Novel recombinase enzymes enable isothermal amplification of DNA / RNA in a consumer-friendly device

Abstract

This disclosure provides recombinase enzymes that enable isothermal amplification of DNA or RNA. Also provided are consumer-friendly diagnostic devices in which the isothermal amplification occurs and a sample is analyzed. In particular, the recombinase enzymes and described diagnostic devices are used to detect inter alia, infectious diseases or non-infectious disease.

Description

Docket No.: 070439.01841 NOVEL RECOMBINASE ENZYMES ENABLE ISOTHERMAL AMPLIFICATION OF DNA / RNA IN A CONSUMER-FRIENDLY DEVICE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. §119(e) of the earlier filing date of U.S. Provisional Patent No.63 / 587,587, filed on October 3, 2023, which is hereby incorporated by reference in its entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] This application contains a Sequence Listing which has been submitted electronically in xml format and is hereby incorporated by reference in its entirety. Said xml copy, created on September 12, 2024, is named 070439_01841_SeqList.xml and is 9,698 bytes in size. FIELD OF THE INVENTION

[0003] The present invention relates to novel recombinase enzymes for molecule detection. In particular, the invention relates to devices, systems, and methods for detecting the presence, absence, and / or quantity of molecules within a sample. BACKGROUND OF THE INVENTION

[0004] Antigen tests enable the detection of a particular viral antigen to diagnose current viral infection. An example of such tests are SARS-CoV-2 antigen tests. Antigen tests (“rapid tests”) obtain results in a shorter period of time than PCR tests, however, antigen tests do not achieve the same degree of accuracy as PCR tests. Specifically, PCR tests can be up to 10 times more sensitive than antigen tests. However, quick and early detection of infectious disease is necessary for patients to obtain treatment and prevent further spread of the disease. In addition to the fact that PCR tests take a longer period of time to obtain results than antigen tests, PCR tests also require extensive laboratory equipment and trained laboratory staff. Thus, rural areas and developing countries may not readily have access to PCR tests.

[0005] Another alternative to PCR tests are nucleic acid amplification technologies (NAATs) that enable rapid and accurate diagnostic testing. NAATs involve PCR and 1 162542436.1Docket No.: 070439.01841 isothermal amplification. There are various types of isothermal amplification technologies (e.g., Loop-mediated isothermal amplification (LAMP), Recombinase polymerase amplification (RPA), Rolling circle amplification (RCA), Strand displacement amplification (SDA) and Nucleic acid sequence-based amplification (NASBA) that each use different enzymes. In contrast to traditional PCR, isothermal amplification technology (IAT) does not require temperature changes and is both highly specific and fast. Notably, IAT is comparable with PCR in terms of amplification efficiency and can even surpass it.

[0006] Despite the advances in diagnostics, there remains a need to develop readily accessible rapid diagnostic tests for infectious diseases and non-infectious diseases (such as cancer or autoimmune diseases). SUMMARY OF THE INVENTION

[0007] In one aspect, this disclosure provides novel recombinase enzymes for isothermal amplification of DNA or RNA. In another aspect this disclosure provides consumer- friendly diagnostic devices in which the isothermal amplification occurs and a sample is analyzed.

[0008] In one aspect, this disclosure provides a recombinase polymerase amplification composition comprising: a) a mixture of at least three enzymes selected from the group comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6; b) a DNA polymerase; and c) a set of primers comprising a first and a second primer.

[0009] In some embodiments, three enzymes comprise the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 4, and SEQ ID NO: 6. In some embodiments, three enzymes comprise the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 5.

[0010] In some embodiments, the DNA polymerase is a eukaryotic or a prokaryotic polymerase. 2 162542436.1Docket No.: 070439.01841

[0011] In some embodiments, the composition further comprises a reaction buffer. In some embodiments, the reaction buffer comprises 50 mM Tris pH 7.0-9.0, 100 mM potassium acetate, 100 mM potassium chloride, a crowding agent, 2 mM DTT (dithiothreitol), 3-20 mM ATP, 50 mM creatine phosphate, 14 mM magnesium acetate, and / or 14 mM magnesium chloride.

[0012] In some embodiments, the primer set comprises tags that decrease the amplification time. In some embodiments, the tags are FAM (fluorescein).

[0013] In some embodiments, the composition is combined with a sample of DNA or RNA. In some embodiments, the composition and sample are heated at a reaction temperature of about 22 °C to about 45 °C for at least 5 minutes.

[0014] In some embodiments, the reaction product is analyzed with a lateral flow test.

[0015] In some embodiments, an at-home diagnostic kit comprises the composition.

[0016] In one aspect, provided is an amplification method to detect a disease, the method comprising: a) contacting a sample of DNA or RNA with a mixture of at least three enzymes with a set of primers, wherein the enzymes are selected from the group comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6; b) heating the mixture of at about 22 °C to about 45 °C for a given period of time to obtain a reaction product; and c) detecting the reaction product with a lateral flow test.

[0017] In some embodiments, the three enzymes are selected from the group comprising: a) the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 4, and SEQ ID NO: 6; or b) the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 5.

[0018] In some embodiments, the mixture is heated for at least 5 minutes.

[0019] In some embodiments, the method further comprises a crowding agent in step and a DNA polymerase in step a).

