WO2021248053A2 - Dispositif de diagnostic du virus sars-cov-2 au point de soins et procédés d'utilisation de celui-ci - Google Patents

Dispositif de diagnostic du virus sars-cov-2 au point de soins et procédés d'utilisation de celui-ci Download PDF

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WO2021248053A2
WO2021248053A2 PCT/US2021/035977 US2021035977W WO2021248053A2 WO 2021248053 A2 WO2021248053 A2 WO 2021248053A2 US 2021035977 W US2021035977 W US 2021035977W WO 2021248053 A2 WO2021248053 A2 WO 2021248053A2
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nucleotide sequence
sample
composition
seq
primer
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PCT/US2021/035977
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WO2021248053A3 (fr
Inventor
Chang Hee Kim
Jacob Jerome TOKAR
Fernando R. MOREIRA
Xiao Jiang
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Godx, Inc.
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Publication of WO2021248053A2 publication Critical patent/WO2021248053A2/fr
Publication of WO2021248053A3 publication Critical patent/WO2021248053A3/fr
Priority to US18/049,552 priority Critical patent/US20230160025A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes

Definitions

  • coronavirus disease (COVID-19) pandemic caused by SARS-CoV-2 has exposed major vulnerabilities in our nation's healthcare system, including its inability to quickly develop and administer rapid and accurate tests for the infectious agent.
  • the first FDA-approved tests administered by the Centers of Disease Control and Prevention (CDC) and New York State Department of Health, which are based on reverse transcription polymerase chain reaction (RT- PCR), are unacceptably slow and expensive in view of the rapid spread of the virus. These tests require that samples are sent away to a laboratory for analysis. As a result, it takes 3 to 5 days from the time these tests are requested to receive results. Additionally, the test requires 6 hours of extensive labor and expensive instruments that are limited in availability.
  • RNA kits which are currently in short supply or backordered, causing bottlenecks in testing. Further, these kits also require specialized equipment (i.e a microcentrifuge or a vacuum manifold). In total, the currently available RT-PCR test could cost up to $1200.00 per patient.
  • One aspect of the invention is a composition for loop-mediated isothermal amplification of a target nucleotide sequence of SARS-CoV-2.
  • the composition for loop-mediated isothermal amplification of a target nucleotide sequence of SARS-CoV-2 comprises a F3 primer comprising nucleotide sequence TTGGTGCAGGTATATGCG (SEQ ID NO: 12) (5' - 3'); a B3 primer comprising nucleotide sequence ACATTGTACAATCTACTGATGTC (SEQ ID NO: 13) (5' - 3'); a FIP primer comprising a Flc nucleotide sequence TAGGCAATGATGGATTGACTAGCTA (SEQ ID NO: 14), a F2 nucleotide sequence TTATCAGACTCAGACTAATTCTCC (SEQ ID NO: 15), and a spacer therebetween (5 1 - 3'); and a BIP primer comprising a Blc nucleotide sequence AACTCTATTGCCATACCCACAAAT (SEQ ID NO: 16), a B2 nucleotide sequence TTGGTCATAGACACTGGTAG (SEQ ID NO:
  • the composition further comprises a loop forward primer comprising a nucleotide sequence CACTACGTGCCCGCCGA (SEQ ID NO: 18) (5' - 3') and a loop backward primer comprising a nucleotide sequence TTTACTATTAGTGTTACC (SEQ ID NO: 19) (5' - 3').
  • the target nucleotide sequence comprises SEQ ID NO: 70.
  • the composition for loop-mediated isothermal amplification of a target nucleotide sequence of SARS-CoV-2 comprises a F3 primer comprising nucleotide sequence GATTTTTGTGGAAAGGGCTATC (SEQ ID NO: 1) or
  • TTTTTGTGGAAAGGGCTATC (SEQ ID NO: 2) (5' - 3'); a B3 primer comprising nucleotide sequence CAAACCAGTGTGTGCCAT (SEQ ID NO: 3) (5' - 3'); a FIP primer comprising a Flc nucleotide sequence AGGGACATAAGTCACATGCAAGAA (SEQ ID NO: 4) and a F2 nucleotide sequence TTCTTATGTCCTTCCCTCAGT (SEQ ID NO: 5) and a spacer therebetween (5' - 3'); and a BIP primer comprising a Blc nucleotide sequence
  • the composition further comprises a loop forward primer comprising a nucleotide sequence GACTACACCATGAGGTGCTG (SEQ ID NO: 8) (5' - 3') and a loop backward primer comprising a nucleotide sequence CATTTGTCATGATGG AAA AG (SEQ ID NO: 9) (5' - 3').
  • the target nucleotide sequence comprises SEQ ID NO: 69.
  • Another aspect of the invention provides nucleic acids prepared from the use of any of the primers disclosed herein.
  • Another aspect of the invention is a method for detecting SARS-CoV-2.
  • the method may comprise contacting any of the primers or compositions disclosed herein with a sample under conditions sufficient for loop-mediated isothermal amplification of the target nucleotide sequence of SARS-CoV-2 and detecting the presence or absence of an amplification product of the target nucleotide sequence.
  • the diagnostic device may comprise at least one reaction chamber comprising any of the primers or compositions disclosed herein and a detection device that provides a readout that indicates whether the target nucleic acid is present in the sample.
  • kits for detecting SARS-CoV-2 may comprise any of the primers or compositions disclosed herein.
  • FIG. 1 shows a schematic of the diagnostic method described herein.
  • FIG. 2 shows a schematic of reverse transcription loop-mediated isothermal amplification (RT-LAMP).
  • FIG. 3 shows a plot of RT-LAMP amplification of 250 and 125 copies per reaction of synthetic RNA targets.
  • NTC non-template control.
  • FIG. 4 shows the lateral flow assay detection readout following RT-LAMP amplification of 1000 and 200 copies per reaction of synthetic RNA targets.
  • NTC non-template control.
  • FIG. 5 shows a schematic depicting an exemplary point-of-need diagnostic device.
  • This design includes a lysis cartridge that is used to lyse the sample, forming a lysate solution.
  • the nucleic acid binding stage (Slider 1) is initially positioned directly beneath the sample inlet so it can collect and bind nucleic acids from the lysate solution.
  • the lysate seeps through a nucleic acid binding substrate positioned at the bottom of a funnel built into Slider 1.
  • the substrate binds nucleic acids and filters out large particulate.
  • the user slides Slider 1 from position one - beneath the sample inlet - to position two.
  • Slider 1 is slid from position two to position three where it may pierce a blister pouch containing elution buffer. In position three, Slider 1 is positioned over the eluate inlet of the amplification stage (Slider 2) in such a way that when the elution buffer flows through the substrate of Slider 1, it delivers the eluate to the eluate inlet of Slider 2.
  • the eluate then flows through microchannels to reaction chambers of Slider 2, which contain a nucleic acid amplification reagent.
  • three of the reaction chambers contain the necessary enzymes and primers to amplify diagnostic markers that are indicative of the presence of a pathogen, and the fourth chamber contains primers to amplify human Amylase, ActB or RNAseP, as a control. Amplification is allowed to proceed for approximately 15-20 minutes. Then, the user moves Slider 2 from its initial position to its final position where Slider 2 may pierce a blister pouch containing lateral flow assay (LFA) running buffer.
  • LFA lateral flow assay
  • the running buffer forces the amplicons to flow from the reaction chambers through microchannels that converge at the output port of Slider 2, positioned directly beneath the collection pad of the diagnostic device (LFA).
  • LFA diagnostic device
  • FIG. 6 shows a schematic depicting an exemplary point-of-need kit.
  • the platform consists of a sample collection tube, a transfer pipette or swab, a lysis cartridge, a disposable processing/detection device, and a reusable handheld controller.
  • FIG. 7 shows a schematic depicting an exemplary lysis cartridge.
  • the lysis cartridge may be configured to filter out particulate and chemical inhibitors to improve nucleic acid recovery and amplification.
  • FIG. 8 shows a schematic depicting an exemplary nucleic acid binding stage.
  • the stage consists of a collection funnel, an outlet where the nucleic acid binding substrate is affixed, and a sponge ramp that compresses and drains reagent sponges.
  • FIG. 9 shows a schematic depicting an exemplary nucleic acid purification device that utilizes laminated glass microspheres
  • a Schematic diagram of the testing design and lamination process. The lamination process is performed by sandwiching the microspheres between two glass fiber membranes. The glass fiber membrane-microspheres sandwich is then sandwiched between two pieces of thermoplastic film. The final layered assembly is laminated by passing through a heat laminator. The laminated microspheres are adhered to the bottom of the 3D-printed funnel for testing nucleic acid isolation
  • c A photograph demonstrating analyte filtration using food dye.
  • FIG. 10 shows a schematic depicting reagent delivery using sponges.
  • the shelf-life reagent reservoirs are pierced and drained into sponges for temporary storage as they await their turn in the protocol sequence.
  • the sponge ramp gently compresses the sponges, draining reagents into the funnel of the binding stage.
  • FIG. 11 shows a schematic depicting an exemplary protocol in which the handheld device is used to analyze a sample
  • Sample is lysed in the lysis cartridge, which is a pierce-bottom tube
  • b The bottom of the lysis cartridge is pierced as it is inserted into the device, draining the lysate into the processing funnel.
