Molecular diagnostic device

Abstract

The present invention relates to membranes for amplification and detection of a nucleic acid and methods of manufacturing thereof. The present invention further relates to diagnostic devices and systems comprising said membranes.

Description

[0001] MOLECULAR DIAGNOSTIC DEVICE

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to membranes for amplification and detection of a nucleic acid and methods of manufacturing thereof. The present invention further relates to diagnostic devices and systems comprising said membranes.

[0004] BACKGROUND TO THE INVENTION

[0005] Molecular diagnostics have remained challenging to implement in low and middle-income countries (LMIC) primarily due to cost considerations and infrastructure requirements.

[0006] Classical PCR-based methods require sophisticated and costly instruments, sophisticated fluidics and a need for temperature cycling. Furthermore, such diagnostics often require coldchain transport and storage of reagents meaning they do not find utility outside of laboratory environments in LMIC settings.

[0007] Isothermal nucleic acid amplification methods are able to amplify target nucleic acids from trace levels to very high and detectable levels within a matter of minutes without the need for thermal cycling and associated instrumentation. Such isothermal methods include Recombinase Polymerase Amplification (RPA), as described in WO 2003 / 072805 and WO 2021 / 094746. WO 2021 / 094746 describes variant RPA methods referred to further herein as ‘mRPA’ which utilize reaction components tagged with intrinsically disordered regions. RPA methods can allow users to detect a particular sequence in trace amounts.

[0008] Attempts have been made to deliver molecular diagnostics at low cost. However, to the best of the present inventors’ knowledge, no platform has achieved the <$10 cost required to significantly improve access to diagnostics in LMIC-settings. Multiple groups have attempted to develop paper-based nucleic acid amplification tests (NAATs) using diverse isothermal methods including LAMP, RPA, NASBA and many more. These typically suffer from low sensitivity, poor manufacturability, complex electronics, power-intensive heating requirements, the need for separate amplification and detection steps (e.g. involving the needfor liquid handling steps, specialist laboratory equipment, etc.) all of which limit their impact in providing access to affordable molecular diagnostics.

[0009] For example, attempts have been made to employ lateral flow-based detection of RPA products (see e.g. WO 2019 / 055780). However, this approach has drawbacks in that: (i) it requires preparation of a lateral flow strip comprising immobilized capture antibodies; and (ii) an RPA amplification step must be performed separately in a different container prior to transfer of the sample to the lateral flow strip for detection.

[0010] It is an object of the present invention to address at least some of the problems noted above.

[0011] SUMMARY OF THE INVENTION

[0012] The present inventors have developed an in-situ amplification-detection approach involving RPA amplification coupled with a colorimetric end-point detection. This approach is performed using a membrane as the reaction matrix. A capillary flow-based fluidic channel can be provided which can obviate the need for active fluidics thus reducing cost as having no additional power requirements or need for additional parts to drive the fluid through the device.

[0013] It is also hypothesized that power requirements to heat a membrane in contact with a heater, rather than heating liquid in a reaction well, are likely to be lower thus also reducing cost . In addition, the reagents needed to perform the amplification-detection reaction can be applied to the membrane and dried enabling simplified production, storage and transport. Further advantages of the present invention include the use of cheaper heating methods e.g. PTC heaters, as well as potentially high-volume, lateral-flow strip like manufacture on an automated basis.

[0014] Accordingly, the present invention provides a membrane for amplification and detection of a nucleic acid, the membrane comprising:

[0015] a sample receiving zone; and

[0016] a reaction zone in fluid communication with the sample receiving zone,wherein the reaction zone comprises a dried reaction mix comprising:

[0017] (i) a recombinase polymerase amplification (RPA) composition;

[0018] (ii) a cleavage molecule; and

[0019] (iii) a probe for detecting the target nucleic acid.

[0020] The present invention also provides a diagnostic device comprising a membrane according to the present invention and a housing, wherein the membrane is positioned inside the housing, optionally wherein the housing comprises:

[0021] a first opening configured to allow a liquid sample to be applied to the sample receiving zone;

[0022] a second opening configured to permit viewing of a detectable signal in the reaction zone; and

[0023] optionally a third opening configured to permit viewing of a detectable signal in the control zone, if present.

[0024] The present invention further provides a diagnostic system comprising:

[0025] one or more diagnostic devices according to the present invention; and

[0026] a base station comprising:

[0027] a housing comprising an engagement portion configured to receive one of said diagnostic devices; and

[0028] a heater configured to heat at least a portion of a diagnostic device received in the engagement portion,

[0029] optionally wherein the housing comprises a plurality of engagement portions, each configured to receive one of said diagnostic devices and a heater configured to heat at least a portion of each of the received diagnostic devices.

[0030] The present invention further provides a method of manufacturing a membrane according to the present invention, the method comprising:

[0031] providing an aqueous reaction mix which comprises: (i) an RPA composition; (ii) a cleavage molecule; and (iii) a probe for detecting the target nucleic acid;

[0032] providing a first sheet of an absorbent material;

[0033] dispensing the aqueous reaction mix onto the first sheet;drying the first sheet to obtain a first sheet comprising a dried reaction mix; providing an adhesive backing layer;

[0034] laminating the first sheet onto the adhesive backing layer to provide a reaction zone; providing a second sheet of an absorbent material;

[0035] laminating the second sheet onto the adhesive backing layer to obtain a membrane assembly comprising a sample receiving zone in liquid communication with the reaction zone.

[0036] DESCRIPTION OF THE DRAWINGS

[0037] The present invention will now be described, by way of non-limitative example only, with reference to the accompanying drawings, in which:

[0038] Figures 1 A-1D show membranes according to embodiments of the present invention.

[0039] Figures 2A and 2B show, schematically, the configuration of a membrane assembly according to an embodiment of the present invention.

[0040] Figure 3 shows a membrane assembly according to an embodiment of the present invention and individual membranes prepared from said membrane assembly.

[0041] Figures 4A-4C show various view of a diagnostic device according to an embodiment of the present invention.

[0042] Figures 5A and 5B show a diagnostic system according to an embodiment of the present invention.

[0043] Figure 6 shows the results of a test carried out with a membrane according to an embodiment of the present invention.

[0044] Figure 7 shows the results of a test carried out with a diagnostic device according to an embodiment of the present invention.Figures 8A-8D show the results of a test carried out with a membrane according to an embodiment of the present invention.

[0045] Figures 9A-9C show the results of a further test carried out with a membrane according to an embodiment of the present invention.

[0046] Figure 10 shows the results of a test carried out to determine sensitivity of three different arrangements of membranes according to embodiments of the present invention.

[0047] Figure 11 shows the results of a screen for suitable surfactant and polymer combinations for use in embodiments of the present invention.

[0048] Figure 12 shows the results of a test carried out to determine the sensitivity of two arrangements of membranes according to embodiments of the present invention.

[0049] Figure 13 shows the results of tests carried out with membranes according to embodiments of the present invention constructed from different materials.

[0050] Figure 14 is a flow-chart depicting a method performed using a diagnostic device according to embodiments of the present invention.

[0051] DETAILED DESCRIPTION

[0052] Definitions

[0053] It is to be understood that the specific membranes, diagnostic devices, diagnostic systems and methods of manufacturing described and defined herein are exemplary and non-limiting and may be adapted or tailored to the specific needs of the user. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

[0054] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example,references to an entity such as “a molecule”, “a polypeptide” and so on, includes two or more such entities.

[0055] Where a range of numbers is presented as being “between” a lower value and an upper value, the range is to be interpreted as including the upper and lower values. For example, a range of between 22 mM to 50 mM, or between about 22 mM to about 50 mM, should be interpreted as including the values of 22 mM and 50 mM or the values of about 22 mM and about 50 mM.

[0056] Nucleic acid

[0057] As used herein, the term nucleic acid encompasses DNA and RNA. “DNA” refers to deoxyribonucleic acid and derivatives thereof, the molecule that carries most of the genetic instructions used in the development, functioning and reproduction of all known living organisms and many viruses. Most DNA molecules consist of two biopolymer strands coiled around each other to form a double helix. The two DNA strands are known as polynucleotides and are composed of simpler units called deoxynucleotides. Each deoxynucleotide is composed of a nitrogen-containing nucleobase - cytosine (C), guanine (G), adenine (A), or thymine (T) - as well as a monosaccharide sugar called deoxyribose and a phosphate group. The deoxyribonucleotides are joined to one another in a chain by covalent (phosphodiester) bonds between the sugar of one deoxyribonucleotide and the phosphate of the next, resulting in an alternating sugar-phosphate backbone. According to base pairing rules (A with T, and C with G), hydrogen bonds bind the nitrogenous bases of the two separate polynucleotide strands to make double-stranded DNA. The specific order of the monomers, i.e. the order of the deoxyribonucleotide bases linked to the sugar / phosphate-backbone, is called the DNA-sequence. As used herein, the term “RNA” refers to ribonucleic acid and derivatives thereof. RNA is typically a single-stranded nucleic acid molecule, and is a polynucleotide composed of simpler units called ribonucleotides. Each ribonucleotide is composed of a nitrogen-containing nucleobase - cytosine (C), guanine (G), adenine (A), or thymine (T) - as well as a monosaccharide sugar called deoxyribose and a phosphate group. The ribonucleotides are joined to one another in a chain by covalent (phosphodiester) bonds between the sugar of one ribonucleotide and the phosphate of the next. The specific order ofthe monomers, i.e. the order of the ribonucleotide bases linked to the sugar / phosphate-backbone, is called the RNA-sequence.

