WO2022031570A1 - Détection rapide non invasive de maladies respiratoires - Google Patents

Détection rapide non invasive de maladies respiratoires Download PDF

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Publication number
WO2022031570A1
WO2022031570A1 PCT/US2021/044119 US2021044119W WO2022031570A1 WO 2022031570 A1 WO2022031570 A1 WO 2022031570A1 US 2021044119 W US2021044119 W US 2021044119W WO 2022031570 A1 WO2022031570 A1 WO 2022031570A1
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WIPO (PCT)
Prior art keywords
collection
collection tube
membrane
mouthpiece
patient
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PCT/US2021/044119
Other languages
English (en)
Inventor
Mithun SINHA
Chandan K. Sen
Ish K. GULATI
Debanjan Mukherjee
Original Assignee
The Trustees Of Indiana University
Research Institute At Nationwide Children's Hospital
The Regents Of The University Of Colorado
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Application filed by The Trustees Of Indiana University, Research Institute At Nationwide Children's Hospital, The Regents Of The University Of Colorado filed Critical The Trustees Of Indiana University
Publication of WO2022031570A1 publication Critical patent/WO2022031570A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/08—Detecting, measuring or recording devices for evaluating the respiratory organs
    • A61B5/097—Devices for facilitating collection of breath or for directing breath into or through measuring devices
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/141—Feedstock

Definitions

  • Active respiratory infections are typically diagnosed directly by swab (i.e. oropharyngeal (OP), nasal mid-turbinate, and anterior nares swabs) or indirectly by blood based assays (i.e. blood based antibody assays). These diagnostic methods may require specific instrumentation and equipment to detect the virus in the collected samples. Such testing is invasive and time consuming to process.
  • swab i.e. oropharyngeal (OP), nasal mid-turbinate, and anterior nares swabs
  • blood based assays i.e. blood based antibody assays
  • Saliva specimen for detection for SARS-COV-2 is non-invasive. However, this specimen type might require additional dilution or pretreatment due to its viscosity. Also, viral RNA might be more difficult to detect in this specimen type, due to high RNase activity present in saliva. The current state-of-the-art, thus, calls for innovative approaches towards non-invasive rapid testing modalities.
  • One aspect of the present disclosure is directed to the collection and detection of virus particles in exhaled air to diagnose respiratory viral infection, and more specifically to non-invasive collection apparatuses and methods of using such devices to collect and detect viral pathogens.
  • a collection apparatus for use in a non-invasive screening of a patient for the presence of respiratory pathogenic viral particles.
  • the collection apparatus is used to collect air-borne and droplet virus particles from the breath of a patient, wherein the collected material is subsequently recovered and analyzed for the presence of viral particles.
  • collection apparatus comprises a collection tube, a mouthpiece assembly, and a collection disk module.
  • the collection tube extends about an axis and defines an inlet and an outlet at the two respective ends of the collection tube so the inlet and an outlet spaced apart axially from one another.
  • the mouthpiece assembly is coupled to the inlet of the collection tube and is configured to be arranged in a mouth of a patient to receive.
  • the mouthpiece assembly in one embodiment further comprises structural elements to prevent air projected into the mouthpiece assembly from exiting back out through the inlet of the collection tube.
  • the mouthpiece further comprises a check valve to prevent air projected into the mouthpiece assembly from exiting back out through the inlet of the collection tube.
  • the collection tube end comprises a collection disk module.
  • the collection disk module comprises a porous filter matrix layer that filters air that is passed through the collection apparatus prior to exiting through the outlet of the collection tube.
  • the porous filter matrix layer is selected to have a pore size sufficiently small to prevent passage of viral particles.
  • the pore size of the matrix is an integer selected from the range of about 20 nm to about 500 nm.
  • this filter layer is a HEPA filter layer.
  • the collection disk module comprises a membrane.
  • the membrane is a nitrocellulose membrane coupled to the collection disk module.
  • the nitrocellulose membrane may be configured to collect and immobilize air-borne virus particles in the respiratory droplets exhaled from the patient through the mouthpiece and into the collection tube.
  • the collection disk module may be removable from the outlet of the collection tube to ease the removal of the membrane/nitrocellulose membrane from the collection apparatus for the subsequent isolation of virus particles immobilized on the membrane.
  • the collection tube may be sealed with an end cap to prevent air leakage through the tube post-collection.
  • the collection tube is sealed with an end cap and the device is cooled to condense the exhaled air trapped in the device that may contain virus particles.
  • the sealing end cap when present, can be removed from the device to assist in the recovery of the collection disk module.
  • exhaled air in such patients will not be volitional and the device is connected to a ventilator device using an adaptor to project air from the patient’s lungs into the collection apparatus of the present disclosure.
  • the interior space defined by the axial extending walls of the collection tube have a conical shape.
  • the conical shape may be configured to concentrate the exhaled air from the patient towards the nitrocellulose membrane on the collection disk module so as to collect a maximum sample of air-borne virus particles on the collection disk module including for example a nitrocellulose membrane.
  • the diameter of the interior space defined by the walls of the collection tube at the inlet is larger (e.g., 5X, 3X, 2X or 1.5X larger) than the diameter of the interior space defined by the walls of the collection tube at the outlet, with the diameter of the interior space defined by the walls of the collection tube decreasing as one moves from the inlet of the collection tube to the outlet of the collection tube.
  • the mouthpiece assembly includes a mouthpiece and a check valve.
  • the mouthpiece may be configured to be arranged in a mouth of a patient.
  • the check valve may be arranged to extend between and interconnect the inlet of the collection tube and the mouthpiece.
  • the check valve may be configured to allow the exhaled air from the patient through the inlet of the collection tube and block backflow through the inlet of the collection tube.
  • the mouthpiece may be removed and disposed after sample collection.
  • the mouthpiece assembly further comprises a cover slit.
  • the cover slit may be located between the check valve and the mouthpiece.
  • the cover slit is configured to receive a non-porous cover strip, wherein insertion of the cover strip into the cover slit of the collection apparatus provides a seal that prevents passage of air through the check valve and into the collection tube.
  • the cover strip prevents exposure of exhaled samples to the outside, after the sample collection is completed, and the mouthpiece is removed.
  • the collection apparatus further includes a hydrophobic coating on the inner surface of the walls defining the interior space of the collection tube.