  • the binding membrane attached to the funnel output binds nucleic acids, such as DNA, as the lysate drains through. Wash and elution buffers are subsequently drained through the membrane, with the eluate wicking into a microchannel (paper or capillary) rather than the waste pad.
  • a microchannel paper or capillary
  • FIG. 12 shows a schematic depicting how the integrated diagnostic platform can be adapted to a direct-to-amplification protocol
  • a Sample is mixed in dilution buffer prior to heat lysis. The heat used for sample lysis is initiated when the protocol is started in the instrument program.
  • the progression of lysate fluid and rehydration of reaction compounds is known to take a specific amount of time, which will be represented in the timing of the automated program (d):
  • the reaction chamber is connected to another microchannel via a magnetic valve, wicking amplicons towards the sample pad of the lateral flow assay.
  • the magnetic valve is actuated by a magnet housed in the handheld instrument.
  • One mode of magnetic valve actuation is to mount a magnet to a lead screw connected to a stepper motor. Being highly controllable and precise, the movements of the stepper motor-lead screw mechanism will occur at specifically programed times built into the instrument software.
  • a sequence of automated electromagnets can be used in place of a magnet mounted to a stepper motor-lead screw mechanism.
  • the running buffer blister is then burst, driving amplicons through the detection strip for the user to read and interpret results.
  • FIG. 13 depicts and exemplary diagnostic device
  • the exemplary device is assembled from 3D printed parts, paper microchannels, and a LFA strip
  • e The sliding channel is moved into active position (yellow arrow), connecting the reaction chamber to the secondary microchannel.
  • Amplicons wick through the secondary channel and into the LFA strip, providing a positive or negative readout.
  • FIG. 14 illustrates the interpretation of lateral flow assay results. Examples of positive, negative, and invalid results are shown.
  • FIG. 15 shows a visual representation of T and C lines generated for a positive control (left) and a negative control (right) on LFA strips.
  • FIG. 16 is a schematic depiction of the diagnostic kit workflow described in Example 6.
  • the inventors provide a novel diagnostic test for SARS-CoV-2.
  • the test is simple, rapid, and low-cost. This test can provide results within a 35-minute visit to the doctor's office, allowing for faster isolation and treatment of infected patients.
  • viral RNA is amplified using reverse transcription loop-mediated isothermal amplification (RT- LAMP), and is detected using an amplicon detection assay, such as a lateral flow assay (LFA).
  • RT-LAMP is an isothermal amplification method that is performed at a constant temperature (i.e., 60-65 °C).
  • the diagnostic test of the present invention can be performed using (1) a simple water bath or heat-block or (2) a point-of-need device fitted with a heating element.
  • this test does not require the use of an expensive thermal cycler instrument, it can be performed in a wider range of settings than conventional PCR-based methods.
  • the test can be performed in community hospitals, primary care offices, mobile clinics, and simple physician's office laboratories (POL) that are equipped with a with a basic water bath or heater.
  • POL physician's office laboratories
  • the test may also be run in settings such as homes, nursing homes, workplaces, meat processing plants, prisons, and rapid screening centers without sending samples to a centralized laboratory.
  • this test will be particularly valuable in resource-limited settings in the developing world.
  • the diagnostic test of the present invention is also high throughput since multiple tests can be set up and run without waiting for instruments to finish running a limited number of samples.
  • the test can be run as needed, without waiting to batch samples for loading into an instrument.
  • this technology should immediately increase the overall testing capacity of a facility because there is no lead time for purchasing instruments.
  • the diagnostic test may further comprise an RNA purification and/or isolation step, which may be accomplished using microspheres. See FIG. 1.
  • the test may also be performed directly on upper respiratory specimens, such as nasopharyngeal (NP) swabs, without RNA extraction, which allows this technology to side-step shortages in RNA extraction kits.
  • Direct testing without RNA extraction is made possible by inactivation of RNases in the sample, e.g., using an inactivation buffer and heat lysis of the virus.
  • RT-LAMP Reverse transcriptase loop-mediated isothermal amplification
  • RT-LAMP is a one-step nucleic acid amplification method that is commonly used for the identification of pathogens.
  • RT-LAMP uses reverse transcriptase to synthesize complementary DNA (cDNA) from RNA sequences and amplify the sequence for detection. The cDNA is then efficiently amplified using DNA polymerase, generating roughly 10 9 copies per hour.
  • RT-LAMP requires at least four primers that each recognize distinct target sequences within the template strand, including two "internal primers,” i.e., a forward internal primer (FIP) and backward internal primer (BIP) and two "outer primers," i.e., a forward 3 (F3) primer and a backward 3 (B3) primer. See steps 2-6 of FIG. 2.
  • BIP primers contain two parts: (1) a portion referred to as “Blc” that is identical to a target sequence (referred to as Blc) that is positioned internally within the template strand, and (2) a portion referred to as “B2” that is complementary to a target sequence (referred to as B2c) that is positioned 3’ to Blc within the template strand.
  • FIP primers contain: (1) a portion referred to as “Flc” that is identical to a target sequence (referred to as Flc) that is positioned internally within the template strand, and (2) a portion referred to as “F2” that is complementary to a target sequence (referred to as F2c) that is positioned 3’ to Flc within the template strand.
  • the outer primers, B3 and F3, anneal to target sequences that are positioned externally relative to the other target sequences within the template strand.
  • the B3 primer anneals to a target sequence (referred to as B3c) that is 3’ to B2c in the template strand
  • the F3 primer anneals to a target sequence (referred to as F3c) that is 3’ to F2c in the template strand.
  • This design ensures that when DNA synthesis is initiated from the outer primers, it displaces the DNA strands synthesized using the internal primers.
  • the FIP/F3 primers are designed to anneal to a DNA template that is complementary to the DNA template annealed by the BIP/B3 primers, as each of these sets is used to form one end of a dumbbell-shaped DNA product (described below).
  • the FIP/F3 primers anneal to the DNA copy produced using the BIP primer
  • the BIP/B3 primers anneal to the DNA copy produced using the FIP primer.
  • an internal primer e.g BIP
  • This cDNA strand is then displaced from the template strand when synthesis (by DNA polymerase) is initiated from its paired outer primer (e.g., B3).
  • the single-stranded cDNA product forms a self-hybridizing loop structure due to the inclusion of a reverse complementary sequence in the internal primer sequence (e.g, Blc - Bl).
  • This annealing and replacement cycle repeats on the opposite end of the target sequence (initiated, e.g., by FIP and F3).
  • the resulting product is a short dumbbell-shaped structure that serves as a seed for exponential LAMP amplification.
  • an additional set of "loop primers” can be used to accelerate LAMP amplification.
  • the loop primers i.e., a loop forward (LF) and a loop backward (LB)
  • LF loop forward
  • LB loop backward
  • amplification products grow and form long concatemers, each with more sites for initiation. This results in rapid accumulation of double-stranded cDNA and amplification byproducts that can be detected by a variety of methods.
  • FIG. 2 illustrates the RT-LAMP amplification process in greater detail.
  • the sample solution is mixed with a reaction solution comprising all the necessary components for this reaction and is incubated at a constant temperature between 60-65°C.
  • the B2 region of a BIP primer anneals to the B2c sequence within the target RNA, and cDNA is synthesized by reverse transcriptase.
  • a B3 primer anneals to the B3c sequence within the target RNA, and a new cDNA is synthesized by reverse transcriptase while the cDNA strand formed using the BIP primer is concurrently released.
  • the F2 region of a FIP primer anneals to the F2c sequence on the opposite end of the released cDNA.
  • a DNA strand is synthesized off of the FIP primer by a DNA polymerase with strand displacement activity.
  • a F3 primer anneals the F3c sequence, and a new DNA strand is synthesized while the DNA strand formed using the FIP primer is concurrently released.
  • the DNA strand synthesized using the F3 primer forms a double stranded DNA with the DNA strand synthesized using the BIP primer.
  • the DNA strand formed using the FIP primer (which was released in step 5) contains complementary sequences at both ends, it self-anneals to form a dumbbell-like DNA structure.
  • This structure becomes the starting structure for LAMP amplification.
  • the dumbbell-like DNA structure is quickly converted into a stem-loop structure by self-primed DNA synthesis, which unfolds the loop at the 5' end.
  • the BIP primer anneals to the single-stranded region in the stem-loop DNA to initiate DNA synthesis, which releases the previously synthesized strand.
  • the released strand forms a stem-loop structure at the 3' end because it contains complementary Flc and FI regions.
  • the dumbbell-like structure initiates self- primed DNA synthesis starting from the 3' end of the FI region.
  • the FIP primer anneals to the F2c region and initiates DNA synthesis.
  • the resulting FlP-linked DNA strand is released by the strand displacement of self-primed DNA synthesis.
  • the same dumbbell-like structure (7) is formed.
  • a FIP (or BIP) primer anneals to the single stranded F2c (B2c) region of the structure formed in step 9 (or step 12), and DNA synthesis releases double stranded DNA.
  • This amplification method produces structures of various sizes that consist of alternating inverted repeats of the target sequence on the same strand.
  • compositions are Compositions:
  • the present invention provides compositions for amplification of a target nucleotide sequence of SARS-CoV-2 by RT-LAMP.
  • the target nucleotide sequence may be any RNA sequence within the SARS-CoV-2 genome.
  • the target sequence is “SARS-CoV-2- specific”, meaning that it is not present in the human transcriptome or in genome/transcriptome of other common pathogens.
