WO2022040270A1 - Processes to detect coronaviruses - Google Patents

Abstract

Processes and devices are disclosed that detect RNA and DNA in a sample, including without limitation RNA from coronaviruses, with said processes generating a fluorescent signal if a segment of a specific RNA molecule is present in a sample.

Description

PROCESSES TO DETECT CORONAVIRUSES

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Patent Application No. 16/996,154, filed August 18, 2020, which is a continuation-in-part of U.S. Patent Application No. 15/826,126, filed November 29, 2017, now U.S. Patent No. 10,920,267, which claims the benefit of U.S. Provisional Application No. 62/427,868, filed November 30, 2016. U.S. Patent Application No. 16/996,154 is also a continuation-in-part of U.S. Patent Application No. 16/168,349, filed October 23, 2018, which is a continuation-in-part of U.S. Patent Application No. 15/826,126, filed November 29, 2017.

The Sequence Listing for this application is labeled “Seq-List.txt” which was created on August 18, 2021 and is 11 KB. The entire content of the sequence listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to processes for detecting DNA and RNA molecules, especially those that arise from infectious diseases, in particular RNA viruses. More specifically, it concerns the detection of the 2019-nCoV coronavirus, known by other names, including the CO VID-19 virus. This invention provides compositions that allow rapid detection of this virus without temperature cycling, working under conditions that allow detection of specified amounts of virus and its variants with very low times to surpass signal thresholds, including with unprocessed samples.

DESCRIPTION OF THE RELATED ART

Methods that detect small numbers of nucleic acid molecules (which include DNA molecules and RNA molecules, collectively "xNA" molecules) from pathogens and other biological agents are useful in diagnostics, research, and biotechnology. In general, the number of xNA molecules that a method must detect to be useful are too few for them to be detected directly. Accordingly, detection methods often begin with an amplification step.

Amplification means a process that yields many product xNA molecules, where production of those molecules requires a starting xNA sequence, a "target" or "analyte". Generally, the product xNA molecules ("amplicons") contain within them one or more segments of DNA whose sequence corresponds to the sequence of a part of the target xNA, or its Watson-Crick complement. These segments arise by polymerase-catalyzed copying of the xNA molecule. However, useful amplification methods often incorporate additional segments into the amplicons, whose sequences arise from tags on primers.

Classically, amplification is done using the polymerase chain reaction (PCR) [Saiki et al. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487-491]. Here, a “forward primer” that is substantially (meaning over 90%) Watson-Crick complementary to a pre-selected region of a DNA target is annealed to the target to form a duplex. Next, the primer-target complex is incubated with a polymerase and the appropriate 2’ -deoxynucleoside triphosphates to yield a Watson-Crick complementary DNA molecule; the target and its complement, as it is formed, are bound in a double stranded double helix. The double strand is then “melted” by heating, typically above 75° C, to give the two complementary DNA strands in single stranded form. The mixture is then cooled so that the original target binds to a second forward primer, while its complement binds to a “reverse primer”, which is designed to bind to a preselected segment downstream in the product DNA molecule. Then, polymerase extension is repeated, with both primers extended to give full-length products, again as duplexes (now two in number). The results are multiple copies of a segment of the target molecules between the primer binding sites, as well as multiple copies of the complement. In asymmetric PCR, the ratio of these two primers is different from unity. Non-target sequences can be added to the amplicons from tags on the 5'- ends of those primers.

Temperature cycling to separate strands in PCR is undesirable in many applications, including those that amplify target xNA for its rapid detection at entrances to public spaces. Thus, the art contains many methods that seek amplification methods that do not need temperature cycling. These include “recombinase polymerase amplification” (RPA) [Piepenburg, O. et al. (2006) DNA Detection using recombination proteins. PLoS Biol 4 (7): e204], rolling circle amplification (RCA), NASBA, helicase-dependent amplification (HD A) [Tong et al. (2011) Multiple strategies to improve sensitivity, speed and robustness of isothermal nucleic acid amplification for rapid pathogen detection. BMC Biotechnol. 11 Art. No: 50][Lemieux et al. (2012) Near instrument-free, simple molecular device for rapid detection of herpes simplex viruses: Expert Review Molec. Diagnostics 12, 437-443] and "loop amplification" (LAMP), among others. These are called “isothermal amplification” methods. One isothermal amplification method is "loop-mediated isothermal amplification" (LAMP) [Kubota et al. (2013) Patent Application Pub. No.: US 2013/0171643 Al Kubota et al. (43) Pub. Date: Jul. 4, 2013 (54) Sequence Specific Real-Time Monitoring Of Loop- Mediated Isothermal Amplification (LAMP)]. LAMP comprises a reaction involving one or more LAMP primers that bind in a Watson-Crick sense to the target xNA. As illustrated in Fig. 1, LAMP may employ six primers that bind by Watson-Crick complementarity to eight distinct regions in the target. The primers are designated internal primers (FIP and BIP), outer primers (F3 and B3), and loop primers (LB and LF).

LAMP is initiated by adding internal primers (FIP or BIP) that anneal by Watson- Crick complementarity to regions (F2c or B2c) within the target xNA analyte. An outer primer (F3 or B3) then hybridizes to its priming site (F3c or B3c) on the target xNA and initiates the formation of self-hybridizing loop structures by strand invasion of the DNA sequences already extended from the internal primers (FIP and BIP). The resulting dumbbell structure then becomes a seed for exponential LAMP amplification by a strand displacing polymerase. Synthesis of product is further accelerated by loop primers (LF and LB), designed to hybridize in oligonucleotide segments between F1c and F2; these are called Bic and B2, respectively, in Fig. 1.

LAMP reactions may be run isothermally. Here, temperatures are fixed between 60° C and 70° C, sometimes slightly lower or slightly higher. The amplicons are concatemers of the targeted region, and may fold to form "cauliflower-like structures" with multiple loops.

One challenge of LAMP is visualization of products formed. Classically, progress of LAMP was followed by measuring the turbidity in the reaction mixture arising from precipitating by-product magnesium pyrophosphate. In real-time analysis, creation of LAMP products may be monitored by adding intercalating dyes to the mixture. Such dyes include SYBR Green® or EvaGreen®. When double-stranded DNA is formed, these dyes bind and, once bound, fluoresce.

These processes do not allow the sequence of the DNA product to be confirmed. Thus, the formation of other products unrelated to the target can give a false positive signal.

Alternative approaches to detect LAMP products are specific for the target. These include molecular beacons (see [Yaren et al. (2016) A norovirus detection architecture based on isothermal amplification and expanded genetic systems. J. Virol. Meth. 237, 64-71] incorporated herein in its entirety by reference). However, use of molecular beacons for monitoring LAMP in real-time is problematic, as stem structures may not be stable at temperatures where LAMP is run, although it may be used for end-point detection of LAMP amplicons. Further, beacon detection fails when the target is present in a crude sample, such as a nasal swab, saliva, or urine.

An alternative way of visualizing LAMP products uses an "assimilating probe" [Kubota et al. (2011) Fret-based assimilating probe for sequence-specific real-time monitoring of loop-mediated isothermal amplification (LAMP). Biol. Eng. Trans. 4, 81-100]. This adds to the LAMP mixture two DNA strands that hybridize over parts of their segments by Watson-Crick complementarity. The first strand has a fluorescence quenching moiety covalently attached at its 3' end; the second strand of the assimilating probe has a fluorophore covalently attached at its 5 ’-end. When the two strands are hybridized, quencher and fluorophore come together, stopping fluorescence.

To work, this assimilating probe must also have a single stranded region attached to the 3'-end of the fluorescently tagged oligonucleotide. This priming sequence is complementary to a selected segment of the target analyte xNA. The second strand and the first strand added to LAMP are preferably in a ratio of 1 : 1, although Kubota et al. (2011) teach that the ratio in a mix may be lower. The preferred concentration of assimilating probes is about 0 μM to about 1 μM .

In assimilating probe LAMP, the priming region of the fluorescently tagged oligonucleotide is extended by a strand-displacing DNA polymerase or reverse transcriptase, with the target xNA used as a template. During LAMP, primer extension from reverse primers reads through the primer on the fluorescently tagged oligonucleotide, and then the segment of DNA from the fluorescently tagged oligonucleotide itself. This read-through displaces the oligonucleotide that bears the quencher. This separates the fluorescent species from the quenching species, allowing fluorescence to be observed and measured from the fluorescently tagged oligonucleotide that has been "assimilated" into the LAMP products.

The visualization process of Kubota et al. (2011) has limitations. A mixture of concatemers is what becomes fluorescent, not a single product. This means that the fluorescence is not present in a single molecule that can be separated, captured and observed directly.

Further, the two strands in the assimilating probe are held together by Watson-Crick pairing between standard nucleotides [Kubota et al. (2011)]. As natural biological samples contain many xNA molecules built from natural nucleotides, these can invade the duplex of the assimilating probe, separating fluorophore and quencher even absent amplification, giving false positives.