[0058] Amino acid

[0059] As used herein, the term “amino acid” refers to any natural or synthetic amino acid, that is, an organic compound comprising carbon, hydrogen, oxygen and nitrogen atoms, and comprising both amino (-NH2) and carboxylic acid (-COOH) functional groups. Typically, the amino acid is an a-, P-, y- or 5-amino acid. The amino acid may be one of the twenty-two naturally occurring proteinogenic a-amino acids. Alternatively, the amino acid may be a synthetic amino acid selected from a-Amino-n-butyric acid, Norvaline, Norleucine, Alloisoleucine, t-leucine, a-Amino-n-heptanoic acid, Pipecolic acid, a,P-diaminopropionic acid, a,y-di aminobutyric acid, Ornithine, Allothreonine, Homocysteine, Homoserine, P-Alanine, P-Amino-n-butyric acid, P-Aminoisobutyric acid, y-Aminobutyric acid, a-Aminoisobutyric acid, isovaline, Sarcosine, N-ethyl glycine, N-propyl glycine, N-isopropyl glycine, N-methyl alanine, N-ethyl alanine, N-methyl P-alanine, N-ethyl P-alanine, isoserine, a-hydroxy-y-aminobutyric acid, Homonorleucine, O-methyl-homoserine, O-ethyl-homoserine, selenohomocysteine, selenomethionine, selenoethionine, Carboxyglutamic acid, Hydroxyproline, Hypusine, Pyroglutamic acid, aminoisobutyric acid, dehydroalanine, P-alanine, y-Aminobutyric acid, 5-Aminolevulinic acid, 4-Aminobenzoic acid, citrulline, 2,3-diaminopropanoic acid, 3 -aminopropanoic acid, hydroxytryptophan, selenohomocysteine, a-aminoglycine and diaminoacetic acid, 2,3-diaminopropionic acid, a,y-diaminobutyric acid, amino-2-keto-butyric acid, 4-acetylphenylalanine and formylglycine, azidolysine, azidoornithine, azidonorleucine, azidoalanine, azidohomoalanine, 4-azidophenylalanine and 4-azidomethylphenylalanine, homoallylglycine, 4-ethynylphenylalanine, 4-propargyloxyphenylalanine, propargylglycine, 4-(2-propynyl)proline, 2-amino-6-({[(lR,8S)-bicyclo[6.1.0]non-4-yn-9-ylmethoxy]carbonyl}amino)hexanoic acid and homopropargylglycine. An amino acid which possesses a stereogenic centre may be present as a single enantiomer or as a mixture of enantiomers (e.g. a racemic mixture). The conventional one-letter or three-letter code for amino acid residues is used herein.Peptide and Polypeptide

[0060] As used herein, the terms “peptide” and “polypeptide” are used interchangeably and refer to a biological molecule comprising polymers of amino acid monomers.

[0061] Protein

[0062] Proteins consist of one or more polypeptides arranged in a biologically functional way, often bound to ligands such as coenzymes and cofactors, or to another protein or other macromolecule (DNA, RNA, etc.), or to complex macromolecular assemblies.

[0063] Enzyme

[0064] The term “enzyme” refers to a protein or polypeptide having catalytic activity and having the capability of catalyzing a chemical reaction.

[0065] Tether

[0066] The term “tether” as used herein typically refers to the association between the signalpromoting molecule and an oligonucleotide of a detection probe to be used with the membranes described herein. In all of the specific detection probe embodiments, the signalpromoting molecule is associated with an oligonucleotide of the probe via a linker, and therefore the association between the signal -promoting molecule and the oligonucleotide is indirect, wherein the signal-promoting molecule is not itself directly chemically bonded to the oligonucleotide.

[0067] Attach

[0068] The term “attach” or “attached” as used herein typically refers to the association between the linker and the oligonucleotide of a detection probe described herein or between the linker and the signal-promoting molecule. The associations between the linker and the oligonucleotide and between the linker and the signal-promoting molecule are direct, wherein the linker is directly chemically bonded to the oligonucleotide or signal-promoting molecule.

[0069] Membrane configurations

[0070] Membranes according to embodiments of the present invention comprise: a sample receiving zone; and a reaction zone in fluid communication with the sample receiving zone. Thesample receiving zone is configured such that a liquid sample can be applied thereto. The sample receiving zone may comprise any suitable absorbent membranous material.

[0071] The reaction zone is in fluid communication with the sample receiving zone such that a liquid sample can be transmitted from the sample receiving zone to the reaction zone by capillary action. The reaction zone may comprise any suitable absorbent membranous material.

[0072] The reaction zone comprises a dried reaction mix. Transmission of the liquid sample from the sample receiving zone to the reaction zone rehydrates the components of the dried reaction mix, thereby allowing an amplification-detection reaction, such as an RPA reaction, to proceed.

[0073] In certain embodiments, the reaction zone may comprise a first portion and second portion which are in liquid communication with each other. The first portion may be arranged between the sample receiving zone and the second portion. Alternatively, the second portion may be arranged between the sample receiving zone and the first portion. In one embodiment: (i) the first portion may comprise one or more protein components of the RPA composition, such as an RPA-SSB molecule, a recombinase agent, a polymerase and / or a cleavage molecule; and (ii) the second portion may comprise one or more nucleic acid components of the RPA composition, such as forward and reverse nucleic acid primers for amplification and / or the probe. In another embodiment: (i) the second portion may comprise one or more protein components of the RPA composition, such as an RPA-SSB molecule, a recombinase agent, a polymerase and / or a cleavage molecule; and (ii) the first portion may comprise one or more nucleic acid components of the RPA composition, such as forward and reverse nucleic acid primers for amplification and / or the probe.

[0074] In certain embodiments the membranes further comprise a control zone. The control zone is in fluid communication with the sample receiving zone such that a liquid sample can be transmitted from the sample receiving zone to the control zone by capillary action. The control zone may comprise any suitable absorbent membranous material.The control zone also comprises a dried control mix. Transmission of the liquid sample from the sample receiving zone to the control zone rehydrates the components of the dried control mix, thereby allowing an amplification-detection reaction, such as an RPA reaction, to proceed.

[0075] In certain embodiments the control zone may comprise a first portion and second portion which are in liquid communication with each other. The first portion may be arranged between the sample receiving zone and the second portion. Alternatively, the second portion may be arranged between the sample receiving zone and the first portion. In one embodiment: (i) the first portion may comprise one or more protein components of the RPA composition, such as an RPA-SSB molecule, a recombinase agent, a polymerase and / or a cleavage molecule; and (ii) the second portion may comprise one or more nucleic acid components of the RPA composition, such as forward and reverse nucleic acid primers for amplification and / or the probe. In another embodiment: (i) the second portion may comprise one or more protein components of the RPA composition, such as an RPA-SSB molecule, a recombinase agent, a polymerase and / or a cleavage molecule; and (ii) the first portion may comprise one or more nucleic acid components of the RPA composition, such as forward and reverse nucleic acid primers for amplification and / or the probe.

[0076] The membrane may have a laminated arrangement. The reaction zone may be provided on a first membranous layer. The sample receiving zone may be provided on a second membranous layer. The control zone may be provided on a third membranous layer. In addition to the membrane layer(s) itself, the membrane may further comprise one or more of : an adhesive backing layer, a further membranous layer comprising a blank zone in liquid communication with the reaction zone and a further membranous layer comprising a blank zone in liquid communication with the control zone.

[0077] Where the membrane has an adhesive backing layer, a portion of the adhesive backing layer may contact a surface of the first membranous layer, and the second membranous layer contacts an opposing surface of the first membranous layer. Where the membrane comprises a further membranous layer comprising a blank zone in liquid communication with thereaction zone, said further membranous layer may contact said opposing surface of the first membranous layer.

[0078] In embodiments where the membrane comprises a third membranous layer comprising the control zone, a portion of the adhesive backing layer may contact a surface of the third membranous layer, and the second membranous layer contacts an opposing surface of the third membranous layer. Where the membrane comprises a further membranous layer comprising a blank zone in liquid communication with the control zone, said further membranous layer may contact said opposing surface of the first membranous layer.

[0079] The membranes described herein rely on capillary action and rehydration of reagents in order to perform an amplification-detection reaction. However, distinct from lateral flow test strips known in the art, the membranes described herein do not rely on immobilized reagents or capture molecules. This means that, in certain embodiments, the membranes described herein do not comprise a test line. In certain embodiments, the membranes described herein do not comprise an immobilized capture molecule, such as an immobilized antibody.

[0080] Figures 1A-1D show, schematically, the structure of various membranes according to embodiments of the present invention.

[0081] Figure 1 A shows a first embodiment of a membrane, in which the membrane comprises a reaction zone 1 comprising a dried reaction mix 2 and a sample receiving zone 3.

[0082] Figure IB shows a second embodiment of a membrane, in which the membrane comprises a reaction zone 1 comprising a dried reaction mix 2, a sample receiving zone 3 and a control zone 5 comprising a dried control mix 4.

[0083] Figure 1C shows a third embodiment of a membrane, in which the membrane comprises: a reaction zone 1 comprising a first portion 2a, at least a part of which comprises a dried reaction mix, and a second portion 2b; and a sample receiving zone 3.Figure ID shows a fourth embodiment of a membrane, in which the membrane comprises: a reaction zone 1 comprising a first portion 2a, at least a part of which comprises a dried reaction mix, and a second portion 2b; a sample receiving zone 3; and a control zone 5 comprising a first portion 4a, at least a part of which comprises a dried control mix, and a second portion 4b.

[0084] As shown in Figures 1 A-1D, the different components of the membranes may be formed on and provided by individual layers. This configuration can allow the different components to be prepared individually (for example as part of a larger sheet and divided from that sheet) and then assembled. One benefit of providing components on separate layers relates to avoidance of premature mixing of reagents prior to addition of a sample to be tested.

[0085] Alternatively, however, two or more of the components may be provided on the same layer as each other. This configuration can result in a more compact device and avoid any potential difficulties with joining the layers; but may require greater control to maintain separation of reagents prior to use.

[0086] Figures 2A and 2B show, schematically, the configuration of a membrane assembly according to an embodiment of the present invention. Figure 2A shows a top-down view of the membrane assembly. Figure 2B shows a side profile of the membrane assembly. The membrane assembly comprises: a first sheet of absorbent material 27 laminated onto an adhesive backing layer 31 to provide a reaction zone 1; a second sheet of absorbent material 28 laminated onto the adhesive backing layer 31 to provide a sample receiving zone 3; a third sheet of absorbent material laminated onto the adhesive backing layer 31 to provide a control zone 5; a fourth sheet of absorbent material 26 laminated onto the adhesive backing layer 31 to provide a first blank zone; and a fifth sheet of absorbent material 30 laminated onto the adhesive backing layer 31 to provide a second blank zone.

[0087] Figure 3 shows a membrane assembly according to an embodiment of the present invention and individual membranes prepared from said membrane assembly.

[0088] Figure 3 A depicts a membrane assembly comprising: a first sheet of absorbent material 27 laminated onto an adhesive backing layer (not shown) to provide a reaction zone 1; a secondsheet of absorbent material 28 laminated onto the adhesive backing layer (not shown) to provide a sample receiving zone 3; a third sheet of absorbent material laminated onto the adhesive backing layer (not shown) to provide a control zone 5; a fourth sheet of absorbent material 26 laminated onto the adhesive backing layer (not shown) to provide a first blank zone; and a fifth sheet of absorbent material 30 laminated onto the adhesive backing layer (not shown) to provide a second blank zone.