  • the present technology is designed to target the RNA sequence encoding the Spike protein (S) of the SARS-CoV-2 virus.
  • the S protein has a 3828 base pair nucleotide sequence that is located near the 3’ end of the SARS-CoV-2 genome.
  • the S protein is produced from a single open reading frame (ORF) and is not the result of frameshifting, as it is the case with other SARS-CoV-2 structural proteins. Additionally, the S protein comprises a furin protease cleavage site (45 base pairs long), which is not found in other coronaviruses such as the severe acute respiratory syndrome (SARS) virus or the Middle East respiratory syndrome (MERS) virus, which may be responsible for the increase in virulence of this isolate (5).
  • SARS severe acute respiratory syndrome
  • MERS Middle East respiratory syndrome
  • the S protein may be divided it into three different segments: (1) a 5’ end S target consisting of 1275 bp, (2) an internal S target (comprising the novel furin cleavage site) consisting of 1275 bp, and (3) a 3’ end S target consisting of 1275 bp.
  • One set of the RT-LAMP primers disclosed herein target the 3' end S target (primer set 1), and the other set targets the internal S target (primer set 2). Accordingly, the present technology includes compositions comprising one or more primers according to Table 1.
  • composition may comprise primer set 1: (i) SEQ ID NO: 1 or SEQ ID NO: 2, (ii) SEQ ID NO: 3, (iii) SEQ ID NO: 10, and (iv) SEQ ID NO: 11; or primer set 2: (i) SEQ ID NO: 12, (ii) SEQ ID NO: 13, (iii) SEQ ID NO: 20, and (iv) SEQ ID NO: 21.
  • An exemplary target sequence comprises UUGGU GCAGG UAUAU GCGCU AGUUA UCAGA CUC AG ACUAA UUCUC CUCGG CGGGC ACGUA GUGUA GCUAG UCAAU CCAUC AUUGC CUACA CUAUG UCACU UGGUG CAGAA AAUUC AGUUG CUUAC UCUAA UAACU CUAUU GCCAU ACCCA CAAAU UUUAC UAUUA GUGUU ACCAC AGAAA UUCUA CCAGU GUCUA UGACC AAGAC AUCAG UAGAU UGUAC AAUGU (SEQ ID NO: 70), which corresponds to a portion of the RNA sequence encoding the Spike protein at positions 1089-1313.
  • Primer set #2 is effective for amplifying this target sequence or is DNA analog.
  • the FIP primer comprises two subsequences: a Flc sequence comprising T AGGC AAT GAT GGATT GACT AGCT A (SEQ ID NO: 14) and a F2 sequence comprising TTATCAGACTCAGACTAATTCTCC (SEQ ID NO: 15) with a spacer therebetween.
  • the BIP primer comprises two subsequences: a Blc sequence comprising AACTCTATTGCCATACCCACAAAT (SEQ ID NO: 16) and a B2 sequence comprising TTGGTCATAGACACTGGTAG (SEQ ID NO: 17) with a spacer therebetween.
  • the primer set 2 composition may also include loop primers one or more primers included in Table 2.
  • the composition comprises both SEQ ID NO: 18 and SEQ ID NO: 19.
  • An exemplary target sequence comprises GAUUU UUGUG GAAAG GGCUA UCAUC UUAUG UCCUU CCCUC AGUCA GCACC UCAUG GUGUA GUCUU CUUGC AUGUG ACUUA UGUCC CUGCA CAAGA AAAGA ACUUC ACAAC UGCUC CUGCC AUUUG UCAUG AUGGA AAAGC ACACU UUCCU CGUGA AGGUG UCUUU GUUUC A A AUG GCACA CACUG GUUUG (SEQ ID NO: 69), which corresponds to a portion of the RNA sequence encoding the Spike protein at positions 573-760.
  • Primer set #1 is effective for amplifying this target sequence or is DNA analog.
  • the FIP primer comprises two subsequences: a Flc sequence comprising AGGGACATAAGTCACATGCAAGAA (SEQ ID NO: 4) and a F2 sequence comprising TTCTTATGTCCTTCCCTCAGT (SEQ ID NO: 5) with a spacer therebetween.
  • the BIP primer comprises two subsequences: a Blc sequence comprising
  • the primer set 1 composition may also include loop primers, including one or more primers included in Table 2.
  • the composition comprises both SEQ ID NO: 8 and SEQ ID NO: 9.
  • the term “spacer” refers to a nucleic acid sequence that is used to link two polynucleotide sequences.
  • the spacer is a TTTT linker.
  • one or more of the primers included in the composition contains a region that is at least partially complementary to the target nucleotide sequence of SARS-CoV-2.
  • the term “complementary” refers to the ability of a nucleic acid molecule to bind to (i.e., hyrbridize with) another nucleic acid molecule through the formation of hydrogen bonds between specific nucleotides (i.e., A with T or U and G with C), forming a double-stranded molecule.
  • the phrase “substantially complementary” can refer to at least 85%, 87%, 88%, 89%, 90%, 92%, 94%, 96%, or 98% complementarity.
  • the target nucleotide sequence that is amplified using the compositions disclosed herein is part of the sequence encoding the SARS-CoV-2 spike (S) protein.
  • the primers provided in Table 1 contain regions that are 100% identical and others that are 100% complementary to the 2019 Spike gene of SARS-CoV-2, and show no cross-reactivity with other coronaviruses or other viral and bacterial pathogens. This evaluation was conducted by aligning the Primer or probe against the Basic Local Alignment Search Tool (BLAST) from the National Center for Biotechnology Information (NCBI).
  • BLAST Basic Local Alignment Search Tool
  • compositions of the present invention may comprise one or more primers that are labeled to allow for detection of an amplification product prepared by RT-LAMP.
  • label is used herein to refer to a chemical moiety that is attached (i.e., covalently or non- covalently) to a primer.
  • the label may be used to capture and/or detect the RT-LAMP amplification products.
  • detectable labels include, for example, a fluorescent label, a chemiluminescent label, a quencher, a radioactive label, an antibody, biotin, and gold.
  • the forward internal primers (FIP) and backward loop primers (BIP) may contain 5' labels (e.g, FAM or biotin), such that the resulting amplification products can be detected by lateral flow assay (LFA) using, e.g, anti-FAM antibody and streptavidin-labeled latex beads.
  • LFA lateral flow assay
  • the backward internal primer (BIP) and loop forward (LF) primers may contain 5' labels.
  • the composition comprises one or more components selected for RT-LAMP amplification.
  • exemplary components include, without limitation, deoxynucleotide triphosphates (dNTPs), a polymerase enzyme, a reverse transcriptase, a buffer solution for loop- mediate isothermal amplification, human control primers, an enzyme co-factor, a positive control nucleic acid, a detergent, an inactivation buffer, a dilution buffer, universal transfer medium (UTM), viral transfer medium (VTM), or dye.
  • an “inactivation buffer” is a buffer than inactivates RNases in a sample.
  • Suitable inactivation buffers include those that comprise an effective amount of one or more of TCEP, guanidium hydrochloride, and EDTA.
  • Suitable dilution buffers include those that comprise an effective amount of citrate.
  • compositions disclosed herein may additionally include components for the loop-mediated isothermal amplification of a control nucleotide sequence from the subject.
  • the compositions my also include primers and reagents for the LAMP of human genes as a test control.
  • Exemplary control genes include, without limitation, Human Ribonuclease P30 ( RRP30 ), Human Albumin (HA), Human Actin Beta ( HAB ), Human Actin Gamma (HAG), Human Tubulin Alpha (HTB), Human saliva Amylases variant #1 and Variant #2 (AMY1AV1 or AMY1AV2 ), Ribonuclease P (RNAseP), Apolipoprotein LI (ApoLl), or Glyceraldehyde 3- phosphate dehydrogenase (GAPDH).
  • RRP30 Human Ribonuclease P30
  • HAB Human Actin Beta
  • HAB Human Actin Gamma
  • HTB Human Tubulin Alpha
  • AMD Human saliva Amylases variant #1 and Variant #2
  • RNAseP Ribonuclease P
  • Apolipoprotein LI ApoLl
  • Glyceraldehyde 3- phosphate dehydrogenase Glyceraldehyde 3- phosphate dehydrogen
  • This nucleic acid comprises in order from 5’ to 3’: Flc, F2, a nucleotide sequence substantially complementary to Flc (i.e., FI), Blc, B2, and a nucleotide sequence substantially complementary to Blc (i.e., Bl).
  • Flc and FI hybridize and Blc and Bl hybridize, such that the nucleic acid forms a dumbbell structure.
  • This dumbbell structure contains multiple sites for the initiation of synthesis. As amplification proceeds from these multiple initiation sites, the products grow and form long catcatemers, (i.e., amplification products) that can be detected by a variety of methods. See Steps 8-12 above.
  • the present invention also provides methods and assays for detecting SARS-CoV-2.
  • the methods may comprise contacting any of the compositions described herein with a sample under conditions sufficient for loop-mediated isothermal amplification of the target nucleotide sequence of SARS-CoV-2 and detecting the presence or absence of an amplification product of the target nucleic acid.
  • Conditions sufficient for amplification of the target nucleotide may comprise an effective temperature of from 55-70 °C and/or an effective amplification time of between 20-60 minutes.
  • the sample may be obtained from a subject having or suspected of having a SARS-CoV- 2 infection.