A displaceable architecture that releases a fluorescently tagged species was reported for primers that carried a quencher by Tanner et al. [Tanner et al. (2012) Simultaneous multiple target detection in real-time loop-mediated isothermal amplification. BioTechniques 53, 81-89]. However, these prime internally to the loop regions. Tanner does not teach a process where a fluorophore-releasing probe primes by Watson-Crick complementarity into the loop regions.

Further, neither Tanner (2012) nor Kubota (2012) teach the use of nonstandard nucleotides in the tag regions that hold together the fluorescently labeled oligonucleotide and the quencher oligonucleotide. Here, "nonstandard nucleotides refers to nucleotides built from an artificially expanded genetic information system" (AEGIS) (Fig. 12). AEGIS components have nucleobase analogs with their hydrogen bonding groups are shuffled. This creates new orthogonally binding nucleobase pairs, which cannot hybridize to any natural xNA in any biological sample.

US Patent 10920267 changed the architecture of the process by placing the fluorescent species on the displaced oligonucleotide, the quencher on the priming oligonucleotide, and the primer at the 3'-end of the displaceable probe priming on the loop region of an amplifiable structure, rather than on the target analyte itself. This allows the fluorescent species to be a single molecule whose sequence is unrelated to the sequence of the target analyte, and to be released only after the amplification fully starts. This, in turn allows it to be captured, even while the amplification is occurring. This signal sequence is also not spread over many amplicons.

In this "displaceable probe LAMP” (DP-LAMP), the two components of the reverse displaceable probe may optionally hybridize via pairing with nonstandard nucleotides AEGIS. The advantages of this are several. AEGIS:AEGIS pairing prevents invasion of the displaceable probe by natural nucleic acids, preventing false positives in complex biological mixtures. This allows the displaced fluorescent probe to be captured in real time, even as amplification occurs.

Also incorporated herein in their entirety by reference are:

Yaren et al. (2016) A norovirus detection architecture based on isothermal amplification and expanded genetic systems. J. Virol. Methods 237, 64-71. Yaren et al. (2016) Standard and AEGIS nicking molecular beacons detect amplicons from the Middle East Respiratory Syndrome coronavirus, J. Virol. Methods 236, 54-61.

Yaren et al. (2017) Point of sampling detection of Zika virus within a multiplexed kit capable of detecting dengue and chikungunya. BMC Infect. Diseases 17.1, 293.

As useful as DP-LAMP is, it suffers limitations. In particular, the time for sufficient signal to be generated to cross a threshold (the Ct times, expressed in minutes; this may also be called a Tt, time for a signal to cross a threshold) must be as short as possible to give a useful test, especially in kits, devices, and kiosks that incorporate the ability to do DP-LAMP. In general, the more target, the lower the Ct. In general, the lower the Ct, the more useful the test. Further, a Ct measured on pure isolated RNA cannot be directly compared to a Ct measured on crude sample; the time spent purifying RNA is far longer than the time to cross a fluorescence threshold.

Comparison of Ct values is difficult, since they depend on the amount of target, and reporting need not be uniform. As reference, Rabe-Cepko reports a Ct of 10 min on pure RNA (300 copies per reaction) [Rabe & Cepko (2020) SARS-CoV-2 detection using isothermal amplification and a rapid, inexpensive protocol for sample inactivation and purification. Proc. Natl. Acad. Sci. 117(39), 24450-58]. The Ellington group reports a Ct of 15-20 min on RNA and gblock templates, using blocked probes on 10,000 copies of template per assay [Bhadra et al. (2020). High-surety isothermal amplification and detection of SARS- CoV-2, including with crude enzymes. bioRxiv. 2020.2004.2013.039941. doi: 10.1101/2020.04.13.039941]. The Sherlock method reports Ct values >20 min on 20 fM template [Joung et al. (2020) Point-of-care testing for COVID-19 using SHERLOCK diagnostics. medRxiv. 2020:2020.05.04.20091231]. Anahtar: did colorimetric detection only, evidently giving a Ct of 30 min with 625 copies of RNA per assay [Anahtar et al. (2020) Clinical assessment and validation of a rapid and sensitive SARS-CoV-2 test using reverse- transcription loop-mediated isothermal amplification. medRxiv. 2020:2020.05.12.20095638]. Dao- Anders reports a Ct of 30 min for 10,000 copies of RNA [Dao et al. (2020) Screening for SARS-CoV-2 infections with colorimetric RT-LAMP and LAMP sequencing. medRxiv. 2020:2020.05.05.20092288].

Further, LAMP Ct values can be sensitive to changes (10% or greater) in concentrations of some LAMP components and to the purity of those components. Thus, it is inventive to deliver a specific set of components that meet Ct metrics applied to a specific target. BRIEF SUMMARY OF THE INVENTION

This invention offers specific compositions that detect COVID-19 and its variants with remarkable speed, with Ct values 5-10 minutes with 10000 copies of purified COVID- 19 RNA, and 10-15 minutes with 1000 copies of COVID-19 RNA, obtained surprisingly from targets in a nasal swab or saliva. Further, a second LAMP targeting human RNase P RNA can be added to give a two-plex LAMP with similar Ct values. Thus, it supports these capabilities:

(a) Testing may be done at the same site where sampling is done with the individual waiting;

(b) The test need not involve sample preparation other than collection, transfer and dilution;

(c) The test may use mid-turbinate nasal swabs, nasopharyngeal swabs, and saliva.

Thus, this invention enables hand-held and portable devices that allow signal to be generated and observed by a user. The enabled devices optionally comprise (i) a microprocessor that controls (ii) a heater that warms to between 50° C and 70° C (iii) a disposable that contains reagents that generated a fluorescent signal when viral RNA is present, together with (iv) a light that illuminates the region of the disposable that generates the fluorescent signal, and (v) a port that allows the user to visualize the fluorescent signal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A. Displaceable probe DP-RT-LAMP architecture, real-time analysis, observed during the amplification, and end-point visualization, occurring at the end of the amplification. DP-RT-LAMP is initiated by adding primers (FIP or BIP) that anneal to F2c or B2c regions. Outer primer (F3 or B3) then hybridizes to F3c or B3c and initiates formation of self-hybridizing loop by strand invasion of the DNA sequences already extended from the internal primers (FIP and BIP). The resulting dumbbell structure is a seed for exponential LAMP amplification by a strand displacing polymerase. This is further accelerated by loop primers (LF or LB) hybridizing to segments between F1c and F2 or B1c and B2, respectively. Further, priming region of the quencher labeled probe (e.g. LB) is extended by a strand- displacing polymerase, and primer extension from the reverse primers then reads through the primer on the quencher labeled probe, displacing the probe that bears the fluorophore. This architecture differs from standard LAMP in how the signaling element moves in the detection architecture.

FIG. IB. Increase in the fluorescent signal and real-time analysis of the process manifests itself as sigmoidal curve as it would in RT-qPCR using TaqMan probes.

FIG. 1C. In addition to real-time analysis, end-point fluorescence can be visualized using an observation box with blue LED exciting at 470 nm through orange filter (Firebird Biomolecular Sciences LLC, US).

FIG. 2. Limits of detection (LODs) determined for the assay using RNA target obtained by in vitro transcription of a synthetic DNA molecule corresponding to a region of the 2019-nCoV genome, and using the CoV2-W3 primer set. Incubation at 65° C only, without pre-incubation at 55° C. Two traces with no-template control (NTC) superimpose, and give no signal. The 500, 50, 25, and 5 copies give progressively longer times to pass a threshold (Tt). This proves the sensitivity of the test in fully contrived samples.

FIG. 3. Limits of detection (LODs) determined for the assay using full length RNA target (Twist) and the CoV2-W3 LAMP primer set. Assays were run at 65° C for 60 min using the LightCycler® 480. Determined LOD was 100 copies/assay with threshold time (Tt) of 25.31 min. Traces from no-template control (NTC), one copy and 10 copies superimpose and give no signal.

FIG. 4. Limits of detection (LODs) determined for the assay using whole viral SARS- CoV-2 that was heat-inactivated (65° C, 30 min, from BEI Resources). Different enzymes and buffers systems were tested as well as incubation temperature was modified. The determined LOD was 10 copies/assay with a Tt of 16 min using Condition 1.

