Methods and systems for detecting and identifying a virus using a lateral flow assay

Abstract

There is provided a method of detecting and identifying a virus, the method comprising the steps of: receiving a sample in a vessel; admixing a reagent with the sample in the vessel to provide a mixture; incubating the mixture; and loading the mixture onto a lateral flow assay device for detecting one or more target RNA of the virus; and interpreting one or more signals of the lateral flow assay device, the one or more signals for detecting if the virus is present in the sample and, if the virus is present, for determining if the virus belongs to a first subtype. There is also provided systems and kits for same.

Description

METHODS AND SYSTEMS FOR DETECTING AND IDENTIFYING A VIRUS USING A LATERAL FLOW ASSAYCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to and benefit from United States Patent Application Serial No. 63 / 661,149 filed on June 18, 2024, which is hereby incorporated by reference in its entirety.FIELD OF THE DISCLOSURE

[0002] The present disclosure relates generally to methods, systems, and kits for detecting and identifying a virus using a lateral flow assay, and in particular to detecting and identifying one or more target RNA and therefore, detect and identify viruses.BACKGROUND

[0003] Avian influenza viruses (AIV) continuously change and evolve, resulting in unpredictable outbreaks, seasonal epidemics, and significant repercussions on both human and animal health. AIVs are divided into subtypes based on the identification of two proteins located on the surface of the virus: hemagglutinin (HA) and neuraminidase (NA).

[0004] There are 18 known HA subtypes and 11 known NA subtypes. In birds, 16 HA and 9 NA subtypes have been identified. Two additional AIV subtypes, H17N10 and H18N11, have been identified in bats. These variants can exist in various combinations. Avian influenza A viruses, particularly those that infect birds, have evolved into a unique genetic lineage based on the regions where they were initially discovered. Particularly, avian influenza A viruses that were originally found in birds in Asia can be distinguished genetically from avian influenza A viruses found in birds in North America. While wild birds act as a primary reservoir for AIVs, these viruses can also infect a wide variety of animals, including humans, horses, pigs, and other birds such as poultry.

[0005] The avian influenza A virus can be divided into one of two categories: (1) low pathogenicity for avian influenza (LPAI); and (2) high pathogenicity for avian influenza (HP Al). These two categories are based on a virus’ molecular make-up as well as its capacity to spread disease and cause mortality. All AIV subtype infections in wild birds and poultry typically have low pathogenicity, with no obvious clinical symptoms or minimum morbidity. The majority of A(H5) and A(H7) viruses circulating among birds are LPAI A viruses, while a small number of avian influenza A(H5) and A(H7) viruses are categorized as HP Al A viruses. HPAI A(H5) or A(H7) virus infections in galliform poultry exhibit significant morbidity and mortality rates as well as a high propensity for rapid dissemination.

[0006] Some current technologies for detecting viruses involve the detection of viral proteins and peptides. However, these technologies often require certain amounts of the proteins and peptides to be present in samples to facilitate detection and identification of viruses. Accordingly, these technologies may fail to detect and identify viruses in samples containing lower amounts of the proteins and peptides. Thus, qualifying samples for these technologies often contain a certain number of viral units and / or must undergo a longer incubation time to allow enough proteins and peptides to be produced via translation.

[0007] Other current technologies for detecting viruses involve molecular techniques that use realtime reverse transcriptase polymerase chain reaction (rRT-PCR) assays to determine the virus' genome. RNA-based methods, which only require the viral units to undergo transcription, may allow for earlier and more effective viral detection and identification as compared to peptide or protein-based methods, which require the viral units to undergo translation. However, these rRT-PCR diagnostic procedures often require precise execution to succeed and are thus difficult to carry out in the field. Moreover,rRT-PCR procedures can be time consuming and expensive, which can lead to delays in detecting and identifying viruses.

[0008] It is therefore a desire to provide a novel process and system of detecting infectious agents that is rapid, efficient, and cost-effective.SUMMARY

[0009] The present disclosure relates to methods, systems, and kits for detecting and identifying a virus using a lateral flow assay.

