze overnight into Dialysis Buffer and store at −20 °C.