  • the hydrophobic coating may be arranged on the inner surface of the collection tube to prevent virus-laden respiratory droplets from adhering to the inner surface of the collection tube.
  • a method for screening patients for respiratory viral pathogens comprising the steps of (i) collecting respiratory droplets from a stream of air using any of the collection apparatus disclosed herein and (ii) analyzing the collected respiratory droplets.
  • the stream of air is generated by a patient projecting air from their lungs into any of the collection apparatus of the present disclosure.
  • the method of collection comprises contacting a patient’s mouth with a mouthpiece coupled to a mouthpiece assembly of the collection apparatus and having the patient exhale, cough or otherwise project air from their lungs into the collection device.
  • the step of collection further comprises placing the cover strip into a cover slit formed in the mouthpiece assembly to seal the interior space of the collection tube.
  • the collection step further comprises removing the collection disk module or nitrocellulose membrane from the device after respiratory droplets from a stream of air have been collected onto the collection disk module of the collection apparatus.
  • the mouthpiece is removable from the check valve and the mouthpiece is removed from the collection apparatus after the patient has exhaled into the apparatus, and optionally after the insertion of the cover strip.
  • the step of analyzing the material collected on the collection disk assembly or the membrane of the collection apparatus comprises the steps of releasing the bound or entrapped material by washing or extracting the collection disk assembly or the membrane, optionally by washing the membrane with an alcohol.
  • the material recovered from the collection disk assembly or the membrane is extracted and nucleic acids are recovered and subjected to further analysis including for example performing real-time quantitative PCR using viral specific PCR primers to detect viral RNA located within the respiratory droplets exhaled into the apparatus by a patient.
  • the method comprises the steps of removing material from the collection disk assembly or the membrane using standard washing techniques, subjected the released material to protease digestion (e.g., use of RNAse free Proteinase K) and recovering nucleic acids, optionally RNA, using techniques known to the skilled practitioner including ethanol precipitation and/or commercial affinity columns.
  • the method of analyzing material recovered from a patients respiratory droplets comprises performing a lateral flow immunoassay.
  • the method of performing real-time quantitative PCR comprises: (i) placing a membrane/matrix comprising the collected respiratory droplets in a container, (ii) extracting the material bound to the membrane/matrix and optionally contacting the membrane with ethanol, (iii) centrifuging the container to collect viral RNA, (iv) washing the viral RNA with 70% ethanol, (v) re-suspending the viral RNA in RNAse free liquid, (vi) generating cDNA from the viral RNA using a plurality of different primers, and (vii) amplifying the cDNA with a plurality of different primers and analyzing the results.
  • the method of detecting a virus comprises: (i) releasing material bound to a membrane, wherein the membrane is removed from a used collection apparatus; (ii) contacting the material with a first antibody directed against COVID-19 or respiratory infectious agent wherein the first antibody optionally further comprises a gold nanoparticle, and (iii) visualizing the results.
  • the method further comprises contacting the first antibody bound to the material with a second antibody directed against CO VID- 19 or respiratory infectious agent, wherein the second antibody binds to a different epitope on a CO VID- 19 or respiratory infection causing microorganism, optionally wherein the second antibody is linked to a detectable marker.
  • the method further comprises a third antibody directed to a control ligand.
  • multiple primer combinations will be used against the a single or multiple respiratory illness causing microorganisms. Usage of multiple primer combinations will enable:
  • a method comprising (i) collecting respiratory droplets from an air stream in a collection apparatus onto a collection assembly with a membrane and (ii) analyzing the membrane for viral particles.
  • the collection apparatus may comprise a collection tube, a mouthpiece assembly, and a membrane.
  • the step of analyzing comprises isolating RNA from the membrane and performing quantitative PCR, or isolating Viral particles from the membrane and conducting an immunoassay.
  • Fig. 1 is a diagrammatic view of a collection apparatus adapted for collecting air-borne virus particles in a non-invasive screening of a patient.
  • the collection apparatus (10) comprises a collection tube (12) having an inlet (20) and an outlet (22), a mouthpiece assembly (14) coupled to the inlet of the collection tube to receive exhaled air from a patient, and a collection disk module (16) coupled to the outlet of the collection tube wherein the collection disk module comprises a porous filter matrix layer and a membrane (44), optionally wherein the membrane is a nitrocellulose membrane for collecting and trapping air-borne virus particles exhaled by the patient.
  • Fig. 2 is a diagrammatic view of the collection apparatus of Fig. 1 showing the mouthpiece assembly (14) of the collection tube (12) includes a mouthpiece (34) configured to be arranged in a mouth of the patient and a check valve (36) configured to block backflow through the inlet of the collection tube, and showing the collection apparatus further includes a cover strip (18) that may be inserted into a cover slit (42) between the check valve (36) and the mouthpiece (14).
  • Fig. 3 is a diagrammatic view of the collection apparatus of Fig. 2 showing the mouthpiece (14) is detachable from the check valve (36);
  • Fig. 4 is a diagrammatic view of the collection apparatus of Fig. 3 showing the collection disk module (16) is removable from the outlet (22) of the collection tube.
  • FIG. 5 is an exploded view of one embodiment of a collection apparatus of the present invention. More particularly, Fig. 5 shows a device comprising a collection tube (12), a mouthpiece cover strip (18) configured to fit in a mouthpiece cover slit (42), a mouthpiece (14) in fluid connection with the interior space of collection tube (12) at a proximal end of collection tube (12), optionally with a check valve positioned between said mouthpiece (14) and said collection tube (12), an end cap (50) attached to the distal end of collection tube (12), and a collection disk module (16) configured to insert into the distal end of collection tube (12), proximal to end cap (50) and in fluid connection with the interior space of collection tube (12). Collection disk module (16) further comprising support disk (52), filter layer (54) and membrane (56), optionally wherein filter layer (54) is a HEPA filter and optionally wherein membrane (56) is a nitrocellulose membrane.
  • Collection disk module (16) further comprising support disk (52), filter layer (54) and membrane (56), optionally
  • Figs. 6A-6C are a cross-section views of the collection apparatus of Fig. 5 in various modes of assembly.
  • Fig. 6A shows the device ready for receiving a respiratory sample from a patient, wherein mouthpiece (14) is attached, end cap (50) is not present, and air flows through the device an out the distal end of collection tube (12);
  • Fig. 6B shows the device in containment mode after receiving a respiratory sample from a patient, wherein mouthpiece (14) is removed, end cap (50) is sealed on the distal end of collection tube (12) and mouthpiece cover strip (18) is inserted into cover slit (42).