[0089] The formation of a membrane such as that shown in Figure 3 A allows membranes to be formed in bulk as part of a larger sheet / laminate structure and then divided up into individual membranes. This can significantly increase reproducibility / uniformity of individual assay devices as well as increasing the speed and efficiency of membrane production and thus reduce the time and cost to produce individual membranes in bulk.

[0090] Figure 3B shows a membrane created by dividing the membrane assembly shown in Figure 3 A to obtain two or more strips of membrane. Similarly to the membrane shown schematically in Figure IB, the membrane comprises: a reaction zone 1 comprising a dried reaction mix 2; a sample receiving zone 3; and a control zone 5 comprising a dried control mix 4; a first blank zone 6; and a second blank zone 7.

[0091] Figure 3C shows membrane of Figure 3B comprised within a housing 9.

[0092] Membrane materials

[0093] The membranes described herein may be constructed from any suitable absorbent material on to which the reagents for performing the amplification-detection reaction can be adsorbed and dried. The membrane may be a glass-fibre membrane, a polyolefin fibre membrane, or a nitrocellulose membrane. Optionally, the membrane comprises SO 15 polyolefin fibre (Porex). Optionally, the membrane comprises Hi-Flow Plus 120 nitrocellulose membrane (Merck). Optionally, the membrane comprises Grade 8980 glass-fibre membrane (Ahlstrom).

[0094] Surfactants

[0095] The membranes described herein may comprise a surfactant. The surfactant may be selected from the group consisting of a polyvinyl alcohol (PVA), a polyvinylpyrrolidone (PVP), andnonyl phenoxypolyethoxylethanol (NP-40). Suitably, the membrane may be pre-treated with the surfactant. For example, the membrane may be submerged in, or sprayed with, a composition comprising the surfactant. The membrane may then the be subject to a drying step prior to application of the reagents required to perform the amplification-detection reaction. The membrane may be pre-treated with composition comprising between 0.1 and 5% (v / v) surfactant, preferably between 0.3 and 0.8% (v / v) surfactant, more preferably about 0.5% (v / v) surfactant. Optionally, the membrane may be pre-treated with a composition comprising about 0.5% (v / v) PVA. Optionally, the membrane may be pre-treated with a composition comprising about 0.5% (v / v) PVP. Optionally, the membrane may be pre-treated with a composition comprising about 0.5% (v / v) NP-40.

[0096] Reagents for performing amplification and detection of a nucleic acid

[0097] The membranes described herein comprise dried compositions comprising the reagents required for amplification and detection of a target nucleic acid.

[0098] The membranes described herein rely on recombinase polymerase amplification (RPA) to amplify the target nucleic acid. RPA is a method for isothermal amplification of nucleic acids. In general, in a first step of RPA, a recombinase agent is contacted with first and second nucleic acid primers and a recombinase loading protein to form first and second nucleoprotein primers. In general, in a second step, the first and second nucleoprotein primers are contacted with a double stranded template nucleic acid to form a first double stranded structure at a first portion of the first strand of the template nucleic acid, and a second double stranded structure at a second portion of the second strand of the template nucleic acid such that the 3’ ends of the first nucleic acid primer and the second nucleic acid primer are orientated towards each other on a given nucleic acid molecule. In general, in a third step, the 3’ end of the first and the second nucleoprotein primers are extended by polymerase to generate first and second double stranded nucleic acids, and first and second displaced single strands of nucleic acid. A single stranded stabilizing agent is employed to stabilize the first and second displaced single strands of nucleic acid. Generally, the second and third steps can be repeated until a desired degree of amplification is reached.RPA methods are disclosed extensively, e.g., in US 7,270,981, US 7,399,590, US 7,666,598, US 7,435,561 and W02010 / 141940 (the contents of each of these references is hereby incorporated by reference). In addition, for a comprehensive recent review see: Review: a comprehensive summary of a decade development of the recombinase polymerase amplification, Li, J. et al., 2019, Analyst, 144, pp31-67.

[0099] As explained below, certain RPA components may be attached / tethered / tagged to one or more IDR-polypeptides. An IDR-polypeptide comprises an intrinsically disordered region. IDR-polypeptides suitable for use in RPA reactions are described extensively in WO 2021 / 094746A1 (the contents of which are hereby incorporated by reference).

[0100] The reaction zone of the membrane described herein comprises a dried reaction mix comprising: (i) a recombinase polymerase amplification (RPA) composition; (ii) a cleavage molecule; and (iii) a probe for detecting the target nucleic acid. Optionally, the dried reaction mix does not comprise polyethylene glycol (PEG).

[0101] If a control zone is present, said control zone may comprise a dried control mix comprising: (i) an RPA composition; (ii) a cleavage molecule; (iii) a control nucleic acid; and (iv) a control probe for detecting the control nucleic acid. Optionally, the dried reaction mix does not comprise polyethylene glycol (PEG).

[0102] The RPA composition may comprise forward and reverse nucleic acid primers for amplification of the target nucleic acid.

[0103] The RPA composition may comprise an RPA single-stranded DNA-binding protein (RPA-SSB) molecule. The RPA-SSB may be selected from the group consisting of Gp32, E. coli SSB protein, phage T4 Gp32 protein, phage Rb69 Gp32, phage vB EcoM NBGl Gp32, or any functional analog, homolog or derivative thereof, and any combination thereof.

[0104] Preferably, the RPA-SSB is Gp32 or phage vB EcoM NBGl Gp32. Optionally, the RPA-SSB is tagged with one or more functional intrinsically disordered regions (IDRs), preferably as a genetically engineered fusion protein comprising the RPA-SSB and an amino acid sequence comprising or consisting of the one or more functional IDRs.The RPA composition may comprise a recombinase agent. The recombinase agent may be selected from the group consisting of UvsX, T4 UvsX, T6 UvsX, RBI 8 UvsX, E. coli phage wV7 UvsX, Shigella phage CB8 UvsX, Shigella phage Shfl2 UvsX, E.coli phage ARI UvsX, phage vB_EcoM_G4507 UvsX, Shigella phage SHFML-11 UvsX, Escherichia phage vB EcoM DalCa UvsX, E. coli RecA, E. coli RadA, E. coli RadB, E. coli Rad 51 or any functional analog, homolog or derivative thereof, and any combination thereof. Preferably, the recombinase agent is UvsX. More preferably, the recombinase agent is Escherichia phage vB EcoM DalCa UvsX;

[0105] The RPA composition may comprise a recombinase loading protein. The recombinase loading protein may be selected from the group consisting of UvsY, E. coli RecO, E. coli RecR or any functional analog, homolog or derivative thereof, and any combination thereof. Preferably, the recombinase loading protein is UvsY. More preferably, the recombinase loading protein is Escherichia phage STO UvsY.

[0106] The RPA composition may comprise a polymerase. The polymerase may be a eukaryotic polymerase selected from the group consisting of pol-a, pol-P, pol-5, pol-s or any functional analog, homolog or derivative thereof, and any combination thereof. The polymerase may be a prokaryotic polymerase selected from the group consisting of Bacillus stearothermophilus polymerase I large fragment, Bacillus subtilis Pol I large fragment (Bsu polymerase), Listeria monocytogenes DNA polymerase I, S. aureus DNA polymerase I (Sau polymerase), E. coli DNA polymerase I Klenow fragment, 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, or any functional analog, homolog or derivative thereof, and any combination thereof. Preferably, the polymerase is S. aureus DNA polymerase I (Sau polymerase) or Bacillus subtilis Pol I large fragment (Bsu polymerase). The polymerase may be a bacteriophage polymerase selected from the group consisting of bacteriophage T4 gp43 DNA polymerase, T7 DNA polymerase and Phi-29 DNA polymerase, or any functional analog, homolog or derivative thereof, and any combination thereofThe dried reaction mix may comprise a cleavage molecule. The cleavage molecule may be any molecule having phosphatase / exonuclease activity, and which is suitable to effect cleavage at the cleavage site by the action of the molecule’s phosphatase activity and / or exonuclease activity. Such a molecule may additionally intrinsically possess apurinic / apyrimidinic (AP) endonuclease activity. However, the detection probes described herein are structured such that apurinic / apyrimidinic (AP) endonuclease activity cannot mediate cleavage at the cleavage site. The cleavage molecule may be a bacterial Exonuclease III. The cleavage molecule may be E. coli Exonuclease III optionally having the amino acid sequence set forth in SEQ ID NO: 1 as set forth below:

[0107] MKFVSFNINGLRARPHQLEAIVEKHQPDVIGLQETKVHDDMFPLEEVAKLGYNVFYHGQKGH YGVALLTKETPIAVRRGFPGDDEEAQRRI IMAEIPSLLGNVTVINGYFPQGESRDHPIKFPA KAQFYQNLQNYLETELKRDNPVLIMGDMNISPTDLDIGIGEENRKRWLRTGKCSFLPEEREW MDRLMSWGLVDTFRHANPQTADRFSWFDYRSKGFDDNRGLRIDLLLASQPLAECCVETGIDY EIRSMEKPSDHAPVWATFRRAGEFKLAAALEHHHHHH

[0108] The cleavage molecule may be Neisseria gonorrhoeae Exonuclease III optionally having the amino acid sequence set forth in SEQ ID NO: 2 below:

[0109] MHHHHHHMMKITTWNVNSLNVRLPQVQNLLADNPPDILVLQELKLDQDKFPAAALQMMGWHC VWSGQKTYNGVAIVSRSVPQDVHFGLPSLPDDPQRRVIAATVGGVRVINVYCVNGEALDSPK FKYKEQWFAALTEFVRDEMTRHGKLVLLGDFNIAPADADCYDPEKWYEKIHCSSVERQWFQN LLDLGLTDSLRQVHPEGAFYTWFDYRGAMFQRKLGLRIDHILVTSEMAAVLKDVRIDLETRA LERPSDHAPVAAEFDL

[0110] Detection probes

[0111] Disclosed herein are detection probes which are suitable for detecting a target nucleic acid or a control nucleic acid. The detection probes described herein are suitable for use in the membranes of the present invention. Further detection probes are disclosed in GB 2410287.3 (the contents of which are hereby incorporated by reference).