[0010] According to one aspect of this disclosure, there is provided a method of detecting and identifying a virus, the method comprising the steps of: receiving a sample in a vessel; admixing a reagent with the sample in the vessel to provide a mixture; incubating the mixture; and loading the mixture onto a lateral flow assay device for detecting one or more target RNA of the virus; and interpreting one or more signals of the lateral flow assay device, the one or more signals for detecting if the virus is present in the sample and, if the virus is present, for determining if the virus belongs to a first subtype.

[0011] In some embodiments, the step of receiving the sample in the vessel comprises: procuring the sample with a sample collector; and placing the sample collector containing the sample within the vessel.

[0012] In some embodiments, the step of procuring the sample with the sample collector comprises procuring an oral sample with a swabbing tool.

[0013] In some embodiments, the step of admixing the reagent with the sample comprises two or more steps of: lysing cells of the sample with a viral transport medium for releasing RNA from aninterior of the cells of the sample; binding the one or more target RNA with a binding component to form an RNA complex; and adding an enhancement component to the sample for increasing a visibility of the one or more signals of the lateral flow assay device.

[0014] In some embodiments, the reagent comprises the viral transport medium, the binding component, the enhancement component, or any combination thereof. In some embodiments, the binding component comprises a control reagent and an RNA binding reagent.

[0015] In some embodiments, the step of binding the one or more target RNA with the binding component to form the RNA complex comprises binding the one or more target RNA with the RNA binding reagent to form the RNA complex.

[0016] In some embodiments, the RNA binding reagent comprises one or more antisense oligonucleotides comprising a peptide backbone. In some embodiments, the one or more antisense oligonucleotides comprises an antisense oligonucleotide labelled with fluorescein amidite, an antisense oligonucleotide labelled with biotin, the antisense oligonucleotide labelled with digoxigenin, or any combination thereof.

[0017] In some embodiments, the step of binding the one or more target RNA with the binding component to form the RNA complex comprises binding the one or more target RNA with the antisense oligonucleotide labelled with fluorescein amidite, the antisense oligonucleotide labelled with biotin, and / or the antisense oligonucleotide labelled with digoxigenin to form the RNA complex.

[0018] In some embodiments, the control reagent comprises a bacterial protein. In some embodiments, the control reagent comprises streptavidin labelled with gold. In some embodiments, the enhancement component comprises beads. In some embodiments, the enhancement component comprises cysteamine labelled with gold.

[0019] In some embodiments, one or both of the step of admixing a reagent with the sample and the step of incubating the mixture occur at a temperature within a range of about 5°C to about 35°C or about 60°C to about 100°C. In some embodiments, the step of incubating the mixture comprises incubating the mixture at a temperature within a range of about 5°C to about 35°C or about 60°C to about 100°C for an incubation time between about 3 minutes to about 10 minutes. In some embodiments, the step of incubating the mixture comprises incubating the mixture at a temperature within a range of about 5°C to about 35°C or about 60°C to about 100°C for an incubation time of about 5 minutes.

[0020] In some embodiments, the lateral flow assay device comprises one or more control regions and one or more test regions for providing the one or more signals. In some embodiments, the one or more control regions and the one or more test regions comprises antibodies for binding the mixture. In some embodiments, the one or more control regions comprises antibodies for binding the control reagent. In some embodiments, the one or more test regions comprises antibodies for binding the RNA complex.

[0021] In some embodiments, the step of adding the enhancement component to the sample comprises binding the enhancement component with one or both of the RNA complex and the one or more control regions.

[0022] In some embodiments, the lateral flow assay device comprises one or more test strips for providing the one or more signals.

[0023] In some embodiments, the one or more test strips comprises one test strip for providing the one or more signals. In some embodiments, the one or more signals comprises one signal for detecting if the virus is present in the sample and, if the virus is present, for determining if the virus belongs to the first subtype. In some embodiments, the one or more signals comprises: a first signal for detectingif the virus is present in the sample and, if the virus is present, for determining if the virus belongs to the first subtype; and a second signal for determining if the virus belongs to a second subtype. In some embodiments, the one or more signals comprises: a first signal for detecting if the virus is present in the sample; and a second signal for determining if the virus belongs to the first subtype. In some embodiments, the one or more signals further comprises one or more additional signals for determining if the virus belongs to one or more additional subtypes.