  • the sample may be obtained from the upper or lower respiratory tract of the subject.
  • samples may include saliva, sputum, nasal fluid, stool, and sewage.
  • any sample that would potentially contain SARS-CoV-2 can be utilized.
  • Exemplary samples include, without limitation, a saliva sample, bronchoalveolar lavage, a sputum sample, a nasopharyngeal sample, a nasal sample, an oropharyngeal sample, sewage, or a stool sample.
  • the sample is a nasopharyngeal swab, oropharyngeal (throat) swab, mid-turbinate nasal swab, anterior nasal swab, nasopharyngeal wash/aspirate, or nasal aspirate
  • subject refers to mammals and non-mammals.
  • a “mammal” may be any member of the class Mammalia including, but not limited to, humans, non-human primates (e.g ., chimpanzees, other apes, and monkey species), farm animals (e.g, cattle, horses, sheep, goats, and swine), domestic animals (e.g, rabbits, dogs, and cats), or laboratory animals including rodents (e.g, rats, mice, and guinea pigs). Examples of non-mammals include, but are not limited to, birds, and the like.
  • the term “subject” does not denote a particular age or sex.
  • a subject is a mammal, preferably a human.
  • the subject has or is suspected of having a SARS-CoV-2 infection.
  • RT-LAMP of SARS-CoV-2 RNA using labeled primers will generate labeled amplicons, e.g, amplicons labelled with FITC and biotin that can be detected by lateral flow assay (LFA) using anti-FITC antibody and streptavidin-labeled latex beads.
  • LFA lateral flow assay
  • Exemplary methods and devices for detecting the labeled amplicons include those described in US Patent Pub. No. 2009/0181388 and 2011/0229887.
  • the amplicons may be detected visually without instrumentation.
  • the amplicons may be detected using dyes that recognize by-products of the DNA synthesis, including intercalating dyes.
  • dyes include, without limitation, malachite green, calcein, hydroxynaphthol blue, or pH-sensitive dyes.
  • Exemplary dyes and methods are disclosed in Nzelu et al. Acta Trop, 132, 1-6 (2014); Tomita el al. Nat Protoc, 3, 877-882. (2008); Goto et al. Biotechniques, 46, 167-172 (2009); and Tanner et al, Biotechniques, 58, 59-68 (2015).
  • the amplicons may be detected via fluorescence with the use of molecular beacon probes.
  • the amplicons may be detect electrochemically.
  • the methods further comprise extracting RNA from the sample obtained from the subject.
  • the RNA may be extracted by lysing the SARS-CoV-2 virus present in the sample.
  • lysis or “lyse” are used to describe the disruption of cells or viral particles within a sample to gain access to materials within. Methods of lysis include, but are not limited to, chemical lysis, thermal lysis, mechanical lysis, and osmotic lysis.
  • lysate is used to refer to the sample following a lysis procedure.
  • the RNA is extracted using a heat lysis method.
  • the sample may be heated to an effective temperature for an effective amount of time as to disrupt the virus envelope allowing for access to the genomic RNA.
  • the RNA is extracted using a chemical lysis method.
  • the SARS-CoV-2 virus may be contacted with a detergent and/or chaotropic salt to disrupt the viral envelope.
  • the RNA in the lysate is protected from RNases by treatment with an RNase inactivation buffer.
  • Suitable inactivation buffers include reducing agents such as TCEP (tris(2-carboxyethyl)phosphine), chaotropic agents such as guanidium hydrochloride, and chelating agents such as EDTA (ethylenediaminetetraacetic acid).
  • the lysed RNA may be further stabilized using a buffering salt such as sodium citrate.
  • the sample is treated with an RNase inactivation buffer and heat lysis prior to the contacting step.
  • the methods further comprise extracting RNA from the sample prior to the contacting step.
  • RNA may be isolated from within biological samples using standard methods that are well known in the art, including those that rely on organic extraction, ethanol precipitation, silica-binding chemistry, cellulose-binding chemistry, and ion exchange chemistry. Many reagents and kits for RNA extraction are commercially available.
  • the methods further comprise isolating or purifying the extracted RNA.
  • the RNA is isolated or purified using microspheres, as described in International Patent Pub. No WO/2019/183263.
  • the methods described herein may also include the amplification of a control nucleotide sequence that originates from the subject, and detection of the presence of an amplification product generated from the control sequence.
  • the control sequence may comprise part of a human gene.
  • the device comprises a housing with a sample inlet for receiving the sample.
  • the housing contains the following within it: (a) a moveable nucleic acid binding stage comprising a permeable nucleic acid binding substrate and an eluate outlet by which an eluate may exit the binding stage; (b) a wash reservoir containing a wash buffer; (c) an elution reservoir containing an elution buffer; (d) a moveable amplification stage comprising an eluate inlet for receiving the eluate from the nucleic acid binding stage, at least one reaction chamber comprising a nucleic acid amplification reagent, and an amplicon outlet by which an amplified sample may exit the amplification stage; (e) a running buffer reservoir containing a running buffer; and (f) a detection device that provides a readout that indicates whether the target nucleic acid is present in the sample.
  • the nucleic acid binding stage is configured such that it can be positioned to be in fluid communication with the sample inlet, the wash reservoir, the elution reservoir, and the amplification stage via the eluate outlet.
  • the amplification stage is configured such that it can be positioned to be in fluid communication with the running buffer reservoir and the detection device via the amplicon outlet. See FIG. 5 for a schematic illustration of one exemplary device.
  • the term "housing” refers to a rigid casing that encloses and protects the device components.
  • the housing may comprise a sample inlet for receiving a sample or sample lysate.
  • the devices of the present invention are self-contained, limiting the potential for exposure to any harmful or infectious materials found in the sample.
  • the housing also prevents contamination of the testing environment with the highly amplified DNA products from RT-LAMP, which can lead to false positives.
  • the housing may comprise one or more readout windows that allow a user to inspect a readout of a detection device within the housing thereby avoiding the need to open the housing and potentially expose the user to harmful or infectious materials.
  • the nucleic acid binding stage is initially positioned directly beneath the sample inlet to receive the sample and direct it into the permeable nucleic acid binding substrate.
  • This substrate binds nucleic acids and filters out large particulate as the sample seeps through it.
  • the nucleic acid binding substrate can be positioned at the bottom of a funnel built into the nucleic acid binding stage, such that the sample is directed into the substrate.
  • the nucleic acid binding substrate may comprise any permeable material that can bind nucleic acids in a complex matrix and can release the nucleic acids when contacted with an eluent.
  • the substrate binds nucleic acids in a nonspecific manner, allowing it to be used to separate any nucleic acid from a sample without having to be tailored for a specific nucleic acid target.
  • Suitable nucleic acid binding substrates include, without limitation, cellulose membranes, cellulose-coated beads, chitosan membranes, chitosan-coated beads, silica columns, aluminum oxide membranes, and the like.
  • the nucleic acid binding substrate is a silica membrane and/or silica microspheres.
  • borosilicate glass microspheres may be used for DNA extraction as disclosed in International Patent Pub. No. WO/2019/109092, which is incorporated by reference herein.
  • the binding substrate comprises silica microspheres and fiber pads (e.g, paper, silica, cellulose, etc.) or membranes.
  • This substrate combines the advantages of silica- bead-based and paper-based nucleic acid extractions while eliminating their drawbacks.
  • micrometer-sized silica beads are packed between two pieces of fiber pads via a low-cost, readily deployable lamination process (FIG. 9).
  • the laminated beads will bind nucleic acids and allow the use of a paper, or a similar material, with larger pore sizes for fast flow and clogging prevention.
  • the use of fiber pads and lamination not only simplifies the silica beads packing process but also enables the use of capillary forces for liquid transfer.
  • nucleic acids The binding of nucleic acids is achieved using a combination of chaotropic agents, such as guanidium isothiocyanate and ethanol, and a co-precipitant such as glycogen and glycoblueTM.
  • chaotropic agents such as guanidium isothiocyanate and ethanol
  • co-precipitant such as glycogen and glycoblueTM.
  • Exemplary nucleic-acid-capture microspheres and their production and use are described in International Patent Pub. No. WO/2019/109092.
  • the binding stage may comprise a collection funnel, binding membrane, and sponge ramp (FIG. 8).
  • the funnel collects liquids as they are introduced to the binding stage.
  • the lysate drains from the lysis cartridge, through the inlet of the processing/detection device and into the collection funnel.
  • the funnel channels the lysate through the binding substrate, which collects nucleic acids as the lysate passes through.
  • the devices are designed to be operated by simple, limited, manual interventions, such as sliding a movable component or pressing down a collapsible chamber.
  • the device may comprise two moveable stages: the nucleic acid binding stage and the amplification stage.
  • the nucleic acid binding stage is configured to slide between positions of fluid communication with the sample inlet, the wash reservoir, and the elution reservoir
  • the amplification stage is configured to slide between positions of fluid communication with the elution reservoir, the running buffer reservoir, and the detection device.
  • the device is intended to be used with a reusable, handheld controller that moves internal components within the processing device in an automated fashion (FIG. 6).
  • the reusable controller removes user manual steps previously required, simplifying platform operation, which increases success rate by removing variability.
  • the handheld platform is portable so it can be used throughout a single clinic or in mobile field clinics, among other unique diagnostic settings.
