Biopsy Sampling Kit

Abstract

The present invention relates to methods and kits for the collection, preservation, and archiving of analytes of interest present in biopsy samples (biological fluid samples). In particular, the present invention relates to a means for collecting a biopsy sample while simultaneously preserving the analytes of interest in a format that allows for subsequent analysis, such that substantially all pathogens present in the biopsy sample are inactivated.

Description

[Technical Field]

[0001] The present invention relates to collection devices, methods, and kits for the collection, preservation, and archiving of analytes of interest present in biopsy samples (biological fluid samples). In particular, the present invention relates to a means for collecting a biopsy sample such that substantially all pathogens present in the biopsy sample are inactivated while simultaneously preserving the analytes of interest in a format that allows for subsequent analysis. [Background technology]

[0002] Biopsy specimen collection is an essential component of clinical medicine and research. Access to high-quality biopsy specimens, collected and processed in a standardized manner to minimize potential bias or confounding factors, is key to the laboratory testing process.

[0003] The pre-analytical phase of laboratory testing involves steps such as sample collection, transportation, sample preparation, and storage, and can be the source of up to 70% of errors in the entire diagnostic process.

[0004] Furthermore, some biopsy sampling methods require a cold chain, i.e., constant refrigeration, between sample acquisition and testing, which can add significant cost to testing and can prevent the use of such methods if an appropriate cold chain is not available.

[0005] Therefore, there is a need for a biopsy sampling device that simplifies sample collection, transport, sample preparation, and storage, ideally one that allows for easier processing with improved automation.

[0006] Furthermore, the COVID-19 pandemic has highlighted the urgent need for preanalytical methods to ensure that biological samples are of sufficient quality for accurate and reliable testing while minimizing the risk of pathogen transmission between patients, healthcare workers, and laboratory staff.

[0007] During the pre-analytical phase for routine confirmation of COVID-19 cases, collection of the right respiratory specimen from the right anatomical site at the right time was essential for rapid and accurate molecular diagnosis. Furthermore, regular testing was necessary to minimize disease transmission, highlighting the need for methods to ensure patient compliance.

[0008] Nasopharyngeal swabs are commonly used for respiratory virus diagnosis, but collection causes discomfort for patients due to the invasive nature of the procedure, limiting compliance with repeat testing, and poses a significant risk to healthcare workers administering the swabs, as patients may sneeze or cough, potentially spraying pathogens onto healthcare workers.

[0009] Therefore, there is an urgent need for more reliable and less resource-intensive sample collection methods, ideally ones that are amenable to self-collection (e.g., in a user-convenient environment such as home or work), are non-invasive, are easy to self-administer, and do not require cold-chain storage / transportation.

[0010] Furthermore, sample collection methods that support self-collection allow for routine testing for conditions or diseases in their pre-symptomatic or asymptomatic states, such as cancer and dementia diseases such as Alzheimer's or Parkinson's disease. This is particularly relevant when self-collection methods allow for ambient transport of samples to a central laboratory for analysis. Such routine testing may allow for earlier clinical / therapeutic intervention and therefore, optimal patient outcomes.

[0011] For example, neuropathological markers of Alzheimer's disease are thought to appear 10-15 years before overt dementia symptoms, but there are currently no routine tests for presymptomatic Alzheimer's disease.

[0012] Instead, Alzheimer's disease is currently diagnosed using a costly and invasive battery of tests, in addition to clinical assessment, cognitive testing, and MR imaging to rule out other diseases, which does not lend itself to routine testing. For example, laboratory-based tests include testing cerebrospinal fluid (CSF) for key biomarkers of Alzheimer's disease, such as amyloid beta (Aβ), total tau, and phosphorylated tau. However, CSF testing is not ideal because it requires a lumbar puncture, which is risky and invasive and can only be performed by trained professionals.

[0013] Such routine presymptomatic testing for Alzheimer's disease is especially necessary because current Alzheimer's disease medications are known to delay the onset of symptoms and / or slow the progression of symptoms.

[0014] Furthermore, routine testing with non-invasive sampling methods adapted for self-collection can be used as a cost-effective first step before applying more expensive diagnostic tests (i.e., funneling / screening tests), thereby ensuring that only relevant patients progress further down the relevant diagnostic pathway, leading to reduced healthcare costs.

[0015] Saliva sampling has emerged as an attractive alternative to nasopharyngeal swabs because it is non-invasive and easy to self-administer. For example, studies have shown that SARS-CoV-2 can be detected in the saliva of COVID-19 patients with a sensitivity level comparable to that of nasopharyngeal or oropharyngeal sampling.

[0016] Devices for collecting saliva samples for subsequent nucleic acid analysis are known, for example, from US Pat. No. 5,939,259 or US Patent Application Publication No. 2013 / 0289265, which are incorporated herein by reference.

[0017] However, there remains a need for a saliva sampling device that provides a simple, convenient, safe, and reliable pre-analysis while minimizing the risk of pathogen transmission and allowing the use of subsequent high-quality laboratory testing (e.g., nucleic acid amplification tests (NAATs) such as real-time reverse transcription PCR (RT-PCR) assays). Summary of the Invention

[0018] A biopsy sampling device and kit are provided for easily sampling biological fluids, such as saliva, from a subject. Preferably, the collection device also allows the collected biological fluid to be inexpensively and safely transported to an analytical laboratory for detection and analysis.

[0019] Preferably, collection of a biological fluid, such as saliva, allows for analysis of analytes of interest (biomarkers), such as nucleic acids and / or proteins, present in the biological fluid, including identification and / or quantification of the analytes of interest.

[0020] The term nucleic acid refers to all forms of RNA, e.g., mRNA, miRNA, rRNA, tRNA, piRNA, NcRNA, and / or DNA, e.g., genomic DNA, recombinant RNA and / or DNA, and analogs of RNA or DNA, including nucleotide analogs. Suitably, nucleic acids can be single-stranded or double-stranded.

[0021] According to a first aspect of the present invention, there is provided a collection device for collecting and storing an analyte of interest from a biological fluid sample, comprising: the biological fluid sample is suspected of containing an analyte of interest and also contains one or more pathogens; the collection device includes a solid matrix incorporating an inactivation solution adapted to inactivate substantially all of one or more pathogens present in the biological fluid sample and preserve at least a portion of the analyte of interest in a format that allows for subsequent analysis; the inactivation solution comprises a combination of protein denaturants selected from one or more detergents, one or more chaotropic salts, one or more weak bases, one or more chelating agents, and one or more reducing agents; In use, when a biological fluid sample is provided to the solid matrix, substantially all of one or more pathogens in the biological fluid sample are inactivated and at least a portion of the analytes of interest present in the biological fluid sample are preserved in a format that allows for subsequent analysis.

[0022] This can be highly advantageous, as by using an inactivation solution adapted to inactivate substantially all one or more pathogens while preserving at least a portion of the analyte of interest in a format that allows for subsequent analysis (e.g., identification and / or quantification), the collection device minimizes the risk of pathogen transmission to healthcare workers and laboratory personnel while enabling the use of subsequent high-quality laboratory testing of the analyte of interest.

[0023] It will be appreciated that the inactivation solution may be adapted to inactivate substantially all of one or more pathogens over an inactivation period. Preferably, the inactivation solution may be adapted to inactivate substantially all pathogens in a biological fluid sample when the biological fluid sample is provided to the solid matrix within 45 minutes of the biological fluid sample being provided to the collection device. Preferably, within 30 minutes of the biological fluid sample being provided to the collection device. More preferably, within 15 minutes of the biological fluid sample being provided to the collection device. In particular, the inactivation period may be shortened by increasing the concentration and / or strength of one or more chaotropic salts, or the concentration and / or strength of one or more surfactants, or a combination thereof.

[0024] Suitably, the inactivation solution can be adapted to inactivate substantially all pathogens in the biological fluid sample such that at least 90%, at least 95%, at least 99%, at least 99.9, or at least 99.99% of the infectious microorganisms present in the one or more pathogens are inactivated. Preferably, the inactivation solution is adapted to inactivate substantially all of the one or more pathogens present in the biological fluid sample such that no infectious microorganisms (e.g., virions) are detectable.

[0025] As used herein, the term "pathogen" is defined as a bacterium, virus, or other microorganism that can cause disease, and is considered synonymous with "infectious microorganism."

[0026] Preferably, the pathogen is in high titer. As used herein, the term "high titer" means that the amount of infectious pathogen in the sample is significantly greater than (≥ 2-fold, ≥ 5-fold, ≥ 10-fold, or ≥ 50-fold) the minimum infectious dose that causes infection of 50% of the population of animals that can be infected with the pathogen. In humans, this is known as Human ID50 or HID50.

[0027] The term "biological fluid" refers to a sample, in liquid or solid form, suspected of containing any analyte of interest, e.g., nucleic acid, protein, or metabolite, dissolved, suspended, mixed, or otherwise contained therein.

[0028] The term "biological fluid" also refers to samples of physiological and pathological body fluids, e.g., secretions, excretions, exudates and leachates, of humans, animals, plants, bacteria, fungi, plasmids, viruses, parasites, etc., containing the analyte of interest, such as human or animal whole blood, plasma, serum, saliva, lymph, synovial fluid, cerebrospinal fluid, semen, saliva, urine, stool, sputum, vaginal washings, fluids from infectious lesions, any cells or cell components, and any combination thereof.

[0029] As used herein, "suspected of containing" is defined as having the possibility of containing. Preferably, the biological fluid sample contains the analyte of interest. Therefore, subsequent analysis of the analyte of interest may include cases where the analyte of interest is detected and / or quantified. Preferably, the biological fluid sample either does not contain the analyte of interest or has a concentration so low that it is below the level of detection. Therefore, subsequent analysis of the analyte of interest may include cases where the analyte of interest is not detected and / or quantified.

[0030] Preferably, the analyte of interest may be at least one component of a pathogen present in the biological fluid sample. Preferably, the analyte of interest may be a component of the biological fluid sample other than a pathogen.

[0031] Preferably, the analyte of interest can be a molecule that can be analyzed in a sample: a molecule that can be determined / identified or measured / quantified. For example, it can include nucleic acids, polynucleotides, oligonucleotides, proteins, polypeptides, oligopeptides, enzymes, amino acids, receptors, carbohydrates, fatty acids, vitamins, minerals, metabolites, lipids, hormones, cells, any intracellular or extracellular molecules and fragments, viruses, viral molecules and fragments. Preferably, the analyte of interest is a nucleic acid or a peptide / protein. More preferably, the analyte of interest is a nucleic acid.

[0032] Preferably, the analyte of interest may be a nucleic acid, including either DNA or RNA, or both. As used herein, the term "nucleic acid" or "polynucleotide" refers to RNA or DNA that is linear or branched, single-stranded or double-stranded, a hybrid, or a fragment thereof. The term also includes RNA / DNA hybrids.

[0033] Suitably, the analyte of interest may be a protein, polypeptide, oligopeptide, or peptide. Preferably, the protein, polypeptide, oligopeptide, or peptide is capable of aggregation in solution and / or is hydrophobic. More preferably, the protein, polypeptide, oligopeptide, or peptide is prone to aggregation in solution and is hydrophobic.

[0034] As disclosed in Freeman et al., "State of the Science in Dried Blood Spots," Clinical Chemistry, Volume 64, Issue 4, April 1, 2018, pp. 656-679; Amado et al., "One decade of salivary proteomics: Current approaches and outstanding challenges," Clinical Biochemistry 46, pp. 506-517 (2013); Yan et al., "Systematic comparison of the human saliva and plasma proteomes," Proteomics-Clinical Applications, 3, pp. 116-134 (2009); and Pawlik et al., "The Role of Salivary Biomarkers in the Early Diagnosis of Alzheimer's Disease and Parkinson's Disease," Diagnostics 2021, 11, 371 (which are incorporated herein by reference), it is believed that there are over 2,000 analytes in blood or saliva that can be used for diagnostic and / or prognostic purposes. Saliva has been identified as functionally equivalent to serum: for example, one proteomic study found 1939 proteins in saliva and 597 proteins observed in plasma.

[0035] As mentioned above, neuropathological markers of Alzheimer's disease are thought to appear 10 to 15 years before cognitive symptoms appear. Therefore, reliable, simple, and self-administered tests for Alzheimer's disease biomarkers could be useful for routinely screening for the disease well before cognitive symptoms appear. Such routine screening could allow for early therapeutic intervention to minimize and / or delay the onset of symptoms. This may also be true for Parkinson's disease and other neurodegenerative diseases.

[0036] As noted in Pawlik et al., "The Role of Salivary Biomarkers in the Early Diagnosis of Alzheimer's Disease and Parkinson's Disease," Diagnostics 2021, 11, 371, saliva contains numerous biomarkers for Alzheimer's disease and Parkinson's disease. For example, the concentrations of Aβ protein and peptide fragments involved in the amyloid cascade present in saliva are significantly elevated in patients with Alzheimer's disease, in contrast to the decreased concentrations of Aβ protein observed in the spinal fluid of such patients.