[0020] In some embodiments, the disease is an infectious disease or a non-infectious disease. 3 162542436.1Docket No.: 070439.01841

[0021] In some embodiments, the method detects the disease in about less than 20 minutes.

[0022] In some embodiments, the method detects 10 or less copies of DNA or RNA.

[0023] In some embodiments, the sample is added to a cassette that is loaded to a device that heats the mixture of step b).

[0024] In some embodiments, the device displays the results of the reaction product.

[0025] In one aspect, provided is a diagnostic device comprising: a) a microfluidic chip comprising a sample reservoir for a sample of DNA or RNA, a reaction buffer, and a Master Mix; b) a plunger that draws the sample from the reservoir to a syringe; c) a heating module that heats the sample; and d) a lateral flow strip that detects a reaction product.

[0026] In some embodiments, the Master Mix comprises a mixture of at least three enzymes with a set of primers, wherein the enzymes are selected from the group comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

[0027] In some embodiments, the device detects an infectious disease or a non-infectious disease.

[0028] In one aspect, provided is a diagnostic device comprising: a) at least two blister pouches mounted onto a piercing structure of a microfluidic cartridge, wherein the first blister pouch contains a Master Mix and the second blister pouch contains a reaction buffer, and optionally further comprises a flow- restrictor structure within a fluidic pathway; b) a reservoir for a sample of DNA or RNA; c) a heating module to heat the sample; and d) a lateral flow strip that detects a reaction product.

[0029] In some embodiments, the Master Mix is introduced into a circuit by actuating the blister.

[0030] In some embodiments, the reaction buffer is delivered into a circuit by i) actuating the blister or ii) via a vent. 4 162542436.1Docket No.: 070439.01841 BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG.1 shows Coomassie stains 4 different enzymes that were expressed in E. coli and purified via affinity chromatography. Enzyme 4 is a commercially available strand- displacing DNA polymerase. Enzyme 1 is Candidatus Poseidoniales archaeon gp32-like protein (SEQ ID NO: 1), Enzyme 2 is Candidatus Woesearchaeota archaeon UvsX-like protein (SEQ ID NO: 3), and Enzyme 3 is Candidatus Woesearchaeota archaeon UvsY- like protein (SEQ ID NO: 6).

[0032] FIG.2 depicts a Coomassie stain indicating that the enzymes in FIG.1 amplify a 300 base pair sequence of pUC19 (a plasmid cloning vector) between 30 °C to 42 °C with non-specific amplification. No DNA input served as a negative control.

[0033] FIGS.3A-3B are schematic overviews of how the recombinase polymerase amplification (RPA) works in two different methods. FIG.3A illustrates RPA when a reaction buffer is used in combination with enzymes in an aqueous form. FIG.3B illustrates RPA when the enzymes are pre-mixed and lyophilized, which reduces the total time of the method.

[0034] FIG.4 is a general overview of a diagnostic device that uses RPA in which a sample of DNA or RNA is loaded into a cassette and then inserted into standalone hardware that heats the cassette to a specified temperature. The results of the reaction product are then displayed on the device.

[0035] FIG.5 illustrates a variety of samples that can be placed into the cassette of FIG. 4.

[0036] FIG.6 is a schematic of a consumable that uses RPA in which air is displaced with a syringe by actuating a plunger driven by a motor via a mechanical interface between the durable and consumable.

[0037] FIG.7 is a schematic of a blister pouch.

[0038] FIG.8 is a high-level schematic of a consumable using blister pouches. Numbers indicate the order of sample processing, where the sample is inserted into the heating / cooling chamber (1), then moves through the mixing module (2), followed by encountering the reaction buffer (3) and Master Mix (4), before transporting back through 5 162542436.1Docket No.: 070439.01841 the mixing module (5) and heating / cooling chamber (6), finally reaching the lateral flow strip (7).

[0039] FIG.9 is a representation of a spring driven system. The flow is driven by a highly compressed spring connected to a plunger, and controlled by passing through a precision orifice, resulting in an almost constant positive pressure on the fluid. The path the fluid is then distributed with the various valves in the system. A large relative size of the plunger in comparison to the channel size, ensures that a great amount of volume is transported for a relatively slow displacement of the spring.

[0040] FIG.10 illustrates blister delivery using a spring driven mechanism. Master Mix (MM) and buffer are introduced into the circuit by actuating the blister where they are contained. Buffer can also be introduced from a vent. Numbers indicate the order of sample processing, where the sample is processed through the mixing module / geometry (1-3), before transporting to the lateral flow strip (4). LF: lateral flow test.

[0041] FIG.11 shows a chamber mechanism combined with a spring driven mechanism. Master Mix (MM) and buffer are introduced into the circuit by drawing fluid from the chambers they are contained in. This method introduces more valves and an overall more complex system. Numbers indicate the order of sample processing, where the sample is encounters the reaction buffer (1), then the Master Mix (MM), and Sample Reservoir (3) before being transported through the heating chamber (4-5) and the mixing module / heating chamber (5-6), and finally reaching the lateral flow strip (7). LF: lateral flow test.

[0042] FIG.12 illustrates examples of the valves.

[0043] FIGS.13A-13C depict different methods of mixing. A: Passive mixing relying on diffusion; B: Active mixing using magnetic stirrers; C: Active mixing using acoustofluidics.