  • the binding stage or amplification stage may be controlled using a handheld controller and moved using a force, such as mechanical or electromagnetic force. Stage movements may be automated by a single-board microcontroller that is coded to control a small electric motor, such as a stepper motor.
  • the small electric motor may spin a lead screw that is threaded with a moving stage.
  • Features attached to this moving stage such as a magnet or clips or similar physical features, connect with the binding and amplification stage within the processing/detection device.
  • the program installed onto the microcontroller moves the electric motor at specific speeds at specific times, according to the protocol designed to collect, amplify and detect target nucleic acid sequences.
  • the microcontroller, electric motor, and external force application stage are all housed within a reusable device or controller.
  • the controller will be made of polymer material that can be easily decontaminated with standard wipes or spray, preventing cross-contamination and the risk of passing infections from one person to another.
  • the reusable controller is designed to be portable so one can be used throughout a doctor’ s office or in a mobile clinic setting.
  • the reusable controller has a user interface and buttons that allows the user to select a protocol specific to a sample or target type.
  • the heating element that is required for nucleic acid amplification will be powered by the reusable controller.
  • the battery pack that powers the microcontroller and the electric motor also powers the heating element.
  • the microcontroller will command the temperature and reaction time.
  • the heating element could be housed within the controller or within the processing/detection device and connected to the power source via external nodes.
  • the device may comprise one or more reservoirs.
  • the device comprises four buffer reservoirs: the wash reservoir, elution reservoir, rehydration buffer reservoir, and running buffer reservoir.
  • a reservoir is suitably liquid-tight and/or airtight to allow the contents to be maintained for extended periods of time in ambient conditions until needed for use.
  • one of more of these reservoirs comprise a blister pouch (e.g an aluminum foil blister pouch).
  • the reservoir's contents can be accessed when the appropriate stage is positioned to be in fluid communication with the reservoir.
  • the stage may be configured to puncture the blister pouch when the stage is in position to receive the reservoir's contents.
  • Prepackaged buffers can be strategically incorporated along the sliding path of each stage.
  • reservoirs are pierced and drained into sponges for temporary storage as they await their turn in the protocol sequence.
  • the sponge ramp gently compresses the sponges, draining reagents into the funnel of the binding stage (FIG. 10).
  • the sponge ramp of the binding stage may be built up from the collection funnel. The purpose of the ramp is to compress reagent sponges as the binding stage moves along the protocol track.
  • the reagent sponges are a passive way to temporarily store reagents after they are expelled from their shelf-life storage reservoirs.
  • some or all of the shelf-life reagent reservoirs are burst all at once.
  • the reservoirs are pierced and drained into the reagent sponges through mechanical actuation as the processing/detection device is inserted into a reusable controller. From there, the sponges saturate with their respective reagent, holding it until compressed by the sponge ramp. As the ramp moves beneath a sponge that is saturated with a specific buffer, it gently compresses the sponge, draining it of a set volume of liquid into the stage.
  • the drained reagents flow into the collection funnel via gravity and are wicked through the binding membrane via fluidic forces.
  • the wash reservoir may contain any suitable wash buffer for nucleic acid extraction.
  • a “wash buffer” is a substance capable of removing impurities adsorbed onto the surface of the nucleic acid binding substrate.
  • the wash buffer should be selected such that nucleic acids adsorbed onto the nucleic acid binding substrate are not extracted when the wash buffer contacts the substrate.
  • the wash buffer may be selected from water, an alcohol (e.g ., ethanol or isopropanol), medium salt buffer (e.g., lOOmM or 200mM NaCl), or combinations thereof.
  • the wash buffer is ethanol diluted to a concentration of 30% or less by volume.
  • the elution reservoir may contain any suitable elution buffer that is able to separate nucleic acid material from the nucleic acid binding substrate.
  • the elution solution is preferably an aqueous solution of low ionic strength.
  • the elution buffer is water or TE buffer (i.e., Tris-HCl ethylenediamine-tetraacetic acid (EDTA)).
  • EDTA Tris-HCl ethylenediamine-tetraacetic acid
  • the running buffer reservoir may contain any running buffer that is compatible with the detection device.
  • Suitable running buffers include, without limitation, water, phosphate buffered saline (PBS), saline-sodium citrate (SSC) buffer, Tris buffer, MES buffer, HEPES buffer, or any other buffer suitable for buffing at a pH between 7.4 and 8.0.
  • the running buffer is a buffered salt solution containing a detergent, such as 75mM Sodium Borate, pH 7.4, 0.25% Tween 20.
  • the device includes a waste pad positioned beneath the nucleic acid binding stage to absorb liquid ⁇ i.e., sample and buffer) that passes through the nucleic acid binding substrate.
  • the waste pad may take any form and may comprise any absorptive material, provided that it does not disrupt device function.
  • the devices comprise at least one reaction chamber in which a nucleic acid amplification reaction may be performed.
  • the reaction chamber contains a nucleic acid amplification reagent.
  • this reagent comprises the basic factors required for DNA amplification, i.e., a DNA polymerase, one or more primers, and nucleoside triphosphates.
  • the nucleic acid amplification reagent is lyophilized and is reconstituted as the eluate flows into the reaction chamber. Use of a lyophilized reagent is advantageous for a point-of-need device because such reagents remain stable for a long time at room temperature.
  • the outlet of the binding stage is positioned above the inlet of the reaction stage.
  • a fluidic connection is made between the two stages, draining eluate from the binding substrate into a capillary microchannel.
  • the microchannel wicks eluate via capillary action into one or several reaction chambers. This wicking could be done through any sort of hydrophilic microchannel.
  • One such example is a paper microchannel that comes into contact with the bottom of the binding membrane (FIG. 11)
  • the reaction chambers may be pre-packaged with lyophilized reaction reagents, including primers, probes, and magnesium.
  • lyophilized reaction reagents including primers, probes, and magnesium.
  • the rehydration buffer reservoir is pierced, draining into the reaction chamber and mixing all reagents.
  • the heating element begins warming the reaction to the desired temperature for the desired duration.
  • the reaction temperature and duration are specific for the desired nucleic acid target that is being amplified.
  • the reaction chamber is brought into fluidic contact with a capillary microchannel, similar to the previous microchannel (FIG. 11).
  • This downstream microchannel wicks the reaction products (i.e., amplicons) towards the sample pad of the lateral flow assay used for target detection.
  • the running buffer reservoir is pierced.
  • the lateral flow assay then works as any standard lateral flow diagnostic test. The user can read the test lines that arise over the next few minutes and determine results.
  • the device could also be read by an automated reading device that is built into the reusable controller.
  • FIG. 12 Another exemplary embodiment of the invention is illustrated in FIG. 12.
  • This embodiment takes advantage of “direct-to-amplification” assay techniques and does not require nucleic acid extraction, wash, or elution, thus, reducing the number of automated steps.
  • This platform is best suited for samples that are more conducive to direct-to-amplification techniques, such as nasal swabs, nasopharyngeal swabs, oropharyngeal swabs and saliva (FIG. 12A).
  • the sample will be heat lysed within the lysis cartridge in a heating slot built into the reusable controller (FIG. 12B). Following lysis, the tube is transferred to the sample inlet of the diagnostic device.
  • the sample inlet is in direct fluidic connection with the reaction chamber. This is because no nucleic acid extraction, washing, or elution is needed prior to amplification. As before, the cartridge is pierced upon insertion, draining an aliquot of sample into the reaction chamber.
  • the rehydration buffer blister pouch is burst and drained, filling the chamber and mixing reagents (FIG. 12C).
  • the reaction is then heated to an adequate temperature for efficient amplification to occur.
  • the reaction chamber is put into fluidic connection with the lateral flow assay via another wicking channel (FIG. 12D).
  • the running buffer blister is burst, driving amplicons along the lateral flow assay, through the detection strip.
  • the strip results are then read by the user or an automated strip reader. Medical action may be taken based on the strip results.
  • the diagnostic device may be configured to facilitate communication of health information.
  • health information may include, for example, location information that allows for tracking, reporting and surveillance of the pandemic.
  • the test data may be incorporated into the electronic medical records of the patient for improved patient treatment and follow-up.
  • barcodes or other indicia may be imprinted on the diagnostic device, allowing the user to photograph the device and results with the barcode in clear view. This photograph can be uploaded to electronic medical record programs, providing diagnostic information to medical workers and/or providing location information for tracking and surveillance.
  • the diagnostic device includes a Bluetooth-enabled strip reader or other communication-enabled strip reader.
  • the diagnostic device may allow for the test data to be geotagged and uploaded automatically when connected to a phone or computer via Bluetooth or another communication protocol.
  • DNA polymerase refers to an enzyme capable of catalyzing the formation of DNA.
  • the DNA polymerase is advantageously a strand-displacing polymerase (i.e. a polymerase with the ability to displace downstream DNA encountered during synthesis).
  • exemplary strand-displacing DNA polymerases include phi29, Bst, Bsm, Bsu, and Klenow fragment.
  • a “primer” is a nucleic acid designed to bind via complementarity to sequences that flank the target sequence in the template nucleic acid. During amplification, polymerases extend primers. The primer’s binding site should be unique to the target sequence with minimal homology to other sequences to ensure specific amplification of the intended target sequence.
  • the nucleic acid amplification reagent may comprise at least one primer that is detectably labeled.