  • Fig. 7 is a cross-section view of the collection apparatus of Fig. 5 showing the flow path and operating principles for the multifluidic design for viral sample collection from exhaled air.
  • Fig. 8 is a flowchart listing the steps of one embodiment of collecting respiratory droplets from a patient using the collection apparatus of the present disclosure, and analyzing the respiratory droplets trapped by the nitrocellulose membrane to determine the infection status of the patient.
  • Figs. 9A-9D provide illustrations of one embodiment of the collection apparatus.
  • Fig. 9A provides a lateral view of the device without mouthpiece (14);
  • Fig. 9B provides an angled view showing the device wall clarity and flow path;
  • Fig. 9C provides a lateral view showing hand-held form factor;
  • Fig. 9D provides a lateral view and an exploded view of the collection disk module.
  • purified and like terms relate to the isolation of a molecule or compound in a form that is substantially free of contaminants normally associated with the molecule or compound in a native or natural environment. As used herein, the term “purified” does not require absolute purity; rather, it is intended as a relative definition.
  • purified polypeptide is used herein to describe a polypeptide which has been separated from other compounds including, but not limited to nucleic acid molecules, lipids and carbohydrates.
  • isolated requires that the referenced material be removed from its original environment (e.g., the natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide present in a living animal is not isolated, but the same polynucleotide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • treating includes prophylaxis of the specific disorder or condition, or alleviation of the symptoms associated with a specific disorder or condition and/or preventing or eliminating said symptoms.
  • an "effective” amount or a “therapeutically effective amount” of a drug refers to a nontoxic but enough of the drug to provide the desired effect.
  • the amount that is “effective” will vary from subject to subject or even within a subject overtime, depending on the age and general condition of the individual, mode of administration, and the like. Thus, it is not always possible to specify an exact “effective amount.” However, an appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
  • patient without further designation is intended to encompass any warm blooded vertebrate domesticated animal (including for example, but not limited to livestock, horses, cats, dogs and other pets) and humans and includes individuals not under the direct care of a physician.
  • carrier means a compound, composition, substance, or structure that, when in combination with a compound or composition, aids or facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of the compound or composition for its intended use or purpose.
  • a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.
  • inhibitor refers to a decrease in an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.
  • a collection apparatus (10) is adapted for non-invasive screening of a patient to collect respiratory droplets possibly containing air-borne virus particles.
  • the collection apparatus (10) is used for screening for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes the coronavirus disease 2019 (CO VID-19).
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • CO VID-19 coronavirus disease 2019
  • Exemplary embodiments of one collection apparatus (10) is shown in Figs. 1-4.
  • a flowchart describing the steps of one method of collecting respiratory droplets using the collection apparatus (10) and analyzing the respiratory droplets to determine the infection status of the patient is shown in Fig. 8.
  • the collection apparatus (10) includes a collection tube (12), a mouthpiece assembly (14), a collection disk module, and an end-cap (16) as shown in Figs. 1-7.
  • the collection tube (12) extends about an axis (11) and is shaped to define an inlet (20) and an outlet (22) wherein said outlet (22) is spaced apart axially from the inlet (20).
  • the mouthpiece assembly (14) is coupled to the inlet (20) of the collection tube (12) and is configured to be arranged in a mouth of the patient. The patient will exhale air through the mouthpiece wherein said apparatus (10) optionally further includes structures to block backflow of the exhaled air.
  • the collection disk module (16) has a nitrocellulose membrane (44) coupled to the outlet (22) of the collection tube (12). The nitrocellulose membrane (44) is configured to collect and trap air- borne virus particles in the respiratory droplets exhaled from the patient through the mouthpiece assembly (14) and the collection tube (12).
  • Active viral infections are typically diagnosed by swab or blood assays and require a specific instrumentation and equipment to detect the virus in the collected samples. Such testing is invasive and time consuming to process. During a viral pandemic, like that of CO VID- 19, it may be important to test large groups of people quickly and efficiently in order to forecast and control the spread of the viral disease. Patients may also prefer non-invasive testing to invasive testing, and non-invasive testing may also be a preferred avenue for certain populations, such as pediatric populations. However, non-invasive screening methods may be challenging for viruses like COVID-19 that may have low viral loads in asymptomatic patients.
  • the collection apparatus (10) provides a way to screen patients non-invasively and collect maximum respiratory droplets/particles from the patient to later be used in detecting the virus.
  • the collection apparatus (10) controls the path of the exhaled air from the patient to collect a large sample volume with maximum containment.
  • the collection tube (12) has a conical shape as shown in Figs. 1-4. Note that while the embodiments shown in Fig. 1-4 show collection tube (12) as having an overall exterior conical shape, alternative embodiments are envisioned wherein the thickness of the walls of collection tube (12) is increased extending down axis (11) moving from inlet (20) to outlet (22) such that the exterior of collection tube (12) is cylindrical in shape but the walls of collection tube (12) define an interior conical shaped space.
  • the conical shape of the interior space of collection tube (12) is configured to concentrate the exhaled air from the patient towards the nitrocellulose membrane (44) so as to collect maximum respiratory droplets/particles from the patient on the nitrocellulose membrane (44), wherein said droplets/particles may contain viral particles.
  • the inlet (20) of the collection tube (12) has a first cross-sectional area and the outlet (22) of the collection tube (12) has a second cross-sectional area.
  • the second cross-sectional area of the outlet (22) is smaller than the first cross-sectional area of the inlet (20).
  • the conical shape of the collection tube (12) is configured to allow the patient to cough or forcefully exhale into the collection apparatus (10). Coughing or forcefully exhaling into the device may allow a higher viral load because of the forced expiration thereby possible increasing the collection of virus particles.
  • the collection apparatus (10) may allow 3-4 exhaled breathes (one exhaled breath is about 400-500 milliliters in an adult).
  • the collection tube (12) is further shaped to include an outer surface (24) and an inner surface (26) as shown in Figs. 1-4.
  • the outer (24) and inner surfaces (26) extend between the inlet (20) and the outlet (22) of the collection tube (12) over the length of the collection tube (12).
  • the inner surface (26) defines a passageway (30) of the collection tube (12).
  • the collection tube design further enables two phases of sample collection onto the membrane in the collection disk module.