[0044] FIGS.14A-14B show the heating mechanism of the device. A: Overview of the heating module; B: Schematic of the flow of the hot and cold fluid through the channel inlet. 6 162542436.1Docket No.: 070439.01841 DETAILED DESCRIPTION OF THE INVENTION

[0045] This disclosure relates to novel recombinase enzymes that enable isothermal amplification of DNA or RNA. In particular, the described recombinase enzymes are used in Recombinase Polymerase Amplification (RPA).

[0046] Recombinase Polymerase Amplification is an isothermal amplification technology (IAT) that involves comprises three enzymes: 1. a recombinase (T4 UvsX), 2. a ssDNA binding protein (SSBP), named gp32, and 3. the Bsu DNA polymerase or another strand-displacing polymerase. UvsX as well as other DNA-binding proteins or cofactors can form nucleic acid-protein complexes with single-stranded DNA. Note that UvsX is used in combination with its accessory protein, UVsY and gp32 to form D-loop recombination structures to initiate amplification. The complex can then hybridize with the complementary region of the target DNA to further complete the strand replacement reaction. T4 gp32 is an SSBP that is necessary for DNA replication and repair. The Bsu DNA polymerase has strand replacement activity (See Wang et al. Biosensors (Basel). 2023.12(2): 160).

[0047] RPA begins when T4 UvsX binds to primers in the presence of ATP and a crowding agent (a high molecular polyethyleneglycol), forming a recombinase-primer complex. The crowding agent prevents spontaneous recombinase-primer disassembly that occurs in the presence of the single stranded binding proteins needed for the amplification. The complex then interrogates double-stranded DNA seeking a homologous sequence and promotes strand invasion by the primer at the cognate site. The displaced DNA strand is stabilized by single-stranded binding proteins. Then the recombinase disassembles and a strand displacing DNA polymerase (e.g., large fragment of Bacillus subtilis Pol 1, Bsu) binds to the 3′ end of the primer to elongate it in the presence of dNTPs. This process undergoes cyclic repetition, resulting in the amplification of a given target (e.g., DNA or RNA). RPA operates at temperatures ranging from 22 °C to 45 °C. In particular, RPA can amplify as few as 1-10 DNA target copies in under 20 minutes (See Lobato and O’Sullivan. Trends Analyt. Chem.2018.98: 19-35). 7 162542436.1Docket No.: 070439.01841

[0048] RPA can be used to detect an infectious disease or a non-infectious disease in a subject. As used herein, the term “subject” refers to an animal, preferably a mammal such as a human.

[0049] In some embodiments, the non-infectious disease is cancer or an autoimmune disease. Notably, RPA can be used to detect any nucleic acid. In some embodiments, the cancer is selected from the group comprising anal cancer, bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, lung cancer, lymphoma, multiple myeloma, ovarian cancer, pancreatic cancer, renal transitional cell cancer, skin cancer, synovial sarcoma, and thyroid cancer. In some embodiments, the autoimmune disease is selected from the group comprising graves’ disease, hashimoto thyroiditis, multiple sclerosis, rheumatoid arthritis, sjögren syndrome, lupus, and inflammatory bowel disease.

[0050] In some embodiments, the infectious disease is selected from the group comprising coronavirus, influenza, rhinovirus, herpes simplex virus, a fungal infection, and a bacterial disease.

[0051] This disclosure also relates to consumer-friendly diagnostic devices in which the isothermal amplification occurs and a sample is analyzed via a lateral flow test. In some embodiments, the sample is placed into disposable cassettes that are inserted into standalone hardware that heats the reaction and displays the result of the reaction. In some embodiments, a consumable is provided that uses air displacement with a syringe to cause the sample to contact a Master Mix reservoir. In some embodiments, the consumables involve blister pouches. In some embodiments, the device uses a spring driven mechanism to drive the fluid into various valves in the system. In some embodiments, a chamber mechanism is used in combination with a spring driven mechanism. Recombinase Enzymes

[0052] As used herein, “recombinases” refer to RPA enzymes that pair oligonucleotide primers with homologous sequences in template double-stranded nucleic acid. Thus, DNA synthesis occurs at specific points in a template double-stranded nucleic acid. 8 162542436.1Docket No.: 070439.01841

[0053] In some embodiments, the recombinases are derived from the archaea kingdom. In some embodiments, the recombinases derived from the archaea kingdom are SSBP, UvsX and UVsY. In some embodiments, the recombinases are modified (e.g., mutated) to improve their efficiency in recombinase polymerase amplification. In some embodiments, the modified recombinases facilitate decreased time to detect a sample of interest. In some embodiments, the modified recombinases can be used to detect a sample in less than 20 minutes such as 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, or any amount of time in-between. In some embodiments, recombinases are used to generate a reaction product via RPA that is applied to a lateral flow test.