  • Exemplary labeling moieties include, without limitation, a gold nanoparticle, a protein binding ligand, a hapten, an antigen, a fluorescent compound, a dye, a radioactive isotope, and an enzyme.
  • the nucleic acids are amplified using a labeled primer set (comprising a forward and a reverse primer), generating amplification products with tagged primers at both ends for easy detection via a lateral flow device.
  • Primers may further comprise nuclease cleavage sites and/or blockers ( e.g ., phosphoramidite blocker) to provide amplification specificity.
  • the eluate may be subjected to a reverse transcription reaction to generate cDNA prior to amplification (i.e., reverse transcription polymerase chain reaction).
  • the nucleic acid amplification reagent may comprise a reverse transcriptase and at least one suitable primer.
  • a "reverse transcriptase” is an RNA-dependent DNA polymerase. The reverse transcriptase initiates synthesis of a DNA transcript using the RNA as a template, forming a single-stranded cDNA.
  • a double-stranded DNA molecule may be produced from the cDNA using a DNA polymerase. Either the single-stranded cDNA or the double-stranded DNA prepared from reverse transcription may serve as the input for the subsequent amplification reaction.
  • Standard methods of nucleic acid amplification require (1) high temperatures to increase reaction kinetics and expedite primer-target annealing and (2) expensive laboratory equipment, such as heating blocks, centrifuges, bead-beaters, magnetic beads, and/or volume dispensing robots.
  • an amplification method must be free of such requirements.
  • the devices of the present invention may be configured such that the nucleic acid amplification step is performed isothermally, without the use of a thermocycler.
  • “isothermally” or “under isothermal conditions” means that reaction is conducted at a relatively constant temperature.
  • the reaction is conducted with temperature fluctuations less than ⁇ 10°C, ⁇ 5°C, or ⁇ 2°C.
  • the amplification methods are performed without any equipment requiring a power supply to provide source heat for the amplification reaction.
  • the methods are performed at a temperature below 70°C, 65°C, 60°C, 55°C, 50°C, 65°C, or 40°C. In certain embodiments, the methods are performed at a temperature below 37°C.
  • the methods may be performed at a temperature between 20°C and 70°C, 20°C and 65°C, 20°C and 60°C, 20°C and 55°C, 20°C and 50°C, 20°C and 45°C, 20°C and 40°C, or 20°C and 37°C.
  • the annealase ICP8 and, optionally, a helicase or a nickase/polymerase may be included to accelerate the reaction.
  • ICP8 is derived from the herpesvirus DNA replication system. This annealase promotes efficient replication of the viral genome during host cell infection by stabilizing single-stranded DNA (ssDNA) and recruiting various factors necessary for replication. Specifically, ICP8 binds ssDNA, samples ssDNA for base pairing, and anneals two ssDNA molecules. Thus, ICP8 can be used to promote the annealing of DNA primers to their complementary targets during an amplification reaction.
  • ICP8 allows the reaction to be performed at a lower temperature with increased specificity.
  • the ICP8 used with the present invention may be from any available source, including from any herpesvirus or another closely related virus.
  • the ICP8 may be derived from chelonid herpesvirus 5, a type of herpesvirus that infects the Hawaiian green sea turtle, which has an internal body temperature of 20-25°C. Since ICP8 lacks helicase function, helicases or nucleases/polymerase must be added to the reaction to generate ssDNA for ICP8 to sample. Any suitable helicase or nuclease/polymerase may be used with the methods of the present invention. Exemplary helicases include, without limitation, UvrD, RecBCD, BLM, WRN, and RecQ. Exemplary nucleases include UL12, nickases, and restriction enzymes.
  • reaction conditions and enzyme components are standard practice for one of skill in the art to optimize reaction conditions and enzyme components to achieve particular reaction goals (i.e., sensitivity, specificity, speed, and efficiency at a given temperature). For instance, conditions such as primer length, melting temperature (Tm), and GC content, reaction buffering conditions (e.g., pH, salt concentrations, dNTP concentrations), and crowding agents (e.g, PEG) can be varied.
  • reaction buffering conditions e.g., pH, salt concentrations, dNTP concentrations
  • crowding agents e.g, PEG
  • the nucleic acid amplification reagent may further comprise additional components, including cofactors, buffering agents, amplification enhancers, or any combination thereof.
  • a “cofactor” is a substance other than the substrate that is essential for the activity of an enzyme.
  • the cofactor may be magnesium, which functions as a cofactor for a variety of polymerases.
  • the cofactor may be introduced to the amplification reaction as a salt, e.g, MgSCri or MgCh.
  • a “buffering agent” comprises a weak acid or base used to maintain the acidity (pH) of a solution near a chosen value after the addition of another acid or base.
  • the buffering agent may be selected from Tris-HCl, (NH4)2S04, or KC1.
  • an “amplification enhancer” is a substance that may enhance amplification specificity, efficiency, consistency, and/or yield.
  • Exemplary amplification enhancers include dimethyl sulfoxide, glycerol, formamide, polyethylene glycol, N,N,N- trimethylglycine (betaine), bovine serum albumin, tetramethylammonium chloride, a detergent, or combinations thereof.
  • the detergent is a nonionic detergent such as Tween 20 or Triton X-100.
  • Any detection device that provides a readout that indicates whether a target nucleic acid is present in a sample may be used with the present invention.
  • the presence of the target nucleic acid may be detected by any suitable method or assay technique, including, without limitation, a binding assay, a colorimetric assay, an electrophoretic assay, a fluorescence assay, a turbidity assay, an electrochemical assay, and the like.
  • Detection devices may provide an analog or digital readout.
  • the detection device is a lateral flow device.
  • a “lateral flow device” is a porous device capable of detecting the presence of a target nucleic acid sequence traversing a series of beds.
  • Lateral flow devices typically comprise (a) a sample loading area at one end; (b) an area comprising a detectably labelled probe, wherein said detectably labelled probe is not bound to the lateral flow device and is capable of wicking across the lateral flow device; (c) an area comprising a capture probe, wherein said capture probe; and (d) absorbent material, wherein the absorbent material wicks an aqueous sample across the lateral flow device when the aqueous sample is added to the sample loading area.
  • the lateral flow device comprises a sample loading area, an amplification area, a solid support, an absorbent sample pad, or any combination thereof.
  • the test results may be displayed using lateral flow assay (LFA) strips, which provide a readout similar to that of a pregnancy test strip.
  • the strips comprise a capture probe for the target nucleic acid sequence, wherein said capture probe is immobilized on the lateral flow device in a region referred to as the "test area".
  • the test area can be in any form with well-defined boundaries, such as a dot, or a strip.
  • the capture probe may be immobilized on the lateral flow device by covalent coupling or affinity binding.
  • the capture probe is attached to the lateral flow device by biotin: streptavi din affinity binding.
  • the capture probe is capable of specifically hybridizing to part of the target DNA sequence, separate from the detector probe sequence to which the detectably labelled primer will bind.
  • the LFA strip may comprise multiple probe-capture lines designed to capture different target sequences.
  • the amplified RNA or DNA are dual-labeled using two primers that have a biotin label on one primer and a second label (e.g a FITC, DIGO or TAMRA tag) on the other reverse primer.
  • the streptavidin conjugated AuNPs/latex bead (the colorimetric moiety) will bind to the biotin side of the amplicons while the tag molecule on the other side is captured by an antibody (anti-FITC or anti-DIGO or anti-TAMRA, respectively) attached to the strips.
  • the rest of the streptavidin conjugated AuNPs/latex bead will be captured by the biotin control line on the LFA.
  • the detection device is configured such that detection is accomplished by visual inspection, either with or without additional instrumentation.
  • results can be quantified by imaging and analysis with a computer.
  • the result can be scanned with a smartphone and electronically sent to a clinician, for example, with a computer that has an Adobe Acrobat grayscale converter or an Image J image processing software to quantify the visible light signal from a gold nanoparticle.
  • a color wheel for visualization of positive tests may be utilized.
  • the devices further comprise a heating element.
  • the heating element is advantageously portable and does not require electricity.
  • the heating element comprises a battery-powered, cell-phone powered, or solar battery powered heating film.
  • the heating element may use a reversible or irreversible exothermic chemical reaction to generate heat.
  • kits comprising any of the compositions or the point- of-need diagnostic devices disclosed herein and further comprising one or more of a lysis cartridge, a lysis agent, a collection device, a heating element, and a handheld controller. These components may be included to ensure that use of the kit is safe, simple, and hands-off. This is of particular importance when the samples used with the present invention may contain harmful or infectious materials.
  • the composition for loop-mediated isothermal amplification is provided in a lyophilized form within the kits.
  • the composition may be provided as a dried pellet that is formulated as shown in Table 18.
  • kits include a collection device.
  • the collection device may comprise any suitable device for containing the sample, such as a container, specimen jar, tube, syringe, needle, bag, specimen collection paper, or swab.
  • the collection device comprises a Puritan® HydraFlock swab or a Pixel nasal self-collection swab, which are designed for absorption and retention of cellular material. Since these swabs can hold approximately 250 pL of sample when fully saturated, their use standardizes sample input without requiring any measurement or transfer of infectious liquids.
  • the collection device may be a spit tube with or without RNA preservatives. Exemplary spit tubes include, without limitation, Spectrum Solutions LLC SDNA-1000 Saliva Collection Device and OraSure Technologies' Omnigene Oral OM-505 saliva collection device.
  • kits include a lysis cartridge comprising a lysis agent.