  • the primary or active phase can occur when exhaled air samples are directed towards the membrane, and sample gets collected upon impact with membrane.
  • the secondary or passive phase can occur post-exhalation, when trapped or recirculating air as well as condensates can indirectly impact the membrane and lead to sample collection.
  • the collection apparatus further includes a hydrophobic coating (32) applied to the inner surface (26) of the collection tube (12) as shown in Figs. 1-7.
  • the non-sticking and hydrophobic coating (32) is arranged on the inner surface (26) of the collection tube (12).
  • the hydrophobic coating (32) is configured to prevent virus-laden respiratory droplets from adhering to the inner surface (26) of the collection tube (12).
  • the hydrophobic coating (32) may comprise a coating of non-polar repellant materials in a silicon dioxide base and may include nanoparticles.
  • the coating may include oleophobic components to repel lipids, which make up the outer casing of viral RNA.
  • the mouthpiece assembly (14) includes a mouthpiece (34) and a check valve (36) as shown in Figs. 1-7.
  • the mouthpiece (34) is configured to be arranged in the mouth of the patient to receive exhaled air from the patient there through.
  • the check valve (36) is arranged to extend between and interconnect the inlet (20) of the collection tube (12) and the mouthpiece (34).
  • the check valve (36) is configured to allow the exhaled air from the patient through the inlet (20) of the collection tube (12), but block backflow out of the collection tube (12) back through the mouthpiece (34). Blocking backflow through the inlet (20) of the collection tube (12) helps minimize the exposure of anyone handling the collection apparatus (10) to the respiratory particles from the patient that may have virus particles.
  • the mouthpiece (34) is configured to be detachable from a casing (38) of the check valve (36). In this way, the mouthpiece (34) may be disposed directly after use by the patient.
  • the mouthpiece (34) may be configured to cover the mouth and nose of the patient. In such embodiments, the patient may not be able to blow exhaled air through the mouthpiece (34) into the collection tube (12). Therefore, in some embodiments, the mouthpiece (34) may be one of a ventilator inline circuit, a tracheostomy circuit, an endo tracheal tube, and a CPAP/oxygen mask covering both the mouth and the nose of the patient.
  • the casing (38) of the check valve (36) is shaped to include a cover slit (42) as shown in Figs. 1-4.
  • the cover slit (42) is located between the mouthpiece (34) and the check valve (36).
  • the cover slit (42) is configured to receive a cover strip (18) included in the collection apparatus (10) as shown in Figs. 2-4.
  • the cover strip (18) may be inserted into the cover slit (42) to seal an outer surface (40) of the check valve (36) from anyone handling the collection apparatus (10).
  • the cover (18) may comprises polymer resin material, and may comprise Polymethyl Methacrylate (PMMA) in some embodiments.
  • the cover (18) may be made of material comprising other 3D print resin substrates, such as Acrylobutadiene Styrene (ABS) or Polylactic Acid (PLA).
  • ABS Acrylobutadiene Styrene
  • PLA Polylactic Acid
  • the collection disk module (16) includes the nitrocellulose membrane (44) and a rigid substrate (46) as shown in Figs. 1-4.
  • the nitrocellulose membrane (44) is arranged on one side (48) of the rigid substrate (46) and faces the passageway (30) of the collection tube (12).
  • the rigid substrate (46) provides structure to the nitrocellulose membrane (44) so that the nitrocellulose membrane (44) may be removed from the collection tube (12) and handled without directly contacting the nitrocellulose membrane (44).
  • the patient exhales or coughs through the mouthpiece (34) into the collection tube (12) so that the respiratory air/droplets possibly containing virus particles pass through the collection disk module (16).
  • the virus particles in the respiratory droplets from the patient are immobilized by the nitrocellulose membrane (44).
  • the collection disk module (16) is removable from the outlet (22) of the collection tube (12) as shown in Fig. 4.
  • the removable collection disk module (16) allows the immobilized virus particles on the nitrocellulose membrane (44) to be accessed for analysis and detection.
  • the collection apparatus (10) may be 3D printed.
  • the collection apparatus (10) may be 3D printed as a single component.
  • the components of the collection apparatus (10) may be printed separately and assembled to form the collection apparatus (10).
  • other manufacturing processes may be used.
  • the collection tube (12) may comprise polypropylene polymer resin material and may comprise Polymethyl Methacrylate (PMMA) materials in the illustrative embodiment.
  • material comprising other 3D print resin substrates such as Acrylonitrile Butadiene Styrene (ABS) or Polylactic Acid (PLA).
  • the collection tube (12) may comprise compliant components to mitigate operation scenarios where the pressure differential across the check valve (36) becomes too high for the patient to exhale into the collection tube (12). To minimize the pressure differential, the collection tube (12) may comprise compliant components to reduce the internal air pressure in the passageway (30).
  • the collection apparatus (10) may be configured to be collapsible.
  • the collection apparatus (10) may be folded or collapsed so that the collection apparatus (10) may be stacked for storage before being recycled or discarded.
  • the collection apparatus (10) may further include a cooling element (50) as suggested in Fig. 1.
  • the cooling element (50) may be arranged around the outer surface (24) of the collection tube (12).
  • the cooling element (50) may be configured to cool the passageway (30).
  • the cooling element (50) may be an internal cooling unit in an external casing for storing the collection apparatus (10) prior to extraction of the nitrocellulose membrane (44) for viral detection.
  • a set of collection apparatuses (10) may be contained in the external casing.
  • the cooling element (50) may be configured to cool with conduction or convection cooling methods.
  • a method comprising (i) collecting respiratory droplets from a stream of air in a collection apparatus (10).
  • the stream of air is provided by a subject.
  • the subject is a mammal.
  • the mammal is a human.
  • the human is patient.
  • the patient is suspected of having a viral infection.
  • the viral infection is suspected of being CO VID-19.
  • the patient places their mouth on the mouthpiece (34) of the collection apparatus (10).
  • the method of collection comprises contacting the mouth of the patient with the mouthpiece (34) coupled to the mouthpiece assembly (14) of the collection apparatus (10).
  • the patient may exhale into the mouthpiece (34) of the collection apparatus (10).
  • the patient exhales or coughs into the mouthpiece (34) at least one time.
  • the patient exhales or coughs into the mouthpiece (34) several times, ideally three to four times, to ensure enough respiratory particles are collected on the nitrocellulose membrane (44).