[0054] In some embodiments, the recombinases comprise the amino acid sequences shown in Table 1. 9 162542436.11481A0P VP.L ENKE DLV F DNI TD A FE TIVPTY VEVKKIDS AM9FKVD34 RNPTSNRIKP LDTASKRSMMI EVID S ANR RTSDIL0VAS DV EEVSELEAEDLSQ GVR S LD REYG MP TADE P TDK70V DSYELVEAL EVDLY:.oNtekcoD . A N Ddednarts-elgnisotgnidnibevorp.mi se ostadni ebddmaoceerrefwo dseenA D oADKEELEYDEKQFKSLAFEKKK LGITQSLcilnr ni Fe eudmTEQ SGK WF GSG FEQWFQ DAFADEQVIKSFFA I DAD KM qnA MDR LDTKLAS PKRD K F AYNGV TQSK GGNFVPETAIePYF F EMAPYPASMAI AsuR LA MT SMTddi nca -2 a- - - X adl3 t2 ao3 tosX vasvoosenl pgibnsu) epag ) eaU ) toU )t ai–nie 1sut hc–nie 1suhc–nie 2suea–nie 2misandonoto emad ranoto et rmadanoto etahcnoto eAid e rie e rie e r m dir e r m. di edi a p y s a p y s a p y a a p y1celama neashc ezndna eohc ezndneohc ezndnechc eznb oniNCoPrakilE( CWrakilE(aC WrakilE(aCaPrakilE(a T m A:O e5tQN]oE5SD 10 NI 1 2 3 4.6304[245261148TI1L Y0 T.9M K QKI3GI4HL0G7YE0S EY:Q.oF RERY ANQMIIKteNLNkALQcoY QLD KEEI FELIR NTEEKD MIYDDESLQL ST IT WY YEIFKYPM K AY V EK QKII LYSDLE LPQ N NNDI QSGEKNK MY KI VF LM KLSVGLVLKD SAEKIA ESEKG L A ADYIQEDF F TKDDEFFHMMPK T HRKLRPN KK SLTEAWEHAD DTEW A DENYQ PASLIVA N VXIQI1XA1LG PKHKAN EIE LARDDI LKD DXIRKKSNAD NESEKKW AKW L DAGGKVEGRVG GG R KFVEYN QY IV K YDIV KYSEAIGE L E SLSAPD DEEWKEWKL K E V DKLVL XV VVENR TRG A V M YGEM Y D - Y - asatY tv oe svsoUut eaa–ni )e3sauhU c– ni )3dhicnroteteor mardaneteeoeor mdanea p yzidsea p yzaca hc e n naohc e nC PrakilE( CWrakilE(5 6 1.634245261Docket No.: 070439.01841

[0056] In some embodiments, the recombinases are modified by methods known in the art including, but not limited to site-directed mutagenesis.

[0057] In some embodiments, the strand-displacing DNA polymerase is a eukaryotic or a prokaryotic polymerase. Examples of eukaryotic polymerases include pol-alpha, pol-beta, pol-delta, pol-epsilon, and mutants or fragments thereof, or combinations thereof. Examples of prokaryotic polymerase include E. coli DNA polymerase I (e.g., Klenow fragment), Bacillus stearothermophilus polymerase I large fragment, bacteriophage T4 gp43 DNA polymerase, Phi-29 DNA polymerase, T7 DNA polymerase, Bacillus subtilis Pol I, Staphylococcus aureus Pol I, E. coli DNA polymerase I, E. coli DNA polymerase II, E. coli DNA polymerase III, E. coli DNA polymerase IV, E. coli DNA polymerase V, and mutants or fragments thereof, or combinations thereof.

[0058] In some embodiments, the recombinases amplify DNA or RNA with non-specific amplification between about 30 °C to about 42 °C such as about 30 °C, about 31 °C, about 32 °C, about 33 °C, about 34 °C, about 35 °C, about 36 °C, about 37 °C, about 38 °C, about 39 °C, about 40 °C, about 41 °C, about 42 °C, or any temperature in- between. Recombinase Polymerase Amplification Composition

[0059] In some embodiments, provided is a recombinase polymerase amplification composition that comprises the recombinases described herein. The composition further contains a set of primers that amplify the target nucleic acid sequence. In some embodiments, there is a first primer and a second primer. The first primer can be complementary to the target nucleic acid sequence. The second primer can comprise a first portion that is complementary to the target nucleic acid sequence and a second portion that is different from the target nucleic acid sequence at one or more positions. The terms “first” and “second” are only used to distinguish one element from another element and should not be constructed to limit the disclosure.

[0060] The primers can comprise sequences that are complementary to a target nucleic acid sequence of interest or that differ from the target nucleic acid sequence of interest at 12 162542436.1Docket No.: 070439.01841 one or more positions. In some embodiments, the target nucleic acid sequence of interest corresponds to that of infectious diseases (e.g., a virus, bacteria, fungus, etc.) or non- infectious diseases such as cancers or autoimmune diseases.

[0061] In some embodiments, the primer set comprises tags that decrease the amplification time due to the fact that the tags facilitate visualization of the reaction via lateral flow chromatography. In some embodiments, the forward or reverse primer is labeled with FAM (fluorescein). In further embodiments, the primer set uses a biotin- labeled probe for product visualization. The biotin-labeled probe can be captured with streptavidin.

[0062] In some embodiments, the recombinase polymerase amplification composition can be combined with fluorescent probes for real-time detection.

[0063] In some embodiments, the primers are between 20 to 35 bases in length such as 20 bases, 21 bases, 22 bases, 23 bases, 24 bases, 25 bases, 26 bases, 27 bases, 28 bases, 29 bases, 30 bases, 31 bases, 31 bases, 32 bases, 33 bases, 34 bases, or 35 bases. In some embodiments, the primers have a GC content that is above 30% and below 70% such as 31%, 40%, 45%, 50%, 55%, 60%, 65%, 68%, 69%, or any amount in-between. The primers can be designed using standard methods known in the art (for example, using the NCBI Primer-Blast website: ncbi.nlm.nih.gov / tools / primer-blast / ).