FIG. 5A-FIG. 5E. Limit of detection on LAMP primers using heat-inactivated SARS-COV-2 or human RNA (for internal control) (A) Real-time analysis of CoV2-W3 primer set (targeting S gene) showed that LOD was 10 copies of RNA/assay. (B) End-point visualization of LAMP products with primer set CoV2-W3 using SafeBlue Illuminator/Electrophoresis System. (C) End-point visualization of LAMP products with primer set CoV2-W3 using a hand-held observation box with integrated blue LED and orange filter (Firebird Biomolecular Sciences LLC). (D) Real-time analysis of CoV2-v2-4 primer set (targeting N gene) showed an LOD of 25 copies of RNA/assay. (E) Real-time analysis of internal control RNaseP-2 primer set (targeting human RNase P gene) showed that LOD was 44 copies of human RNA/assay. FIG. 6A. Sampling work-flow and results output. Dry nasal swabs are used as sampling method. Swabs are first eluted in a sample preparation buffer and aliquots from that are added into DP-RT-LAMP mixtures. End-point results are visualized by fluorescence of fluorescein excited by the emission of a blue LED and an orange filter. Saliva is mixed with a sample preparation buffer, and an aliquot is added into the DP-RT-LAMP mixture. End-point results are visualized using the same method for nasal swab sampling. Saliva is added to paper that has been covalently modified with quaternary ammonium salts (Q-paper). Q-paper carrying the saliva sample is directly introduced into DP-RT-LAMP mixture without further manipulation. End-point fluorescent signal is visualized using blue LED and orange filter. Note that the square of Q-paper is observable, but does not compromise the analysis.

FIG. 6B. In addition to end-point visualization, DP-RT-LAMP experiments were also run in real-time using Genie® II (Optigene, UK).

FIG. 7A-FIG. 7D. Optimization of sampling methods and fluorescence visualization with presently preferred methods. (A) Four different methods were evaluated for nasal swab sampling, including (i) TE elution, (ii) the method of Rabe-Cepko et al., (iii) a method combining Cepko with Chelex-100, and (iv) a process without a heating step. Heat- inactivated SARS-CoV-2 isolate was spiked into nasal swab elutions and each method’s sensitivity was determined. Purified RNA control was included as a reference. (B) For saliva sampling, five methods were evaluated: (i) crude saliva without any treatment, (ii) the Cepko method, (iii) the Cepko method coupled with Chelex and a heat-step, (iv) the same without a heating step, and (v) deposition of saliva on Q-paper and its direct introduction into DP-RT- LAMP. A purified RNA control was included as a reference. (C) End-point visualization of finalized methods: Nasal swab and one of the saliva sampling methods uses buffer solution containing 1 mM Na citrate pH 6.5, 2.5 mM TCEP, 1 mM EDTA, 10 mM LiCl and 15% Chelex-100. LODs for both samplings were determined to be 100 copies/assay. (D) End- point visualization of saliva deposited on Q-paper and its direct use in DP-RT-LAMP reaction. The LOD was 100 copies/assay using Q-paper.

FIG. 8A-FIG. 8C. Further evaluation of presently preferred sampling methods and sensitivity analysis with contrived samples using heat-inactivated SARS-CoV-2 template from BEI. (A) Varying amounts of RNA was spiked into nasal swab samples. 500 copies of RNA were detected with consistency and RNase P gene was used as sampling control. (B) and (C) Varying amounts of RNA was spiked into saliva samples or saliva that was deposited onto Q-paper, respectively. 200 copies of RNA were detected with consistency and RNase P gene was detected successfully.

FIG. 9A-FIG. 9B. Analysis of inhibitory effects of saliva as a sample. DNA was used as the spike-in template and Tt values were determined in the absence (A) or presence (B) of saliva.

FIG. 10A-FIG. 10F. (A-C) Biplexed detection of SARS-COV-2 RNA and RNase P (internal control). Varying amounts (10⁵, 10⁴, 10³, 10², 10 and 5 copies) of heat-inactivated SARS-CoV-2 (BEI resources) spiked with 440 copies of purified human RNA. Fluorescence signals from three channels were recorded every 30 seconds using LightCycler® 480. Corresponding Tt values were shown on the table. Channel 483-533 (A and D) is specific for SARS-CoV-2 RNA, channel 523-568 (B and E) can detect signals from both targets (ladder formation manifests itself), and channel 558-610 (C and F) is specific for RNase P. (D-F) 10⁶ and 10³ copies of SARS-CoV-2 RNA was spiked into processed nasal swab samples and analyzed simultaneously using three fluorescent channels. Corresponding Tt values were shown on the table. The complexity of the curve when the competing viral amplification takes place involves a complex kinetic relationship as resources specific to the viral amplification (primer set) are consumed, allowing the positive control to again compete for common resources (e.g. dNTPs, enzymes).

FIG. 11A-FIG. 11C. (A) Workflow of lyophilization first involves the removal of glycerol from commercial enzymes. This is done by replacing enzyme storage buffer with its glycerol free version via ultrafiltration. The next step combines 10X primer mix and dNTPs with dialyzed enzymes. The mixture is then frozen (liquid N2) and lyophilized for 4-6 hours. (B) Lyophilized reagents were activated by supplementing lyophilized reagents with rehydration buffer, and templates containing SARS-CoV-2 RNA or contrived nasal/saliva samples were added to DP-RT-LAMP mixture was analyzed on Genie II and Tt values were determined. (C) End-point fluorescence was visualized using blue LED and orange filter.

FIG. 12. Artificially expanded genetic information system in a preferred embodiment.

FIG. 13. Self-avoiding molecular recognition system (SAMRS), in a preferred embodiment.

FIG. 14. A schematic showing, without limitation, the essential elements of a hand- held personal coronavirus detector. A cartridge (Fig. 15, for example) is inserted into the device. Pressing an "on button" starts a microprocessor to direct a heating element to maintain the liquid at 65 ± 5° C for a pre-selected period of time. During that time, an isothermal process amplifies an oligonucleotide, allowing the solution to allow the generation of unquenched fluorescence up excitation with a light. In this example, a blue 410 nm emission LED generates the fluorescence of fluorescein, whose fluorescence is observed through an orange filter. With a microprocessor of more sophistication, variable temperature and temperature cycling may optionally be performed.

FIG. 15. The cartridge used, without limitation, in the hand-held device in Fig. 14. The inventive steps include the use of Q-paper to receive saliva from the tongue of an individual who may be infected with the coronavirus; a sample of the saliva and the coronavirus that it may contain is thus delivered to the Q-paper. An inventive feature is to have optionally the Q-paper to carry a flavor, to encourage effective deposition of the saliva. A lid is then screwed on the tube, said lid carrying a protruding device that punctures a ball containing buffer required for the isothermal amplification processes. The bottom of the tube contains reagents that are required for the amplification, including without limitation, enzymes, oligonucleotides, and triphosphates. Also inventive is to incorporate into the device a thermosetting plastic that ensures that, after heating at 65 ± 5° C for a pre-selected period of time, the tube can no longer be opened.

FIG. 16. The processes of the instant invention may be executed in a kiosk that can deliver liquid. As an inventive step that simplifies workflow, the sample, including without limitation saliva, may be collected on a ball of size pre-selected to fit within a tube. In this implementation, the reagents are lyophilized in the tube, and liquid containing the other reagents is delivered by the kiosk. The kiosk has one or more slots that accept the tube after it receives a sample. The kiosk then delivers liquid to said tube, such that the combination of materials in the tube and in the kiosk-delivered liquid are sufficient to amplify the target DNA or RNA. The kiosk is further equipped with elements that warm a region of the tube to a temperature between 50 and 70° C, preferably 65° C. The amplification generates a fluorescent signal when viral RNA is present in the sample. A light in the kiosk illuminates the region of the tube that generates the fluorescent signal, and the kiosk contains an element that detects the fluorescent signal.

FIG. 17. The processes of the instant invention may optionally also be executed in a kiosk that can deliver liquid that is refrigerated so that the enzymes, triphosphates, and other unstable components required for the amplification are delivered by the kiosk. Other innovative features are described as elsewhere, including the collection of saliva on a ball of pre-selected size. That ball too may be optionally flavored, so as to encourage saliva collection. The ball may as well be semiporous, most preferably on its outer layers, to provide space into which saliva liquid carrying virus may enter. Also optionally, the ball may carry on or near its surface, material that selectively binds to the coronavirus, including without limitation an aptamer that binds the coronavirus, or an antibody that binds the coronavirus.

In the latter case the liquid preferably washes the viral RNA from the surface of the tube at an elevated temperature.

FIG. 18. Real-time monitoring of DP-LAMP reaction as fluorescence intensity vs. time (min). For SARS-CoV-2, signal is detected between 5 to 15 min and for internal control targeting RNase P, signal is detected between 12-20 min for both nasal swabs and saliva samples.

FIG. 19. Determination of LOD and threshold times in saliva samples spiked with heat-inactivated SARS-CoV-2 in bi-plexed fashion. Two ratios of CoV-2 primer mixes were tested (Top) Doubled the amount of 10X primer mix for CoV-2 (3.5 μL) and used 1.75 μL of 10X RNasePv2-3 set. LOD is ~80 template copies/μL within 20 min. Internal control RNasePv2-3 primer set generates signal within 18 minutes. (Bottom) Halved the amount of 10X primer mix for CoV-2 (1.75 μL) and used 1.75 μL of 10X RNasePv2-3 set. LOD is -300 template copies/μL within 20 min. Internal control RNasePv2-3 primer set generates signal within 18 minutes.