[0037] Without wishing to be bound by theory, the Aβ in the patient's saliva 1-42 Elevated levels of Aβ are believed to be indicative of Alzheimer's disease. 1-42 is one or more of the following: -Aβ protein, -Aβ-42, -Aβ-40, Aβ-34, and / or -Aβ-20.

[0038] Preferably, the above-mentioned Aβ 1-42 Alzheimer's disease is considered to be present when the patient's saliva contains one or more of the proteins at a concentration that is 1.5 times or more the average concentration found in the saliva of individuals without Alzheimer's disease (negative controls). Preferably, one or more Aβ1-42 The protein concentration is 1.5 to 3 times the concentration of the negative control, and more preferably about 2.45 times the concentration of the negative control.

[0039] In particular, Aβ-42 and Aβ-40 are involved in amyloidosis and are insoluble / hydrophobic, whereas Aβ-34 and Aβ-20 are involved in amyloid clearance and are soluble.

[0040] Additionally or alternatively, the total tau protein concentration in the saliva of Alzheimer's disease patients is unchanged, but the ratio of salivary phosphorylated tau (p-tau) concentration to total salivary tau protein (t-tau) concentration is indicative of Alzheimer's disease and / or frontotemporal dementia. Preferably, a salivary p-tau:t-tau ratio greater than 1.2 times the average concentration found in the saliva of individuals without Alzheimer's disease (negative control) is considered indicative of Alzheimer's disease and / or frontotemporal dementia. Preferably, p-tau is any of the following: a) residues S400, T403 and S404; b) residue S396, or c) S404 residue.

[0041] More preferably, the p-tau is at residue S396.

[0042] Suitable additional biomarkers for Alzheimer's disease that can be detected in saliva include glial fibrillary acidic protein, lactoferrin, and neuronal damage marker (NFL). For example, a salivary lactoferrin concentration of less than 7.43 μg / mL is considered a sign of either Alzheimer's disease or amnestic mild cognitive impairment.

[0043] Preferably, in testing for Alzheimer's disease using the collection device of the present invention, the biofluid sample is analyzed for a combination of the above neuropathological markers, thereby improving accuracy.

[0044] Preferably, the analytes of interest may be stored on the collection device for the time necessary to transport the sample of biological fluid, or a portion thereof, from the collection source to the location where subsequent analysis will occur, preferably for minutes, hours, days, months, or longer.

[0045] The temperature conditions under which biological specimens can be stored in the collection device provided by the present invention are not limited. Typically, samples are transported and / or stored at ambient or room temperature, for example, about 10°C to about 50°C, preferably about 15°C to about 25°C. Preferably, samples are stored in a cool environment. For example, for short-term storage, samples can be refrigerated at about 2°C to about 10°C. In yet another example, samples can be refrigerated at about 4°C to about 8°C. In another example, for long-term storage, samples can be frozen at about -80°C to about -10°C. In yet another example, samples can be frozen at about -50°C to about -20°C. Furthermore, the collection device is preferably stored under dry or desiccated conditions and / or in an inert atmosphere.

[0046] Preferably, the collection device is capable of collecting multiple types of biological fluids, for example, fecal and saliva samples from the same subject.

[0047] Preferably, the collection device can be used to collect multiple types of biological fluids. This can be highly advantageous for enabling more accurate or sensitive testing or for monitoring the progression of an infection. For example, during the recent COVID-19 pandemic, nasopharyngeal and oropharyngeal swabs were the recommended specimen types for COVID-19 diagnostic testing. Antibody response tests using plasma and serum and ELISA-based assays for detecting IgM / IgG antibodies are also available, and dried blood spots can be suitably used for such ELISA tests. Furthermore, combinations of saliva and other biological fluids can also be used, such as saliva and feces, to monitor the progression of a disease, such as COVID-19, in a patient.

[0048] Advantageously, the collection device allows for the collection of multiple dried biological fluid specimens in a home environment. Even more advantageously, the collection device can be used to conveniently collect multiple types of biological fluid specimens from a subject / patient with no or reduced risk of biohazardous infection transmission between the patient and healthcare personnel.

[0049] Preferably, the solid matrix can be an absorbent and / or adsorbent material that does not irreversibly bind to nucleic acid.Preferably, the solid matrix can include cellulose material, porous glass, woven porous polymer, non-porous polymer, or a combination thereof.Preferably, the solid matrix can be a non-dissolving matrix, such as cellulose, particularly SF cellulose, that can receive and retain saliva samples containing nucleic acid.Such solid matrix is ​​also known as "filter paper".Preferably, the solid matrix can be a porous flat or sheet material.

[0050] It will be appreciated that the solid matrix (especially the non-dissolving filter solid matrix) has the ability to easily and rapidly adsorb and / or absorb biological fluids and efficiently and accurately release the analytes of interest. In preferred embodiments, each solid matrix is ​​capable of adsorbing and / or absorbing at least 0.05 ml, 0.1 ml, 0.15 ml, or preferably 0.2 ml or more of biological fluid suspected of containing the analyte of interest.

[0051] The collection device can include one or more designated areas, each comprised of a solid matrix configured to receive, adsorb, and / or absorb a biological fluid sample. When multiple designated areas are provided, this allows for obtaining multiple types of biological fluid specimens while avoiding cross-contamination during sample collection, storage, and transport. These may be color-coded to indicate to a user the biological fluid provided in each area. Preferably, different designated areas may include different components. For example, a particular designated area may include means for assisting in the drying of a particular biological fluid. Additionally or alternatively, a particular designated area may include means for removing or binding components of a biological fluid deposited on that area.

[0052] Preferably, each of the one or more designated areas may include a matrix-retaining portion, such as a card frame or a polystyrene or plastic backing / support layer. Each matrix-retaining portion may substantially surround and / or support the solid matrix, holding the solid matrix in a fixed position relative to the remainder of the collection device. Preferably, each of the one or more designated areas may include a portion that encases or covers the solid matrix after sample collection. Preferably, the portion that encases or covers the solid matrix may be a sleeve portion. Preferably, the portion that encases or covers the solid matrix may be one or more flaps that can move from a first position to at least a second position to alter access to the solid matrix.

[0053] Suitably, the "backing layer" or "support layer" may be formed from a suitable substrate, such as a polymeric or plastic material, paper, plastic, metal foil, a laminate containing metal foil, a metallized film, glass, a silicon oxide coating, and an aluminum oxide coating, a liquid crystal polymer layer, and a layer of a nanocomposite material, a metal or metal alloy, and an acrylic. Suitably, any non-porous material may be used to form the backing layer.

[0054] Preferably, the collection device comprises a backing layer and an intermediate layer containing one or more apertures, each covered by a solid matrix attached to the backing layer. When the collection device contains multiple apertures, each covered by a solid matrix, the solid matrices are separated from one another to avoid cross-contamination between different biological fluid types.

[0055] Preferably, when the collecting device includes a back layer and an intermediate layer, the collecting device can further include a printable surface layer (top layer) having a replica of one or more apertures in the back layer. The surface layer is fixed, for example, glued, to the top of the intermediate layer so that the one or more apertures are aligned, thereby exposing the solid matrix(es) to air on both the front and back sides. The surface layer may be made of, but is not limited to, paper, plastic, metal foil, laminates including metal foil, metalized film, glass, silicon oxide coating, aluminum oxide coating, liquid crystal polymer layer, and nanocomposite material, metal or metal alloy, and acrylic layer, etc. In a preferred embodiment, the surface layer is made of paper suitable for printing and lamination. Important information can be printed on the front side of the card.

[0056] Suitably, the one or more detergents may be selected from the group comprising or consisting of ionic detergents such as sodium dodecyl sulfate (SDS), deoxycholic acid, cholic acid and sarkosyl, and non-ionic detergents such as the Triton family (octoxynol, e.g., Triton X100, Triton X-114), Nonidet P-40 (NP-40), Igepal® CA-630, and the Tween family (e.g., Tween-20 and Tween-80).

[0057] Suitably, the one or more chaotropic salts may be selected from the group comprising or consisting of guanidinium salts (e.g., guanidinium isothiocyanate (GITC), guanidine thiocyanate, guanidine hydrochloride), sodium iodide, sodium perchlorate, sodium thiocyanate and potassium iodide.

[0058] One or more weak bases are included for pH buffering purposes and may be selected from the group including or consisting of 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris), 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), citrate buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and phosphate buffer. One skilled in the art will recognize that the pH of the selected buffer is typically in the range of 3 to 8.

[0059] One or more chelating agents are included to bind the divalent metal ions magnesium and calcium, and to bind transition metal ions, particularly iron. Suitably, the one or more chelating agents may be selected from the group including or consisting of ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), and 8-hydroxyquinoline (8HQ).

[0060] Suitably, the one or more reducing agents may be selected from the group comprising or consisting of dithiothreitol (DTT), dithioerythritol (DTE), L-glutathione (GSH), and tris(2-carboxyethylphosphine hydrochloride (TCEP) and 2-mercaptoethanol (2-ME).

[0061] When the solid matrix comprises cellulose (e.g., filter paper), the solid matrix incorporating the inactivation solution may comprise at least 50% by weight cellulose and 12-40% by weight of one or more chaotropic salts. Without wishing to be bound by theory, it is believed that this concentration of chaotropic salts is effective in inactivating substantially all pathogens in a biological fluid sample while preserving analytes of interest in the biological fluid sample in a form that allows for subsequent identification and / or quantification.

[0062] When the solid matrix comprises cellulose (e.g., filter paper), the solid matrix incorporating the inactivation solution can comprise at least 50% by weight cellulose, 12-40% by weight of one or more chaotropic salts, 1-5% by weight of one or more surfactants, 0.5-1.5% by weight of one or more weak bases, 0.1-0.6% by weight of one or more chelating agents, and 0.2-0.7% by weight of one or more reducing agents. Without wishing to be bound by theory, this combination of components within these ranges is believed to be particularly effective for inactivating substantially all pathogens in a biological fluid sample while simultaneously preserving analytes of interest in the biological fluid sample in a format that allows for subsequent identification and / or quantification.

[0063] Preferably, the inactivation solution comprises or consists of guanidinium isothiocyanate (GITC) (also known as thiocyanic acid and guanidine (1:1)), sodium dodecyl sulfate (SDS), octoxynol (also known as Triton X-100), Tris base (also known as trometamol), ethylenediaminetetraacetic acid (EDTA), and dithiothreitol (also known as DL-1,4-dithiothreitol). Without wishing to be bound by theory, it is believed that this combination of ingredients within these ranges is highly effective in inactivating substantially all pathogens in a biological fluid sample while simultaneously preserving analytes of interest in the biological fluid sample in a format that allows for subsequent identification and / or quantification.

[0064] When the solid matrix comprises cellulose (e.g., filter paper), the solid matrix incorporating the inactivation solution may comprise at least 50 wt% cellulose, 12-40 wt% thiocyanic acid and guanidine (1:1), 1-3 wt% sodium dodecyl sulfate, 0.5-2 wt% octoxynol, 0.5-1.5 wt% trometamol, 0.1-0.6 wt% ethylenediaminetetraacetic acid, and 0.2-0.7 wt% DL-1,4-dithiothreitol.

[0065] Preferably, the inactivation solution is capable of lysing cells and denaturing some or all proteins while protecting the analyte of interest from degradation, such as by protecting the nucleic acids within the lysed cells from nucleases, thereby effectively inactivating substantially all pathogens in a biological fluid sample while simultaneously preserving the nucleic acids for future detection and analysis.

[0066] Suitably, if the analyte of interest is a nucleic acid, it can be released after collection and storage so that it can be amplified by conventional techniques such as polymerase chain reaction (PCR) or other techniques that use DNA and / or RNA.

[0067] Advantageously, the inactivation solution is capable of lysing cells and denaturing proteins while protecting other proteins or peptides (analytes of interest) from degradation and aggregation, thereby effectively inactivating substantially all pathogens in a biological fluid sample while simultaneously preserving the proteins or peptides for future detection and analysis.

[0068] Preferably, if the analyte of interest is a protein or metabolite, it can be released after collection and storage, allowing it to be detected by conventional techniques such as enzyme-linked immunosorbent assay (ELISA), mass spectrometry, or other techniques for detecting proteins or metabolites. Because inactivation solutions can denature proteins, for techniques that rely on antibodies, it is desirable for the antibody to have binding specificity for an epitope exposed when the protein / peptide analyte of interest is denatured. If such an antibody is not available, other techniques, such as mass spectrometry, are suitable. Additionally or alternatively, the protein / peptide analyte of interest can be reconstituted in solution by incubation in a buffer containing a solubilizing agent such as CHAPS, e.g., PBS buffer containing 0.45% CHAPS.

[0069] Once the biological fluid sample has been absorbed and / or adsorbed onto the solid matrix and dried, the collection device, or portions thereof, can be safely sent for analysis using standard mail systems at ambient temperatures and over a wide range of humidity levels. Advantageously, any analytes of interest present on the solid matrix can then be eluted or redissolved from the solid matrix.