  • the lysis cartridge provides a simple, hands-off means to lyse cellular components within the sample.
  • the lysis cartridge may be configured to allow for direct insertion of a sample ( e.g ., via a swab or needle). To promote lysis, the operator may be instructed to cap and shake the cartridge.
  • a “lysis agent” is a composition capable of breaking down or disrupting a cellular membrane or virus envelope. Ideally, the lysis agent results in efficient cell lysis without the use of any equipment, such as a heating block or vortex.
  • Suitable lysis agents include, without limitation, chaotropic salts (e.g., guanidine thiocyanate, alkali metal perchlorates, alkali metal iodides, Nal, alkali metal trifluoroacetates, alkali metal trichloroacetates, alkali metal thiocyanates, urea, guanidine HC1, guanidine thiocyanate, guanidium thiosulfate, and thiourea), lytic enzymes (e.g, beta glucurondiase, glucanase, glusulase, lysozyme, lyticase, mannanase, mutanolysin, zymolase, cellulase, lysostaphin, pectolyase, and streptolysin O), and detergents (e.g, sodium dodecyl sulfate (SDS), IG
  • the lysis cartridge can be used with samples that require particulate filtration, such as stool or sewage (FIG. 7).
  • the lysis cartridge may make use of a small pore filter, such as a filter membrane or a porous foam filter.
  • a small pore filter such as a filter membrane or a porous foam filter.
  • the filter can also be embedded with resin, activated charcoal or something similar for the removal of non-particulate, chemical contaminants.
  • Complex samples, such as stool contain reaction inhibitors like bile salts. The removal of such inhibitors can greatly improve nucleic acid recovery and amplification reaction efficiency.
  • kits may also include primers and reagents for detecting a control gene.
  • control gene or primers may be any of those disclosed herein.
  • NAAT nucleic acid amplification test
  • RNA is extracted using silica microspheres, amplified using reverse transcriptase loop-mediated isothermal amplification (RT- LAMP), and detected by lateral flow assay (LFA) (FIG. 1).
  • RT- LAMP reverse transcriptase loop-mediated isothermal amplification
  • LFA lateral flow assay
  • the 10X LAMP primer mix for SARS-CoV-2 contains 16 mM each of FIP and BIP, 4 pM of F3 and B3, and 2 pM of LF and LB.
  • the sequence of the primers is listed in Table 10.
  • RT-LAMP primer set 1 for SARS-CoV-2 detection FITC-tagged FIP and biotin-tagged LB primers are only used to replace non-tagged FIP and LB primers for lateral flow detection.
  • Each 25 m ⁇ RT-LAMP reaction mix contains 2.5 m ⁇ 10X primer mix, 2.5 m ⁇ 10X RT-LAMP reaction buffer (Empirical Bioscience), 3.5 m ⁇ lOmM each dNTPs mix (Empirical Bioscience), 6 m ⁇ lOOmM MgSCri (Empirical Bioscience), 0.5 m ⁇ 15,000 units/ml WarmStart® RTx Reverse Transcriptase (New England Biolabs), 1 m ⁇ 8,000 units/ml Bst DNA polymerase (Empirical Bioscience), 0.5 m ⁇ 25 mM ROX reference dye (Empirical Bioscience), 1.25 m ⁇ 20X EvaGreen dye (Empirical bioscience), and 2 m ⁇ of target.
  • the volume
  • RNA target diluted in water or human saliva are used for testing.
  • Human saliva is diluted 10 times using a sample dilution buffer, containing 0.1 mM sodium citrate, 2 mM EDTA to chelate divalent metal cations and inactivate RNAses, and 2.5 mM TCEP.
  • Tris(2- carboxyethyl)phosphine (“TCEP”) is used to denature RNA-degrading enzymes or RNAses by reducing disulfide bonds in proteins including RNAses. This increases the stability of the RNA and also increases the sensitivity of the assay. This treatment in addition to the heat lysis also improves the safety of the assay for the user by the inactivation of the virions.
  • the diluted saliva is further incubated at 95 °C for 10 min using a heat block before adding to the RT-LAMP reaction mix.
  • the RT-LAMP reaction is carried out at 65 °C for up to 1.5 hr using the QuantStudio ® 3 Real-Time PCR System (ThermoFisher Scientific).
  • the RT-LAMP mix in 200 m ⁇ individual PCR tubes are incubated at 65 °C degrees for 25 min using a water bath. After the incubation, the result is read using the lateral flow assay in EasyNAT ® disposable nucleic acid lateral flow assay detection device (Ustar Biotechnologies) following the manufacture’s instruction.
  • Synthetic RNA targets of 250 copies and 125 copies per reaction were spiked into the RT-LAMP reaction mix. As is shown in FIG. 3, 5/5 of the 250 copies/reaction group and 3/5 of the 125 copies/reaction group were detected within 25 min of incubation. 0/5 of the non-template control was detected in the one-hour incubation period.
  • RT-LAMP reaction mix was prepared and incubated as described above.
  • An enzyme-free control without sample and enzyme was prepared to check for non-specific binding of the tagged primers. As is shown in FIG. 4, 0/1 of the enzyme-free control and 0/3 of the non-template control was detected, and 2/3 of the 200 copies/reaction and 3/3 of the 1000 copies/reaction were detected after the 25 min incubation period.
  • Example 2 RT-LAMP amplification and LFA detection of SARS-CoV-2 in nasopharyngeal swab samples
  • the 10X LAMP primer mix for SARS-CoV-2 contains 16 mM each of FIP and BIP, 4 pM of F3 and B3, and 2 pM of LF and LB.
  • the sequences of the primers are listed in Table 11.
  • the 10X LAMP primer mix for the human control i.e., a positive control based on detection of the human amylase gene
  • the sequences of the primers for detection of this control are listed in Table 12.
  • Each 30 m ⁇ COVID RT-LAMP master mix contains 5m1 10X primer mix, 5 m ⁇ 10X RT- LAMP reaction buffer (Empirical Bioscience), 7 m ⁇ lOmM each dNTPs mix (Empirical Bioscience), 3.5 m ⁇ lOOmM MgS04 (Empirical Bioscience), 1 m ⁇ 15,000 units/ml WarmStart® RTx Reverse Transcriptase (New England Biolabs), and 1.75 m ⁇ 8,000 units/ml Bst DNA polymerase (Empirical Bioscience). The volume is toped up to 30 m ⁇ using molecular-biology- grade water.
  • Each 30 m ⁇ human control LAMP master mix contains 5m1 10X primer mix, 5 m ⁇ 10X RT- LAMP reaction buffer (Empirical Bioscience), 7 m ⁇ lOmM each dNTPs mix (Empirical Bioscience), 3.5 m ⁇ lOOmM MgSCri (Empirical Bioscience), and 2 m ⁇ 8,000 units/ml Bst DNA polymerase (Empirical Bioscience).
  • the volume is toped up to 30 m ⁇ using molecular-biology- grade water.
  • the inactivation buffer contains 1 M TCEP, 0.25 M guanidinium hydrochloride, and 0.125M EDTA at pH 7.6.
  • the dilution buffer contains 1 mM sodium citrate at pH 6.5.
  • the negative control mix contains 17.85 mM TCEP, 4.46 mM guanidium hydrochloride, 2.23 mM EDTA, and
  • the positive control mix contains 17.85 mM TCEP, 4.46 mM guanidium hydrochloride, 2.23 mM EDTA, 0.89 mM sodium citrate, and 200 copies/m ⁇ synthetic SARS-CoV-2 RNA (Twist Bioscience) at pH 6.5.
  • RNA from contrived samples Contrived samples were made by adding 2 m ⁇ of 1.125 X 10 4 copies/m ⁇ of synthetic SARS-CoV-2 RNA into 20 m ⁇ of inactivation buffer immediately followed by adding 100 m ⁇ of COVID- 19-negative nasopharyngeal swab sample in UTM (Universal Transport Medium) to prevent RNA degradation by RNase.
  • the RNA, inactivation buffer, and sample were mixed by pipetting five times.
  • the inactivated sample was incubated at 95°C degrees for 5 min using a heat block to mimic the virus lysis step. After incubation, 1 ml of dilution buffer was added to the sample and mixed by pipetting five times.
  • a 20 m ⁇ aliquot of diluted sample was then added to the 30 m ⁇ COVID RT-LAMP master mix and mixed by pipetting five times. Another 20 m ⁇ aliquot of sample was added to the 30 m ⁇ human control LAMP master mix and mixed by pipetting for five times.
  • a positive control and a negative control COVID RT-LAMP reaction mix were made by mixing a 20 m ⁇ of positive control mix or negative control mix respectively with a 30 m ⁇ COVID RT-LAMP master mix by pipetting five times.
  • the prepared COVID RT-LAMP reaction mix and human control LAMP reaction mix were incubated at 65°C for 25 min using a water bath. After the incubation, the test result was read using the lateral flow assay in EasyNAT® disposable nucleic acid lateral flow assay detection device (Ustar Biotechnologies) following the manufacture’s instruction.
  • the COVID- 19 test was able to detect 19/20 of the contrived samples at 225 copies/m ⁇ synthetic SARS-CoV-2 RNA, and 20/20 human control test were positive for the nasopharyngeal swab sample in UTM.