[0112] The probe for detecting a target nucleic acid and / or the probe for detecting a control nucleic acid may comprise:A. one or two single-stranded oligonucleotides each comprising a sequence which is complementary to a target nucleic acid or the control nucleic acid; and

[0113] B. a repressible cleavage-dependent signalling system which:

[0114] a. comprises a signal-promoting molecule tethered to the oligonucleotide(s) via at least one linker, wherein the at least one linker is attached to the signalpromoting molecule and to a chemical group of the oligonucleotide(s) and thereby defines a cleavage site on the oligonucleotide(s);

[0115] b. provides a detectable signal which:

[0116] i. is dependent upon cleavage at the cleavage site by the cleavage molecule, and

[0117] ii. following hybridisation of the oligonucleotide(s) with the target sequence, is not dependent upon:

[0118] 1. cleavage of the oligonucleotide(s) at an apurinic / apyrimidinic site (abasic site) of the oligonucleotide(s), e.g. at a 3 '-side of an abasic site and / or at a 5'-side of the abasic site, or at a site comprising an abasic furan; or

[0119] 2. cleavage of the oligonucleotide(s) at a mismatch site; and c. is configured so that when the probe is present in the test solution:

[0120] i. when the oligonucleotide(s) is not hybridised with the target sequence, cleavage at the cleavage site by the cleavage molecule is inhibited and production of a detectable signal is thereby repressed; and ii. upon hybridisation of the oligonucleotide(s) with the target sequence, the cleavage molecule cleaves at the cleavage site, thereby producing a detectable signal.

[0121] In the detection probes described herein, a signal-promoting molecule is attached to the oligonucleotide(s) by one or more linkers. The linker(s) is not attached to an abasic nucleotide or an abasic residue. The linker(s) is not attached to a chemical group of an abasic nucleotide or abasic residue. The linker(s) is not attached to the oligonucleotide(s) at a nucleotide or residue position adjacent to a position in the oligonucleotide(s) which is occupied by an abasic nucleotide or an abasic residue. By adjacent, it is meant within 1, 2, 3,4, 5, 6, 7, 8, 9 or 10 nucleotide or residue positions in the oligonucleotide(s) from a position which is occupied by an abasic nucleotide or an abasic residue.

[0122] The signal-promoting molecule may be tethered to the oligonucleotide(s) via at least one linker which is attached to a chemical group of the oligonucleotide(s) and which thereby defines a cleavage site on the oligonucleotide, wherein the cleavage site is susceptible to cleavage mediated by phosphatase or exonuclease activity when contacted with a cleavage molecule having phosphatase or exonuclease activity. Conversely, the signal-promoting molecule may be tethered to the oligonucleotide(s) via at least one linker which is attached to a chemical group of the oligonucleotide(s) and which thereby defines a cleavage site on the oligonucleotide, wherein the cleavage site is not susceptible to cleavage mediated by apurinic / apyrimidinic (AP) endonuclease activity if contacted with a cleavage molecule having apurinic / apyrimidinic (AP) endonuclease activity.

[0123] The probes described herein may generate a detectable signal via cleavage of the probe at the cleavage site, wherein cleavage occurs by action of phosphatase or exonuclease activity, and not by apurinic / apyrimidinic (AP) phosphatase activity.

[0124] Provided that the cleavage site is susceptible to cleavage mediated by phosphatase or exonuclease activity, and not to cleavage mediated by apurinic / apyrimidinic (AP) endonuclease activity, the linker(s) defining the cleavage site may be attached to the nucleotide or residue of the oligonucleotide at a phosphate group of the nucleotide or residue. The linker may be attached to the sugar of the nucleotide or residue of the oligonucleotide. The linker may be attached to the base of the nucleotide or residue of the oligonucleotide.

[0125] When the probe is present in a test solution comprising a recombinase polymerase amplification (RPA) single-stranded DNA-binding protein (RPA-SSB), a cleavage molecule and the target nucleic acid:

[0126] 1. when the oligonucleotide(s) is not hybridised with the target nucleic acid, cleavage at the cleavage site by the cleavage molecule is inhibited and production of a detectable signal is thereby repressed; and2. upon hybridisation of the oligonucleotide(s) with the target nucleic acid, the cleavage molecule cleaves at the cleavage site, thereby producing a detectable signal.

[0127] The provision of a detectable signal following hybridisation of the oligonucleotide with the target sequence is not dependent upon a cleavage mechanism that is dependent upon the presence of an apurinic / apyrimidinic nucleotide or residue (also referred to as an abasic site or an apurinic / apyrimidinic (AP) site) or a cleavage mechanism that is dependent upon the presence of a mismatch in the oligonucleotide; e.g. :

[0128] 1. is not dependent upon cleavage of the oligonucleotide(s) at an apurinic / apyrimidinic site (abasic site) of the oligonucleotide(s), e.g. at a 3 '-side of an abasic site and / or at a 5'-side of the abasic site, such as at a position in the oligonucleotide(s) occupied by an abasic residue or an abasic nucleotide;

[0129] 2. is not dependent upon cleavage of the oligonucleotide(s) at a mismatch site; or

[0130] 3. is not dependent upon cleavage of the oligonucleotide(s) at a site comprising an abasic furan, such as tetrahydrofuran (THF).

[0131] Accordingly, the probes described herein are not compatible with the action of the apurinic / apyrimidinic (AP) endonuclease activity of Formamidopyrimidine DNA glycosylase or Exonuclease III.

[0132] The probes described herein can be configured to achieve these functional attributes. For example, the linker which attaches the signal-promoting molecule to the oligonucleotide and which defines the cleavage site is not attached to an abasic residue or an abasic nucleotide of the oligonucleotide, e.g. the sugar molecule of an abasic nucleotide. The linker which attaches the signal-promoting molecule, such as a cofactor for an enzyme, to the oligonucleotide and which defines the cleavage site may not be attached to the oligonucleotide at a position adjacent to a position in the oligonucleotide which is occupied by an abasic residue or an abasic nucleotide.

[0133] The probes described herein are suitable for use in RPA reactions because they fail to provide a detectable signal when the oligonucleotide is not hybridised with the target nucleic acid.However, when the oligonucleotide is hybridised with the target nucleic acid e.g. during RPA cycles, a detectable signal can be produced.

[0134] RPA reactions require the use of an RPA single-stranded DNA-binding protein (RPA-SSB) molecule, typically Gp32. The RPA-SSB will bind to the oligonucleotide of the probe of the invention since the probe comprises a single-stranded portion comprising a sequence which is complementary to a target nucleic acid sequence of interest. The RPA-SSB will dissociate from the oligonucleotide of the probe when the single-stranded portion of the probe hybridises with the target nucleic acid sequence to form a double-stranded structure. A signal-promoting molecule is attached to the oligonucleotide(s) of the probe via at least one linker comprising a cleavage site. Without being bound by theory, the inventors believe that when the oligonucleotide is not hybridised with the target sequence, binding of the RPA-SSB to the oligonucleotide prevents the cleavage molecule from gaining access to the cleavage site. Consequently, no detectable signal is produced. However, when the oligonucleotide is hybridised with the target nucleic acid, the RPA-SSB is dissociated from the oligonucleotide, thus allowing the cleavage molecule to gain access to the cleavage site. Consequently, a detectable signal is produced.

[0135] The cleavage molecule which initially performs cleavage at the cleavage site is an enzyme having phosphatase and / or exonuclease activity, as further described and defined herein. Cleavage at the cleavage site may occur by hydrolysis of the phosphomonoester bond which attaches a phosphate group to the remainder of the oligonucleotide, preferably the terminal phosphate group at the 3’ terminal end of the oligonucleotide. Without wishing to be bound by theory, cleavage at the cleavage site provides a detectable signal depending upon the nature of the signal-promoting molecule and how the signal-promoting molecule is linked to the oligonucleotide.

[0136] The signal-promoting molecule may be a polypeptide which comprises or consists of a betagalactosidase alpha peptide, and which is attached to the oligonucleotide by one or two linkers. If two linkers are used, a first linker may be attached to the polypeptide at a first amino acid position of the polypeptide and the second linker may be attached to the polypeptide at a second amino acid position of the polypeptide. The first linker may beattached to a terminal phosphate group of the oligonucleotide, e.g. at the 3’ terminal end, and the second linker may be attached to the oligonucleotide at a different position, such as attached to the nucleobase of the 3’ terminal nucleotide. Such a probe does not provide a detectable signal when it is not hybridised to the target nucleic acid. This is because the RPA-SSB prevents the cleavage molecule from gaining access to the cleavage site. However, when the probe is hybridised to the target nucleic acid sequence of interest, the probe will provide a detectable signal. This is because the RPA-SSB no longer prevents the cleavage molecule from gaining access to the cleavage site. In such a situation, cleavage at the cleavage site by the cleavage molecule (phosphatase / exonuclease) will separate the first linker from the oligonucleotide. This can allow the signal-promoting molecule (beta-galactosidase alpha peptide) to come into contact with beta-galactosidase omega fragment (omega peptide) in solution to form beta-galactosidase holoenzyme which can for example, catalyse the conversion of orthonitrophenyl-P-D-galactopyranoside (ONPG) to ortho-nitrophenol (ONP); or the conversion of chlorophenol red-P-D-galactopyranoside (CPRG) to chlorophenol red. Without wishing to be bound by theory, the inventors believe that a single cleavage reaction to separate the first linker from the oligonucleotide is required to allow the beta-galactosidase alpha peptide to come into contact with beta-galactosidase omega fragment to form betagalactosidase holoenzyme which can then act upon its substrate to yield a detectable product, such as for example chlorophenol red. In such a situation, the signal-promoting molecule (beta-galactosidase alpha peptide) may remain tethered to the oligonucleotide (via the second linker), i.e. is liberated relative to the oligonucleotide, and can still produce a detectable signal. A second cleavage reaction by the same cleavage molecule may occur to separate the second linker from the oligonucleotide. In such a situation, the signal-promoting molecule (beta-galactosidase alpha peptide) may be separated from the oligonucleotide and produce the detectable signal.

[0137] A signal-promoting molecule in and of itself is generally not intended to be detected, but rather it acts as an activator of a downstream process; for example, a signal -promoting molecule may be a polypeptide which is a component of an enzyme complex, a domain of an enzyme, a fragment of an enzyme or an enzyme cofactor.A non-limiting exemplary embodiment of such a polypeptide is beta-galactosidase alpha peptide (alpha peptide) which is described below. It will be appreciated that any other suitable alternative polypeptide which is a component of an enzyme complex, a domain of an enzyme, a fragment of an enzyme or an enzyme cofactor may also be used, simply by adapting the principles of probe structure described herein, and the discussion below concerning alpha peptide applies mutatis mutandis to any other suitable alternative polypeptide.