  • the patient exhales into the mouthpiece (34) for between about 1 second and about 10 seconds. In some embodiments, the patient exhales into the mouthpiece (34) for at least about 1 second. In some embodiments, the patient exhales into the mouthpiece (34) for at least about 3 seconds.
  • the step of collection further comprises placing the cover strip (18) into the cover slit (42) formed in the mouthpiece assembly (14). Once the patient has exhaled into the apparatus (10), the cover strip (18) is inserted into the cover slit (42) to seal the outer surface (40) of the check valve (36). Sealing the outer surface (40) of the valve (36) prevents exposing anyone handling the apparatus from contacting any residue left on the apparatus (10).
  • the collection step further comprises removing the mouthpiece (34) from the check valve (36).
  • the mouthpiece (34) may then be removed and disposed of in a hazardous waste container.
  • the collection tube (12) with the collection tube end cap (50) coupled to the outlet (22) may then be transported to the next location for testing in some embodiments.
  • the collection step further comprises removing the collection disk module (16) from the outlet (22) of the collection apparatus (10). In other embodiments, once the sample is collected, the collection disk module (16) may be removed from the outlet (22) of the collection tube (12). The remaining components of the apparatus (10), i.e. the collection tube (12), cover strip (18), and check valve (36) may then be disposed of in a hazardous waste container.
  • the method further comprises analyzing the collected respiratory droplets on the membrane (44). In one embodiment, the step of analyzing comprises performing real-time quantitative PCR on viral RNA located within the respiratory droplets.
  • the general principles and conditions for amplification of nucleic acids using PCR are well known in the art, the details of which are provided in numerous references including United States Patent No. 4,683,195, United States Patent No. 4,683,202 and United States Patent No. 4,965,188, all to Mullis, et al., and all of which are specifically incorporated herein by reference.
  • the method of performing real-time quantitative PCR comprises: (i) placing a membrane comprising the collected respiratory droplets in a recovery container, (ii) contacting the membrane with ethanol, (iii) centrifuging the recovery container to collect viral RNA, (iv) washing the viral RNA with 70% ethanol, (v) re-suspending the viral RNA in RNAse free liquid, (vi) generating cDNA from the viral RNA using a plurality of different primers, and (vii) amplifying the cDNA with a plurality of different primers and (viii) analyzing the results.
  • the method includes using a plurality of different primers each configured to amplify various viral genes of the virus to be detected.
  • Typical PCR methods use a single primer to generate cDNA from the viral RNA and amplify the cDNA.
  • a random hexamer primer is employed for generating cDNA. Random hexamers are combinations of 4 nucleic acids in various combinations to prime the cDNA formation.
  • Thermo Scientific Random Hexamer Primer (catalog # SO 142) could be used per the manufacturer’s instructions.
  • RNA virus particle detection may increase the sensitivity of the real-time quantitative PCR.
  • the primers for amplifying the cDNA are selected from the viral genome. In some embodiments, the primers bind to the minor groove.
  • the recovery container is configured to be centrifuged. In illustrative embodiments, the recovery container is a microcentrifuge tube. In some embodiments, the membrane is a nitrocellulose membrane (44).
  • the amount of ethanol added to the recovery container comprising the removed nitrocellulose membrane is about 500 pL. In some embodiments, the membrane (44) is allowed to stay in contact with the ethanol for about an hour.
  • the viral RNA is allowed to air dry after the step of washing.
  • the RNAse free liquid is RNAse free water.
  • the viral RNA is re-suspended in about 20 pL of RNAse free water.
  • the step of performing rt-qPCR for RNA virus particle detection does not require the phenol based organic isolation process.
  • the primers for generating the cDNA is M-MuLV 1st strand cDNA synthesis kit (Thermo scientific).
  • the method of analyzing the respiratory droplets comprises performing a lateral flow assay.
  • the method of detecting an analyte using a lateral flow assay is well known in the art.
  • United States Patent Serial No. 5,141,850 is specifically incorporated by reference in its entirety.
  • the Universal Lateral Flow Assay system manufactured and sold by Abeam may be used to perform the lateral flow assay.
  • the Universal Lateral Flow assay system (abeam # ab270537) may be employed.
  • two antibodies or aptamers that bind to different epitopes on a COVID-19 viral particle is employed.
  • the antibodies sold by Abeam# ab273835, clone 3C9 & abeam# ab31951, clone 4A6C9 could be employed for capturing and detecting providing a positive result on the test line of the lateral flow assay.
  • LFA Lateral Flow Assay development: In an LFA assay, coated antibody or aptamer is immobilized on the conjugate pad. A primary antibody or aptamer against target antigen (human coronavirus) is immobilized over the test line. A secondary antibody or probe against labeled conjugate antibody/aptamer is immobilized at control zone. Intensity of color at the test line corresponds to the amount of target analyte and is measured with an optical strip reader or visually inspected.
  • target antigen human coronavirus
  • a method of detecting CO VID-19 comprises: (i) extracting material bound to a membrane, wherein the membrane is removed from a used collection apparatus 10; (ii) contacting the material with a first antibody directed against CO VID- 19, wherein the first antibody further comprises a gold nanoparticle, and (iii) visualizing the results.
  • the method further comprises contacting the first antibody bound to the material with a second antibody directed against CO VID- 19, wherein the second antibody binds to a different epitope on a COVID-19 viral particle.
  • the method further comprises a third antibody directed to a control ligand.
  • a method comprising (i) collecting respiratory droplets from an air stream in the collection apparatus 10 and (ii) analyzing the membrane (44) for viral particles.
  • the step of analyzing comprises performing real-time quantitative PCR or lateral flow assay.
  • the primers are selected from the viral genome.