  • Example 3 Standard operating procedure for RT-LAMP amplification and LFA detection of SARS-CoV-2 in nasopharyngeal swab samples
  • NP swab in transport media such as the UTM Viral Transport (COPAN Diagnostics Inc, CAT# 3C047N), BD UVT Viral Transport Collection Kit (BD CAT# 220531) or an equivalent, should be used as sample input.
  • a positive control and a negative control should be run at the beginning and the end of each batch of testing.
  • test result should be ready within 1 min of the insertion into the device.
  • Control samples that can be used with this protocol a) A "no template" (negative) control is needed to ensure that there is no amplicon contamination in the environment that can result in false positives, and the master mix components and amplification are not causing primer dimerization that can lead to sample-independent false amplification and is used before and after of each batch of testing series and/or shift together with actual COVID- 19 sample tests. It is a mixture of inactivation buffer, dilution buffer, and molecular-biology-grade water.
  • a positive template control of synthetic SARS-CoV-2 RNA (MT007544.1, Twist Bioscience) with a concentration of 200 copies/ pL (equivalent to 10X LOD when added to the control reaction tubes) is needed to ensure that the reverse transcription and amplification are taking place, and that the primers are working and amplifying at the lowest level of detection.
  • the positive control covers the whole genome of SARS- CoV-2 and contains all the targets of the probes used in the assay. Positive control test is used before and at after of every testing series and or shift.
  • An extraction control is not needed since the test does not require RNA extraction.
  • a specimen control of human amylase gene is needed to ensure that the polymerase is functional and that the sample does not contain enough of any LAMP inhibitor to inhibit the reaction, and that sufficient quantities of the human sample was obtained to amplify gene in the human NP swab sample and is used with every sample tested.
  • the internal control is amplified and detected as a separate reaction in a separate lateral flow assay contained in a cartridge and is run on every sample in parallel to the RT-LAMP test for the virus.
  • the T line detects SARS-CoV-2 RNA in the case of the COVID-19 test, and detects human DNA in the case of the human specimen control test ( see FIG. 14).
  • Negative Control The “no template” negative control should have a visible C line, and no visible T line (see FIG. 15)
  • the positive template control should have a visible C line and a visible T line (see FIG. 15)
  • the COVID test may be packaged as a kit comprising one or more of the following:
  • the SARS-CoV-2 RT-LAMP reaction mix and the control RT- LAMP reaction mix are provided as dried (e.g., lyophilized) pellets.
  • the pellets are formulated as shown in Table 18. Table 18. Formulations of prepared reaction mixes
  • the test takes approximately 35 minutes from start of test to test read out, as detailed below. This is a medium throughput test that requires no sophisticated equipment. One operator is able to perform up to 10 tests per hour in batches. Specimen transport to results will vary from site to site and it is dependent on the technical team conducting the assay. Ideally, the tests will be conducted in series of at least 30 tests at the time to increase throughput and time to result. The test is ideal for rapid, one-off testing without the need for batching e.g. at a rural hospital or urgent care before a procedure. 1) Adding sample to lysis/storage buffer and heat lysing: 5 minutes
  • Example 4 Performance evaluation of the COVID-19 diagnostic test described in Example 3 1. Limit of Detection (LoD) - Analytical Sensitivity:
  • the limit of detection (LOD) of the COVID-19 test were determined to be the lowest concentration of a synthetic SARS-CoV-2 RNA (Twist Synthetic SARS-CoV-2 RNA, MT007544.1) spiked into clinical COVID-19 negative nasopharyngeal swab (NP) matrixes that can be detected 19 out of 20 replicates.
  • a preliminary determination of the LOD was performed by two three-fold dilutions of the synthetic RNA targets into negative NP swabs with three replicates in each dilution group.
  • the detection result of the preliminary LOD determination is summarized in Table 19 and the full data set can be found in Appendix 2.
  • the lowest diluted concentration (225 copies/pL) that can be detected 3/3 replicates was further confirmed by the successful detection of 19/20 replicates to be the LOD of the COVID-19 test, as indicated in Table 19.
  • Inclusivity analytical sensitivity : The inclusivity of the COVID-19 test primers was evaluated using in silico analysis using NC_045512 from GenBank as a reference for SARS-CoV-2. In total, 22,427 complete length SARS-CoV-2 genomes were downloaded from the NCBI database (October 23, 2020) and aligned. Before analysis, 18 sequences were excluded due to ambiguous nucleotides in target region, which reduced the total to 22,409 sequences for inclusivity check. Results are summarized in Table 20.
  • B2 bat adenovirus (Sequence ID: KX961096.1, highest human adenovirus is 60% similarity)
  • LF Mycobacterium tuberculosis , Bordetella pertussis , Bacillus anthracis , Staphylococcus epidermidis , Staphylococcus aureus
  • LB Influenza A (H6N1 Sequence ID: EU049951.1, next highest influenza A sequence is 72% similarity), Streptococcus pneumoniae , Candida albicans)
  • Nasal allergy spray Triamcinolone acetonide 10 %(v/v) 3/3 0/3 (Nasocort)
  • Saliva collection buffer 0.1 mM Sodium Citrate and 2 mM EDTA at pH 9.6, stored in 800 pL single use aliquots in 1.5 mL RNase-free Eppendorf tubes
  • a 10X primer mix will be made in single use aliquots (100 pL) for each primer set as follows:
  • Exemplary lysis buffers include the following:
  • the buffers may be pH adjusted, for example to 6.5.
  • Inactivation buffer 1 M TCEP, 0.25 M Guanidium hydrochloride, and 0.125 M EDTA at pH 7.6, stored in 20 pL single use aliquots in 1.5 mL RNase-free Eppendorf tubes
  • Tube 1 used for sample preparation, contains drop of Inactivation Buffer
  • Tube 2 contains Dilution Buffer
  • Tubes with lyophilized amplification reagents o Tube 3: contains COVID-19 Reaction chemicals o Tube 4: contains Specimen Control Reaction chemicals
  • Dropper A 100 pL
  • Dropper B general purpose
  • Dropper C 40 pL
  • Sample input should only be nasal or NP swab in UTM or equivalent virus transport media. Protocol:
  • Step 1 Prepare kit and lab space
  • the Fire7 tablet purchased from GoDx will have the miniPCR App (Ampylus LLC) pre installed. Assay heating protocols, GoCOVIDx Program 1 and Program 2, will already be loaded into the miniPCR App. Within the miniPCR App, connect the tablet to the machine via BluetoothTM. Larger 1.5 mL tubes (Tube 1) fit into center channel junctions of miniPCR machine. Smaller 0.2 mL tubes (Tube 3 & 4) fit into the miniPCR tube slots.
  • Step 2 Inactivate and heat lyse
  • Droppers A and C are specifically designed to transfer specific volumes via the Pipette Tube. Extra liquid in the overflow bulb is OK. There is no need to squeeze the overflow bulb.]
  • Step 3 Dilute lysate • Squeeze bulb of Dropper B then immerse tip into Tube 2 liquid. Release pressure on bulb to pick up entire volume and dispense into Tube 1 by moving tip into Tube 1 and squeezing the bulb.
  • Each Tube 3 & 4 contains a dried-down pellet of reaction chemicals for either COVID-19 or Specimen Control (check tube label).
  • the dried down pellet will dissolve when liquid is added to the tube in the following steps.
  • Step 5 Incubate amplification reactions
  • Tube 3 Place Tube 3 into one EasyNAT device and Tube 4 into the other.
  • Tube 3 Target determines COVID-19 diagnosis
  • Tube 4 Target confirms adequate patient sampling was performed.
  • Example 7 Exemplary diagnostic device.
  • FIG. 13 An exemplary diagnostic device and its use is illustrated in FIG. 13.
  • the pieces of the device may be fabricated using SLA 3D printing (Form2, FormLabs), although those of skill in the art can prepare the device and components using other methods.
  • Lysed sample is added to the inlet (FIG. 13C). As shown, the lysed sample is added via micropipette, but the lysed sample may be drained directly from a pierce-bottom sample tube, removing the need for liquid handling equipment. Lysate is wicked along the paper microchannel into the reaction chamber. Once the sample wicks into the reaction chamber, reaction reagents are mixed in, setting up the isothermal amplification (FIG. 13D).
  • the reaction reagents may be lyophilized and stored within the reaction chamber when packaged until ready for use.
  • the dry reagents will mix with the sample lysate following the addition of rehydration buffer, which will drain from a reagent blister reservoir.
  • the sliding channel is moved from inactive position (far end of its slot) to active position (near end of its slot), providing fluidic connection between the reaction chamber and the LFA strip (FIG. 13E).
  • the channel may be moved manually moved or automatically moved with the use of a controller.
  • the amplicons wick through the sliding channel into the secondary paper microchannel all the way to the sample pad of the LFA strip. From there the sample wicks through the diagnostic strip, providing either a positive or negative readout (FIG. 13F).

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Abstract

Cette invention concerne un dispositif de diagnostic du virus SARS-COV-2 au point de soins, trousse et procédés d'utilisation de celui-ci. Le dispositif comprend une composition pour l'amplification isotherme médiée par boucle d'une séquence nucléotidique cible du SARS-CoV-2, qui comprend des amorces pour l'amplification RT-LAMP de la séquence cible.
PCT/US2021/035977 2020-06-04 2021-06-04 Dispositif de diagnostic du virus sars-cov-2 au point de soins et procédés d'utilisation de celui-ci WO2021248053A2 (fr)

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