FIG. 20. (left) Representative HPLC of a mixture of relatively pure components in the presently preferred ratio with impurities less than 10% in each component, giving the results in Example 10. (right) Representative HPLC of a mixture of components in the presently preferred ratio with impurities equal or greater than 10% in each component, giving results in Example 10.

DESCRIPTION OF THE INVENTION

The displaceable probe LAMP (DP-LAMP, US Patent 10920267) is used, with extensive experimentation required to components and their relative concentrations to generate a Ct substantially less than 20 minutes, and less than 15 minutes, with 1000 targets. In its implementation to detect COVID-19, it is initiated by a reverse transcriptase (RT), DR- RT-LAMP. In its general form, before extensive experiments discovered specific probe sequences, specific purities, and specific component relative concentrations, DP-RT-LAMP uses six primers binding eight distinct regions within a target RNA (Fig. 1A). It runs at constant temperatures from 62° C to 72° C, and uses a reverse transcriptase and a DNA polymerase with strong strand displacing activity (most preferably, a Bst DNA polymerase). During its initial stages, forward and backward internal primers (FIP and BIP), with outer forward and backward primers (F3 and B3), form a double loop structure. This becomes the seed for subsequent LAMP. Amplification rate is further improved by loop primers (LF and LB), designed to bind single stranded regions of the loops. These yield concatemers with multiple repeating loops.

In DP-RT-LAMP (as in DP-LAMP), signal is created by a displaceable probe, a short DNA carrying a 3'-fluorophore that is displaced from a complementary oligonucleotide as the desired amplification is completed. That complementary oligonucleotide has a 5'-quencher, and carries a tag that is a primer that binds to one of the loops in the initial LAMP double loop structure (Fig. 1A). Thus, each probe is delivered to the amplification mixture as a target-sequence-independent double-strand probe region and a single-stranded target-priming region.

In the DP architecture, in the absence of target, no fluorescence is observed due to quenching of fluorophore by a quencher in the undisplaced duplex. In the presence of target, the single-stranded portion of the quencher probe binds to the target and is extended. Further polymerase extension by reverse primers displaces the quencher strand from the fluorescently labeled strand, allowing the emission of fluorescence and its analysis in real-time. As a consequence of the displacing process, "S-shaped" curves appear in a plot of fluorescence versus time, similar to RT-PCR and similar Ct (or Tt-threshold time) analyses (Fig. IB).

The displaceable probes can have sequences that have no substantial similarity to the sequence of any portion of the target analyte. This allows the fluorescently tagged displaced probe to be captured, either during or after the amplification. The fluor is preferably fluorescein (FAM), but any of a wide range of fluors in the art may be used. In the preferred embodiments, fluorescein signal with 10000 target molecules emerges in ca. 20 min and visible to human eye. In the most preferred embodiments, signals emerge in less than 15 minutes.

Signals may be visualized in an observation box that uses a blue LED to excite the fluorescein, and an orange filter to allow the emission light to be observed without interferences with the excitation light (Fig. 1C). Fluorescence from other fluors, as known in the art, can be observed using excitation light and filters appropriate for other fluors.

Several fluors can be used with several targets. In one implementation, FAM was used for SARS-CoV-2 detection and the internal control targeting the human RNase P RNA uλ_ex-λ_em=495 nm-520 nm, color observed with excitation at 470 nm, green). JOE was used for RNase P gene for multiplexed LAMP experiments (λ_ex-λ_em=529 nm-555 nm, excitation at 470 nm, yellow) as well as Cy5 (λ_ex-λ_em=648 nm-668 nm). Iowa Black FQ (Iowa Black RQ for Cy5) was used as a common quencher with absorption range of 420-620 nm (500-700 nm for Iowa Black RQ).

This process was used to measure the sensitivity of a specific DP-RT-LAMP primer set (CoV2-W3, SEQ ID NO: 1 through SEQ ID NO: 8) that had been selected from three trial sets that targeted the spike region of the CO VID-19 virus genome. RNA targets were prepared by transcription of a DNA template (~230 nt). Varying concentrations of RNA was used to determine assay sensitivity, following the procedure of [Glushakova, et al. (2017) Detection of chikungunya viral RNA in mosquitoes on cationic (Q) paper based on innovations in synthetic biology. J. Virol. Methods 246, 104-111][Yaren et al. (2016), op.

With this target, limits of detection (LODs) were 5 copies/assay, giving a threshold time (Tt, equivalent of Ct) of 22.5 min (Fig. 2, Table 1). When the 230mer RNA target was replaced by the complete RNA genome (Twist Biosciences, SARS-CoV-2 RNA), the sensitivity dropped to 100 copies/assay with Ct = 25.3 min (Fig. 3, Table 1).

Table 1. Sensitivity measurements in initial conditions

Extensive experimentation was then done to explore the sensitivity of the assay and the Ct that it delivered by changing conditions. These included:

(a) adding a second reverse transcriptase (SuperScript IV (SSIV) to the WarmStart reverse transcriptase (WS-RTx, NEB) already present,

(b) changing the reaction buffer,

(c) adding random hexamers (12 μM ),

(d) adding excess reverse primer (B3 primer), and

(e) varying the incubation temperature (Table 2, Table 3). Table 2. Improvement in the LOD using CoV2-W3 primer set and full-length RNA template

(Twist) by modifying DP-RT-LAMP conditions.

* Sporadic NTC issue.

Samples were first incubated at 55° C for 10 min, then at 65° C for 50 min.

Each reaction mixture was pre-incubated at 55° C for 10 min to ensure formation of sufficient cDNA by the warm start RT. This was then followed by incubation at 65° C. Table 3. Variety of DP-RT-LAMP components tested with full length RNA target (Twist).

These modifications improved sensitivity with the full-length COVID-19 RNA genome; LODs improved to 10 copies/assay. This compares favorably with SARS-CoV-2 colorimetric assay from New England Biolabs, which has a reported LOD of 500 copies/assay [Zhang et al. (2020) Rapid Molecular Detection of SARS-CoV-2 (COVID-19) Virus RNA Using Colorimetric LAMP. medRxiv. 2020:2020.02.26.20028373. doi: 10.1101/2020.02.26.20028373]. However, use of 5X SSIV buffer gave fluorescent signal in absence of target (no template controls, NTCs). This drove the choice of the presently preferred conditions that (i) use the original buffer, (ii) WS-RTx as the only reverse transcriptase, (iii) in the presence of random hexamers, and (iv) or excess B3. These conditions gave no "NTC problem" up to 60 minutes, with an LOD of 10 copies/assay. Refining these conditions further, better Ct values were observed with excess B3 than with random hexamers. Therefore, excess B3 primer was used in further DP-RT-LAMP experiments.

Table 4. Presently preferred conditions for the DP-RT-LAMP

Developing DP-RT-LAMP with high sensitivity and low Ct with heat-inactivated virus isolate The LOD was assessed using authentic, non-synthetic virus that had been heat- inactivated (SARS-CoV-2 isolate, inactivated at 65° C for 30 minutes, BEI Resources). This was also used to "spike" sample from nasal swabs and saliva samples. Three conditions previously tested for the synthetic RNA (Twist Bioscience) were again tested for the BEI target. The best sensitivity (10 copies/assay) was achieved with "Condition 1", using the NEB isothermal amplification buffer, excess B3 primer, and WS-RTx with incubation at 55° C for 10 min (initially), followed by further incubation at 65° C 50 min (Fig. 4 and Table 4). With the current modifications in DP-RT-LAMP protocol, another DP-RT-LAMP primer set was designed to target the N gene of SARS-CoV-2. We also designed a DP-RT- LAMP primer set to target the human RNase P gene. Detection of the amplicon from human RNAse P was intended to serve as an internal control to assess the adequacy of the sample collection.

The primer set targeting S gene (CoV2-W3) gave an LOD of 10 copies/assay in 16 min; the fluorescence signal arising from fluorescence was excited at 470 nm (typically an LED) and visualized through an orange filter to block the excitation light (Fig. 5A, 5B, and 5C). The primer set targeting the N gene (within the BEI sample) had an LOD of 25 copies/assay within a 12 min Tt. The system targeting the human RNase P gene had an LOD of 44 copies/assay, within a 16 min Tt (Fig. 5D and 5E). Threshold times were compared to RT-qPCR where N gene and RNase P gene was detected in multiplex format. For this comparison, Ct values were converted to corresponding Tt values; DP-RT-LAMP was found to be very similar to multiplex RT-qPCR in terms of sensitivity, but was also faster than RT- qPCR. Comparative values are in Table 5 and Table 6.