  • Primer Sequence Combinations for Human Coronaviruses SARS-CoV2 and 299E employed include the following:
  • CISCoVlF TAC GGC GCC GAT CTA AAG TC (SEQ ID NO: 1)
  • CISCoVIR CCA TCA GGG CCA CAG AAG TT
  • ClSCoV2F CGT ACG TGG CTT TGG AGA CT
  • ClSCoV2R ACC ATG AGG TGC AGT TCG AG
  • ClSCoV3F TCG AAC TGC ACC TCA TGG TC
  • ClSCoV3R AGA TCG GCG CCG TAA CTA TG
  • ClSCoV4F ATC TGT GTG GCT GTC ACT CG (SEQ ID NO: 7)
  • ClSCoV4R AGG GAC AAG GCT CTC CAT CT (SEQ ID NO: 8)
  • C2SCoVlF TGA GCC AGT GCT CAA AGG AG (SEQ ID NO: 9)
  • C2SCoVlR CGC CAA TAA GCC ATC CG (SEQ ID NO: 10)
  • C2SCoV2F TGG CCA TGG TAC ATT TGG CT (SEQ ID NO: 11)
  • C2SCoV2R GAC TCC TTT GAG CAC TGG CT (SEQ ID NO: 12)
  • C2SCoV3F GAC GGT TCA TCC GGA GTT GT (SEQ ID NO: 13)
  • C2SCoV3R CAG TAC GCA CAC AAT CG(SEQ ID NO: 14)
  • C3SCoVlF ACC CGC AAT CCT GCT AAC AA (SEQ ID NO: 15)
  • C3SCoVlR CAA GCA AAG CAA GA (SEQ ID NO: 16)
  • C3SCoV2F CAA GCC TTA CCG CAG AGA CA (SEQ ID NO: 17)
  • C3SCoV2R TAG CCC ATC TGC CTT GTG TG (SEQ ID NO: 18)
  • C3SCoV3F ATG GGT TGC AAC TGA GGG AG (SEQ ID NO: 19)
  • C3SCoV3R ACG AGA GGC TTG ACT GC (SEQ ID NO: 20)
  • C3SCoV4F CTA CGC AGA AGG GAG CAG AG (SEQ ID NO: 21)
  • C3SCoV4R TCA AGC AAA GCA AG (SEQ ID NO: 22).
  • C1C229E1F CTG CAA CCG TGT GAC ACT TG (SEQ ID NO: 23)
  • C1C229E1R TGA TTG CCA TGC AGA CCC AT (SEQ ID NO: 24)
  • C1C229E2F TGT GCA TGG TGA TGC TTT GC (SEQ ID NO: 25)
  • C1C229E2R CCG GTC CAA TCA CCA ACA GA
  • C1C229E3F AGC AAG CTG GTG CTG GTA TT (SEQ ID NO: 27)
  • C1C229E3R CAT GGC CAG ACG ACA CTC AT (SEQ ID NO: 28)
  • C2C229E1F GCT CGT AGT ACC AG (SEQ ID NO: 29) C2C229E1R: TGG CCT TGA CCT TCA TC (SEQ ID NO: 30) C2C229E2F: TGG GTA TTG GCG GTC CTA GA (SEQ ID NO: 31) C2C229E2R: TGA GCA GTT TCA GGG TCG TC (SEQ ID NO: 32) C2C229E3F: TCC AAC ACT ACG CAG GA CAC (SEQ ID NO: 33) C2C229E3R: CCG GTC ACA TGT ACA GCC AT (SEQ ID NO: 34) wherein F stands for Forward primer and R stands for reverse.
  • the lateral flow assay employs antibodies directed against COVID-19.
  • a first antibody is a capture antibody directed to COVID-19 and further comprises a gold nanoparticle and a second antibody is a detection antibody and directed a different epitope of COVID-19.
  • the capture antibody is conjugated to Ulfa-Tag, and the detection antibody is conjugated to 42nm GOLD.
  • the antibodies are abeam# ab273835, clone 3C9 & abeam# ab31951, clone 4A6C9.
  • an apparatus adapted for non-invasive collection of respiratory droplets contained in exhaled air from a patient comprising a collection tube that extends about an axis, the collection tube shaped to include an inlet and an outlet, wherein the outlet is spaced apart axially from the inlet, a mouthpiece assembly coupled to the inlet of the collection tube and configured to be arranged in the mouth of a patient to receive exhaled air from the patient there through, and block backflow through the inlet of the collection tube, and a collection disk module comprising a nitrocellulose membrane coupled to the outlet of the collection tube, and configured to collect respiratory droplets contained in exhaled air from the patient through the mouthpiece and the collection tube and immobilize any air-borne virus particles present in said respiratory droplets onto said nitrocellulose membrane, wherein the walls of said collection tube define an interior conical shape configured to concentrate the exhaled air from the patient towards the nitrocellulose membrane so as to collect a maximum sample of air-borne virus particles on the nitrocellulose membrane
  • the apparatus of embodiment 1 wherein said mouthpiece assembly comprises a mouthpiece configured to be arranged in a mouth of a patient and a check valve arranged to extend between, and interconnect the inlet of the collection tube and the mouthpiece, wherein said check valve is configured to allow the exhaled air from the patient through the inlet of the collection tube and block backflow through the inlet of the collection tube.
  • the apparatus of embodiment 1 or 2 wherein the mouthpiece assembly further comprises a cover slit located between the check valve and the mouthpiece, said cover slit configured to receive a cover strip included in the collection apparatus to seal an outer surface of the check valve.
  • the apparatus of any one of embodiments 1-3 is provided, wherein the collection disk module is configured to be removable from the outlet of the collection tube.
  • the apparatus of any one of embodiments 1-4 is provided, wherein collection apparatus is 3D printed.
  • the apparatus of any one of embodiments 1-5 wherein the apparatus further comprises a hydrophobic coating applied on an inner surface of the collection tube to prevent virus-laden respiratory droplets from adhering to the inner surface of the collection tube.
  • the apparatus of any one of embodiments 1-6 wherein the collection apparatus consists of disposable materials.
  • the apparatus of any one of embodiments 1-7 is provided, wherein the collection disk module is configured to include a HEPA filter layer to filter air coming out of the collection tube.
  • the apparatus of any one of embodiments 1-7 is provided, wherein the collection tube comprises an end cap that can be used to seal the container at the collection end, and prevent leakage of sample.
  • a method of collecting respiratory droplets from a patient comprises a) providing a collection apparatus in accordance with any one of embodiments 1-7; b) contacting the mouth of the patient with the mouthpiece of said collection apparatus and having patient project air form their lungs into the mouthpiece assembly; and c) removing said membrane from the collection apparatus after step b) is completed.
  • a method of embodiment 8 is provided, wherein the collection step further comprises placing a cover strip into a cover slit formed in the mouthpiece assembly after step b) but prior to step c).
  • a method of any one of embodiments 8-9 is provided, wherein the mouthpiece is removed from the collection tube to assist removal of the membrane.
  • a method of any one of embodiments 8-10 is provided, further comprising the step of screening for virus particles in the respiratory droplets collected on the membrane.