[0070] Preferably, where the analyte of interest is a component of at least one pathogen, the collection device can be used to test for the presence, absence, or amount of a viral or bacterial agent, particularly a viral or bacterial pathogen. Preferably, the viral agent can be selected from coronavirus, influenza virus, norovirus, rabies (lyssavirus), human papillomavirus, Epstein-Barr virus, herpes simplex virus, hepatitis virus, particularly hepatitis C virus, monkeypox virus, and HIV.

[0071] Suitably, when the analyte of interest is a component of at least one pathogen, the collection device can be used, for example, for microbiome profiling or identification, to test for the presence, absence, or quantity of bacterial agents associated with bacterial dysbiosis, periodontitis, dental caries, diabetes, obesity, metabolic disorders, cancer, CVD, immune-related systemic diseases.

[0072] Preferably, if the analyte of interest is a biomarker for a neurodegenerative disease, the collection device can be used to diagnose the neurodegenerative disease. Preferably, the diagnosis is an initial screening / filtering process prior to a more comprehensive and / or reliable diagnostic method. Preferably, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, other memory disorders, Huntington's disease, motor neuron disease, multiple system atrophy, and progressive supranuclear palsy. More preferably, the neurodegenerative disease is either Alzheimer's disease or Parkinson's disease.

[0073] Preferably, the inactivation solution is capable of effectively inactivating high titers of SARS CoV 2 viral particles such that there are no detectable viral particles within 45 minutes of providing the biological fluid sample to the collection device.

[0074] Advantageously, RNA can be recovered from SAR CoV 2 viruses dried onto a solid matrix and used for RT PCR detection of viral RNA.

[0075] Preferably, the collection device may comprise a sample receiving portion, an identification tag portion, and a cover portion. Preferably, the identification portion and at least a portion of the cover portion may be folded over to cover the sample receiving portion. Preferably, at least a portion of the identification portion may be removable and retainable by a user. Preferably, the sample receiving portion may include an identification tag, for example a barcode.

[0076] Preferably, the collection device may comprise a card frame having a solid matrix portion comprised of filter paper that has been pretreated with an inactivating solution to inactivate viral pathogens and stabilize RNA for downstream analytical procedures.

[0077] Preferably, the collection device may include a card frame having a solid matrix portion comprised of filter paper that has been pretreated to inactivate viral and / or bacterial pathogens and stabilize RNA for downstream analytical procedures. Preferably, the collection device may be pretreated to stabilize DNA for downstream analytical procedures.

[0078] Preferably, the collection device may include at least two flaps movable from a first position to a second position, where in the first position the filter paper is accessible for providing a sample thereon and in the second position the filter paper is inaccessible for providing a sample thereon. Preferably, text information for a user may be provided on the collection device. Preferably, the collection device may include a unique device identification (UDI) number. Preferably, the collection device may include at least two copies of the unique device identification (UDI) number, where a first copy of the unique device identification (UDI) number is detachable from the collection device. For example, the first copy may be provided on a first portion of the collection device, which is joined to a second portion of the collection device via a frangible attachment.

[0079] Preferably, the sample receiving portion contains a dye, such as Alizarin Red S, that indicates the presence and spread of saliva in the sample receiving portion. Preferably, the dye indicates the drying process of saliva onto the collection device. Preferably, the dye may be provided in the collection device in a range of 0.01-0.05%.

[0080] Preferably, recovery of the analyte for testing from the collection device can be performed using an extraction kit from a third party vendor, such as Qiagen. Preferably, recovery can be performed using Trizol LS, QIAamp Viral RNA Mini Kit, and appropriate buffers. Preferably, detection can be performed according to known clinical tests.

[0081] Preferably, RNA recovery from the solid matrix can be performed using Trizol RNA extraction.

[0082] For example, Trizol RNA extraction and downstream RT-PCR can be performed using the techniques described in Corman VM, Landt O, Kaiser M, Molenkamp R, Meijer A, Chu DK, et al., Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill. 2020;25, which is incorporated herein by reference.

[0083] Preferably, two 4.5 mm disks of solid matrix, or sections ranging from approximately 2 mm to 8 mm in length and width, can be transferred to an Eppendorf tube containing Trizol LS (375 μL) and OptiPro (125 μL). For extraction, the sample is agitated on a shaker, and the RNA is extracted with chloroform. The RNA is then purified with the RNA Clean and Concentrator-5 kit according to the manufacturer's instructions and eluted with nuclease-free water. The eluted RNA can be used for downstream qPCR purposes, as exemplified below.

[0084] Suitably, recovery of the analyte for testing from the collection device may be carried out using the method of any of the second, third or fourth aspects of the invention.

[0085] According to a second aspect of the present invention, there is provided a method of detecting nucleic acids provided from dried biological fluid from a collection device, comprising: the collection device includes a solid matrix, e.g., filter paper, that has been pretreated with an inactivation solution adapted to inactivate substantially all of one or more pathogens present in the biological fluid sample and preserve at least a portion of the analyte of interest in a format that allows for subsequent analysis; the inactivation solution comprises a combination of protein denaturants selected from one or more detergents, one or more chaotropic salts, one or more weak bases, one or more chelating agents, and one or more reducing agents; The method is: - washing a portion of the solid matrix containing the dried biological fluid with a pre-wash buffer at room temperature to form a pre-dried biological fluid, wherein the pre-wash buffer 60% to 80% ethanol, and an inactivation solution comprising a combination of protein denaturants selected from one or more detergents, one or more chaotropic salts, one or more weak bases, one or more chelating agents, and one or more reducing agents; - washing the portion of the solid matrix containing the previously dried biological fluid with a first washing solution, the first washing solution comprising at least 70% ethanol; - optionally washing the portion of the solid matrix containing the previously dried biological fluid with a second washing solution at room temperature, the second washing solution being at least 95% ethanol, which preferably reduces the drying time of the stored nucleic acids on the solid matrix; - eluting the analyte of interest from the solid matrix by incubating the solid matrix with RNAase-free water; Includes:

[0086] Preferably, the collection device is the collection device of the first aspect.

[0087] Preferably, the dried biological fluid is a biological fluid sample that was previously suspected of containing an analyte of interest and that contains a pathogen.

[0088] Preferably, the dried biological fluid is produced by providing a biological fluid sample to a solid matrix, whereby substantially all pathogens in the biological fluid sample are inactivated and at least a portion of the analytes of interest present in the biological fluid sample are preserved in a format that allows for subsequent analysis, and then drying the biological fluid sample to provide the dried biological fluid.

[0089] Advantageously, the method allows for the extraction of nucleic acids such as RNA, DNA and RNA / DNA.

[0090] Without wishing to be bound by theory, it is believed that the pre-wash step serves to solubilize dried components and remove protein aggregates and cellular debris from the sample. The first wash step is believed to remove salts and impurities that may interfere with analyte detection techniques, such as polymerase chain reaction or other nucleic acid detection techniques. The second wash step is believed to aid in drying the sample.

[0091] Preferably, the pre-wash solution comprises: -Approximately 60% to 80% ethanol, preferably approximately 70% ethanol, an inactivation solution comprising a combination of protein denaturants selected from one or more detergents, one or more chaotropic salts, one or more weak bases, one or more chelating agents, and one or more reducing agents; Includes:

[0092] Preferably, the one or more detergents may be selected from the group consisting of ionic detergents such as sodium dodecyl sulfate (SDS), deoxycholic acid, cholic acid and sarkosyl, and non-ionic detergents such as the Triton family (octoxynol, e.g., Triton X100, Triton X-114), Nonidet P-40 (NP-40), Igepal® CA-630 and the Tween family (e.g., Tween-20 and Tween-80).

[0093] Suitably, the one or more chaotropic salts may be selected from the group consisting of guanidinium salts (e.g., guanidinium isothiocyanate (GITC), guanidine thiocyanate, guanidine hydrochloride), sodium iodide, sodium perchlorate, sodium thiocyanate, and potassium iodide.

[0094] One or more weak bases are included for pH buffering purposes and may be selected from the group consisting of 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris), 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), citrate buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and phosphate buffer. Those skilled in the art will recognize that the pH of the selected buffer is typically in the range of 3 to 8.

[0095] One or more chelating agents are included to bind the divalent metal ions magnesium and calcium, and to bind transition metal ions, particularly iron. Suitably, the one or more chelating agents may be selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), and 8-hydroxyquinoline (8HQ).

[0096] Suitably, the one or more reducing agents may be selected from the group consisting of dithiothreitol (DTT), dithioerythritol (DTE), L-glutathione (GSH), and tris(2-carboxyethylphosphine hydrochloride (TCEP) and 2-mercaptoethanol (2-ME).

[0097] When the solid matrix comprises cellulose (eg, filter paper), the solid matrix incorporating the inactivation solution may comprise at least 50% by weight cellulose and 12-40% by weight of one or more chaotropic salts.

[0098] When the solid matrix comprises cellulose (e.g., filter paper), the solid matrix incorporating the inactivation solution may comprise at least 50% by weight cellulose, 12-40% by weight of one or more chaotropic salts, 1-5% by weight of one or more surfactants, 0.5-1.5% by weight of one or more weak bases, 0.1-0.6% by weight of a chelating agent, and 0.2-0.7% by weight of a reducing agent.

[0099] Preferably, the inactivation solution comprises or consists of guanidinium isothiocyanate (GITC), sodium dodecyl sulfate (SDS), octoxynol, Tris base, ethylenediaminetetraacetic acid (EDTA), and dithiothreitol.

[0100] Preferably, the inactivation solution comprises or consists of 1:1 thiocyanic acid and guanidine, sodium dodecyl sulfate, octoxynol, trometamol, ethylenediaminetetraacetic acid, and DL-1,4-dithiothreitol, preferably in the range of 12-40% thiocyanic acid and guanidine (1:1), 1-3% sodium dodecyl sulfate, 0.5-2% octoxynol, 0.5-1.5% trometamol, 0.1-0.6% ethylenediaminetetraacetic acid, and 0.2-0.7% DL-1,4-dithiothreitol.

[0101] Preferably, the pre-wash solution is about 70% ethanol, 10%-20% RNAse-free water, and 10%-20% inactivation solution.

[0102] Preferably, the first wash solution is about 70% ethanol and 30% RNAse-free water. Preferably, the second wash solution is about 95% ethanol.

[0103] The volume of wash buffer depends on the size of the matrix that needs to be processed. For example, if two 4.5 mm disks are punched out of the solid matrix and washed before nucleic acid elution, approximately 400 μl is used. If one 4.5 mm disk is used, the volume of pre-wash solution can be reduced to 200 μl. The amount of analyte present in the biological sample to be measured determines the amount of matrix that needs to be used (e.g., one, two, or three disks). Depending on the biological sample being analyzed, two washes may be optimal, three washes may be optimal, or four or more washes may be necessary. The following protocol has been optimized for both saliva and fecal samples. For saliva only, two wash steps have been found to work well. A two-minute wash time is recommended because it shortens the time for each cycle, although a five-minute wash time can also be used. Gentle shaking is advantageous to ensure uniform contact of the solution with the solid matrix. Drying at ambient conditions is usually sufficient for 10 to 30 minutes. To elute RNA, room temperature is preferred, but the temperature can be raised to 50°C (over 10-30 minutes). If only DNA is to be eluted, the temperature can be raised to 95°C (over 10-30 minutes). Note that temperatures above 95°C are not optimal for eluting RNA, as RNA degrades at 95°C. To elute both RNA and DNA, temperatures between 10-50°C are preferred. Elution times vary depending on the temperature; longer elution times may be required at room temperature.

[0104] Preferably, in certain embodiments, the dedicated analyte extraction area or areas of the solid matrix can be removed and washed as follows: - Wash 3 times with 400 μl pre-wash buffer at room temperature, incubating for 2 minutes at each wash step with shaking at 450 rpm. - Wash 3 times with 200 μl of First Wash Solution. Incubate for 2 minutes at each wash step with shaking at 450 rpm. - Wash 3 times with 100 μl of second wash solution at room temperature, incubating each wash step for 2 minutes with shaking at 450 rpm.

[0105] Preferably, each washing step may be performed multiple times, for example, two or three times. Preferably, the solid matrix may be incubated between washing steps, and preferably, the solid matrix may be washed with shaking, for example, at 450 rpm, for any or all washing steps.

[0106] Preferably, the washed matrix is ​​allowed to dry at room temperature for 30 minutes before eluting the nucleic acid from the solid matrix by incubating the solid matrix with 100 μl of RNAase-free water in a water bath at 50° C. for 60 minutes.

[0107] Advantageously, the methods of the present invention can be performed without the need for centrifugation, which is particularly advantageous when automation of sample processing is desired. Advantageously, the methods of the present invention can be performed more quickly than conventional methods such as Trizol extraction.

[0108] Preferably, the biological fluid may be saliva dried onto a solid matrix, such as filter paper. Preferably, the biological fluid may be faeces dried onto a solid matrix, such as filter paper. Preferably, the biological fluid may be a combination of saliva and biological fluid provided to different areas of the collection device. Preferably, the biological fluid may be fluid from a lesion.

[0109] Additionally, optionally, the method may include recovering nucleic acids from the saliva sample provided by the collection device. Preferably, the method may further include detecting nucleic acids provided in the saliva sample.