[0064] In some embodiments, the recombinase polymerase amplification composition further comprises a crowding agent. As used herein, a “crowding agent” refers to an agent that enhances the interaction of the polymerase with the nucleic acid during recombinase polymerase amplification or otherwise influences the kinetics of the recombinase binding to the nucleic acid. In some embodiments, the crowding agent is selected from the group comprising polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), Ficoll, and dextran. In some embodiments, the crowding agent has a molecule weight of less than 200,000 daltons. In some cases, the crowding agent has a molecular weight of at least 1 kilodalton, at least 2 kilodaltons, at least 3 kilodaltons, at least 4 kilodaltons, at least 5 kilodaltons, at least 6 kilodaltons, at least 8 kilodaltons, or at least 10 kilodaltons. In some embodiments, the crowding agent is present in an amount of about 0.5% to about 15% weight to volume (w / v) such as about 13 162542436.1Docket No.: 070439.01841 0.5% w / v, about 1% w / v, about 5% w / v, about 10% w / v, about 15% w / v or any amount in-between.

[0065] In some embodiments, the recombinase polymerase amplification composition is lyophilized. In some embodiments, the lyophilized composition reduces the overall time of the reaction (FIGS.3A and 3B). In some embodiments, the composition comprises a reaction buffer. In some embodiments, the reaction buffer comprises 50 mM Tris pH 7.0- 9.0, 100 mM potassium acetate, 100 mM potassium chloride, a crowding agent, 2 mM DTT (dithiothreitol), 3-20 mM ATP, 50 mM creatine phosphate, 14 mM magnesium acetate, and / or 14 mM magnesium chloride. Consumer-Friendly Diagnostic Devices / Kits

[0066] In some embodiments, the recombinase polymerase amplification composition described herein is used with a consumer-friendly diagnostic device to detect inter alia, an infectious disease or a non-infectious disease (such as cancer or an autoimmune disease). In some embodiments, provided is an at-home diagnostic kit comprising the recombinase polymerase amplification composition described herein. Advantages of the diagnostic devices described herein are that they are non-invasive, affordable, provide rapid results, have high sensitivity, and broad applicability (e.g., detect infectious diseases or non-infectious diseases.).

[0067] The following types of operations are considered when designing the diagnostic device: media reservoirs and actuators, valving, mixing, and heating.

[0068] In some embodiments, a sample is placed into a disposable cassette along with the RPA composition described herein. The cassette is inserted into standalone hardware that heats the reaction and displays the results (FIG.4). There are various cassette / sample configurations that can be used, including, but not limited to a separate lancet, an integrated lancet, a collection cup with a swab, etc. (FIG.5).

[0069] In one aspect, provided is a diagnostic device comprising: a) a microfluidic chip comprising a sample reservoir for a sample of DNA or RNA, a reaction buffer, and a Master Mix; b) a plunger that draws the sample from the reservoir to a syringe; 14 162542436.1Docket No.: 070439.01841 c) a heating module that heats the sample; and d) a lateral flow strip that detects a reaction product.

[0070] In some embodiments, the Master Mix comprises a mixture of at least three enzymes with a set of primers, wherein the enzymes are selected from the group comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

[0071] In some embodiments, the reaction buffer comprises 50 mM Tris pH 7.0-9.0, 100 mM potassium acetate, 100 mM potassium chloride, a crowding agent, 2 mM DTT (dithiothreitol), 3-20 mM ATP, 50 mM creatine phosphate, 14 mM magnesium acetate, and / or 14 mM magnesium chloride.

[0072] In some embodiments, the diagnostic device uses a plunger to draw the sample from a reservoir to a syringe (FIG.6). A plunger then pushes the sample to come into contact with the Master Mix reservoir. The mixture is then heated and tested with a lateral flow strip.

[0073] In some embodiments, the diagnostic device comprises a consumable that contains blister pouches (FIGS.7 and 8). As used herein a “blister pouch” is a package consisting of a cavity, pocket or bubble made from clear plastic and a backing (such as a card or paperboard). An actuation force for each blister can be applied individually (one valve on or other valves off) to ensure better flow rate. Blister pouches can contain a reaction buffer or a Master Mix as described herein.

[0074] In some embodiments, a blister mechanism of delivery is determined by calculating the mass flow rate from the blister pouch to the channel: ^ Calculate channel area A (A=π (di / 2)^2) ^ Assume actuation force (F) and for how much time (t) is applied on the blister ^ Calculate the maximum mass of the liquid inside the channel (m=d*A*l) ^ d = liquid density ^ L = channel length ^ Calculate acceleration (a) from first Newton’s law ^ Derive velocity from acceleration (initial velocity is needed) ^ Calculate mass flow rate by mdot = r * A * V 15 162542436.1Docket No.: 070439.01841 ^ r = liquid density ^ A = area ^ V = velocity

[0075] In one aspect, provided is a diagnostic device comprising: a) at least two blister pouches mounted onto a piercing structure of a microfluidic cartridge, wherein the first blister pouch contains a Master Mix and the second blister pouch contains a reaction buffer, and optionally further comprises a flow-restrictor structure within a fluidic pathway; b) a reservoir for a sample of DNA or RNA; c) a heating module to heat the sample; and d) a lateral flow strip that detects a reaction product.