  • a method of any one of embodiments 8-12 wherein material bound to the membrane is extracted by contacting the membrane with ethanol; and collecting the extracted material by centrifuging the recovery container, optionally wherein the isolated RNA is washed with 70% ethanol and re-suspend in RNAse free liquid.
  • a method of embodiments 14 wherein the detection step further comprises contacting the first antibody bound to the material with a second antibody directed against CO VID- 19, wherein the second antibody binds to a different epitope on a CO VID-19 viral particle.
  • a method of any one of embodiments 14 and 15 is provided wherein the second antibody is labeled.
  • a method of collecting respiratory droplets from an air stream in a collection apparatus comprising a collection tube shaped to define an inlet and an outlet spaced apart from the inlet, a mouthpiece assembly coupled to the inlet of the collection tube, and a membrane coupled to the outlet of the collection tube configured to collect and immobilize air-borne virus particles, and analyzing the membrane for viral particles by performing one of real-time quantitative PCR and lateral flow assay.
  • a method of embodiments 17 comprises (i) placing the membrane comprising the collected respiratory droplets in a recovery container, (ii) contacting the membrane with ethanol, (iii) centrifuging the recovery container to collect viral RNA, (iv) washing the viral RNA with 70% ethanol, (v) re-suspending the viral RNA in RNAse free liquid, (vi) generating cDNA from the viral RNA using a plurality of different primers, and (vii) amplifying the cDNA with the plurality of different primers and analyzing the results.
  • the analyzing step comprises (i) extracting material bound to the membrane, (ii) contacting the material with a first antibody directed against
  • the analyzing step further comprises contacting the first antibody bound to the material with a second antibody directed against CO VID- 19, wherein the second antibody binds to a different epitope on a COVID-19 viral particle.
  • coated antibody or aptamer is immobilized on the conjugate pad.
  • a primary antibody or aptamer against target antigen human coronavirus
  • a secondary antibody or probe against labeled conjugate antibody/aptamer is immobilized at control zone. Intensity of color at the test line corresponds to the amount of target analyte and is measured with an optical strip reader or visually inspected.
  • the advantage of this kit is its adaptability to any pair of capture and detection antibodies, which permits detection of any type of antigen.
  • the capture antibody is conjugated to Ulfa-Tag
  • the detection antibody is conjugated to 40nm GOLD, both of which require only 30 seconds to set up ensuring its PoC application.
  • Subjects can exhale into the device (10) through a disposable mouthpiece (34). Both normal exhalation and forceful exhalation may be possible to process.
  • Exhaled air can pass through the mouthpiece (34) into a one- way valve (36) enclosed within an outer casing (38) that is contiguous with the collection vial body (12).
  • a small slot (42) may be placed between the mouthpiece (34) and the casing (38), where the mouthpiece (34) detaches and a small cover (18) fits and seals the outer surface (40) of the valve (36) to minimize exposure amongst health care workers handling the collection vial (12).
  • Valve (36) can be designed to sustain one-way trans-valvular opening pressure into the collection tube (12) for multiple exhalations to allow for sufficient sample collection.
  • Inner wall (26) of the collection tube (12) can be coated with a repellant or hydrophobic coating (32) to prevent virus particles from adhering to the wall (26), increasing their suspension time in the collection tube (12).
  • Sample collection onto membrane of collection disk module can occur in two modes. The primary or active mode of collection can occur when exhaled air samples into the collection tube directly impacts membrane and leads to sample collection. The secondary or passive mode of collection can occur when post-exhalation circulating trapped air sample, and any condensate, indirectly collects on the membrane.
  • EXAMPLE 3 Real-time Quantitative PCR
  • human coronavirus 229E can be blown through a collection apparatus (10), viral particles bound to nitrocellulose membrane (44) may be reverse transcribed.
  • Viral particles can be detected through quantitative real time PCR assay. To validate the ability to detect coronavirus from nitrocellulose membrane (44) on the collection apparatus (10), different titers of virion particles are blown into the collection apparatus (10). Sensitivity determination: To increase the sensitivity of the reaction multiple primers can be used during the real-time PCR. In order to avoid false negatives, TaqmanTM based detection technique was used containing a minor groove binding (MGB) primer. The sequence for forward and reverse primer combinations for SARS-CoV2 are provided as SEQ ID NOs 1-34.
  • the virus embedded nitrocellulose membrane (44) is placed in a microcentrifuge tube. About 500 pL of ethanol was added to the tube, allowed to stay for an hour and centrifuged at 10,000Xg. Ethanol can dissolve the nitrocellulose membrane (44) and precipitate the viral RNA. The viral RNA is washed in 70% Ethanol and air-dried. The resultant RNA can be suspended in 20 pL of RNase free water.
  • the cDNA was synthesized using M-MuLV first strand cDNA synthesis kit (Thermo scientific). The resulting cDNA could be stored in -20 °C or applied to virus PCR detection directly. Specific human coronavirus (229E) primers were used for PCR amplification to detect viruses.