[0138] The enzyme beta-galactosidase can be separated into two inactive fragments of different sizes, which on their own are incapable of hydrolysing a beta-galactosidase substrate (or a chromogenic analogue). The smaller fragment is referred to as the alpha peptide, alpha fragment, alpha domain or enzyme donor. The alpha peptide is about 100 amino residues in length. The larger fragment is referred to as the omega peptide, omega fragment, omega domain, or enzyme acceptor. The omega fragment is about 900 amino residues in length. When the alpha and omega fragments are combined, the beta-galactosidase enzyme is reconstituted and is active. Catalytic activity of beta-galactosidase can then be detected.

[0139] The nucleotide position(s) on the oligonucleotide(s) at which alpha peptide is tethered are not critical, provided that at least one linker attaching alpha peptide comprises a cleavage site and the probe is capable of operating according to the RPA-SSB-mediated repressible / activatable mechanism of action described herein and is suitable for use in an RPA reaction.

[0140] A user can readily structure a probe such that at least one linker attaching alpha peptide to the oligonucleotide comprises a cleavage site which is cleaved, by the action of a cleavage molecule having phosphatase / exonuclease activity, when the probe is in double-stranded form and hybridised to the target sequence of interest in a test solution comprising a RPA-SSB, and conversely such that cleavage by the cleavage molecule does not occur when the probe is in single-stranded form and not hybridised to the target sequence of interest in a test solution comprising a RPA-SSB. These criteria can readily be tested empirically according to methods described herein.Preferably, alpha peptide is tethered to the oligonucleotide(s) via at least one linker which is attached to the terminal phosphate group at the 3’ terminal end of the probe. Accordingly, this linker comprises the cleavage site which includes the terminal phosphate group, and cleavage is thereby affected by the action of a cleavage molecule which is any molecule having phosphatase / exonuclease activity. Alternatively, the alpha peptide may be tethered to the oligonucleotide(s) via at least one linker which is attached to another chemical group and / or at another nucleotide position of the oligonucleotide(s), provided that the probe is structurally arranged such that cleavage at the cleavage site is affected by the action of a cleavage molecule which is any molecule having phosphatase / exonuclease activity and the probe is suitable for use in an RPA reaction.

[0141] The detection probe may be a probe wherein the polypeptide is a beta-galactosidase alpha peptide. The detection probe may be a probe wherein the polypeptide is a beta-galactosidase alpha peptide having an amino acid sequence as set forth below:

[0142] SLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNK (SEQ ID NO: 3)

[0143] MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEK (SEQ ID NO: 4)

[0144] Preferably the first linker is attached to the first amino acid residue of the sequence at the N-terminal end of the polypeptide (underlined, typically via (but not limited to) the addition of an azide on the N-terminus i.e. coupling of azidoacetic acid) and the second linker is attached to the second (underlined) amino acid residue of the sequence in the direction proximal to the C-terminal end.

[0145] Preferably, the N-terminal amine of the peptide is reacted with azidoacetic acid to add the azide to the N-terminus. Alternative methods may involve the reaction of longer carbon chains or PEG based linkers. Further alternative methods may involve the reaction of bromo / iodo acetic acid with the N-terminus to conjugate the peptide with an oligo-SH.

[0146] The detection probe may have the structure:

[0147]

[0148] wherein * is the terminal phosphate group at the 3’ end of the oligonucleotide; LI is the first linker; L2 is the second linker; and SEQ ID NO: X is SEQ ID NOs: 3 or 4, wherein in each sequence the underlined K residue or the underlined C residue is attached to LI and the other underlined residue is attached to L2, or wherein in each sequence the underlined K residue or the underlined C residue is attached to L2 and the other underlined residue is attached to LI .

[0149] Detectable signal

[0150] As discussed above, the signal-promoting molecule of the detection probe may be a polypeptide which is a component of an enzyme complex, a domain of an enzyme, a fragment of an enzyme or an enzyme cofactor. The detectable signal may be produced in a reaction which is dependent upon either the partial or complete release of the polypeptide from the detection probe.

[0151] Where the signal-promoting molecule is an alpha peptide, the detectable signal may be produced in a reaction which is dependent upon the enzymatic activity of beta-galactosidase mediated by the presence of alpha peptide of the detection probe. The detectable signal may be generated by a method comprising:

[0152] 1) contacting the alpha peptide of the probe with beta-galactosidase omega fragment (omega peptide) to form beta-galactosidase holoenzyme; and2) detecting the presence of beta-galactosidase holoenzyme in solution.

[0153] The step of detecting the presence of beta-galactosidase holoenzyme may comprises: a colorimetric assay, a fluorescence assay, a chemiluminescence assay, a bioluminescence assay or an electrochemical assay.

[0154] Where the detectable signal is dependent on a colorimetric assay for beta-galactosidase holoenzyme, the reaction zone and the control zone, if present, may comprise a chromogenic substrate. The chromogenic substrate may be selected from the group consisting of:

[0155] 1. chlorophenol red-P-D-galactopyranoside (CPRG);

[0156] 2. 5-Bromo-6-chloro-3-indolyl-P-D-galactopyranoside (Magenta-Gal);

[0157] 3. 5-Bromo-3-indolyl P-D-galactopyranoside (Bluo-Gal);

[0158] 4. orthonitrophenyl-P-D-galactopyranoside (ONPG);

[0159] 5. 5-bromo-4-chloro-3-indolyl-P-D-galactopyranoside;

[0160] 6. 6-Chloro-3-indolyl-beta-D-galactopyranoside and optionally nitroblue tetrazolium salt (NBT);

[0161] 7. 3,4-Cyclohexenoesculetin P-D-galactopyranoside and Fe3+;

[0162] 8. 5-Iodo-3-indolyl-P-D-galactopyranoside; and

[0163] 9. 1 -Methyl -3-indolyl-P-D-gal actopy ranosi de .

[0164] Beta-galactosidase holoenzyme may be detected by:

[0165] 1. reacting beta-galactosidase holoenzyme with chlorophenol red-P-D-galactopyranoside (CPRG) in solution, thereby forming a chromophore reaction product chlorophenol red, and detecting the presence of chlorophenol red, optionally by measuring the absorbance of the solution at 570 to 595 nm, e.g. 575 nm;

[0166] 2. reacting beta-galactosidase holoenzyme with 5-Bromo-6-chl oro-3 -indolyl-P-D-galactopyranoside (Magenta-Gal) in solution, thereby forming a magenta precipitate reaction product 5,5' dibromo-6,6'-dichloro-indigo; and detecting the presence of 5,5' dibromo-6,6'-dichloro-indigo, optionally by measuring the absorbance of the solution at 565 nm;

[0167] 3. reacting beta-galactosidase holoenzyme with 5-Bromo-3-indolyl P-D-galactopyranoside (Bluo-Gal) in solution, thereby forming a blue precipitate reaction product 5,5'-dibromo-indigo; and detecting the presence of 5,5'-dibromo-indigo;4. reacting beta-galactosidase holoenzyme with orthonitrophenyl-P-D-galactopyranoside (ONPG) in solution, thereby forming a chromophore reaction product ortho-nitrophenol (ONP), and detecting the presence of ortho-nitrophenol (ONP), optionally by measuring the absorbance of the solution at 420 nm;

[0168] 5. reacting beta-galactosidase holoenzyme with 5-bromo-4-chloro-3-indolyl-P-D-galactopyranoside in solution, thereby forming a blue precipitate reaction product 5,5'-dibromo-4,4'-dichloro-indigo; and detecting the presence of 5, 5'-dibromo-4, d'-dichloroindigo;

[0169] 6. reacting beta-galactosidase holoenzyme with 6-Chl oro-3 -indolyl-beta-D-galactopyranoside in solution, optionally together with nitroblue tetrazolium salt (NBT), thereby forming a precipitate reaction product 6,6' dichloro indigo; and detecting the presence of 6,6' dichloro indigo;

[0170] 7. reacting beta-galactosidase holoenzyme with 3,4-Cyclohexenoesculetin P-D-galactopyranoside in the presence of Fe3+in solution, thereby forming a black precipitate reaction product, which is a complex formed of 2 molecules of 3,4-Cyclohexenoesculetin (2,3-dihydroxy-7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-one) and oneFe3+; and detecting the presence of the black precipitate reaction product; or

[0171] 8. reacting beta-galactosidase holoenzyme with 5-Iodo-3-indolyl-P-D-galactopyranoside in solution, thereby forming a purple precipitate reaction product 5,5'-diiodo indigo; and detecting the presence of 5,5'-diiodo indigo, optionally by immunoblotting, or by measuring the absorbance of the solution at 575 nm; or

[0172] 9. reacting beta-galactosidase holoenzyme with l-Methyl-3-indolyl-P-D-galactopyranoside in solution, thereby forming a green precipitate reaction product 1,1'-dimethyl isoindigo; and detecting the presence of l,l'-dimethyl isoindigo.

[0173] Diagnostic devices and systems

[0174] The present invention also provides diagnostic devices comprising a membrane as described herein positioned inside a housing. The housing may be constructed from any suitable material, such as a formable thermoplastic, for example cyclic olefin (e.g. TOPAS COC, Europlex COC), PET or polyester. The housing may be 3D printed, or may be produced by a process of thermoforming, hydroforming, injection moulding, or lamination of multiple layers (as described for example in GB 2622895). The housing may be configured to protect themembrane, e.g. during transportation and storage. The housing may comprise one or more further components of the diagnostic device.

[0175] In some embodiments, the housing comprises a first opening configured to allow a liquid sample to be applied to the sample receiving zone. The housing may also comprise a second opening configured to permit viewing of a detectable signal in the reaction zone. If a control zone is present in the membrane, the housing may also comprise a third opening configured to permit viewing of a detectable signal in the control zone.

[0176] The diagnostic devices may comprise one or more further components. For example, the diagnostic device may comprise one or more peelable layers. Alternatively, or additionally, the diagnostic device may comprise one or more spacer layers and / or one or more protective foil layers.

[0177] An example diagnostic device 10 is shown in Figures 4A-13C. Figure 4A shows an expanded view of the layers of the diagnostic device 10. Figure 4B shows a perspective view of the assembled diagnostic device 10. Figure 4C shows a top-down and side view of the diagnostic device 10 with the dimensions indicated.

[0178] The diagnostic device of Figure 4 comprises a membrane 18 positioned within a plurality of layers that collectively form a housing. The diagnostic device shown in Figure 4 further comprises one or more peelable layers 11, one or more protective foil layers 12, 17, one or more spacer layers 13, a heater 14, a battery 16, and an insulating layer 15 arranged between the heater and the battery.