[0076] In some embodiments, the Master Mix comprises a mixture of at least three enzymes with a set of primers, wherein the enzymes are selected from the group comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. In some embodiments, the reaction buffer comprises 50 mM Tris pH 7.0-9.0, 100 mM potassium acetate, 100 mM potassium chloride, a crowding agent, 2 mM DTT (dithiothreitol), 3-20 mM ATP, 50 mM creatine phosphate, 14 mM magnesium acetate, and / or 14 mM magnesium chloride.

[0077] In some embodiments, the Master Mix is introduced into a circuit by actuating the blister. In some embodiments, the reaction buffer is delivered into a circuit by i) actuating the blister or ii) via a vent.

[0078] In some embodiments, the diagnostic device uses a spring driven system to drive the fluid (FIG.9). For example, the flow of the fluid can be driven by a highly compressed spring connected to a plunger, and controlled by passing through a precision orifice, resulting in an almost constant positive pressure on the fluid. The path the fluid is then distributed with the various valves in the system. A large relative size of the plunger in comparison to the channel size, ensures that a great amount of volume is transported for a relatively slow displacement of the spring. 16 162542436.1Docket No.: 070439.01841

[0079] In some embodiments, the diagnostic device uses a spring driven system in combination with blister delivery (FIG.10). This device comprises a series of valves. Additionally, this device comprises two main paths: 1. no pre-heating and 2. pre-heating. In both cases, there is a mandatory step of adding and mixing the Master Mix. In some embodiments, there is mandatory step of adding and mixing a reaction buffer. In some embodiments, the Master Mix comprises a mixture of at least three enzymes with a set of primers, wherein the enzymes are selected from the group comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. In some embodiments, the reaction buffer comprises 50 mM Tris pH 7.0-9.0, 100 mM potassium acetate, 100 mM potassium chloride, a crowding agent, 2 mM DTT (dithiothreitol), 3-20 mM ATP, 50 mM creatine phosphate, 14 mM magnesium acetate, and / or 14 mM magnesium chloride.

[0080] In some embodiments, the diagnostic device uses a spring driven system in combination with a chamber mechanism (FIG.11). In this device, Master Mix and reaction buffer are introduced into the circuit by drawing fluid from the chambers in which they are contained. This method introduces more valves and an overall more complex system. Additionally, this device comprises two main paths: 1. no pre-heating and 2. pre-heating. In both cases, there is a mandatory step of adding and mixing the Master Mix. In some embodiments, there is an optional step of adding and mixing a buffer. Furthermore, in this device all fluids are driven from a single source.

[0081] In preferred embodiments, the diagnostic device comprises a chip as the consumable with a fluidic network and various components enabling flow movement and operations (mixing, heating). Operations on the chip can be made by small actuators. In some embodiments, the channels between the modules can be 100 µm x 100 µm ducts. In some embodiments, the chip integrates existing microfluidic techniques when suitable.

[0082] In some embodiments, the diagnostic device integrates various techniques used for the above-mentioned operations (e.g., valving (FIG.12), mixing (FIGS.13A-13C), heating (FIGS.14A-14B), etc.) within a low-cost and reliable chip that interfaces with a durable system. 17 162542436.1Docket No.: 070439.01841 Definitions

[0001] As used herein and in the appended claims, the singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise.

[0002] As used herein, the terms “including,” “comprising,” “containing,” or “having” and variations thereof are meant to encompass the items listed thereafter and equivalents thereof as well as additional subject matter unless otherwise noted.

[0003] As used herein, the phrases “in one embodiment,” “in various embodiments,” “in some embodiments,” and the like are used repeatedly. Such phrases do not necessarily refer to the same embodiment, but they may unless the context dictates otherwise.

[0004] As used herein, the terms “and / or” or “ / ” means any one of the items, any combination of the items, or all of the items with which term is associated.

[0005] As used herein, the term “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection. Exceptions can occur if explicit disclosure or context clearly dictates otherwise. All publications mentioned herein are incorporated herein by reference in their entireties. EXAMPLES

[0083] It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments or examples disclosed, but it is intended to cover modifications that are within the spirit and scope of the present invention as defined by the appended claims. Example 1. Generation of Novel Recombinase Enzymes (SSBP, UvsX and UvsY)

[0084] Recombinase enzymes derived from the derived archaea kingdom, and the gp32- like proteins were modified by the insertion of phenylalanines and tyrosines. The recombinases were expressed in E. coli and purified via affinity chromatography (FIG. 1). Enzymes 1-3 and a DNA polymerase and a strand displacing DNA polymerase (e.g., any strand displacing DNA polymerase) were used in recombinase polymerase 18 162542436.1Docket No.: 070439.01841 amplification to amplify a sequence of pUC19 (plasmid cloning vector). The sequences of the primer set used in the reaction are shown in Table 2.