Table 5. Performance of DP-RT-LAMP for virus and human targets

Table 6. Performance of standard RT-PCR for virus and human targets

Simple sample preparation of nasal swabs and saliva samples

For a test to rapidly identify carriers who present a risk, sample preparation must be minimal, and instrumentation must be "field-deployable". Several have sought low sample preparation workflows, as RNA purification from biological samples is time consuming and timely delivery of test results can be impaired with limited supplies of sample purification kits [Rabe & Cepko (2020) op cit.][Bhadra et al. (2020) op cit.] [Anahtar et al. (2020) op cit.][Dao et al. (2020) op. cit.].

To meet these specifications, three protocols for SARS-CoV-2 were tested. The behavior of the virus itself defines the sampling procedure. False negatives arising from defective sampling are often as problematic as (or more problematic than) false negatives arising from failure of the assay. Fortunately, the life cycle of SARS-CoV-2 appears to allow simple sampling, with even mid-turbinate sampling being adequate, as well as saliva sampling [Broughton et al. (2020) CRISPR-Cas 12-based detection of SARS-CoV-2. Nature Biotechnology. 2020;38(7):870-4.] [Srivatsan et al. (2020) Preliminary support for a “dry swab, extraction free” protocol for SARS-CoV-2 testing via RT-qPCR. bioRxiv. 2020:2020.04.22.056283].

Therefore, a preferred protocol uses dry mid-turbinate or anterior nasal swabbing as a collection method, and relies on the positive control targeting human RNase P to ensure that the collection was adequately aggressive. Post sampling, swabs were eluted in various elution/ inactivation buffers. An aliquot from the elution solution was added directly to the DP-RT-LAMP mixture, and analyzed in real-time and by visualization of end-point fluorescence (Fig. 6A). Spiked saliva (with saliva alone as the negative control) diluted with concentrated inactivation buffer (1 : 100 ratio of buffer to saliva) was also used. An aliquot of the resulting mixture was added to the DP-RT-LAMP mixture and analyzed similarly (Fig. 6A)

Alternatively, saliva can be placed on "Q-paper", a cellulose filter paper that carries quaternary ammonium groups. Q-paper has been previously used to capture arboviral RNA from single mosquitoes after a drop of ammonia is added to the carcasses [Yaren et al. (2017) op. cit.]. In this work, the Q-paper holding the viral RNA could be added directly to the DP- RT-LAMP mixture without any sample preparation. The fluorescence can be analyzed in real-time or by end-point visualization, again using blue LED excitation with fluorescence observed through an orange filter (Fig. 6A). The fluorescence can also be seen in a hand-held observation box.

Real-time analyses were performed on a (Roche LightCycler® 480). Performance was equally satisfactory when readout was done on a portable Genie® II instrument, from Optigene. Genie® II processes 16 samples simultaneously using the FAM-channel (483-533 nm). The data outputs are similar to those obtained with the more expensive real-time PCR instrument. Genie® II offers positive/negative results with Tt values as good as obtained with the PCR instrument, but at a fraction of the cost and useable in the lobby of a workplace, a courtroom, or a school (Fig. 6B).

Presently preferred workflows for 2019-nCoV (COVID-19) are, with preferred volumes:

(a) Dry nasal swabs are the sample method, obtained by mid-turbinate swabbing.

(b) Swabs are eluted in a sample preparation buffer, typically 50-1000 μL.

(c) An aliquot (typically 4 to 10 μL, maximum 25 μL) from the sample preparation buffer holding the eluate is added to a DP-RT-LAMP mixture.

(d) The mixture is heated at 65° C for 15-30 minutes.

(e) End-point results are visualized using blue LED and orange filter.

As an alternative presently preferred workflow, saliva (typically 100-1000 μL) is spit into a tube, and a sample (typically 5-10 μL) is added directly to a portion of sample preparation buffer briefly. Here, aliquot of that mixture is added to the DP-RT-LAMP mixture (typically 25-100 μL). End-point results are visualized as with the nasal swab samples.

As an alternative presently preferred workflow, saliva (typically 10-50 μL) is placed onto a small square (typically 3-5 mm) of quaternary ammonium modified paper (Q-paper). The Q-paper coated with saliva is directly introduced into DP-RT-LAMP mixture (typically 50-200 μL) without further manipulation. End-point fluorescent signal is visualized using blue LED and orange filter, as before. The Q-paper square is observable, but does not hinder the analysis.

As an alternative presently preferred workflow, to replace end-point visualization, DP-RT-LAMP experiments are also run in real-time using a Genie® II (Optigene, UK) instrument. This allows the appearance of fluorescence arising from the displaced probes to be visualized as the amplification proceeds. Representative curves are shown in various drawings. Validation of DP-RT-LAMP assay with contrived nasal swabs

Having established work flow parameters, various elution/inactivation buffers with or without a heat step gave a presently preferred protocol. Fig. 7A summarizes methods used to process mid-turbinate or nasal anterior swabs. TE (Tris-HC1 pH 7.0, 1 mM EDTA) as an elution buffer gave LODs

1000 copies/assay, with Ct values of

30 min. The procedure of Rabe and Cepko [op. cit.] was used, with swabs eluted in buffer with NaOH, TCEP and EDTA, incubated (95° C 5 min), and spiked with known of BEI template. These gave 100 copies/assay LODs (Tt

23 min).

Despite its promise, this approach did not give reproducible results when nasal swabs were spiked with inactivated virus prior to the 95° C heating step. A similar problem was observed when same buffer was combined with Chelex-100, in a workflow that incorporated two heating steps, one at 56° C for 15 min and a second at 95° C for 5 min. Dao Thi et al. [op. cit.] also report similar results when nasal swab elution mixtures were spiked with RNA, and then heated (95° C, 5 min). However, treatment of clinical samples using the method developed by Rabe and Cepko [op. cit.] with heating at 95° C for 5 min did not cause a decrease in assay sensitivity.

To further simplify sampling work-flow, NaOH was replaced with sodium citrate (in a pH range of 5 to 7, preferably pH 6.5) and added TCEP, EDTA, LiCl and Chelex-100. Swabs were eluted at room temperature without any additional heating step. Here, 100 copies of viral RNA were detectable per assay within 16-18 min. In addition to real-time analysis, end-point fluorescent images were also visible to human eye at 100 copies/assay (Fig. 7C).

The sensitivity with nasal swab samples was analyzed using contrived nasal swabs. Here, 500 RNA copies/assay were detected consistently at 100%. Ca. 200 copies/assay were detected with 50% efficiency, and 100 copies of RNA/assay were detected at 20% efficiency. The internal control that targets the RNase P gene was detected at 100%, indicating that the sample collection was sufficiently aggressive (Figs. 8A and 8D).

Validation ofDP-RT-LAMP assay with contrived saliva samples

Crude saliva was first added to DP-RT-LAMP without any treatment, with a saliva:LAMP reaction mixture ratio of 1 :5. As shown in Fig. 7B, 1000 copies of RNA were detectable only after 50 min. This suggested that crude saliva was not useable as a sample on its own. Suspecting that RNA might be rapidly degraded in saliva, saliva samples spiked with DNA were tested (Fig. 9). Here again, the emergence of the signal was substantially delayed, even though the delay was not as large as with the analogous RNA. We then tried nasal elution buffers in more concentrated form. Here, saliva (100 μL) was treated with 100X buffer (0.25 M TCEP, 0.1 M EDTA, 0.1M NaOH or Na citrate, 1 μL) with or without 15% Chelex-100, and with or without a heating step. Multiple runs showed the most reliable results with inactivation buffer containing TCEP, EDTA, sodium citrate, LiCl and Chelex- 100; the LOD was - 100 copies/assay. Fluorescent signals obtained from positive samples were clearly differentiable from those arising from samples lacking target (Fig. 7C).

As an alternative to this saliva sampling method, saliva was absorbed on to Q-paper, which was placed after a brief time (5 min) at room temperature (to simulate how processing might occur in a workplace lobby) directly into the DP-RT-LAMP mixture. Analogous to what is seen with mosquito carcasses [Yaren et al. 2017, op. cit.\, 100 copies of viral RNA were detected by spotting SARS-CoV-2 RNA onto saliva coated Q-paper and directly amplified by DP-RT-LAMP. Visualization of positive signals was done in the 3-D printed box (Fig. 7D).

Sensitivities of assays with two saliva samplings were analyzed using contrived saliva samples. Here, 200 copies of RNA/assay could be detected by both methods with 100% detection rate with a small sample size (5 cases); 100 copies of RNA were detected with 50% efficiency using 100X inactivation solution, and with 40% efficiency using Q-paper for sampling. Additionally, an internal control targeting RNase P gene was detected at 100% in both methods (Fig. 8A-C).