[0179] The diagnostic devices described herein may be standalone devices that can be used at the point of care without the need for any additional equipment such as an external heater and power source. Thus, in certain embodiments the diagnostic device comprises a heater. The heater may be a positive temperature coefficient (PTC) heater. The heater may extend the entire length of the membrane. Alternatively, the heater may be aligned with the reaction zone. Where the membrane comprises a control zone, the heater may comprise a first heater aligned with the reaction zone and a second heater aligned with the control zone.To power the heater, the diagnostic device may further comprise a battery. The battery may be a printed battery.

[0180] As shown in the embodiment in Figure 4, the diagnostic device may comprise an insulating layer 15 arranged between the heater 14 and the battery 16.

[0181] Alternatively, in other embodiments, the diagnostic devices do not comprise any electronic components, e.g. the diagnostic devices do not comprise a battery or a heater. Such diagnostic devices have advantages in terms of lower cost and ease of manufacture.

[0182] However, devices require an external heat source in order to perform amplification-detection reactions, such as RPA amplification-detection reactions, at temperatures above that of the ambient environment. Accordingly, embodiments of the present invention also provide a diagnostic system comprising a diagnostic device such as those already described (and which do not comprise a battery or a heater) and a base station. An example diagnostic system according to an embodiment of the present invention is shown in Figures 5A and 5B.

[0183] Figure 5 shows a diagnostic system comprising one or more diagnostic devices 10 and a base station 100 which comprises a comprising a plurality of engagement portions 102, each configured to receive one of said diagnostic devices. Figure 5A shows a view of one embodiment of a diagnostic system 100. Figure 5B shows a view of the diagnostic system with a further attachment which further comprises an attachable Eppendorf tube holder 114 for one more Eppendorf tubes 116.

[0184] The base station 100 has a heater 104 associated with each of the engagement portions 102 (which may be a single heater for the whole base station 100, or, preferably, an individual heater for each of the engagement portions 104) configured to heat at least a portion of each of the received diagnostic devices.

[0185] The base station 100 also has an indicator 106 associated with each engagement portion which can be used to show one or more statuses of the engagement portion, including one ormore of whether the engagement portion is empty, occupied, heating, and / or finished a heating (or other processing cycle).

[0186] The base station 100 also has a USB-C port 108, which may be used as a power supply to the base station 100 and / or to transfer data to / from the base station (for example to apply settings to the internal firmware in the base station such as a length of time and temperature for heating or other processing cycles). This can allow a single base station to be manufactured which can be used for different analysis purposes, with the particular settings for each analysis purpose either being set in advance and the appropriate setting chosen or activated by a control signal, or allowing a user to set particular settings by signal provided through the USB-C port 108.

[0187] The base station 100 also has an on / off indicator 110 and a power button 112.

[0188] The base station 100 may further comprise LED and photosensor pairs associated with each engagement portion which can be used to determine presence of absence of a coloured product, which exists only if the sample applied to the device contains the target nucleic acid sequence. The base station may transmit measurement results for each diagnostic device to an electronic record system. Each diagnostic device may carry a unique indicium, such as a quick response (QR) code, or linear bar code, that can be associated with a patient sample, thus permitting test results to be attached to an individual patient record.

[0189] Methods of manufacture

[0190] Embodiments of the present invention also provide methods of manufacturing a membrane or membrane assembly such as those described herein.

[0191] In outline, a method of manufacturing a membrane or membrane assembly according to an embodiment of the present invention comprises the steps of:

[0192] a) providing an aqueous reaction mix which comprises: (i) an RPA composition; (ii) a cleavage molecule; and (iii) a probe for detecting the target nucleic acid;

[0193] b) providing a first sheet of an absorbent material;

[0194] c) dispensing the aqueous reaction mix onto the first sheet;d) drying the first sheet to obtain a first sheet comprising a dried reaction mix;

[0195] e) providing an adhesive backing layer;

[0196] f) laminating the first sheet onto the adhesive backing layer to provide a reaction zone; g) providing a second sheet of an absorbent material;

[0197] h) laminating the second sheet onto the adhesive backing layer to obtain a membrane assembly comprising a sample receiving zone in liquid communication with the reaction zone.

[0198] Where the membrane or membrane assembly to be manufactured comprises a control, the method comprises the further steps of

[0199] a) providing an aqueous control mix which comprises: (i) an RPA composition; (ii) a cleavage molecule; (iii) a control nucleic acid; and (iv) a control probe for detecting the control nucleic acid;

[0200] b) providing a third sheet of an absorbent material;

[0201] c) dispensing the control mix onto the third sheet;

[0202] d) drying the third sheet to obtain a third sheet comprising a dried control mix;

[0203] e) laminating the third sheet onto the adhesive backing layer such that membrane assembly further comprises a control zone in liquid communication with the sample receiving zone.

[0204] Where the membrane or membrane assembly to be manufactured comprises one or more blank zones, the method may comprise the further steps of

[0205] a) providing one or more further sheets of an absorbent material;

[0206] b) laminating the one or more further sheets of absorbent material onto the adhesive backing layer such that the membrane assembly further comprises a first blank zone in liquid communication with the reaction zone and optionally a second blank zone in liquid communication with the control zone, if present.

[0207] As described elsewhere herein, the method may manufacture membrane assemblies which are in sheet form and which can be subsequently divided into two or more strip membranes which are membranes according to embodiments of the present invention.More details of example methods of manufacturing membranes and membrane assemblies according to embodiments of the invention are given in the examples below.

[0208] The sheets of absorbent material used in the method described herein may comprise any suitable absorbent material on to which the reagents for performing the amplification and detection reaction can be adsorbed and dried. The sheets of absorbent material may comprise glass-fibre membrane, a polyolefin fibre membrane, or a nitrocellulose membrane.

[0209] Optionally, the sheets of absorbent material may comprise SO 15 polyolefin fibre (Porex). Optionally, sheets of absorbent material may comprise Hi-Flow Plus 120 nitrocellulose membrane (Merck). Optionally, sheets of absorbent material may comprise Grade 8980 glass-fibre membrane (Ahlstrom). The first, second, third, and one or more further sheets of absorbent material described above may comprise the same material. Alternatively, the first, second, third, and one or more further sheets of absorbent material described above may comprise different materials.

[0210] The adhesive backing layer may be made from any suitable material onto which the sheets of absorbent material may be laminated. For example, the adhesive backing layer may comprise a suitable solvent-based pressure-sensitive adhesive or a suitable UV-cured adhesive applied to a suitable solid support to provide an adhesive backing layer with suitable thermal transfer and thickness properties.

[0211] The present invention is further illustrated by the following examples that, however, are not to be construed as limiting the scope of protection. The features disclosed in the foregoing description and in the following examples may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.

[0212] EXAMPLES

[0213] The following Examples are provided to illustrate the invention but not to limit the invention.

[0214] Example 1 - Preparation of amplification-detection membraneReagent preparation

[0215] Reaction mixes were prepared to the final concentrations indicated to the required volume for reagent dispensing as below:

[0216] 1) Reaction mix for detection of Influenza B nucleic acid (proteins were manufactured in LacZ free system):

[0217] a. 70 mM Tri cine pH 7.5

[0218] b. 6.56 mM KOAc

[0219] c. 0.011% Tween 20

[0220] d. 0.88 mM dNTPs

[0221] e. 0.018 mg / mL Epidermidis Polymerase (Pol)

[0222] f. 0.10 mg / mL Creatine Kinase (CK)

[0223] g. 0.30 mg / mL T4 UvsX S64V

[0224] h. 0.060 mg / mL T4 UvsY

[0225] i. 0.025 mg / mL Exonuclease III

[0226] j . 0.12 mg / mL His-MBP-TEV-Omega

[0227] k. 1.25% 2-hydroxypropyl-y-cyclodextrin

[0228] l. 3% Trehalose

[0229] m. 2.5mM ATP

[0230] n. 20mM Phosphocreatine

[0231] o. 0.8 pM FluBNS_F2_35 forward primer

[0232] p. 0.8 pM FluBNS_R22_39 reverse primer

[0233] q. 0.02 pM FluB probe 1

[0234] r. 0.76 mg / mL T4 Gp32 HRP1

[0235] Primers

[0236] Forward: FluBNS_F2_35 ATGGCCATCGGATCCTCAACTCACTCTTCGAGCG (SEQ ID NO: 5)

[0237] Reverse: FluBNS_R22_39 TCTCCCTCTTCTGGTGATAATCGGTGCTCTTGACCAAAT (SEQ ID NO: 6)FluB Probe 1 CAAATTGGGATAAGACTCCCACCGCAGTTTCAGCTGCTCGAATTGGCTT[dT-C2- DBCO-C2]-[C3-DBCO-C2]-Alpha-50(-6)*

[0238] Alpha-50(-6) denotes amino acid sequence of:

[0239] SLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNK (SEQ ID NO: 3)

[0240] 2) Control mix for detection of beta-2-microglobulin nucleic acid used an internal control (proteins were manufactured in LacZ free system):

[0241] a. 70 mM Tri cine pH 7.5

[0242] b. 6.56 mM KOAc

[0243] c. 0.011% Tween 20

[0244] d. 0.88 mM dNTPs

[0245] e. 0.018 mg / mL Epidermidis Polymerase (Pol)

[0246] f. 0.10 mg / mL Creatine Kinase (CK)

[0247] g. 0.30 mg / mL T4 UvsX S64V

[0248] h. 0.060 mg / mL T4 UvsY

[0249] i. 0.025 mg / mL Exonuclease III

[0250] j . 0.12 mg / mL His-MBP-TEV-Omega

[0251] k. 1.25% 2-hydroxypropyl-y-cyclodextrin

[0252] l. 3% Trehalose

[0253] m. 2.5mM ATP

[0254] n. 20mM Phosphocreatine

[0255] o. 0.8 pMB2M-Al_Fl forward primer

[0256] p. 0.8 pM B2M-A1_R5 reverse primer

[0257] q. 0.02 pM B2M probe 1

[0258] r. 0.76 mg / mL T4 Gp32 HRP1

[0259] s. 40 copies / pL B2M synthetic DNA

[0260] PrimersForward: B2M-A1 F1 GAGATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGC (SEQ ID NO: 8)

[0261] Reverse: B2M-A1 R5

[0262] GACTTTCCATTCTCTGCTGGATGACGTGAGTAAAC (SEQ ID NO: 9)

[0263] B2M Probe 1

[0264] GCTCGCGCTACTCTCTCTTTCTGGCCTGGAGGCTA[dT-C2-DBCO-C2]-[C3-DBCO-C2]-Alpha-42*

[0265] * Alpha-42 denotes amino acid sequence of:

[0266] MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEK (SEQ ID NO: 4)

[0267] Reagent dispensing

[0268] Glass-fibre material (Ahlstrom 8980) was cut to 24 mm length x 5 mm width per strip for dispensing of influenza B and B2M mRPA mixes prepared as described above. Reagent dispensing was performed using a Biodot XYZ3210 dispensing platform. A 5x10 array of 924.5 nL droplets was dispensed per strip onto separate 8980 reagent pads for influenza B and B2M. Lengths of dispensed material were dried in a desiccating oven for 45 minutes at 40°C, 2% relative humidity. Dispensed materials were then stored at room temperature in Mylar bags with desiccant until laminate assembly.