[0110] Preferably, the method uses RT PCR detection of SARS CoV 2 viral RNA, as known in the art. Preferably, nucleic acids can be recovered from the collection device of the invention (e.g., the filter paper portion of the solid matrix containing dried saliva) using a QIAamp Viral RNA kit (Qiagen) according to the manufacturer's instructions.

[0111] Suitably, SARS CoV-2 detection can be performed using any suitable nucleic acid detection technique, for example, by qPCR using the E-Serbeco assay and the LightCycler® 96 system.

[0112] Preferably, the reaction setup includes TaqMan TM Fast Virus 1-Step Master Mix, E_Sarbeco_F (Seq ID No1: 5´-ACAGGTACGTTAATAGTTAATAGCGT-3´), E_Sarbeco_R (Seq ID No2: 5´-ATATTGCAGCAGTACGCACACA-3´), E_Sarbeco_P (Seq ID No3: FAM-5´-ACACTAGCCATCCTTACTGCGCTTCG-3´-BHQ1), and input total RNA are used. 1 ~10 5 A standard curve can be generated using a standard panel containing Twist Synthetic SARS-CoV-2 RNA controls in RNA copies / mL. Thermal cycling is performed using a LightCycler® 96 system at 55°C for 10 minutes for reverse transcription, followed by 95°C for 3 minutes, followed by 45 cycles of 95°C for 15 seconds and 58°C for 30 seconds. RNA titers (copies / mL) can be calculated by interpolating the cycle threshold (Ct) of the standard curve generated from the standard panel using the LightCycler® software.

[0113] Preferably, a portion of the solid matrix and an aliquot of the buffer solution can be incubated together at room temperature for 30 minutes at 50°C. Preferably, the incubated portion of the solid matrix and an aliquot of the buffer solution can be mixed with ethanol (96-100%). Preferably, the portion of the solid matrix and the buffer solution mixed with ethanol can be centrifuged. Preferably, the washed matrix and solution can be transferred to a QIAamp Mini column or the like. Preferably, the RNA eluate from the column is used in an RT-PCR reaction or stored at -20°C until further use.

[0114] Preferably, the analytical sensitivity of the method for detecting nucleic acids from the dry sample provided by the collection device is comparable to the sensitivity of the wet sample.

[0115] According to a third aspect of the present invention there is provided a method of detecting an analyte of interest provided from a dried biological fluid from a collection device, wherein the analyte of interest is a protein, polypeptide, oligopeptide or peptide; the collection device includes a solid matrix, e.g., filter paper, that has been pretreated with an inactivation solution adapted to inactivate substantially all of one or more pathogens present in the biological fluid sample and preserve at least a portion of the analyte of interest in a format that allows for subsequent analysis; the inactivation solution comprises a combination of protein denaturants selected from one or more detergents, one or more chaotropic salts, one or more weak bases, one or more chelating agents, and one or more reducing agents; The method is: - eluting the analyte of interest from the solid matrix by incubating at least a portion of the solid matrix comprising the dried biological fluid with an elution buffer, and then removing the elution buffer, wherein the elution buffer comprises: one or more buffer salts, one or more solubilizing agents, and one or more protease inhibitors Includes:

[0116] Preferably, the collection device is the collection device of the first aspect.

[0117] Preferably, the dried biological fluid is a biological fluid sample that was previously suspected of containing an analyte of interest and that contains a pathogen.

[0118] Preferably, the dried biological fluid is produced by providing a biological fluid sample to a solid matrix, whereby substantially all pathogens in the biological fluid sample are inactivated and at least a portion of the analytes of interest present in the biological fluid sample are preserved in a format that allows for subsequent analysis, and then drying the biological fluid sample to provide the dried biological fluid.

[0119] Preferably, the method may further comprise the step of briefly washing the solid matrix with a pre-elution buffer prior to eluting the analyte of interest from the solid matrix, which may or may not have the same composition as the elution buffer, but one or more buffer salts, one or more solubilizing agents, and One or more protease inhibitors Includes:

[0120] Washing with pre-elution buffer is carried out to wet the solid matrix and remove microparticles.Preferably, washing can include providing the solid matrix with pre-elution buffer, then briefly vortexing the mixture, microcentrifuging, and incubating at room temperature for 5 minutes while shaking on a tabletop shaker at 5500 rpm.Then, aspirating the supernatant to remove microparticles from the solid matrix.

[0121] Preferably, elution comprises applying an elution buffer to the solid matrix and incubating with agitation on a tabletop shaker at 5500 rpm for 1 hour at 37° C. The mixture is then briefly vortexed, microcentrifuged, and the supernatant aspirated to provide a solution comprising the analyte of interest, wherein the analyte of interest is a protein, polypeptide, oligopeptide, and / or peptide.

[0122] Preferably, the protein, polypeptide, oligopeptide or peptide is capable of aggregation in solution and / or is hydrophobic. More preferably, the protein, polypeptide, oligopeptide or peptide is prone to aggregation in solution and is hydrophobic.

[0123] When the concentration of peptides / proteins in saliva is low (e.g., on the order of pg / µL), providing more solid matrix and / or dried biofluid allows for collection of an appropriate amount of analyte. For example, if 100 µL of saliva is supplied to the collection device of the present invention, five 6 mm diameter disks of solid matrix (equivalent to approximately 45 µL of saliva) are sufficient to elute Aβ-42 and / or Aβ-40 at a concentration appropriate for ELISA assays. The amount of buffer used to elute the analytes of interest from the solid matrix is ​​also important, as it affects the final concentration. In the above example, approximately 150 µL of elution buffer is adequate to elute Aβ-42 and / or Aβ-40 at a concentration appropriate for ELISA assays.

[0124] Preferably, the elution buffer comprises: buffer salts providing a pH between 6 and 9, preferably between 6.5 and 8.5; 0.1-0.6% of one or more solubilizers and 1-3% protease inhibitor Includes:

[0125] Preferably, the buffer salt may be phosphate buffered saline (PBS) or Tris buffered saline (TBS). Preferably, the buffer salt is PBS at a pH of about 7.4.

[0126] Suitably, the one or more solubilising agents may be selected from the group consisting of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate (CHAPSO), and / or thioflavin (such as thioflavin T or thioflavin S). Preferably, the solubilising agent is CHAPS and / or thioflavin S.

[0127] CHAPS and CHAPSO are non-denaturing zwitterionic detergents used to solubilize biological macromolecules such as proteins, particularly when those biological macromolecules are poorly soluble or insoluble in aqueous solutions due to their inherent hydrophobicity.

[0128] Thioflavins prevent protein / peptide aggregation, especially amyloid aggregation, and bind to amyloid fibrils but not amyloid monomers.

[0129] Preferably, the protease inhibitor is a PLAAC-protease inhibitor such as pepstatin, leupeptin, antipain, aprotinin, and chymostatin (Roche Cat. No: 11836170001, Halt TM Protease inhibitor cocktail (Thermo Scientific TM ), Pierce TM Protease inhibitor cocktail (Thermo Scientific TM ), Halt TM Protease and phosphatase inhibitor cocktail (Thermo Scientific TM ), Pierce TM Protease and phosphatase inhibitor cocktail (Thermo Scientific TM) and PMFS protease inhibitor (Thermo Scientific TM ) may be selected from the group consisting of

[0130] Preferably, the elution buffer further comprises: one or more chelating agents, one or more carrier proteins, one or more phosphatase inhibitors, and one or more biocides Contains one or more of the following:

[0131] Suitably, the one or more chelating agents may be selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), and 8-hydroxyquinoline (8HQ). Preferably, the chelating agent is EDTA.

[0132] Suitably, the one or more biocides may be selected from the group consisting of ProClin and sodium azide. Preferably, the biocide is ProClin.

[0133] Suitably, the one or more carrier proteins may be selected from the group consisting of bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLA). Preferably, the carrier protein is BSA.

[0134] In a preferred embodiment, the elution buffer comprises: buffer salts to provide a pH of approximately 7.4, 0.1-0.6% of one or more solubilizers, 1-3% of one or more protease inhibitors, 0.3 to 1.5% of one or more chelating agents, 0.05 to 2% of one or more carrier proteins, and 0.03 to 0.07% of one or more biocides Includes:

[0135] In a further preferred embodiment, the elution buffer comprises: PBS, which provides a pH of 7.4 0.01-0.1% of one or more non-denaturing zwitterionic surfactants, 0.1-0.5% thioflavin, 1-3% of one or more protease inhibitors, 0.3 to 1.5% of one or more chelating agents, 0.05-2% of one or more carrier proteins, 0.03 to 0.07% of one or more biocides Includes:

[0136] In a further preferred embodiment, the elution buffer comprises: PBS, which provides a pH of 7.4 0.01-0.1% CHAPS, 0.1-0.5% Thioflavin S, 1-3% protease inhibitors, 0.3~1.5% EDTA, 0.05-2% BSA, 0.03-0.07% ProClin Includes:

[0137] Preferably, the method may further comprise detecting the analyte of interest. Suitable methods are known in the art, such as ELISA. However, the inactivation solution may denature the protein. Preferably, the denatured protein can be refolded using methods known in the art.

[0138] When protein denaturation occurs, for any technology that relies on antibodies, it is desirable that the antibody have binding specificity to an epitope exposed when the protein / peptide analyte of interest is denatured. However, such antibodies may not be known. Therefore, alternative means, such as mass spectrometry, may be required to detect the analyte of interest.

[0139] According to a fourth aspect of the present invention there is provided a method of detecting an analyte of interest provided from a dried biological fluid from a collection device, wherein the analyte of interest is a protein, polypeptide, oligopeptide or peptide; the collection device includes a solid matrix, e.g., filter paper, that has been pretreated with an inactivation solution adapted to preserve at least a portion of the analyte of interest in a form that allows for subsequent analysis; the inactivation solution comprises one or more protease inhibitors and one or more solubilizing agents; The method is: - eluting the analyte of interest from the solid matrix by incubating at least a portion of the solid matrix comprising the dried biological fluid with an elution buffer and then removing the elution buffer, the elution buffer comprising: one or more buffer salts, one or more solubilizing agents, and one or more protease inhibitors Includes:

[0140] Advantageously, this can be used to enable detection methods that would otherwise be affected by protein denaturation, for example detection methods that rely on antibody technology where the antibodies have binding specificity for epitopes that are not exposed when the protein / peptide is denatured.

[0141] Preferably, the collection device is capable of inactivating substantially all pathogens present in the biological fluid sample upon drying to provide a dry biological fluid.

[0142] Preferably, the inactivation solution does not contain detergents or chaotropic salts. Otherwise, the statements that apply to the third aspect also apply to the fourth aspect.

[0143] According to a further aspect of the present invention, there is provided a collection device for collecting and storing an analyte of interest from a biological fluid sample, comprising: the biological fluid sample is suspected of containing an analyte of interest and also contains a pathogen; the analyte of interest is a protein, polypeptide, oligopeptide or peptide; the collection device includes a solid matrix, e.g., filter paper, that has been pretreated with an inactivation solution adapted to preserve at least a portion of the analyte of interest in a form that allows for subsequent analysis; the inactivation solution comprises one or more protease inhibitors and one or more solubilizing agents; In use, when a biological fluid sample is provided to the solid matrix, at least a portion of the analytes of interest present in the biological fluid sample are preserved in a format that allows for subsequent analysis.

[0144] Preferably, the collection device is capable of inactivating substantially all pathogens present in the biological fluid sample upon drying.

[0145] Preferably, the inactivation solution does not contain detergents or chaotropic salts, and it will be appreciated that this will result in fewer pathogens being inactivated than the collection device of the third aspect. Otherwise, the statements applicable to the first aspect also apply to this further aspect.

[0146] Preferably, the collection device of this further aspect is used in the method of the fourth aspect.

[0147] According to a fifth aspect of the present invention, there is provided a collection device according to the first aspect of the present invention; a. A pipettor for dripping the biofluid into the collection device; b. Biofluid collection cup; c. Bags, such as Ziploc or biohazard marked bags; d. desiccants, and e. Return envelope for storing and transporting dried biological fluid samples to the designated laboratory for analysis. and at least one of:

[0148] Preferably, the kit also comprises: a. Instructions for use to guide specimen collection; b.Gloves, c. Towelette d.Temperature strips, e. Cleaning agents and f. Disposal container for the kit It may also include at least one of:

[0149] Preferably, the collection kit includes a collection device that further includes a sample identification tag, such as a barcode.

[0150] According to a sixth aspect of the present invention, there is provided a method of using the collection device of the first aspect of the present invention for at least one of microbiome profiling, genotyping purposes, disease diagnosis, treatment selection, and disease severity determination.

[0151] If desired, saliva, blood, or fecal samples can be collected. As will be appreciated, the nucleic acids obtained using the collection devices described herein can be used for a variety of clinical applications.

[0152] Preferably, the collection device of the first aspect of the present invention can be used with the method of any of the second, third or fourth aspects to perform at least one of diagnosing a disease, selecting a treatment, and determining the severity of a disease.