[0085] Table 2. Nucleic acid sequences of primers. SEQ Name Nucleic Acid Sequence ID T G [00pUC19 DNA between 30 °C -40 °C with non-specific amplification (FIG.2). Example 2. Use of Novel Recombinase Enzymes in a User-Friendly Diagnostic Device

[0087] The recombinase enzymes identified in Example 1 are used in a user-friendly diagnostic device to detect an infectious disease or non-infectious disease (such as cancer or an autoimmune disease) in a subject. These enzymes are lyophilized and combined with a sample (such as blood, saliva, etc.). The sample and lyophilized mix are contained in a disposable cassette that is inserted into standalone hardware that heats the reaction to the desired temperature for RPA. The reaction product is tested in a lateral flow test. The entire process takes about 15 minutes (FIG.3B). The standalone hardware displays the results of the test, allowing the subject to easily obtain the results at their convenience. Optionally, the hardware can share the results with the subject’s medical provider.

[0088] Sequence Listing

[0089] Note: Amino acids in bold and underlined were added to improve binding to single-stranded DNA.

[0090] SEQ ID NO: 1 - Candidatus Poseidoniales archaeon – gp32-like protein (Enzyme 1) MSTFADMKKQRKSNLTSLIKETEKITSPNVFGDTDDRFWRPELDKSGNGYAVVRFLPAPD GEDLPWARMWNHGFQGPGGWYIENSLTTLGQYDPVSEHNYQLWNSGYEANKEVYRK 19 162542436.1Docket No.: 070439.01841 QKRRLNYTANVYIIKDPAHPENEGQVKFYRFGKKIFDKINDLMNPEFEDESPVNPFDLWE GANFKMKIRKIEGFSNYDKSEFETPTALLDDEARLEEIWKSEFSLKELVSTDKFKTFEELK IKLDKVLGLGTENIPPAFSLPKDKTEDVPFDGGVPIQTTPKPAAESEDNDEEALGYFQKLA ETA

[0091] SEQ ID NO: 2 - Candidatus Woesearchaeota archaeon – gp32-like protein (Enzyme 1) MSFSDLKKQSSLGSLTNKLVKEVEKMNNSGGSGADDRLWKPEMDKSGNGYAVIRFLPAP EGEDLPWVKLYSHAFQGPGGWYIENSLTTIGGYDPVGELNYELWNSGYESDKDVYRKQ KRKLSYYANIYVVKDPANPQNEGGVFLYKFGKKIFDKIMDAMQPEFEDETPINPFDFWQ GANFKLKLKKVAGFWNYDSSEFDRQAPLLDDDDALEALWKKQYSLSAFTAADQFKTY DELKKRLDYVLGSKSTRNVAQEETEYDNYAATEQKSVSEEDVMQKLEDSYKASKQVES TSTADDDDPMSYFSKLADS

[0092] SEQ ID NO: 3 - Candidatus Woesearchaeota archaeon – UvsX-like protein (Enzyme 2) MNDFLKDVIKETGNEYASLASEGVVGGDVESFIDTGSYAFNALLSGSIYGGLPGS RITAIAGEAATGKTFFALGICKHFLDKDKDAGVIYFESENAVSKDMLEKRGIDSSR VVIMPVATVQEFRLQAIKIIDKYLEQEKDKRKPIMFVLDSLGMLSTTKEMEDTAE GKETRDMTRSQIVKSAFRVLTLKLGQAKVPMIMTNHTYDVIGSMFPQKEMGGGS GLKYAASSIVYLGKKKEKDGTEVVGNIVHCKNYKSRITKENAMVDVRLTYTKGL DQHYGLLDLAEEAGIFSKVSTRYELPDGSKQYAKTINNEPEKYFKKEILDKIDEYT RKKFTYGTEE

[0093] SEQ ID NO: 4 - Candidatus Pacearchaeota archaeon – UvsX-like protein (Enzyme 2) MNDFLSDLVKTLGDEHTTIASDEKSSAEFSGTVDTGSYVLNAVLSGSIYGGVPDNKVTV FAGETATGKTFFVLGVVSQFLKNNPEGSVMYFDTESAVTNEMMQSRGIDTRRVIKSEPD TIQKFRHTALQTIDFQISQDKEERRPMMMILDSLGQLSSTKEIEDTAKGDETRDMTKAQI LKATFRVLNLKLAKANVPLLVCNHVYDVVGAYYPTKEMSGGSGLKYSASTIAMLSKR KERDSDKEIVGNIITVKMEKSRLSKENKQVEVLLTYDKGLDRYYGLIDMAVDAGIFKKL 20 162542436.1Docket No.: 070439.01841 SNRIELPDASKHYASKIYGNPKEYFTTDIMERIEKYANQEFQYGGMTTVEVEPEVVVDEE KEGSAETLTMIDADVE

[0094] SEQ ID NO: 5 - Candidatus Pacearchaeota archaeon – UvsY-like protein (Enzyme 3) MNLEDLKEEAKKDLPILNHEHMEQESFKNQIIKPKWLEYKTNFEQLLIMRRSEHQKLYR EKWEYYGGKSDAKVYAARPFDIKVLKNDLQMYIQSDDEILELQNKIAYYELIIKYIEGV VKSIDNRGWDIRHAQDWKKFEAGMI

[0095] SEQ ID NO: 6 - Candidatus Woesearchaeota archaeon– UvsY-like protein (Enzyme 3) MTLDELKVQVANDLKVNDERLDTESLKNQELYAKYLEIKSNFELLMYKAKGDYKILYR XKWEYYGGKAXAXIXETKPFDLKVLKSDLSVYIESDEDIIKLENKIVYLETVIKYVDGVL KSIASRGWDIKNAIQWKNFEAGLM

[0096] SEQ ID NO: 7- Forward primer CCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATC

[0097] SEQ ID NO: 8- Reverse primer GGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCG 21 162542436.1

Claims

Docket No.: 070439.01841 CLAIMS What is claimed is:

1. A recombinase polymerase amplification composition comprising: a) a mixture of at least three enzymes selected from the group comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6; b) a DNA polymerase; and c) a set of primers comprising a first and a second primer.