Table 7. Performance of assay with whole coronavirus (BEI Resources)

Table 8. Variety of DP-RT-LAMP components tested with full length RNA target (Twist).

Multiplex detection of SARS-CoV-2 and RNase P

An assay robust for workplace entrance use must incorporate a signal to indicate that sampling is sufficiently aggressive. The DP architecture allows simultaneous detection of viral RNA and human RNase P gene in single tube. To show this, we spiked varying amounts of viral RNA into 440 copies of purified human RNA. Using the LightCycler® 480 and three fluorescent channels, 10 copies of SARS-CoV-2 RNA could be detected in the presence human RNA in two-plex format when equal amount of the two (virus and human) 10X LAMP primer sets were present.

When viral RNA was present in higher amounts, the signal for the positive control was delayed to 32.5 minutes, instead of appearing 21-23 min. This is presumed to reflect the two amplification processes competing for some of the same LAMP amplification resources.

A similar degree of sensitivity was achieved when viral RNA was ~ 1000 copies/assay (Figs. 10A-C). We spiked 10⁶ and 10³ copies of viral RNA into processed nasal swab samples and ran multiplexed assays. When 10⁶ copies of RNA were present, the RNase P gene was not detected, since the viral amplifications consumed LAMP resources (e.g. dNTPs). Thus, the presence of the virus signal itself proves that the LAMP amplification is working; a separate positive control is not needed. In the presence of 1000 copies of RNA, both targets were detected efficiently with Tt value of RNase P being very similar to its value without viral RNA (Figs. 10E-F).

Lyophilization ofDP-RT-LAMP reagents For public space use, reagents used must robustly survive transport and storage in amateur hands. Accordingly, lyophilized reagent mixtures were prepared and their performance tested.

The presently preferred process to create lyophilized reagents begins by removing glycerol from enzymes as delivered by various suppliers (e.g. New England Biolabs). For this, an ultrafiltration column with a lOkDa cut-off limit was loaded with DP-RT-LAMP enzymes. The enzyme storage buffer containing glycerol was exchanged with glycerol-free version enzyme storage buffer. Then, 10X primers mix and dNTPs were added. The mixture was lyophilized for 4 to 6 hours, leaving dry reagents as a white fluffy powder (Fig. 11 A). Dry reagents were activated by rehydration buffer containing necessary salts and detergents and varying amount of viral RNA (1000 to 100 copies/assay). Alternatively, rehydration was done with contrived nasal and saliva samples (1000 copies of spiked RNA). Tt values were similar to their non-lyophilized versions (Fig. 11B) and clear visual fluorescent signals were recorded in all cases (Fig. 11C).

The device that reads the generated fluorescent signals

A schematic of the presently preferred implementation of the device invention is shown in FIG. 14. The personal coronavirus detector resembles an electronic cigarette. In the presently preferred implementation, it is battery powered. It contains a slot into which can be placed a disposable cartridge that, in its presently preferred implementation, performs a DP- RT-LAMP based on a sample of saliva. The disposable generates, in the presently preferred implementation, green fluorescence in 15-20 min if the saliva contains coronaviral RNA at a level sufficient that the user creates a forward infection risk for an environment that (s)he enters. In the presently preferred implementation, that fluorescence is observed via illumination with a 410 nm blue LED, and visual or cell phone capture of the fluorescence seen through an orange filter.The utility of this device is apparent, as it will allow individuals to enter a dentist office, board an airplane, enroll for a semester at a university, or enter a workplace, with enhanced confidence that they will not contaminate fellow travelers, students, or workers. Its output may serve as an entry badge to public spaces. Also useful, the device may be used by individuals who have COVID who are self-quarantining. Through a cell phone app, they will deliver to epidemiologists who are working remotely daily reports of symptoms and viral loads. This will allow such individuals to constitute a distributed research lab to build a database of information about coronavirus biology. EXAMPLES

These examples illustrate several presently preferred embodiments of the instant invention. Alternatives may be substituted as is presently understood in the art.

Example 1. Presently preferred of the DP-RT-LAMP primers and displaceable probes

LAMP primers and strand displaceable probes were purchased from Integrated DNA Technologies (IDT, Coralville, IA) (Table 9). Strand-displaceable probes were 5’-quencher labeled with Iowa Black-FQ (IBFQ). Fluorescently labeled displaceable probes partially complementary to the quencher labeled probes were 3 ’-labeled with FAM. Alternatively, for multiplexing purposes, internal control probes targeting the human RNase P gene were 5’- labeled with IBFQ and 3 ’-labeled with JOE.

Underlined sequences are double strand segments of strand-displacing probes. FAM was used for SARS-CoV-2 detection and internal control targeting RNase P gene (λ_ex- λ_em=495 nm-520 nm, color observed with excitation at 470 nm, green), JOE was used for RNase P gene for multiplexed LAMP experiments (λ_ex-λ_em=529 nm-555 nm, color observed with excitation at 470 nm, yellow). Iowa Black FQ was used as a common quencher with absorption range of 420-620 nm. This quencher is typically used with fluorophores that emit in green to pink range of the visible spectrum. Presently preferred primers are listed in Table 9. Substantially identical primers are those that do not differ by more than two nucleotides within them, or by more than two nucleotides in length. The standard nucleotides in these primers may be replaced by their SAMRS equivalents.

Example 2. Targets to validate the invention with SARS-CoV-2 RNA as the target IVT RNA fragment preparation

Target RNA was generated from synthetic DNA fragments of the viral genes of interest. Synthetic DNA gene fragments were ordered from IDT as gBlocks. An initial PCR introduced the T7 promoter. Next, 150 nM of PCR product was used in T7 RNA transcription reaction (50 μL total volume); the reaction mixture was incubated at 37° C for 16h. DNA templates were removed by digestion with DNase I, the mixture was phenol-CHCL extracted, and the RNA was recovered by EtOH precipitation. The product RNA was quantified using a Nanodrop UV spectroscopy, and reference materials with known concentrations were prepared in serial dilutions in TE buffer (10 mM Tris pH 7.0, 1 mM EDTA) and aliquots were stored at -80° C.

Fully synthetic SARS-CoV-2 RNA

Synthetic SARS-CoV-2 RNA Control was from Twist Bioscience (MT007544.1, 1x10⁶ RNA copies/μL). It was used for initial limit of detection (LOD) studies. Appropriate dilutions were made in 1 mM Na citrate pH 6.5, 0.4 U/μL RNase inhibitor (NEB, Ipswich, MA) and aliquots were stored at -80° C.

Heat-inactivated SARS-CoV-2 isolate

Authentic SARS-CoV-2, isolate USA-WA 1/2020, was obtained through BEI Resources (cat no. NR-52286, 1.16x10⁹ genome equivalents/mL). This virus has been inactivated by heating at 65° C for 30 minutes. Target dilutions were made in 1 mM Na citrate pH 6.5, 0.4 U/μL RNase inhibitor (NEB, Ipswich, MA) supplemented with 0.15 ng/μL human RNA and aliquots (100 μL) were stored at -80° C. This target was used to determine final LODs and spike-in experiments where minimal sample preparation methods were sought for nasal swab and saliva sampling.

Example 3. The presently preferred DP-RT-LAMP assay for 2019-nCoV

12.5 μL of 2X WarmStart LAMP master mix (NEB) was combined with 2.5 μL of 10X LAMP primer set, 1 μL of excess B3 primer (300 μM ), 0.175 μL of dUTP (100 mM, Promega), 0.5 μL of Antarctic Thermolabile UDG (lU/μL, NEB), 0.5 μL of RNase inhibitor (40U/μL, NEB), 2 μL of template RNA or inactivated virus isolate, and 6 μL of nuclease-free water (or briefly processed nasal/saliva samples) to bring the final reaction volume to 25 μL.

10X LAMP primer set consists of 16 μM each of FIP and BIP, 2 μM each of F3 and B3, 5 μM LF (or LB for CoV2-v2-4 set), 4 μM LB (or LF for CoV2-v2-4 set), 150 nM quencher-bearing probe, and 100 nM of fluorophore-bearing probe. Reactions were monitored in real-time using either a LightCycler® 480 (Roche Life Science, US) or a Genie® II (Optigene, UK) instrument. 8-strip PCR tubes were first incubated at 55° C for 10 min followed by incubation at 65° C for 45-60 min. During the 65° C incubation, fluorescence signal was recorded every 30 seconds using FAM/SYBR channel of the instrument. End- point observation of the fluorescence signal generated by strand displaceable probes was enabled by blue LED light (excitation at 470 nm) through orange filter of SafeBlue Illuminator/ Electrophoresis System, MBE-150-PLUS (Major Science, US) or 3D printed observation box (Firebird Biomolecular Sciences, US). Example 4. The preferred process from nasal swabs

Mid-turbinate and anterior nasal swab sampling

Clean WIPE Swab, 3” Semi-Flexible bulb tip (HT1802-500, Foamtec International) is used for nasal sampling. Each nostril is swabbed for at least 10 seconds using the same swab. The swab is placed in sterile 15 mL falcon tube and stored at 4° C until processing.