[0153] For example, the collection devices and / or methods of the present invention can be used in connection with infectious diseases, various types of cancer (such as breast cancer, prostate cancer, pancreatic cancer, lung cancer, colon cancer, etc.), and neurodegenerative diseases such as Alzheimer's disease and other memory disorders, Parkinson's disease, Huntington's disease, motor neuron disease, multiple system atrophy, and progressive supranuclear palsy.

[0154] An embodiment of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0155] [Figure 1] FIG. 1 is a top view of a collection device of the present invention showing the sample receiving portion (b), the cover portion (a), and the identification tag portion (c). [Figure 2] FIG. 2 is a bottom view of the collection device of the present invention. [Figure 3] 10A and 10B are diagrams showing the folding direction of at least a portion of the identification portion (c) and the cover portion (a) when the identification portion (c) and the cover portion (a) are folded to cover the sample receiving portion (b). [Figure 4] Figure 1 shows the analysis of the presence or absence of SARS CoV 2 viral RNA according to an already established RT PCR procedure using the N1 and N2 assays in the analytical laboratory. [Figure 5] Table 1. [Figure 6] Table 2. [Figure 7] FIG. 1 shows the mean DI score per sample and preparation method with standard deviation bars. [Figure 8] FIG. 1 shows the average abundance score for each probe and preparation method across all samples. [Figure 9] Schematic diagram of the collection device unfolded (A) and folded (B). [Figure 10] FIG. 1 shows a collection device with holes and apertures suitable for distinguishing between different biofluid types. [Figure 11] FIG. 1 shows a filter paper solid matrix provided (eg, adhered) to a backing or support layer of a biocollection device. [Figure 12] 12A and 12B illustrate preferred embodiment designs for collecting four different types of biological fluid specimens. Figure 12A illustrates an example of a backing layer with appropriate holes and apertures. Figure 12B illustrates a filter paper solid matrix adhered to the backing layer to avoid cross-contamination of different types of biological fluid specimens. Figure 12C illustrates an example of a surface layer with an exact replica of the holes and apertures as in Figure 12A and side flaps. Figure 12D illustrates an example of a surface layer adhered to the top of the middle layer of a collection device. [Figure 13]Figure 13A shows an example of an unprinted collecting device, Figure 13B shows an example of a printed collecting device, and Figure 13C shows an example of a folded collecting device. [Figure 14] Figure 14A shows several designs of collection devices that demonstrate their flexibility for customization depending on the application and purpose. Figure 14B shows several designs of collection devices that demonstrate their flexibility for customization depending on the application and purpose. [Figure 15] Figure 15A is a schematic diagram of a collection device, and Figure 15B is a diagram illustrating the step of drying the resulting biofluid on a flat surface after the biofluid has been absorbed into the collection device. [Figure 16] FIG. 1 shows the efficacy of amplification using Trizol RNA extraction (T), the cleaning and extraction method of the present invention (H), and QiaGen viral RNA extraction (Q). [Figure 17] FIG. 11 shows extrapolated viral RNA copy numbers from Hvidovre dilution data using Trizol RNA extraction (T), the inventive cleaning and extraction method (H), and QiaGen viral RNA extraction (Q). [Figure 18] FIG. 1 shows the concentrations of amyloid fragments Aβ-40 and Aβ-34 obtained from saliva samples using the collection device of the present invention, where the concentrations were measured using two different analytical platforms: Mesoscale (MSD) and SIMOA (SR-X). DETAILED DESCRIPTION OF THE INVENTION

[0156] Saliva was collected by passive drool collection, with two drops of saliva placed in the center of the sample-receiving portion of the collection device. The sample-receiving portion contains filter paper with an indicator dye that clearly indicates the spread of saliva upon application. A colored indicator dye, such as a pink indicator dye that turns yellow / white and encircles the area containing the dried saliva sample with a red ring, can be used. Once visually confirmed, the collection device was allowed to air dry for at least 45 minutes. The card was then folded and placed in a biohazard-marked aluminum bag containing a desiccant. The aluminum bag was then placed in a return envelope and sent to the designated laboratory for analysis. Each collection device contains a unique barcode ID for tracking and tracing purposes. This unique barcode ID can be used to access results from the reference laboratory.

[0157] Example 1 - Stability study As background for the following example: At the analytical stage, real-time reverse transcription PCR (RT-PCR) assays remain the molecular test of choice for etiologic diagnosis of pathogens such as SARS-CoV-2, while antibody-based techniques are used as ancillary tools. Viral genes targeted in such assays include the N, E, S, and RdRP viral genes. To avoid potential cross-reactivity with other endemic coronaviruses and potential genetic drift of SARS-CoV-2, at least two molecular targets are included in routine confirmatory assays.

[0158] A collection device of the present invention was prepared by providing filter paper as the solid matrix, which was treated by immersion in the following solutions: Buffer component concentration GITC 4M SDS 2% Triton X-100 1% Tris HCl (pH 7.6) 100 mM EDTA 10mM DTT 20mM Alizarin Red 1mM

[0159] The filter paper was then dried to provide a solid matrix incorporating the inactivation solution within the following definition: at least 50 wt% cellulose, 12–40 wt% thiocyanic acid and guanidine (1:1), 1–3 wt% sodium dodecyl sulfate, 0.5–2 wt% octoxynol, 0.5–1.5 wt% trometamol, 0.1–0.6 wt% ethylenediaminetetraacetic acid, and 0.2–0.7 wt% DL-1,4-dithiothreitol.

[0160] This was subsequently used in a stability study, which was carried out under various temperature conditions during collection, drying, storage, and transport to the laboratory for testing. The stability of the collection device was investigated considering delayed transport time (7 days), normal postal time (3 days), hot summer conditions (40°C and 50°C), and a winter profile.

[0161] A mixture of saliva and SARS-CoV-2 virus stock dilutions, requiring 30–31 cycles of amplification, was spotted onto filter paper and allowed to dry for 45 minutes in a BSL3 laboratory. The filter paper was then shipped on dry ice (-80°C) to an analytical laboratory for analysis. Triplicate filter paper discs were incubated at 4°C, room temperature, 40°C, and 50°C for 3 and 7 days. For the winter profile, triplicates were incubated at -20°C for 16 hours, followed by room temperature (20°C) for 4 hours, followed by -20°C for 2 hours, followed by 4°C for 40 hours, and then 20°C for 6 hours. Samples were analyzed for the presence of SARS-CoV-2 viral RNA using a RT-PCR procedure using the N1 and N2 assays established in the analytical laboratory.

[0162] The results (Figure 4) suggest that SARS-CoV RNA on collection devices is very stable when exposed to winter profiles. The RNA is very stable at both 4°C and room temperature when incubated for 3 and 7 days. When RNA-containing collection devices were incubated at 40°C for 3 days (2 cycles) and 7 days (6 cycles), an increased number of cycles was required. This suggests that RNA becomes unstable at 40°C when exposed for very long periods. No clear amplification was observed at 50°C, suggesting that viral RNA is not stable at this temperature. Additional studies are needed to determine RNA stability in the 40°C-50°C temperature range and to determine how long RNA can be exposed at these temperatures.

[0163] Example 2 - Storage stability To determine the shelf life of the collection device of the present invention, collection devices were prepared along the lines of that of Example 1. These were then stored for 3, 6, 12, and 24 months, and the assays of Example 1 were repeated along with the infectivity studies.

[0164] Inactivation of SARS-2 CoV2 was determined by viral activity - infectivity of eukaryotic cells by assessing cytopathic effect (CPE) and S (spike) protein immunostaining.

[0165] Evaluation after 3 months based on CPE detection and immunostaining confirmed that the test matrix retained its efficacy and completely inactivated the virus after 45 minutes of contact time, 3 months after manufacture.

[0166] Furthermore, detection of viral RNA by RT PCR was deemed effective.

[0167] A SARS-CoV 2 virus stock (serum-free medium) originally collected from a Danish COVID-19 patient and subsequently propagated in Vero E6 cells was used (passage 39, 7.6 log10 TCID50 / ml). 50 μL of virus stock or 50 μL of a 1:1 mixture of saliva and virus stock was spotted onto filter paper discs (pretreated with inactivation solution or untreated) and incubated for 15, 30, or 60 minutes. Filter eluates were obtained by filter incubation with 1 mL of Vero E6 cell culture medium for 30 minutes followed by shaking. One mL of the 1:50 diluted filter eluate was transferred to cells (Vero E6, confluent T25 flasks). After 30 minutes of incubation, medium was added to each culture to bring the total volume to 4 mL. The 1:50 dilution of the filter eluate did not cause CPE in cell cultures. Cultures were monitored microscopically by CPE detection and S protein immunostaining, as summarized in Table 1 (Figure 5).

[0168] All cultures inoculated with filter paper pretreated with inactivation solution were negative for CPE and S staining, regardless of incubation time (15, 30, and 60 minutes) and addition of saliva. All cultures inoculated with filter paper not pretreated with inactivation solution were positive for CPE and S staining, regardless of incubation time (15, 30, and 60 minutes) and addition of saliva. Control cultures inoculated with saliva alone did not develop CPE. Thus, filter paper containing inactivation solution completely inactivates the SARS-CoV-2 virus within 45 minutes of contact time (15 minutes on filter paper and 30 minutes of eluate incubation time).

[0169] The analytical performance of the collection device was evaluated by spiking saliva with serial dilutions of SARS-CoV-2 virus stock at 10.6 log10 copies / mL and spotting onto 6 mm pretreated filter paper discs (pretreated with inactivation solution) in a BSL-3 facility (Table 2 - Figure 6). The virus-containing filter paper discs were allowed to dry for 45 minutes and then sent to a testing laboratory under conditions mimicking real-world environmental settings. Samples were analyzed for the presence of SARS-CoV-2 viral RNA according to established procedures in each laboratory.

[0170] In the first example, virus-containing filter paper discs were incubated with Trizol LS (375 μL) and OptiPro (125 μL) for 2 hours on a shaker at 250 RPM. RNA was extracted with chloroform in 5PRIME Phase Lock Gel Heavy tubes, purified with the RNA Clean and Concentrator-5 kit according to the manufacturer's instructions, and eluted in 15 μL of nuclease-free water.

[0171] For viral RNA detection, the reaction was performed using TaqMan TM Fast Virus 1-Step Master Mix: Set up using 400 nM E_Sarbeco_F (Seq ID No: 1 5´-ACAGGTACGTTAATAGTTAATAGCGT-3´), 400 nM E_Sarbeco_R (Seq ID No: 2 5´-ATATTGCAGCAGTACGCACACA-3´) and 200 nM E_Sarbeco_P (Seq ID No: 3 FAM-5´-ACACTAGCCATCCTTACTGCGCTTCG-3´-BHQ1) and 2.5 µL of input total RNA.

[0172] To generate a standard curve, a standard panel containing Twist Synthetic SARS-CoV-2 RNA controls at 101 to 105 RNA copies / mL was used. For reverse transcription, a LightCycler® 96 system was used, thermally cycling at 55°C for 10 minutes, followed by 95°C for 3 minutes, followed by 45 cycles of 95°C for 15 seconds and 58°C for 30 seconds. RNA titers (copies / mL) were calculated by interpolating the cycle threshold (Ct) values ​​of the standard curve generated from the standard panel using the LightCycler® software.

[0173] In the second example condition, two different RNA recovery methods were tested. In one method, two 4.5 mm discs were washed as follows: Wash 3 times with 200 μl pre-wash buffer at room temperature, incubating each wash step for 2 minutes with shaking at 450 rpm. Wash three times with 200 μl of a first wash solution containing 70% ethanol, incubating each wash step for 2 minutes with shaking at 450 rpm. Wash three times with 100 μl of a second wash solution containing 95% ethanol at room temperature, incubating each wash step for 2 minutes with shaking at 450 rpm.

[0174] Alternatively, RNA was recovered using the QIAamp Viral RNA Extraction Kit. Each disc was transferred to 560 μl of prepared buffer AVL containing carrier RNA and incubated at room temperature (50°C) for 30 minutes to elute the RNA. The procedure was then carried out according to the manufacturer's instructions (QiaGen). RNA was eluted in 60 μl of RNAase-free water. CFX96Touch TM Five μl of RNA template was used for SARS CoV2 N1 and N2 assay detection by RTPCR using a real-time RT PCR platform (Biorad).

[0175] The washed filters were dried at room temperature for 30 min, and viral RNA was eluted by incubating the filter paper discs with 100 μl of RNAase-free water in a water bath at 50°C for 30 min.

[0176] The inactivation solution is a mixture of 70% ethanol, 10%-20% RNAase-free water, and 10%-20% inactivation solution, which is a combination of a surfactant and a protein denaturant selected from chaotropic salts, weak bases, chelating agents, and reducing agents. Preferably, the inactivation solution contains or consists of thiocyanic acid and guanidine (1:1), sodium dodecyl sulfate, octoxynol, trometamol, ethylenediaminetetraacetic acid, and DL-1,4-dithiothreitol. Preferably, the solid matrix was pre-cleaned using 12-40% thiocyanic acid and guanidine (1:1), 1-3% sodium dodecyl sulfate, 0.5-2% octoxynol, 0.5-1.5% trometamol, 0.1-0.6% ethylenediaminetetraacetic acid, and 0.2-0.7% DL-1,4-dithiothreitol.