  • Primer Sequence Combinations for Human Coronaviruses SARS-CoV2 and 299E employed include the following: Combination 1 Pairs 3,14,16 and 18
  • CISCoVlF TAC GGC GCC GAT CTA AAG TC (SEQ ID NO: 1)
  • CISCoVIR CCA TCA GGG CCA CAG AAG TT
  • ClSCoV2F CGT ACG TGG CTT TGG AGA CT
  • ClSCoV2R ACC ATG AGG TGC AGT TCG AG
  • ClSCoV3F TCG AAC TGC ACC TCA TGG TC
  • ClSCoV3R AGA TCG GCG CCG TAA CTA TG
  • ClSCoV4F ATC TGT GTG GCT GTC ACT CG (SEQ ID NO: 7)
  • ClSCoV4R AGG GAC AAG GCT CTC CAT CT (SEQ ID NO: 8) Combination 2
  • C2SCoVlF TGA GCC AGT GCT CAA AGG AG (SEQ ID NO: 9)
  • C2SCoVlR CGC CAA TAA GCC ATC CG (SEQ ID NO: 10)
  • C2SCoV2F TGG CCA TGG TAC ATT TGG CT (SEQ ID NO: 11)
  • C2SCoV2R GAC TCC TTT GAG CAC TGG CT (SEQ ID NO: 12)
  • C2SCoV3F GAC GGT TCA TCC GGA GTT GT (SEQ ID NO: 13)
  • C2SCoV3R CAG TAC GCA CAC AAT CG (SEQ ID NO: 14)
  • C3SCoVlF ACC CGC AAT CCT GCT AAC AA (SEQ ID NO: 15)
  • C3SCoVlR CAA GCA AAG CAA GA (SEQ ID NO: 16)
  • C3SCoV2F CAA GCC TTA CCG CAG AGA CA (SEQ ID NO: 17)
  • C3SCoV2R TAG CCC ATC TGC CTT GTG TG (SEQ ID NO: 18)
  • C3SCoV3F ATG GGT TGC AAC TGA GGG AG (SEQ ID NO: 19)
  • C3SCoV3R ACG AGA GGC TTG ACT GC (SEQ ID NO: 20)
  • C3SCoV4F CTA CGC AGA AGG GAG CAG AG (SEQ ID NO: 21)
  • C3SCoV4R TCA AGC AAA GCA AG (SEQ ID NO: 22)
  • C1C229E1F CTG CAA CCG TGT GAC ACT TG (SEQ ID NO: 23)
  • C1C229E1R TGA TTG CCA TGC AGA CCC AT (SEQ ID NO: 24)
  • C1C229E2F TGT GCA TGG TGA TGC TTT GC (SEQ ID NO: 25)
  • C1C229E2R CCG GTC CAA TCA CCA ACA GA
  • C1C229E3F AGC AAG CTG GTG CTG GTA TT (SEQ ID NO: 27)
  • C1C229E3R CAT GGC CAG ACG ACA CTC AT (SEQ ID NO: 28)
  • C2C229E1F GCT CGT AGT ACC AG (SEQ ID NO: 29)
  • C2C229E1R TGG CCT TGA CCT TCA TC
  • C2C229E2F TGG GTA TTG GCG GTC CTA GA
  • C2C229E2R TGA GCA GTT TCA GGG TCG TC
  • C2C229E3F TCC AAC ACT ACG CAG GA CAC (SEQ ID NO: 33)
  • C2C229E3R CCG GTC ACA TGT ACA GCC AT (SEQ ID NO: 34)
  • Lateral flow tests are widely used in human health for point of care testing. They may be performed by a healthcare professional or by the patient in a range of settings including the laboratory, clinic or home. They are widely used to control a variety of biomarkers of infectious diseases, metabolic, and functional disorders rapidly and easily.
  • the first immune-chromatographic tests were introduced in the 1980s for pregnancy self-testing. Immuno-chromatography is a key component of the modern methods used to control psychoactive compound oncomarkers, acute infarction markers, allergens, and microorganisms that cause various diseases, as well as in zero-diagnostics to detect antibodies of pathogens in the blood. Results of lateral flow assays can be registered not only by special detectors but also by mobile devices and office equipment.
  • Nitrocellulose membrane bound coronavirus/microorganisms can be detected by lateral flow assay (LFA).
  • LFA lateral flow assay
  • Universal Lateral Flow assay system (abeam # ab270537) along with monoclonal antibody pairs detecting separate epitopes of human coronavirus were used (abeam# ab273835, clone 3C9 & abeam# ab31951, clone 4A6C9).
  • the kit is adaptable to any pair of capture and detection antibodies, which permits detection of multiple types of antigen.
  • the capture antibody is conjugated to Ulfa-Tag
  • the detection antibody is conjugated to 42nm GOLD, both of which require only about 34 seconds to set up ensuring its Point of Care (PoC) application.
  • PoC Point of Care
  • Validation of the assay Positive control; the assay is validated using human coronavirus 229E amplified on human lung fibroblast cells MRC9 (ATCC CCL-212). For negative control, Parker rat coronavirus isolated from rat epithelial cells is used. Sensitivity determination: Post validation of the assay, sensitivity of the assay is determined. For this, virion particles (range 104- 109 virus particles) can be spotted on the membrane (44) and the detection limit can be tested. Next human coronavirus particles from the nitrocellulose membrane (44) were tested.

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Abstract

Un appareil de collecte est conçu pour être utilisé pour collecter des gouttelettes respiratoires à partir d'un flux d'air exhalé par un patient dans un processus de criblage non invasif pour détecter des micro-organismes pathogènes présents chez des patients.
PCT/US2021/044119 2020-08-04 2021-08-02 Détection rapide non invasive de maladies respiratoires WO2022031570A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023205584A1 (fr) * 2022-04-18 2023-10-26 Infinity Biologix Llc Systèmes de collecte de particules virales et leurs utilisations
WO2024082003A1 (fr) * 2022-10-17 2024-04-25 Griffith University Acides nucléiques antiviraux et compositions

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030106555A1 (en) * 2000-02-24 2003-06-12 Euan Tovey Nasal filter and sampler
US20070173731A1 (en) * 2006-01-26 2007-07-26 Neil R. Euliano Breath and Breath Condensate Analysis System and Associated Methods
US20090275015A1 (en) * 2008-04-30 2009-11-05 Aleta Behrman Bonner Non-invasive respiratory rapid diagnosis method
US20140261430A1 (en) * 2013-03-15 2014-09-18 Lucy Carol Davis Facial Mask Apparatus and Method of Making
WO2019178247A1 (fr) * 2018-03-15 2019-09-19 Biolum Sciences Llc Dispositifs de capteur et systèmes de surveillance de marqueurs dans l'haleine

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030106555A1 (en) * 2000-02-24 2003-06-12 Euan Tovey Nasal filter and sampler
US20070173731A1 (en) * 2006-01-26 2007-07-26 Neil R. Euliano Breath and Breath Condensate Analysis System and Associated Methods
US20090275015A1 (en) * 2008-04-30 2009-11-05 Aleta Behrman Bonner Non-invasive respiratory rapid diagnosis method
US20140261430A1 (en) * 2013-03-15 2014-09-18 Lucy Carol Davis Facial Mask Apparatus and Method of Making
WO2019178247A1 (fr) * 2018-03-15 2019-09-19 Biolum Sciences Llc Dispositifs de capteur et systèmes de surveillance de marqueurs dans l'haleine

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023205584A1 (fr) * 2022-04-18 2023-10-26 Infinity Biologix Llc Systèmes de collecte de particules virales et leurs utilisations
WO2024082003A1 (fr) * 2022-10-17 2024-04-25 Griffith University Acides nucléiques antiviraux et compositions

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