[0269] Lamination

[0270] For lamination, additional strips of 8980 glass fibre (2x 10mm length strips and lx 6mm length strips were cut). Materials were laminated onto an adhesive backing layer (Trutransfer / Trufilm) as shown in Figure 2 to prepare membrane assemblies. An example membrane assembly is shown in Figure 3 A. The membrane assemblies were stored pouched with desiccant overnight to allow the adhesive to cure. The following day the membrane assemblies were removed and cut to 5 mm widths using a Biodot CM5000 guillotine. The resulting membranes were stored in Mylar bags with desiccant until required. A single membrane is shown in Figure 3B.Example 2 - Amplification / detection assay

[0271] Amplification-detection membranes were prepared as described in Example 1. To perform the amplification-detection reaction, 154 pL of a solution containing 20 mM magnesium acetate and 1 copies / pL of influenza B synthetic DNA and / or internal control (IC) DNA was applied to sample receiving zone of the membrane. The membranes were placed into a 3D printed housing containing a PTC heater and powered by 2x AAA batteries. The results are shown in Figures 6 and 7 were obtained after a 60-minute incubation.

[0272] The tests leading to the results shown in Figure 6 were carried out as follows: negative control sample in duplicate comprising neither influenza B nucleic acid nor control nucleic acid (-); a single sample comprising 30 copies / uL control nucleic acid only (IC); and a replicate of nine samples comprising 1 copy / uL control nucleic acid and 1 copy / uL of influenza B nucleic acid (IC+B). From top to bottom, the three rows of images show the membranes before application of a sample (T = 0), after a 30-minute incubation with a sample (T = 30), and after a 60-minute incubation with a sample (T = 60).

[0273] The diagnostic device depicted in Figure 7 comprises a membrane according to the present invention arranged inside a housing which comprises a heater powered by 2x AAA batteries. Figure 7 shows the application of sample comprising 30 copies / uL control nucleic acid only (IC) and a sample in duplicate comprising control nucleic acid and 1 copy / uL of influenza B nucleic acid (IC+B).

[0274] Figure 7A shows the results obtained with a diagnostic device in which the entire membrane is exposed for application of the sample and for viewing of a detectable signal in the reaction zone and the control zone. Figure 7B shows the results obtained with a diagnostic device in which the membrane is positioned within a housing and wherein the housing comprises a first opening configured to allow a liquid sample to be applied in the sample receiving zone, a second opening configured to permit viewing of a detectable signal in the reaction zone and a third opening configured to permit viewing of a detectable signal in the control zone.Example 3 - Parallel control

[0275] Figures 8A-8D show the results of a test carried out with a membrane according to an embodiment of the present invention for amplification and detection of a nucleic acid derived from influenza B. The membrane comprised a reaction zone 1, a control zone 5, and a sample receiving zone 3. The membrane can be arranged within a housing having a viewing window positioned as indicated by the overlaid rectangle.

[0276] Figure 8A shows the membrane before application of the sample. Figure 8B shows the membrane after incubation with a sample comprising 1 copy / uL of influenza B nucleic acid and 1 copy / uL of control nucleic acid. Figure 8C shows the membrane after incubation with a sample comprising 40 copies / uL of influenza B nucleic acid. Figure 8D shows the membrane after incubation with a sample comprising 40 copy / uL of control nucleic acid.

[0277] Example 4 - Opposed end control

[0278] Figures 9A-3D show the results of a further test carried out with a membrane according to an embodiment of the present invention for amplification and detection of a nucleic acid derived from influenza B. The membrane comprises a reaction zone 1, a control zone 5, and a sample receiving zone 3. Again, the membrane can be arranged within a housing having a viewing window positioned as indicated by the overlaid rectangle.

[0279] Figure 9A shows the membrane before application of the sample. Figure 9B shows the membrane after incubation with a sample comprising neither influenza B nucleic acid nor control nucleic acid. Figure 9C shows the membrane after incubation with a sample comprising influenza B nucleic acid and control nucleic acid.

[0280] Example 5 - Sensitivity testing

[0281] Figure 10 shows the results of a test carried out to determine sensitivity of three different arrangements of membranes according to embodiments of the present invention. The membranes are labelled as: double (D), single I (S-I), and single II (S-II).D refers to a membrane in which two depositions of reagents have been made. In such membranes, the reaction zone and the control zone (if present) may each comprise a first portion comprising the protein components required to perform the amplification-detection reaction and a second portion comprising the nucleic acid components required to perform the ampl ifi cati on-detecti on reach on .

[0282] S-I and S-II refers to membranes in which a single deposition of reagents on to the absorbent material has been made (S-I and S-II differ in terms of reagent concentrations but are otherwise identical). In such membranes, the reaction zone and the control zone (if present) may each comprise a single portion comprising all of the protein and nucleic acid components required to perform the amplification-detection reaction.

[0283] The tests were carried out using the following sample: a negative control sample comprising neither influenza B nucleic acid nor control nucleic acid (-); and a sample in triplicate comprising 1 copy / uL of influenza B nucleic acid (+B) for each membrane.

[0284] Top to bottom, the rows of images show the membrane before application of a sample (T = 0), after a 40-minute incubation with a sample (T = 40), and after a 60-minute incubation with a sample (T = 60). It was observed that membranes prepared using a single deposition of reagents and a reverse flow geometry provided better control of signal localisation.

[0285] Example 6 - Surfactant and polymer selection

[0286] Figure 11 shows the results of a screen for suitable surfactant and polymer combinations for use in embodiments of the present invention. The figure shows the application of sample in duplicate comprising neither influenza B nucleic acid nor control nucleic acid (-) and a sample in duplicate comprising 1 copy / uL of influenza B nucleic acid (+B) for each membrane format. The figure shows the results for membranes pre-treated with a composition comprising the following surfactants : 0.5% (v / v) PVA, 0.5% (v / v) PVP, or 0.5% (v / v) NP40. A membrane which had not been pre-treated with a surfactant composition was used as a control. Figure 11 A shows the results for membranes comprising nitrocelluloseHF120 polymer (Merck). Figure 1 IB shows the results for membranes comprising S015 polyolefin (Porex).

[0287] Example 7 - Sensitivity testing

[0288] Figure 12 shows the results of a test carried out to determine the sensitivity of two arrangements of membranes according to embodiments of the present invention. Figure 12A shows the results for a membrane prepared using a single deposition of reagents. Figure 12B shows the results for a membrane prepared using two depositions of reagents.

[0289] The figures show the application of sample comprising control nucleic acid only (IC) and a sample in triplicate comprising control nucleic acid and influenza B nucleic acid (IC+B). The figure shows the membrane before application of a sample (T = 0), after a 40-minute incubation with a sample (T = 40), and after a 60-minute incubation with a sample (T = 60). It was observed a single deposition of reagents provides for a lower background signal relative to membranes prepared using a double deposition.

[0290] Example 8 - Materials testing

[0291] Figure 13 shows the results of tests carried out with membranes according to embodiments of the present invention constructed from different materials. Figure 13 shows the results from the application of sample comprising neither influenza B nucleic acid nor control nucleic acid (-), a sample comprising 1 copy / uL of influenza B nucleic acid (+B), and a sample comprising 10 copies / uL of influenza B nucleic acid (++B). The figure shows the devices after a 40-minute incubation. Rows 1-3 show the signal produced by the different membranes cropped to the same region. The membranes constructed from using Porex SO 15 (row 1), HF 120 nitrocellulose membrane (row 2), glass fibre membrane (Ahlstrom grade 8980) (row 3). A lateral flow device (bottom) was tested as a comparison.

[0292] ***

[0293] It should be understood by those skilled in the art that while the present invention has been described with reference to exemplary embodiments, it is not limited to the disclosedexemplary embodiments. Various modifications, combinations, sub-combinations and alterations may occur depending on design requirements and other factors insofar as they are within the scope of the appended claims or the equivalents thereof. Features from any example or embodiment of the present disclosure can be combined with features from any other example or embodiment of the present disclosure.

Claims

CLAIMS1. A membrane for amplification and detection of a nucleic acid, the membrane comprising:a sample receiving zone; anda reaction zone in fluid communication with the sample receiving zone, wherein the reaction zone comprises a dried reaction mix comprising:(i) a recombinase polymerase amplification (RPA) composition;(ii) a cleavage molecule; and(iii) a probe for detecting the target nucleic acid.

2. The membrane of claim 1 further comprising:a control zone in fluid communication with the sample receiving zone wherein the control zone comprises a dried control mix comprising:(i) an RPA composition;(ii) a cleavage molecule;(iii) a control nucleic acid; and(iv) a control probe for detecting the control nucleic acid.

3. The membrane of claim 1 or 2, wherein:the dried reaction mix and / or the dried control mix, if present, does not comprise polyethylene glycol (PEG);said membrane does not comprise a test line; and / orsaid membrane does not comprise an immobilized capture molecule, such as an immobilized antibody.

4. The membrane of any one of the preceding claims having a laminated arrangement, optionally wherein the membrane comprises:an adhesive backing layer;a first membranous layer comprising the reaction zone;a second membranous layer comprising the sample receiving zone;a third membranous layer comprising a control zone;a further membranous layer comprising a blank zone in liquid communication with the reaction zone; and / ora further membranous layer comprising a blank zone in liquid communication with the control zone.

5. The membrane of claim 4, wherein the membrane comprises:an adhesive backing layer;a first membranous layer comprising the reaction zone; anda second membranous layer comprising the sample receiving zone,wherein a portion of the adhesive backing layer contacts a surface of the first membranous layer, andwherein the second membranous layer contacts an opposing surface of the first membranous layer,optionally wherein the membrane comprises a further membranous layer comprising a blank zone in liquid communication with the reaction zone and wherein said further membranous layer contacts said opposing surface of the first membranous layer.