[0177] Preferably, temperatures below 50°C are advantageous for RNA recovery. Temperatures above 50°C are advantageous for DNA recovery. For some downstream applications, for example, when determining the presence of both RNA-based pathogens, such as SARS CoV2 virus, influenza virus, and DNA-based pathogens, such as bacterial or viral pathogens, such as simian varicella virus, varicella zoster virus, or Epstein-Barr virus, the ability to obtain both DNA and RNA from a single sample, such as a dried saliva sample, is advantageous. This allows for simultaneous testing, thus reducing the need to obtain samples and allowing for more rapid screening of subjects.

[0178] Washing with at least the first wash solution was performed by incubating each disc for 2 minutes with agitation (400 RPM). After the wash step, the filter paper discs were air-dried at room temperature for 30 minutes, and the discs were then incubated in 100 μl of RNAase-free water at 500°C for 60 minutes to elute the viral RNA. Alternatively, RNA was recovered using the QIAamp Viral RNA Extraction Kit. Each disc was transferred to 560 μl of prepared buffer AVL containing carrier RNA and incubated at room temperature for 30 minutes at 50°C to elute the RNA. The procedure was then carried out according to the manufacturer's instructions (QiaGen). RNA was eluted with 60 μl of RNAase-free water. CFX96 Touch TM Five microliters of RNA template was used for SARS-CoV-2 N1 and N2 assay detection by RT-PCR using a real-time RT-PCR platform (Biorad). As mentioned above, the Trizol method can also be used for RNA recovery.

[0179] Good amplification curves were observed for the assays tested (Figure 16). The results are summarized in Table 3 and suggest good analytical performance in terms of amplification efficiency, linearity, repeatability, and reproducibility of measurements between different RNA extraction methods and assays. All measurements were in good agreement, with the difference in mean Cq falling within ±1.96 SD.

[0180] Advantageously, by using the recovery methods described herein, both RNA and DNA can be obtained, as opposed to recovery of RNA only, for example, when using the Trizol method or methods using the QIAamp Viral RNA extraction kit.

[0181] In the present disclosure, the pre-wash buffer contains 10-20% of the inactivation solution used for pre-treatment of the filter paper matrix, which advantageously allows for the stepwise removal of contaminants, such as cell debris, detergents, and protein aggregates, from the pre-treated card. The first wash solution removes salts and other contaminants. Some of these contaminants are known to inhibit PCR reactions, so their removal is advantageous before further analysis. The disclosed method is preferably operable using commercially available, readily available automated plate handlers. Furthermore, the disclosed method can be easily automated because it uses only three components: inactivation solution, ethanol, and water.

[0182] [Table 1]

[0183] [Table 2]

[0184] In all cases, target amplification was highly linear and efficient (R2 ≥ 0.98, and amplification efficiency > 93%). The results were highly repeatable and dependent on the assay used. For one assay (N1 assay), the coefficient of variation (CV) observed between replicates was less than 5%, whereas for another assay (E assay), it was 17%, but in all cases it was less than 20%. Furthermore, the results were highly reproducible regardless of differences in RNA extraction method, assay type measured, RT-PCR instrument used, test site facility, or operator. Good agreement was observed between assays, methods, and test site measurements. All data were within ±1.96 SD.

[0185] Example 3 - Virus detection research Clinical details were reviewed for nine patients (sample IDs #73-81) from whom dried saliva samples were collected. For five samples (sample IDs #73-77), oropharyngeal swab-based NAAT results were collected both before and after the collection date (Table 4). For two COVID-19 patients, oropharyngeal (OP) nucleic acid amplification test (NAAT) results were negative (sample IDs #73 and #74). These patients tested positive for OP swab NAAT both 2-3 days before and 4-5 days after the collection date, indicating false negative results on the day of collection. For four samples (sample IDs #78-81), the patients were confirmed positive, but no OP swab NAAT results were available on the day of dried saliva collection. However, in the case of two samples, dried saliva samples were collected from recovering patients 10 days after symptom onset (approximately 15 days after infection).

[0186] [Table 3]

[0187] For detection of SARS CoV RNA, nucleocapsid viral proteins N1 and N2 were amplified as viral targets, and a human RNase P target was amplified as a control to monitor RNA integrity. Detailed RT PCR results are shown in Appendix V. Summary RT PCR results are shown below (Table 5).

[0188] The results suggest high consistency between the gold standard based on dried saliva and swab samples. For samples for which NAAT results from oropharyngeal swabs were not available on the day of dried saliva sample collection, the dried saliva NAAT was consistent with the expected progression of disease, where no or low viral titers in the lower respiratory tract of recovering patients would be expected 15 days after infection. In two patients (#73 and #74), OP NAAT results were positive both 2 days before and 4 days after sample collection but negative on the day of dried saliva collection. Samples were collected 1 day after symptom onset. This may indicate that these patients had low viral shedding in the lower respiratory tract on the day of collection or that OP swab sampling was not performed appropriately. Low viral shedding was confirmed by the dried saliva NAAT result for #74, as the Cq values ​​of the N1 and N2 amplicons were high compared to the other positives (32, 40, and 34, 73, respectively). A high Cq was also observed in case #78, but the OP swab NAAT was unavailable on the day of collection. In one sample (#73), both the OP swab and the dried saliva NAAT were negative on the day of dry saliva collection, but the patient was confirmed COVID-positive. The NAAT result indicated zero or very low viral titers in the respiratory tract on this day, possibly indicating some kind of early response to infection that may have reduced viral titers in this area (Table 6).

[0189] [Table 4]

[0190] Dried saliva collected using the collection device described herein can be used as an alternative collection method to nasopharyngeal / oropharyngeal swabs for detecting active SARS-CoV-2 infection. High consistency of results was observed between dried saliva-based and swab-based NAATs. This also indicates that SARS-CoV-2 RNA is stable for more than six weeks under ambient conditions using the collection device described herein.

[0191] [Table 5-1]

[0192] Example 4 - Assay using multiple biofluids As described below, a study was conducted using the disclosed collection device (HemoDx device) and a nucleic acid recovery kit for gut microbiome analysis and SARS CoV 2 detection. Multiple biological fluids were collected from the same patient using the same device. For example, both saliva and fecal samples were collected from the patient, and the samples were analyzed to determine the presence or absence of SARS CoV 2.

[0193] In this study, bacterial gDNA and viral RNA were extracted from feces using the collection device and methods described herein in combination with the GA-map® Dysbiosis test Lx V2 and the GA-map® COVID-19 Faecal Test Kit.

[0194] Results show that the collection device and method can be used to extract bacterial gDNA and viral RNA from feces. However, to obtain gDNA, the sample must first be diluted.

[0195] Samples were analyzed from the biobank. Three instrument cards were prepared for each sample. A small amount of sample material was smeared onto filter paper pretreated with inactivation solution. The cards were stored at room temperature overnight (3:30 PM to 7:30 AM). For each sample, three 1.5 mL Eppendorf tubes containing two 4.5 mm filter paper discs were processed according to the extraction protocol, yielding 100 μL of nucleic acid eluate.

[0196] Dysbiosis: Three dilutions (undiluted, 1:10, 1:100) of gDNA eluates extracted from four samples using the method described below were analyzed using the GA-map® Dysbiosis test Lx V2. gDNA from this study (HemoDx) and the reference method (GA Service Lab) were analyzed together on the same plate to eliminate run-to-run variability.

[0197] Four samples at three dilutions (undiluted, 1:10, 1:100) were analyzed with the GA-map® COVID-19 Fecal Test Kit using the extraction method described herein. Samples 1 and 2 had previously been confirmed as Covid-19 positive using the reference method of the QIAamp Viral RNA Kit (Qiagen) with the GA-map® COVID-19 Fecal Test Kit. There was no reason to suspect that Samples 3 and 4 were Covid-19 positive.

[0198] Extraction protocol used: In the nucleic acid extraction method described herein, in this study, the filter discs were not transferred to new 1.5 mL Eppendorf tubes after drying at room temperature for 30 minutes, but this did not appear to have any significant effect on the results.

[0199] Quantitative results of the Dysbiosis test: After the 16S rRNA PCR amplification step, Quant-iT was performed on a FLUOstar Omega. TM Quantitative DNA measurements were performed using (Table 1). No amplicons were detected in undiluted samples.

[0200] The undiluted samples did not yield PCR amplicon yields within the expected range, so these samples were not included in downstream analysis. The other two test parameters (1:10 and 1:100 diluted eluates) yielded slightly higher yields than the standards. The average yields for the standards, 1:10 and 1:100, were 30.9, 42.2, and 37.5, respectively.

[0201] Dysbiosis index (DI) scores were calculated based on probe signals read by Luminex 200. The DI index is on a scale of 1 to 5, with DI 1-2 indicating a normal bacterial composition and DI 3-5 indicating a disturbed / dysbiotic microbiota. Decimal DI score results were calculated using an in-house GA bioinformatics tool.

[0202] The results (Fig. 7) show that the scores of the current extraction are overall slightly higher compared to the reference method, but the replicate variability is similar (STD<0.4 for all three parameters).

[0203] Bacterial abundance score: The bacterial abundance score was calculated based on the probe signal read on the Luminex 200 using GA-map® Analyzer software. The abundance score ranged from -3 to +3, with a score of 0 indicating normal / expected levels of the bacterial target, -1 to -3 indicating decreased levels, and +1 to +3 indicating increased levels. An in-house GA bioinformatics tool was also used to interpret the results (Figure 8).

[0204] The results show similar bacterial profiles across extraction methods, but some systematic differences between the reference and our method extractions. Systematic reduction of the extract of the present invention compared to the reference: Bacteroides spp. & Prevotella spp., Dialister invisus & Megasphaera micronuciformis, Alistipes Systematic increase of the extract of the invention compared to the references: Ruminococcus albus & R. bromii, Dorea spp., Anaerobutyricum hallii

[0205] On both plates, the negative and positive controls produced signals (Cq and PFU values) within the expected range. The RP Ct values ​​exceeded the upper limit for all sample replicates at 1:100 dilution. Therefore, the results of these samples were not further analyzed. Furthermore, the RP Ct values ​​exceeded the upper limit for all parameters in all samples for ≥1 replicate. Of the included samples, two were Covid-19 positive (S1 and S2) and two were Covid-19 negative (S3 and S4). The expected N1 and N2 Ct values ​​for samples S1 and S2 were 27 and 31, respectively, with an expected RP Ct value of 32 (original analysis results using the GA-map® COVID-19 stool test in May 2020; data not shown).

[0206] Covid-19 samples were analyzed using a CFX96 C1000 real-time thermal cycler, and the resulting files were prepared using Bio-Rad CFX Maestro software.

[0207] [Table 6]

[0208] [Table 7]

[0209] The results show, as expected, a positive Covid-19 signal in samples S1 and S2 and a negative signal in S3 and S4. However, compared to the original analysis of the same samples, the signal was significantly lower for all three target sequences (N1, N2, and RP), indicating reduced sensitivity compared to the GA-map® COVID-19 stool test.

[0210] For example, it may be advantageous to collect multiple different types of biofluids from different sites on the patient because, for example, in the early days (days 1-8 post-infection) the virus is present in the oral region and detectable using saliva or swabs, whereas in later stages of infection (>day 8, Long Covid) the virus is found in stool samples but not in saliva or swab samples (RTPCR negative). Preferably, a device that can be used to collect different types of samples and stabilize these samples for transport to enable testing provides advantageous flexibility in the use of the collection device.

[0211] Additionally, the same collection device, e.g., card frame, can be used to collect different biological specimens from the same patient, determining the stage of disease, e.g., early if present in saliva and later if present in a stool sample, and collecting both saliva and stool samples for SARs CoV 2, so that both RNA and DNA can be detected using the same device, where appropriate.

[0212] Alternatively, the collection device may be used to collect and stabilize biological samples so that RNA recovered from the collection device can be used for gene expression profiling and microRNA profiling, while DNA recovered from the collection device can be used for genotyping or microbiome analysis. Saliva, blood, or fecal samples can be obtained as needed. As will be appreciated, nucleic acids obtained using the collection devices discussed herein may be used in a variety of clinical applications, such as diagnosing, selecting treatments, and assessing the severity of diseases, including infectious diseases, various types of cancer (such as breast cancer, prostate cancer, pancreatic cancer, lung cancer, and colon cancer), and neurodegenerative diseases such as Alzheimer's disease and other memory disorders, Parkinson's disease, Huntington's disease, motor neuron disease, multiple system atrophy, and progressive supranuclear palsy.

[0213] As shown in Figures 16 and 17, after extraction using the method of the present invention, template formation is at least comparable to other conventional extraction methods used in the art.

[0214] Example 5 - Large-scale double-blind viral testing As an extension of the assay of Example 3, a further study was conducted which demonstrated the use of self-sampling using the collection device of the present invention to determine the presence or absence of SARS CoV 2 using a blind test.