2. The composition of claim 1, wherein three enzymes comprise the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 4, and SEQ ID NO:

6.

3. The composition of claim 1, wherein three enzymes comprise the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO:

5.

4. The composition of claim 1, wherein the DNA polymerase is a eukaryotic or a prokaryotic polymerase.

5. The composition of claim 1, wherein said composition is lyophilized.

6. The composition of claim 1, further comprising a reaction buffer.

7. The composition of claim 6, wherein the reaction buffer comprises 50 mM Tris pH 7.0-9.0, 100 mM potassium acetate, 100 mM potassium chloride, a crowding agent, 2 mM DTT (dithiothreitol), 3-20 mM ATP, 50 mM creatine phosphate, 14 mM magnesium acetate, and / or 14 mM magnesium chloride.

8. The composition of claim 1, wherein the primer set comprises tags that decrease the amplification time.

9. The composition of claim 8, wherein the tags are FAM (fluorescein).

10. An at-home diagnostic kit comprising the composition of claim 1.

11. The composition of claim 1, wherein said composition is combined with a sample of DNA or RNA.

12. The composition of claim 10, wherein said composition and said sample are heated at a reaction temperature of about 22 °C to about 45 °C for at least 5 minutes. 22 162542436.1Docket No.: 070439.01841 13. The composition of claim 12, wherein the reaction product is analyzed with a lateral flow test.

14. An amplification method to detect a disease, the method comprising: a) contacting a sample of DNA or RNA with a mixture of at least three enzymes with a set of primers, wherein the enzymes are selected from the group comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6; b) heating the mixture of at about 22 °C to about 45 °C for a given period of time to obtain a reaction product; and c) detecting the reaction product with a lateral flow test.

15. The method of claim 13, wherein the three enzymes are selected from the group comprising: a) the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 4, and SEQ ID NO: 6; or b) the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO:

5.

16. The method of claim 14, wherein the set primers comprise tags that decrease the amplification time.

17. The method of claim 16, wherein the tags are FAM (fluorescein).

18. The method of claim 14, wherein the mixture is heated for at least 5 minutes.

19. The method of claim 14, further comprising a crowding agent and a DNA polymerase in step a).

20. The method of claim 14, wherein the disease is an infectious disease or a non- infectious disease.

21. The method of claim 14, wherein said method detects the disease in about less than 20 minutes.

22. The method of claim 14, wherein said method detects 10 or less copies of DNA or RNA.

23. The method of claim 14, wherein the sample is added to a cassette that is loaded to a device that heats the mixture of step b). 23 162542436.1Docket No.: 070439.01841 24. The method of claim 23, wherein the device displays the results of the reaction product.

25. A diagnostic device comprising: a) a microfluidic chip comprising a sample reservoir for a sample of DNA or RNA, a reaction buffer, and a Master Mix; b) a plunger that draws the sample from the reservoir to a syringe; c) a heating module that heats the sample; and d) a lateral flow strip that detects a reaction product.

26. The device of claim 25, wherein the Master Mix comprises a mixture of at least three enzymes with a set of primers, wherein the enzymes are selected from the group comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO:

6.

27. The device of claim 25, wherein the reaction buffer comprises 50 mM Tris pH 7.0-9.0, 100 mM potassium acetate, 100 mM potassium chloride, a crowding agent, 2 mM DTT (dithiothreitol), 3-20 mM ATP, 50 mM creatine phosphate, 14 mM magnesium acetate, and / or 14 mM magnesium chloride.

28. The device of claim 25, wherein said device detects a disease that is an infectious disease or a non-infectious disease.

29. A diagnostic device comprising: a) at least two blister pouches mounted onto a piercing structure of a microfluidic cartridge, wherein the first blister pouch contains a Master Mix and the second blister pouch contains a reaction buffer, and optionally further comprises a flow-restrictor structure within a fluidic pathway; b) a reservoir for a sample of DNA or RNA; c) a heating module to heat the sample; and d) a lateral flow strip that detects a reaction product.

30. The device of claim 29, wherein the Master Mix comprises a mixture of at least three enzymes with a set of primers, wherein the enzymes are selected from the 24 162542436.1Docket No.: 070439.01841 group comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO:

6.

31. The device of claim 29, wherein the reaction buffer comprises 50 mM Tris pH 7.0-9.0, 100 mM potassium acetate, 100 mM potassium chloride, a crowding agent, 2 mM DTT (dithiothreitol), 3-20 mM ATP, 50 mM creatine phosphate, 14 mM magnesium acetate, and / or 14 mM magnesium chloride.

32. The device of claim 29, wherein Master Mix is introduced into a circuit by actuating the blister.

33. The device of claim 29, wherein the reaction buffer is delivered into a circuit by i) actuating the blister or ii) via a vent.

34. The device of claim 29, wherein said device detects an infectious disease or a non-infectious disease. 25 162542436.1

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