6. The membrane of claim 5, wherein the membrane further comprises a third membranous layer comprising the control zone,wherein a portion of the adhesive backing layer contacts a surface of the third membranous layer, andwherein the second membranous layer contacts an opposing surface of the third membranous layer,optionally wherein the membrane comprises a further membranous layer comprising a blank zone in liquid communication with the control zone and wherein said further membranous layer contacts said opposing surface of the first membranous layer.

7. The membrane of any one of the preceding claims, wherein the membrane is a glassfibre membrane, polyolefin fibre membrane, or a nitrocellulose membrane.

8. The membrane of any one of the preceding claims, wherein the RPA composition comprises:a. an RPA single-stranded DNA-binding protein (RPA-SSB) molecule;b. a recombinase agent;c. a recombinase loading protein;d. a polymerase; ande. forward and reverse nucleic acid primers for amplification.

9. The membrane of any one of the preceding claims, wherein the reaction zone and the control zone, if present, each comprise a first portion and second portion which are in liquid communication with each other, optionally wherein:(i) (a) the first portion is arranged between the sample receiving zone and the second portion; or(b) the second portion arranged between the sample receiving zone and the first portion; and / or(ii) (a) the first portion comprises one or more protein components of the RPA composition, such as an RPA-SSB molecule, a recombinase agent, a polymerase and / or a cleavage molecule; and / or(b) the second portion comprises one or more nucleic acid components of the RPA composition, such as forward and reverse nucleic acid primers for amplification and / or the probe.

10. The membrane of claim 8 or 9, wherein:a. the RPA-SSB is selected from the group consisting of Gp32, E. coli SSB protein, phage T4 Gp32 protein, phage Rb69 Gp32, phage vB EcoM NBGl Gp32, or any functional analog, homolog or derivative thereof, and any combination thereof, preferably wherein the RPA-SSB is Gp32 or phage vB EcoM NBGl Gp32, optionally wherein the RPA-SSB is tagged with one or more functional intrinsically disordered regions (IDRs), preferably as a genetically engineered fusion protein comprising the RPA-SSB and an amino acid sequence comprising or consisting of the one or more functional IDRs;b. the recombinase agent is selected from the group consisting of UvsX, T4 UvsX, T6 UvsX, RBI 8 UvsX, E. coli phage wV7 UvsX, Shigella phage CB8 UvsX, Shigella phage Shfl2 UvsX, E.coli phage ARI UvsX, phage vB_EcoM_G4507 UvsX, Shigella phage SHFML-11 UvsX, Escherichia phage vB EcoM DalCa UvsX, E. coli Rec A, E. coli RadA,E. coli RadB, E. coli Rad 51 or any functional analog, homolog or derivative thereof, and any combination thereof, preferably wherein the recombinase agent is UvsX, more preferably Escherichia phage vB EcoM DalCa UvsX;c. the recombinase loading protein is selected from the group consisting of UvsY, E. coli RecO, E. coli RecR or any functional analog, homolog or derivative thereof, and any combination thereof, preferably wherein the recombinase loading protein is UvsY, more preferably Escherichia phage STO UvsY; and / ord. the polymerase is selected from the group consisting of:i. a eukaryotic polymerase selected from the group consisting of pol-a, pol-P, pol-5, pol-s or any functional analog, homolog or derivative thereof, and any combination thereof;ii. a prokaryotic polymerase selected from the group consisting of Bacillus stearothermophilus polymerase I large fragment, Bacillus subtilis Pol I large fragment (Bsu polymerase), Listeria monocytogenes DNA polymerase I, S. aureus DNA polymerase I (Sau polymerase), E. coli DNA polymerase I Klenow fragment, 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, or any functional analog, homolog or derivative thereof, and any combination thereof, preferably wherein the polymerase is S. aureus DNA polymerase I (Sau polymerase) or Bacillus subtilis Pol I large fragment (Bsu polymerase); andiii. a bacteriophage polymerase selected from the group consisting of bacteriophage T4 gp43 DNA polymerase, T7 DNA polymerase and Phi-29 DNA polymerase, or any functional analog, homolog or derivative thereof, and any combination thereof.

11. The membrane of any one of the preceding claims, wherein the probe and, if present, the control probe comprises:A. one or two single-stranded oligonucleotides each comprising a sequence which is complementary to a target nucleic acid; andB. a repressible cleavage-dependent signalling system which:a. comprises a signal-promoting molecule tethered to the oligonucleotide(s) via at least one linker, wherein the at least one linker is attached to the signalpromoting molecule and to a chemical group of the oligonucleotide(s) and thereby defines a cleavage site on the oligonucleotide(s);b. provides a detectable signal which:i. is dependent upon cleavage at the cleavage site by the cleavage molecule, andii. following hybridisation of the oligonucleotide(s) with the target sequence, is not dependent upon:

1. cleavage of the oligonucleotide(s) at an apurinic / apyrimidinic site (abasic site) of the oligonucleotide(s), e.g. at a 3 '-side of an abasic site and / or at a 5 '-side of the abasic site, or at a site comprising an abasic furan; or2. cleavage of the oligonucleotide(s) at a mismatch site; and c. is configured so that when the probe is present in the test solution:i. when the oligonucleotide(s) is not hybridised with the target sequence, cleavage at the cleavage site by the cleavage molecule is inhibited and production of a detectable signal is thereby repressed; andii. upon hybridisation of the oligonucleotide(s) with the target sequence, the cleavage molecule cleaves at the cleavage site, thereby producing a detectable signal.

12. The membrane of claim 11, wherein the signal-promoting molecule is a polypeptide, and wherein the detectable signal is produced in a reaction which is dependent upon the polypeptide, optionally wherein the detectable signal is produced in a reaction which is dependent upon the enzymatic activity of the polypeptide.

13. The membrane of claim 11 or 12, wherein the signal-promoting molecule is a betagalactosidase alpha peptide (alpha peptide).

14. The membrane of any one of the preceding claims, wherein the reaction zone and the control zone, if present, comprises a chromogenic substrate, optionally wherein the chromogenic substrate is selected from the group consisting of:chlorophenol red-P-D-galactopyranoside (CPRG);5-Bromo-6-chloro-3-indolyl-P-D-galactopyranoside (Magenta-Gal);5-Bromo-3-indolyl P-D-galactopyranoside (Bluo-Gal);orthonitrophenyl -P -D-gal actopy ranosi de (ONPG);5-bromo-4-chloro-3-indolyl-P-D-galactopyranoside;6-Chl oro-3 -indolyl-beta-D-galactopyranoside and optionally nitroblue tetrazolium salt (NBT);3,4-Cyclohexenoesculetin P-D-galactopyranoside and Fe3+;5-Iodo-3-indolyl-P-D-galactopyranoside; and1 -Methyl -3-indolyl-P-D-gal actopy ranosi de .

15. The membrane of any one of the preceding claims wherein the cleavage molecule is Escherichia coli Exonuclease III, preferably having the amino acid sequence set forth in SEQ ID NO: 1.

16. A diagnostic device comprising a membrane according to any one of the preceding claims and a housing, wherein the membrane is positioned inside the housing, optionally wherein the housing comprises:a first opening configured to allow a liquid sample to be applied to the sample receiving zone;a second opening configured to permit viewing of a detectable signal in the reaction zone; andoptionally a third opening configured to permit viewing of a detectable signal in the control zone, if present.

17. The diagnostic device of claim 16, which further comprises:one or more peelable layers;one or more spacer layers; and / orone or more protective foil layers.

18. The diagnostic device of claim 16 or 17, which further comprises:a heater, optionally a positive temperature coefficient (PTC) heater;a battery; andoptionally an insulating layer arranged between the heater and the battery.

19. The diagnostic device of claim 18, wherein:(a) the heater extends the entire length of the membrane; or(b) the heater is aligned with the reaction zone, optionally wherein the membrane further comprises a control zone, and wherein the heater comprises a first heater aligned with the reaction zone and a second heater aligned with the control zone.

20. A diagnostic system comprising:one or more diagnostic devices according to claim 16 or 17; anda base station comprising:a housing comprising an engagement portion configured to receive one of said diagnostic devices; anda heater configured to heat at least a portion of a diagnostic device received in the engagement portion,optionally wherein the housing comprises a plurality of engagement portions, each configured to receive one of said diagnostic devices and a heater configured to heat at least a portion of each of the received diagnostic devices.

21. The diagnostic system of claim 20, wherein the base station further comprises:an indicator associated with each engagement portion, wherein the base station is configured to adjust the output of the indicator to communicate information about a diagnostic device received in the engagement portion,optionally wherein the information communicated by the indicator includes one or more of:the status of the engagement of the diagnostic device with the base station; and the status of a reaction on the diagnostic device.

22. A method of manufacturing a membrane according to any one of claims 1 to 15, the method comprising:providing an aqueous reaction mix which comprises: (i) an RPA composition; (ii) a cleavage molecule; and (iii) a probe for detecting the target nucleic acid;providing a first sheet of an absorbent material;dispensing the aqueous reaction mix onto the first sheet;drying the first sheet to obtain a first sheet comprising a dried reaction mix; providing an adhesive backing layer;laminating the first sheet onto the adhesive backing layer to provide a reaction zone; providing a second sheet of an absorbent material;laminating the second sheet onto the adhesive backing layer to obtain a membrane assembly comprising a sample receiving zone in liquid communication with the reaction zone.

23. The method of claim 22, which further comprises:providing an aqueous control mix which comprises: (i) an RPA composition; (ii) a cleavage molecule; (iii) a control nucleic acid; and (iv) a control probe for detecting the control nucleic acid;providing a third sheet of an absorbent material;dispensing the control mix onto the third sheet;drying the third sheet to obtain a third sheet comprising a dried control mix; laminating the third sheet onto the adhesive backing layer such that membrane assembly further comprises a control zone in liquid communication with the sample receiving zone.

24. The method of claim 22 or 23, which further comprises:providing one or more further sheets of an absorbent material;laminating the one or more further sheets of absorbent material onto the adhesive backing layer such that the membrane assembly further comprises a first blank zone in liquid communication with the reaction zone and optionally a second blank zone in liquid communication with the control zone, if present.

25. The method of any one of claims 22 to 26, which further comprises:dividing the membrane assembly to obtain two or more strips of membrane.

Images