[0215] The study was conducted by an independent contract research organization and consisted of 100 COVID-19 (SARS CoV-2) positive subjects and 100 COVID-19 (SARS CoV-2) negative subjects.

[0216] As controls, nasopharyngeal and oropharyngeal swab samples were obtained according to ICMR standard of truth recommendations by medical professionals. Patients were then provided with a kit (HemoDx kit) according to the fifth aspect and instructed to provide a saliva sample. The saliva sample was either self-collected at the hospital on the same day or self-collected at home on the same day under ambient conditions, with the collection device mailed to the laboratory's mailing address.

[0217] Participants were instructed to place at least two drops of saliva in a designated area of ​​the collection device and then allow the saliva to dry under ambient conditions for at least 45 minutes before sealing it in an appropriate bag (containing desiccant).

[0218] All samples were blinded by removing patient identifiers and assigning a unique barcode to each study sample, and blinding was not performed by the same individuals who performed subsequent laboratory analyses.

[0219] The presence (and copy number) of SARS CoV-2 was determined using RT-PCR-based detection of SARS CoV-2 RNA using ORF 1 ab and N gene. ARIDIA COVID-19 was used as a positive control and ARIDIA dH2O was used as a negative control.

[0220] Nasopharyngeal and oropharyngeal swab samples were tested using Thermofisher Scientific's Covipath COVID-19 RT-PCR kits, which are designed for the qualitative detection of the ORF 1 ab and N genes of the SARS-CoV-2 genome by real-time Reserve Transcriptase PCR.

[0221] RNA was collected from saliva samples collected using the collection device of the present invention using a QiaGen viral RNA kit according to the manufacturer's instructions, although other RNA collection kits are available.

[0222] RNA recovered from saliva samples collected using the collection device of the present invention was assessed for the presence of SARS CoV-2 as follows: 1. Thaw the necessary reagents, including the positive control, on ice. 2. Prepare one PCR master mix per primer / probe pair: see Table 7. 3. According to the sample setup, 20 μL of RT-PCR master mix was dispensed onto a 96-well PCR plate. 4. 5 μL of template was added to the plate according to the plate setup. a. Positive control: ARIDIA COVID-19 (PosCtr) b. Negative control: ARIDIA dH2O (NTC) 5. The plate was sealed and the samples were loaded onto the IANLONG-GENTIER 48E RT PCR platform. The PCR program is shown in Table 8.

[0223] [Table 7] [Table 8]

[0224] [Table 8] [Table 9]

[0225] Conclusion: A total of 225 participants were enrolled in the trial, but 15 participants failed prescreening and 10 failed screening. Exclusion criteria were that patients had to be over 18 years of age and provide signed, written informed consent.

[0226] Of the remaining participants, 200 were enrolled to use dried saliva spots to detect Covid-19: 100 patients tested positive for Covid and 100 patients tested negative for Covid.

[0227] For three patients, saliva samples were collected 5 days after the nasopharyngeal and oropharyngeal swabs, and for five patients, saliva samples were collected 6 days after the nasopharyngeal and oropharyngeal swabs.

[0228] Seven of the samples tested positive for Covid-19 based on nasopharyngeal and oropharyngeal swabs (RTPCR). However, corresponding saliva samples tested negative using the device of the present invention. Two days after the initial sample collection, these patients were tested again using the collection device of the present invention, with two testing positive and five testing negative.

[0229] No side effects were reported during the study, except for COVID infection among participants.

[0230] sensitivity: RT PCR detection based on the "N" gene: -Nasopharyngeal swab (gold standard): 96% - Dried saliva using the collection device of the present invention: ≥ 95%. RT PCR detection based on "ORF" gene: -Nasopharyngeal swab (gold standard): 90% - Dried saliva using the collection device of the present invention: ≥ 95%.

[0231] Overall, there was 97% consistency in the results observed between dried saliva collected using the collection device of the present invention and nasopharyngeal and oropharyngeal swabs.

[0232] This indicates that saliva sampling using the collection device of the present invention is as reliable / more reliable than nasopharyngeal swabs depending on the assay used for RT PCR detection of COVID-19.

[0233] Specificity: Specificity of tests based on RT PCR detection: -Nasopharyngeal swab (gold standard): 100% -Dried saliva using the collection device of the present invention: 100%

[0234] There was 100% compliance between the swab samples and the HemoDx dried saliva collection device, and 100% compliance with clinical diagnosis - i.e., all healthy controls had negative results using the swab samples and the collection device of the present invention.

[0235] In summary, detection of SARS CoV2 using saliva samples collected using the collection device of the present invention is comparable / superior to the current gold standard of buccal swabs (specificity = 100%, sensitivity = ≥ 95%).

[0236] Example 6 - Extending virus checking As an extension of the assay of Example 3, further studies were designed to assay whether the collection device of the present invention could be used to detect the presence or absence of other respiratory viruses, such as influenza A, influenza B, and respiratory syncytial virus A / B, in dried saliva.

[0237] This is proposed as an equivalence study comparing: a) RT-PCR-based detection of respiratory viruses (other than SARS CoV2) using nasopharyngeal / oropharyngeal swabs (Standard of truth), b) RT PCR-based respiratory virus detection using dried saliva samples collected using the collection device of the present invention.

[0238] At least 30 male or female subjects will have biofluid samples collected on the same day using both nasopharyngeal / oropharyngeal swabs and the collection device of the present invention, where saliva is the biofluid collected.

[0239] These samples are then allowed to dry under ambient conditions for 45 minutes before being transported to the analysis site. In the collection device of the present invention, saliva samples are collected by study participants at home, who then mail the samples to be studied.

[0240] If a subject's nasopharyngeal / oropharyngeal swab tests positive for influenza A, influenza B, or respiratory syncytial virus A / B by RT PCR (at least 10 subjects per condition), the corresponding collection device of the present invention will test for the presence or absence of that virus using RT PCR.

[0241] Without wishing to be bound by theory, it is believed that the collection device of the present invention provides comparable or improved accuracy compared to samples collected by nasopharyngeal / oropharyngeal swabs.

[0242] Example 7 - Testing for Alzheimer's Disease (AD) and Parkinson's Disease (PD) using fecal samples Mutations in two homologous presenilin genes: presenilin 1 (PS1, MIM 104 311) on chromosome 14 and presenilin 2 (PS2, MIM 600 759) on chromosome 1 are the most common and account for more than half of known familial cases of Alzheimer's disease (AD). Mutations in the gene encoding amyloid precursor protein (APP, MIM 104 760) on chromosome 21 are relatively rare. Furthermore, the E4 allele of ApoE is associated with sporadic forms of AD. Testing a subject for the presence of such mutations, along with a positive family history and the early onset (40s and 50s) common to all monogenic forms, should serve as indicators for molecular genetic diagnosis. Preferably, DNA can be isolated from collected biological fluids, such as blood, and tested for the presence of these mutations.

[0243] Parkinson's disease (PD) is inherited in a Mendelian autosomal dominant or autosomal recessive manner in a small number of families. Mutations in the α-synuclein (SNCA) and leucine-rich repeat kinase 2 (LRRK2) genes have been found in late-onset disease, while mutations in parkin (PARK2), ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), PTEN-induced putative kinase 1 (PINK1), and oncogene DJ1 (DJ1) have been found in early-onset disease. Point mutations, duplications, and triplications in the α-synuclein gene, located on chromosome 4, are characteristic of PD and occur in most forms, including rare early-onset familial PD. The gene and gene product have been identified by characterizing monogenic autosomal dominant forms of PD. Some gene products of mutated genes in autosomal dominant forms have been associated with mitochondrial dysfunction, oxidative stress, and mishandling of damaged or abnormal forms of the gene product (e.g., oligomeric α-synuclein). More than 70 mutations in the large parkin gene have been associated with early-onset Parkinson's disease. Mutations in the parkin gene may be responsible for up to 50% of familial cases of autosomal recessive juvenile Parkinson's disease. Another gene, ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), located on chromosome 4, encodes a protein belonging to the deubiquitinase family. The UCH-L1 protein constitutes 1% of brain proteins and its function is estimated to be to recycle ubiquitin by hydrolyzing ubiquitinated peptides. This enzyme plays a role in modifying damaged proteins that would otherwise accumulate to toxic levels within neurons. Two homozygous mutations in the PINK1 gene associated with Parkinson's disease have also been found in Spanish and Italian families. Evidence suggests that the DJ-1 protein is involved in oxidative stress and neurodegeneration. A gradual progression of symptoms with a sustained response to levodopa treatment is a clinical hallmark of DJ-1 Parkinson's disease. It is believed that determining the genetic status of a subject may facilitate understanding of the mechanisms underlying brain neuronal maintenance.

[0244] miRNAs belong to a family of short, single-stranded, 21-22 nucleotide-long non-coding RNAs, constituting approximately 1% of all human genes. They represent the most abundant small RNA class in animals. Altered expression of microRNAs (miRNAs) in many disease states, including neurodegeneration, along with the application of miRNAs to biological fluids in various pathologies, makes miRNAs promising candidates as neurodegenerative disease biomarkers and may lead to the identification of novel therapeutic targets.

[0245] Plasma miRNA biomarkers have been reported to detect MCI. In this study, an initial pool of miRNAs was selected from miRNAs known to be abundant in the brain and neurons. Researchers then identified two sets of biomolecular diagnostic marker pairs: the miR-132 family (miR-128 / miR-491-5p, miR-132 / miR-491-5p, and miR-874 / miR-491-5p) and the miR-134 family (miR-134 / miR-370, miR-323-3p / miR-370, and miR-382 / miR-370) with fairly high sensitivity and specificity (79–100% and 79–95%, respectively). In another longitudinal study, the identified miRNA biomolecular diagnostic marker pairs successfully detected MCI in the majority of patients during the asymptomatic stage, 1–5 years before clinical diagnosis.

[0246] A study using qRT-PCR suggested that the expression levels of miR-1, miR-22-5p, and miR-29 in peripheral blood could distinguish PD patients from healthy subjects, and that miR-16-2-3p, miR-26a-2-3p, and miR30a could distinguish treated from untreated patients. A recent study using next-generation sequencing of whole blood leukocytes found that 16 miRNAs, including miR-16, miR-20a, and miR-320, were significantly altered in PD patients compared with healthy controls.

[0247] Some studies have shown that blood gene expression data can be used to detect Alzheimer's disease and Parkinson's disease early. Transcriptomics typically requires extreme cold-chain logistics (dry ice) to maintain the integrity of mRNA. Advantageously, the present device and method alleviate the need for such cold-chain logistics.

[0248] Gut microbiome characteristics have been associated with certain disorders and diseases, including neurological disorders (Alzheimer's disease and Parkinson's disease). For example, gut microbiota sequencing revealed that the relative abundance of Enterobacteriaceae in the feces of PD patients correlated strongly with the severity of postural instability and gait difficulties compared with controls. Several studies have shown altered correlations between saliva and fecal microbiota, highlighting the potential use of saliva-based screening as an alternative to or in addition to fecal samples in microbiological studies of systemic diseases.

[0249] Example 8 - Testing for Alzheimer's Disease (AD) using peptide biomarkers / analytes of interest A feasibility study was conducted to detect the presence and concentration of peptide fragments Aβ-40 and Aβ-34 (involved in the amyloid cascade) in the saliva of healthy patients. This was carried out to evaluate the feasibility of detecting the presence and concentration of peptide fragments Aβ-40 and Aβ-34 in patients as indicators of Alzheimer's disease.

[0250] Peptide fragments were collected using the method of the third aspect of the invention and stored for subsequent analysis, i.e.: 1. 100 μL of saliva was spotted onto the collection device of the present invention, where the inactivation solution contained 0.45% CHAPS and 0.1% thioflavin. The average diameter of the spot was approximately 20 mm. 2. The saliva was dried at room temperature for approximately 45 minutes and then sent by mail to the laboratory for analysis. 3. Five 6 mm diameter disks of solid matrix containing dried saliva were punched from the collection device and 200 μL of 1×PBS buffer, pH 7.4, containing 0.45% CHAPS and 0.1% thioflavin was added. 4. This was briefly vortexed, microcentrifuged and incubated at room temperature for 5 minutes while shaking at 5500 rpm on a tabletop shaker. 5. The supernatant was then aspirated. 6. Next, add 150 μL of elution buffer to the solid matrix, where the elution buffer contains: Item Concentration 1x PBS pH 7.4 EDTA 0.30% BSA 0.10% Proclin 0.05% Thioflavin S 0.10% Protease inhibitor 2% CHAPS 0.05% 7. This mixture was then incubated for 1 hour at 37°C with shaking at 5500 rpm on a tabletop shaker. 8. The mixture was then briefly vortexed and microcentrifuged, and the supernatant was aspirated to obtain a solution containing the analyte of interest. 9. This was then stored at -20°C until analyzed by immunodetection.