The nasal swab was eluted in 100 μL of buffer solution (1 mM Na citrate pH 6.5, 2.5 mM TCEP, 1 mM EDTA, 10 mM LiCl, 15% Chelex-100) by brief vortexing. Swabs were then removed and elution solution was briefly spun down. 6 μL of sample elution was combined with 2 μL of inactivated virus (BEI resources) or water (no template control) and added to 17 μL of DP-RT-LAMP reaction mixture (12.5 μL of 2X WarmStart LAMP master mix, 2.5 μL of 10X LAMP primer set, 1 μL of excess B3 primer (300 μM ), 0.175 μL of dUTP (100 mM,), 0.5 μL of Antarctic Thermolabile UDG (lU/μL, NEB), 0.5 μL of RNase inhibitor (40U/μL, NEB). Samples were then incubated and analyzed in real-time as described above.

Other methods were tested, included TE buffer (10 mM Tris-HCl pH 7.0, 1 mM EDTA) for eluting nasal swabs. Another method used an inactivation buffer containing 10 mM NaOH, 2.5 mM TCEP and 1 mM EDTA followed by incubation at 95° C for 5 min. This buffer solution was then coupled with 15% Chelex-100 and extended heating (56° C 15 min and 95° C 5 min).

Saliva to DP-RT-LAMP testing

Saliva samples are collected before brushing the teeth or 1 hour after brushing the teeth. A sample of saliva is preferably ~1 mL, collected in sterile 5 mL falcon tube and stored at 4° C until processing and samples processed within 1 hour. Alternatively the saliva may be collected by having a saliva provider suck on a ball. This ball may optionally be semi-porous, or be flavored.

100 μL of 15% Chelex-100 in a 1.6 mL microcentrifuge tube was spun briefly and supernatant was removed. To that, 100 μL of saliva mixed with 1 μL of concentrated sample preparation solution (0.1 M Na citrate pH 6.5, IM LiCl, 0.25 M TCEP, 0.1 M EDTA) was added. Each sample was briefly vortexed and spun down to settle Chelex-100 down. 6 μL of saliva sample was combined with 2 μL of inactivated virus (BEI resources) or water (no template control) and added to 17 μL of DP-RT-LAMP mixture and the reaction was done as previously described. Collecting saliva on Q-paper and DP-RT-LAMP testing

Q-paper preparation

Whatman filter paper (1 gram) was immersed in 50 mL of 1.8% aq. NaOH solution for 10 min. Treated paper was collected by filtration and immersed in aq. EPTMAC (2,3- epoxypropyl) trimethylammonium chloride) solution for 24h at RT. The mass ratio of EPTMAC to filter paper was 0.28 to 1. Cationic (Q) paper was collected by vacuum filtration and neutralized with 50 mL of 1% AcOH. Final product was washed three times with ethanol (96%) and dried at 55° C for Ih. Q-paper sheets were cut into small rectangles (~ 0.5 x 0.2cm) for saliva collection.

Saliva on Q-paper

Q-paper is first dipped into saliva samples and soaked for 5 seconds, then air dried for 5 min. Q-paper containing saliva is directly inserted into 50 μL of DP-RT-LAMP mixture (25 μL of 2X WarmStart LAMP master mix, 5 μL of 10X LAMP primer set, 2 μL of excess B3 primer (300 μM ), 0.35 μL of dUTP (100 mM,), 1 μL of Antarctic Thermolabile UDG (lU/μL, NEB), 1 μL of RNase inhibitor (40U/μL, NEB) and 16 μL of nuclease-free water) and reaction was proceeded as described above.

Example 5. Biplexed DP-RT-LAMP to detect SARS-CoV-2 and RNase P (Internal Control)

BEI 2019-nCoV thermally inactivated virus in human RNA background

Varying amounts (10⁵, 10⁴, 10³, 10², 10 and 5 copies) of heat-inactivated SARS-CoV- 2 (BEI resources) spiked with 440 copies of human RNA, 1.25 μL of 10X CoV2-W3 LAMP primer set (F AM-labeled probe) and 1.25 μL of 10X RNaseP-2 LAMP primer set (JOE- labeled probe) were added to DP-RT-LAMP mixture (25 pl total volume).

Multiplexed reaction mixtures were pre-incubated at 55° C for 10 min, then 65° C for 50 min and the fluorescence signals from three channels were recorded every 30 seconds using LightCycler® 480 (Roche Life Science, US) during 65° C incubation. Channel 483-533 is specific to FAM-labeled SARS-CoV-2 probe, channel 523-568 is an intermediate channel for both FAM- and JOE-probes, and channel 558-610 is specific to JOE-labeled RNase P probe.

BEI 2019-nCoV thermally inactivated virus spiked into nasal swab samples 6 μL of briefly processed nasal swab was combined with 2 μL of heat-inactivated SARS-CoV-2 isolate to give 10⁶to 10³ RNA copies per assay. Nasal samples spiked with targets were added to DP-RT-LAMP mixture containing CoV2-W3 and RNAseP-2 LAMP primers in equal amounts (1.25 μL each of 10X LAMP primer sets) to give a total 25 μL assay volume. Real-time analysis was performed as mentioned above using LightCycler® 480 with three fluorescence channels.

Example 6. Use of lyophilized reagents in DP-RT-LAMP

Dialysis example for 10 LAMP reactions

10 μL of Bst 2.0 WarmStart® DNA Polymerase (8U/μL, NEB), 10 μL of WarmStart® RTx Reverse Transcriptase (15 U/μL, NEB), 5 μL of Antarctic Thermolabile UDG (1 U/μL, NEB), 5 μL of RNase inhibitor (40 U/μL, NEB) was combined with 170 μL of dialysis buffer (10 mM Tris-HCl pH 7.5, 50 mM KC1, 1 mM DTT, 0.1 mM EDTA, 0.1% Triton X-100). 200 μL mixture was then placed in an ultrafiltration membrane (10 kDA cut-off limit, Millipore, Billerica, MA). Samples were centrifuged at 13,000 rpm for 8 min, then further washed with 250 μL of dialysis buffer twice to concentrate glycerol free enzyme mix down to 30 μL. 30 μL of enzyme mix was combined with 25 μL of 10X LAMP primer set, 10 μL of 300 μM B3 primer, 35 μL of dNTP mix (10 mM each of dATP, dCTP, dGTP and 5 mM each of dTTP and dUTP) and 25 μL of IM D-(+)-trehalose. Combined mixture was then distributed into 8- strip PCR tubes as 12.5 μL aliquots. Samples were frozen by liquid nitrogen and lyophilized for 4-6h. Lyophilized reagents were stored at RT and tested within 7 days.

Reconstitution of lyophilized LAMP reagents

6 μL of sample (nasal swab/saliva or RNA template) was mixed 19 μL of reconstitution buffer (2.5 μL 10X isothermal amplification buffer (NEB), 1.5 μL 100 mM MgSCL and 15 μL of nuclease-free water) and added into lyophilized reagents. DP-RT- LAMP reactions were monitored in real-time using Genie II; fluorescence signal was visualized as described above.

Example 7. Biplexed DP-RT-LAMP to detect SARS-CoV-2 and RNase Pv2-3 (Internal Control)

In 25 μL LAMP reaction, 3.5 μL of 10X LAMP mixture (1.75 μL of CoV-2 primer mix with FAM probe and 1.75 μL of RNaseP primer mix with Cy5 probe) was mixed with 12.5 μL of WarmStart LAMP 2X Master Mix (NEB), 0.5 μL of Antarctic thermolabile UDG (1 U/μL), 0.5 μL of RNase Inhibitor, Murine (40 U/μL), 0.2 μL of 100 mM dUTP and 0.25 μL of ET-SSB (500 ng/μL) and 5 μL of sample. (FIG. 18).

Table 10. Present preferred primer concentrations

Example 8. Testing conditions for nasopharyngeal swabs stored in VTM

Swab 1 was eluted in 200 μL Firebird buffer (ImM Sodium Citrate, ImM EDTA, 2.5mM TCEP and lOmM LiCl, 5% Chelex-100) +95° C for 5 minutes. Swab 2 was eluted in 200 μL VTM (Corning Transport Medium 25-500-CV) +95° C for 5 minutes.

Table 11. LAMP conditions: 55° C for 10 minutes then 65° C for 45 minutes

Swab 3 was eluted in ImL of VTM (Corning Transport Medium 25-500-CV) +95° C for 5 minutes. Dilutions were made after the heat step.