[0251] The concentrations of Aβ-40 and Aβ-34 in saliva samples were measured using SIMOA (SR-X), as shown in Figure 18. As mentioned above, these results were obtained from saliva collected from healthy control subjects. Because the concentrations of Aβ-40 and Aβ-34 in the saliva of Alzheimer's disease patients are higher than those of healthy controls, this is considered sufficient for demonstrating the feasibility of the present invention.

[0252] Surprisingly, these peptide fragments dried onto the collection device of the present invention were detectable for at least 6 weeks after collection when the collection device was stored under ambient conditions.

[0253] Advantageously, the collection devices and methods of the present invention allow for the collection of biological fluids and stabilization of analytes so that both DNA and RNA can be analyzed, and the collection devices and methods are useful for gene-based methods and RNA, e.g., miRNA or mRNA for large miRNAs, to determine genotypes, or for gene expression profiling for disease diagnosis or monitoring, treatment selection, and disease severity assessment. It can be advantageous.

[0254] Furthermore, it will be apparent to those skilled in the art that the collection devices and kits for collecting biological fluids, as described herein, can also be used to collect other types of fluids, such as wastewater or sewage, and can be used to track or detect the presence or absence of bacterial or viral pathogens. For example, because viruses are shed in the feces of infected individuals and can be measured in wastewater, the collection devices can be used for monitoring and early detection of SARS CoV2 in wastewater or sewage, allowing for monitoring the spread of pathogens in communities at a local or population level.

Claims

1. 1. A collection device for collecting and storing an analyte of interest from a biological fluid sample, comprising: the biological fluid sample is suspected of containing the analyte of interest and one or more pathogens; the collection device includes a solid matrix incorporating an inactivation solution adapted to inactivate substantially all of the one or more pathogens present in the biological fluid sample and preserve at least a portion of the analyte of interest in a format that allows for subsequent analysis; the inactivation solution comprises a combination of protein denaturants selected from one or more detergents, one or more chaotropic salts, one or more weak bases, one or more chelating agents, and one or more reducing agents; A collection device wherein, in use, when the biological fluid sample is provided to the solid matrix, substantially all of the one or more pathogens in the biological fluid sample are inactivated and at least a portion of the analytes of interest present in the biological fluid sample are preserved in a format that allows for subsequent analysis.

2. 10. The collection device of claim 1, wherein the inactivation solution is adapted to inactivate substantially all of the one or more pathogens within 45 minutes of the biological fluid sample being provided to the collection device.

3. 3. The collection device of claim 1, wherein the inactivation solution is adapted to inactivate substantially all of the one or more pathogens present in the biological fluid sample such that at least 90%, at least 95%, at least 99%, at least 99.9%, or at least 99.99% of the infectious microorganisms present in the one or more pathogens are inactivated.

4. 4. The collection device of claim 3, wherein the inactivation solution is adapted to inactivate substantially all of the one or more pathogens present in the biological fluid sample such that no infectious microorganisms are detectable.

5. 5. The collection device of claim 1, wherein the inactivation solution is adapted to inactivate substantially all of the one or more pathogens if the one or more pathogens are at a high titer.

6. The collection device of any one of claims 1 to 5, wherein the solid matrix is ​​filter paper.

7. The collection device according to any one of claims 1 to 6, wherein the inactivation solution comprises 12 to 40% by weight of a chaotropic salt.

8. 8. A collection device according to any one of claims 1 to 7, wherein one or more of the chaotropic salts are selected from the group consisting of guanidinium salts, such as guanidinium isothiocyanate, guanidine thiocyanate or guanidine hydrochloride, sodium iodide, sodium perchlorate, sodium thiocyanate and potassium iodide.

9. 9. A collection device according to any one of claims 1 to 8, wherein one or more of the surfactants are selected from the group consisting of ionic surfactants such as sodium dodecyl sulfate, deoxycholic acid, cholic acid and sarkosyl, and non-ionic surfactants such as the Triton family, i.e. octoxynols, e.g. Triton X100 or Triton X-114, Nonidet P-40, Igepal® CA-630, and the Tween family, e.g. Tween-20 or Tween-80.

10. 10. The collection device of any one of claims 1 to 9, wherein one or more of the weak bases is selected from the group consisting of 2-amino 2-hydroxymethyl-propane-1,3-diol, 2-(N-morpholino)ethanesulfonic acid, 3-(N-morpholino)propanesulfonic acid, citrate buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and phosphate buffer.

11. 11. The collection device of any one of claims 1 to 10, wherein one or more of the chelating agents is selected from the group consisting of ethylenediaminetetraacetic acid, ethylene glycol tetraacetic acid, and 8-hydroxyquinoline.

12. 12. The collection device of any one of claims 1 to 11, wherein one or more of the reducing agents is selected from the group consisting of dithiothreitol, dithioerythritol, L-glutathione, 2-carboxyethylphosphine hydrochloride, and 2-mercaptoethanol.

13. 13. The collection device of any one of claims 1 to 12, wherein the deactivation solution comprises guanidinium isothiocyanate, sodium dodecyl sulfate, octoxynol, Tris base, ethylenediaminetetraacetic acid, and dithiothreitol.

14. 14. The collection device of any one of claims 1 to 13, comprising at least 50% by weight cellulose and a deactivating solution comprising 12-40% by weight guanidinium isothiocyanate, 1-3% by weight sodium dodecyl sulfate, 0.5-2% by weight octoxynol, 0.5-1.5% by weight Tris base, 0.1-0.6% by weight ethylenediaminetetraacetic acid, and 0.2-0.7% by weight dithiothreitol.

15. A collection device according to any one of claims 1 to 14, wherein the analyte of interest is a nucleic acid or a protein / peptide.

16. 16. A collection device according to any one of claims 1 to 15, wherein the analyte of interest is a component of at least one of the pathogens, and wherein the at least one of the pathogens is a viral agent selected from coronavirus, influenza virus, norovirus, rabies (lyssavirus), human papillomavirus, Epstein-Barr virus, herpes simplex virus, hepatitis virus, in particular hepatitis C virus, monkeypox virus and HIV.

17. 16. The collection device of any one of claims 1 to 15, wherein the analyte of interest is a component of at least one of the pathogens, and the at least one of the pathogens is a bacterial agent associated with microbiome profiling, bacterial dysbiosis, periodontitis, dental caries, diabetes, obesity, metabolic disorders, cancer, CVD, immune-related systemic diseases.

18. A collection device according to any one of claims 1 to 15, wherein the analyte of interest is a biomarker for a neurodegenerative disease, in particular Alzheimer's disease or Parkinson's disease.

19. 19. The collection device of any one of claims 1-18, wherein the inactivation solution is capable of inactivating substantially all SARS CoV 2 viral particles present in the biological fluid sample such that no viral particles are detectable within 45 minutes of providing the biological fluid sample to the collection device.

20. A collection device according to any preceding claim, wherein the biological fluid sample is a saliva sample.

21. 1. A method for detecting an analyte of interest provided from a dried biological fluid from a collection device, wherein the analyte of interest is a nucleic acid, the collection device comprising a solid matrix pretreated with an inactivation solution adapted to inactivate substantially all of the one or more pathogens present in the biological fluid sample and preserve at least a portion of the analyte of interest in a format that allows for subsequent analysis, the inactivation solution comprising a combination of protein denaturants selected from one or more surfactants, one or more chaotropic salts, one or more weak bases, one or more chelating agents, and one or more reducing agents, the method comprising: a. washing a portion of the solid matrix containing the dried biological fluid with a pre-wash buffer at room temperature to form a pre-dried biological fluid, wherein the pre-wash buffer comprises: i. 60% to 80% ethanol, and ii. An inactivation solution comprising a combination of protein denaturants selected from one or more detergents, one or more chaotropic salts, one or more weak bases, one or more chelating agents, and one or more reducing agents. and b. washing the portion of the solid matrix containing the pre-dried biofluid with a first washing solution, wherein the first washing solution comprises at least 70% ethanol; c. Eluting the analyte of interest from the solid matrix by incubating the solid matrix with RNAase-free water; A method comprising:

22. 22. The method of claim 21, further comprising washing the portion of the solid matrix containing the pre-dried biofluid with a second wash solution at room temperature, wherein the second wash solution comprises at least 95% ethanol.

23. 23. The method of claim 21 or 22, wherein eluting the analyte of interest provides both RNA and DNA from the dried biological fluid.

24. The method of any one of claims 21 to 23, wherein the dried biological fluid is saliva or faeces.

25. 1. A method for detecting an analyte of interest provided from a dried biological fluid from a collection device, wherein the analyte of interest is a protein, polypeptide, oligopeptide, or peptide; the collection device comprises a solid matrix pretreated with an inactivation solution adapted to inactivate substantially all of the one or more pathogens present in a biological fluid sample and preserve at least a portion of the analytes of interest in a format that allows for subsequent analysis, the inactivation solution comprising a combination of protein denaturants selected from one or more detergents, one or more chaotropic salts, one or more weak bases, one or more chelating agents, and one or more reducing agents; The method comprises: - eluting the analyte of interest from the solid matrix by incubating at least a portion of the solid matrix containing the dried biological fluid with an elution buffer and then removing the elution buffer, wherein the elution buffer one or more buffer salts, one or more solubilizing agents, and one or more protease inhibitors A method comprising:

26. the elution buffer one or more buffer salts providing a pH between 6 and 9; - 0.1 to 0.6% of said one or more solubilizers, and - 1 to 3% of said one or more protease inhibitors 26. The method of claim 25, comprising:

27. The elution buffer comprises: one or more chelating agents, - one or more carrier proteins, one or more phosphatase inhibitors, and - one or more biocides 27. The method of claim 25 or 26, comprising one or more of:

28. 28. The method of claim 27, wherein the one or more chelating agents are selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), and 8-hydroxyquinoline (8HQ).

29. 29. The method of claim 27 or 28, wherein the one or more carrier proteins are selected from the group consisting of bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLA).

30. 30. The method of any one of claims 27 to 29, wherein the one or more biocides are selected from the group consisting of ProClin and sodium azide.

31. the elution buffer one or more buffer salts providing a pH of about 7.4; - 0.1 to 0.6% of said one or more solubilizers, -1 to 3% of said one or more protease inhibitors; - 0.3 to 1.5% of said one or more chelating agents, - 0.05 to 2% of said one or more carrier proteins, and - 0.03 to 0.07% of said one or more biocides The method of any one of claims 27 to 30, comprising:

32. the elution buffer - PBS providing a pH of 7.4, - 0.01 to 0.1% of one or more non-denaturing zwitterionic detergents, - 0.1 to 0.5% thioflavin, -1 to 3% of said one or more protease inhibitors; - 0.3 to 1.5% of said one or more chelating agents, - 0.05 to 2% of said one or more carrier proteins, - 0.03 to 0.07% of said one or more biocides The method of any one of claims 27 to 31, comprising:

33. the elution buffer - PBS providing a pH of 7.4, - 0.01 to 0.1% CHAPS, - 0.1 to 0.5% Thioflavin S, - 1-3% protease inhibitors, - 0.3 to 1.5% ethylenediaminetetraacetic acid, - 0.05 to 2% BSA, -0.03 to 0.07% ProClin The method of any one of claims 27 to 32, comprising:

34. 34. The method of any one of claims 27 to 33, wherein the method further comprises washing the solid matrix with a pre-elution buffer prior to eluting the analytes of interest from the solid matrix, the pre-elution buffer comprising one or more buffer salts, one or more solubilizing agents, and one or more protease inhibitors.

35. 1. A method for detecting an analyte of interest provided from a dried biological fluid from a collection device, wherein the analyte of interest is a protein, polypeptide, oligopeptide, or peptide; the collection device includes a solid matrix pretreated with a deactivation solution adapted to preserve at least a portion of the analyte of interest in a form that allows for subsequent analysis; the inactivation solution comprises one or more protease inhibitors and one or more solubilizing agents; The method comprises: - eluting the analyte of interest from the solid matrix by incubating at least a portion of the solid matrix containing the dried biological fluid with an elution buffer and then removing the elution buffer, wherein the elution buffer one or more buffer salts, one or more solubilizing agents, and one or more protease inhibitors A method comprising:

36. A collection device according to any one of claims 1 to 20; a. a pipettor for dropping the biological fluid sample into the collection device; b. a biofluid sample collection cup; c. Bags with biohazard symbols, d. desiccant, and e. A return envelope for storing and transporting the biological fluid sample to the designated laboratory for analysis. and at least one of Kit including:

37. 21. A method of using the collection device of any one of claims 1 to 20 for at least one of microbiome profiling, genotyping purposes, disease diagnosis, treatment selection, and disease severity determination.

38. 36. A method of using the collection device of any one of claims 1 to 20 in a) the method of any one of claims 21 to 24, b) the method of any one of claims 25 to 34, or c) the method of claim 35 for at least one of microbiome profiling, genotyping purposes, disease diagnosis, treatment selection, and disease severity determination.

39. 34. A collection device according to any one of claims 1 to 20 for detecting neurodegenerative diseases, in particular Alzheimer's disease and Parkinson's disease, by a) the method according to any one of claims 21 to 24, b) the method according to any one of claims 25 to 34, or c) the method according to claim 35.

Images