Table 12. 55° C for 10 minutes then 65° C for 30 minutes

Example 9. Biplexed DP-RT-LAMP to simultaneously detect SARS-CoV-2 and RNase Pv2-3 (Internal Control) of saliva samples stored in VTM

LAMP assay mixture uses the compositions described in Example 7. For saliva testing, 0.5 mL of saliva sample was mixed with 0.5 ml VTM (Red, Mole Bioscience) and heat inactivated at 95° C for 5 min, then 5X diluted with water. 5 μL of the sample was added to LAMP assay (25 μL total). RNA template (heat inactivated Covid 19, Isolate USA-WA 1/2020 (cat no. NR-52286, BEI Resources) was diluted to obtain 6600, 4356, 2875, 1897,1252, 827, 546 copies/μL. 2.5 μL of each solution was added to each sample. Two 10X primer mixes were tested: A first mix used was 3.5 μL of CoV-2 primer mix and 1.75 μL of RNasePv2-3 set. A second mix use 1.75 μL of each of CoV-2 and RNasePv2-3 primer mix. CoV-2 RNA templates were successfully amplified by both primer mix ratios (FIG. 19). 3.5 μL and 1.75 μL of CoV-2 primer mix detected template concentrations down to ~80 copies/μL and -300 copies/μL within 20 minutes. Internal control RNasePv2-3 primer set generates signal within 18 minutes.

Example 10. Influence of concentration and purity of components on Ct values

The essence of the instant invention are specific primer sequences, purities, and relative concentrations that give presently preferred Ct values of less than 15 minutes with 10000 target molecules. The most preferred values are lower with fewer target molecules. This is not obtained by other isothermal architectures and, in particular, by LAMP. Thus, in the most preferred architecture, with data shown in Table 15, the Ct values are less than 10 minutes with just 1000 targets. The ordinary artisan will understand that lower Ct values will be obtained with more targets, and higher Ct values will be obtained with fewer targets.

Experimentation quantitated the impact of the concentration, purity, and relative amounts of various components of the presently preferred sequences on outcome. In general, increasing the relative amounts of FIP and BIP can dramatically slow down LAMP, increasing Ct to an undesirable amount. Thus, these are called "critical primers".

In contrast, the relative amounts of LB and LF primers can often be doubled without slowing LAMP. The same is true for F3 and B3. Table 13 provides illustrative data. Based on these and other results, this disclosure teaches that the concentration range of the critical components must be constrained to within 20% of the disclosed values, more preferably to within 10%. Table 13. 55° C for 10 minutes then 65° C for 30 minutes targeting plasmid DNA

A similar importance is assigned to purity. FIG. 20 shows representative purities of components, the left panel showing "more pure" materials, and the right panel shows "less pure" materials. Table 14 shows the impact on Ct values of the different purities on a model reaction.

Table 14.

Example 11. Demonstration of low Ct values with one step LAMP on crude samples

Table 15. Ct values for the presently preferred assay on samples

Claims

37 CLAIMS What is claimed is:

1. A process to detect a segment of a target RNA presented in a biological sample, wherein said segment serves as a template, wherein said process comprises a displaceable probe reverse transcriptase loop amplification of said template in an amplification mixture, wherein (a)(1) said template has six regions, in the following order from the 3'-end to the 5'-end, termed F3c, F2c, F1c, B1, B2, and B3,

(a)(2) said amplification mixture is provided an external primer, termed F3, that is substantially Watson-Crick complementary to F3c,

(a)(3) said amplification mixture is provided a first internal primer that has two regions, one F1c towards its 5'-end and the other F2 towards its 3'-end, where the two regions are joined by a linking oligonucleotide, and where F1c is substantially Watson-Crick complementary to F1 and F2 is substantially Watson-Crick complementary to F2c, wherein polymerase-catalyzed extension of said first internal primer generates a first copy that comprises F1c, F2, F1, Bic, B2c, and B3c in the 5'- to 3' direction, wherein F1c is substantially Watson-Crick complementary to F1 , F2 is substantially Watson-Crick complementary to F2c, F1 is substantially Watson-Crick complementary to F1c, Bic is substantially Watson-Crick complementary to B1, B2c is substantially Watson-Crick complementary to B2, and B3c is substantially Watson-Crick complementary to B3, and then (a)(4) said amplification mixture is provided a second external primer, termed B3, which is substantially Watson-Crick complementary to B3c and

(a)(5) said amplification mixture is provided a second internal primer that has two regions, one Bic towards its 5'-end and the other B2 towards its 3'-end, where the two regions are joined by a linking oligonucleotide, and where Bic is substantially Watson-Crick complementary to B1 and B2 is substantially Watson-Crick complementary to B2c, wherein polymerase-catalyzed extension of said second internal primer generates a second copy that comprises Bic, B2, B1, F1c, F2c, and F1 in the 5'- to 3' direction, said second copy can form a structure having two loops, and

(a)(6) said amplification mixture is provided a tagged primer that is a DNA molecule comprising two regions, the first tag region carrying a fluorescence quenching moiety at or near its 5'-end and the second tag region substantially Watson-Crick complementary to a region between B1 and B2 or a region between F1 and F2, and 38

(a)(7) said amplification mixture is provided a displaceable probe that is a DNA molecule having a fluorescent moiety at or near its 3'-end, said displaceable probe being substantially Watson-Crick complementary to the first tag region, wherein said amplification mixture contains a reverse transcriptase, and

(b) said biological sample is delivered as

(b)(1) a nasal swab,

(b)(2) a nasopharyngeal swab,

(b)(3) a sample of saliva,

(b)(4) a sample in viral transport medium and wherein detection comprises the observation of a fluorescent signal arising from the displaceable probe either at the end of the amplification or during the amplification.

2. The process of Claim 1, wherein said target RNA is from CO VID-19 or one of its variants.

3. The process of Claim 2, wherein said fluorescent signal generated from not more than 10000 of said target RNA molecules crosses a threshold in less than 10 minutes.

4. The process of Claim 2, wherein said fluorescent signal generated from not more than 1000 of said target RNA molecules crosses a threshold in less than 10 minutes.

5. The process of Claim 2, wherein time to detection is less than 18 minutes with 100 template molecules per sample.

6. The process of Claims 1 through 5 that includes at least one more set of primers for a second displaceable probe reverse transcriptase loop amplification.

7. The process of Claim 6, wherein the target of a second displaceable probe reverse transcriptase loop amplification is human RNase P.

8. The process of Claims 1 through 7, wherein said primers comprise sequences that are substantially identical to SEQ ID NO: 1 through SEQ ID NO: 60.

9. The process of Claims 1 through 8, wherein one or more of the nucleotides within said primers probe is replaced by a nucleotide analogs shown in Figure 13.

10. The process of Claims 1 through 9, wherein one or more of the nucleotides within said tagged primer and probe are the nucleotide analogs shown in Figure 12.

11. The process of Claims 1 through 10, wherein said incubation temperature is from 50 to 70° C.

12. The process of Claims 1 through 11, wherein excess B3 is added.

13. The process of Claims 1 through 12, wherein the mixture is pre-incubated at 55° C prior to incubation at 60-70° C.

14. The process of Claims 1 through 13, wherein the sample is a nasopharyngeal swab, a nasal swab, or a saliva sample that is extracted with a solution that has a pH of 5 and 7.

15. The process of Claim 14, wherein the swab is subjected to an extraction with a solution that contains Chelex-100 without any heat step.

16. The process of Claims 1 through 15, wherein the LAMP amplification is done in the presence of Q-paper.

17. The process of Claims 1 through 16, wherein the detection is done by direct observation of the fluorescence with the human eye.

18. The process of Claims 1 through 17, wherein the amplification mixture comprises lyophilized reagents.

19. The process of Claims 1 through 18, wherein the oligonucleotide components contain less than 10% impurity.

20. A portable or handheld device to generate and detect a fluorescent signal arising if a segment of an RNA molecule is present in a sample, wherein said device comprises

(a) a slot wherein a disposable is placed,

(b) a microprocessor that controls a heater, wherein

(c) said heater warms a region of said disposable to a temperature between 50 and 70 °C, wherein

(d) said disposable contains reagents generate a fluorescent signal when viral RNA is present,

(e) said device contains a light that illuminates the region of the disposable that generates the fluorescent signal, and

(f) a port allows the user to visualize the fluorescent signal, wherein said fluorescent signal is generated by a process of Claims 1 through 19.

21. A kiosk that holds one or more slots that accepts a tube that contains a sample, and then delivers liquid to said tube and then warms a region of said tube to a temperature between 50 and 70 °C, wherein tube and/or said liquid contains reagents generate a fluorescent signal when viral RNA is present in said sample, and wherein a light in said kiosk illuminates a region of said tube that generates the fluorescent signal, and said kiosk contains an element that detects the fluorescent signal, wherein said fluorescent signal is generated by a process of Claims 1 through 19.

22. The kiosk of Claim 21, wherein said sample is delivered on or near the surface of a ball that has been exposed to saliva.