[0024] In some embodiments, the one or more test strips comprises a plurality of test strips. In some embodiments, the plurality of test strips comprises: a first test strip for providing a first signal of the one or more signals, the first signal for detecting if the virus is present in the sample; and a second test strip for providing a second signal of the one or more signals, the second signal for determining if the virus belongs to the first subtype. In some embodiments, the plurality of test strips comprises: a first test strip for providing a first signal of the one or more signals, the first signal for detecting if the virus is present in the sample and, if the virus is present, for determining if the virus belongs to the first subtype; and a second test strip for providing a second signal of the one or more signals, the second signal for determining if the virus belongs to a second subtype. In some embodiments, the plurality of lateral flow strips comprises one or more additional lateral flow strips for providing one or more additional signals, the one or more additional signals for determining if the virus belongs to one or more additional subtypes.

[0025] In some embodiments, the step of loading the mixture onto the lateral flow assay device comprises loading a plurality of aliquots of the mixture onto a plurality of lateral flow assay test strips. In some embodiments, the step of loading the mixture onto the lateral flow assay device comprises dispensing one or more aliquots of the mixture onto one or more sample reception openings of the lateral flow assay device.

[0026] In some embodiments, the step of interpreting the one or more signals of the lateral flow assay device comprises: confirming that the lateral flow assay device works properly by identifying whether a control signal of the one or more signals is present; confirming that the virus is present within the sample by identifying whether a viral signal of the one or more signals is present; and determining whether the virus belongs to the first subtype by identifying whether a subtype signal of the one or more signals is present.

[0027] In some embodiments, the one or more signals comprises a viral signal for confirming that the virus is present within the sample, a control signal for confirming that the lateral flow assay device works properly, a subtype signal for determining whether the virus belongs to the first subtype, or any combination thereof. In some embodiments, the one or more control regions provides the control signal. In some embodiments, the one or more test regions provides one or both of the viral signal and the subtype signal.

[0028] In some embodiments, the sample comprises an animal sample. In some embodiments, the sample comprises a livestock sample. In some embodiments, the sample comprises an avian sample. In some embodiments, the sample comprises a poultry sample.

[0029] In some embodiments, the virus comprises an influenza virus. In some embodiments, the virus comprises an avian influenza virus.In some embodiments, the one or more target RNA of the virus is transcribed from a matrix gene, a hemagglutinin gene, a neuraminidase gene, or any combination thereof. In some embodiments, the one or more target RNA of the virus is transcribed from a matrix gene and a hemagglutinin gene. In some embodiments, the one or more target RNA of the virus is transcribed from a matrix gene, an H5 gene, an H7 gene or any combination thereof.

[0030] In some embodiments, the antisense oligonucleotide labelled with fluorescein amidite, the antisense oligonucleotide labelled with biotin, and / or the antisense oligonucleotide labelled with digoxigenin bind specifically to RNA from an avian influenza virus. In some embodiments, the antisense oligonucleotide labelled with fluorescein amidite, the antisense oligonucleotide labelled with biotin, and / or the antisense oligonucleotide labelled with digoxigenin bind specifically to RNA transcribed from a matrix gene, an H5 gene, an H7 gene or any combination thereof.

[0031] In some embodiments, the antisense oligonucleotide comprising a peptide backbone binds specifically to RNA from an avian influenza virus. In some embodiments, the RNA from an avian influenza virus comprises RNA transcribed from a matrix gene, an H5 gene, an H7 gene or any combination thereof.

[0032] According to another aspect of this disclosure, there is a provided a system for performing the methods of the present disclosure.

[0033] According to another aspect of this disclosure, there is provided a system for detecting and identifying a virus, the system comprising: a sample preparation assembly for forming a mixture from a sample and a reagent; and a lateral flow assay device for detecting one or more target RNA of the virus within the mixture, wherein the lateral flow assay device displays one or more signals for determining if the virus is present in the sample and, if present, if the virus belongs to a first subtype.

[0034] In some embodiments, the sample preparation assembly comprises a vessel, the reagent, and a sample collector. In some embodiments, the sample collector comprises a swabbing tool. In some embodiments, the reagent comprises a viral transport medium, a binding component, an enhancement component, or any combination thereof. In some embodiments, the viral transport medium comprises one or more components for lysing cells of the sample for releasing RNA from aninterior of the cells of the sample. In some embodiments, the binding component comprises a control reagent and an RNA binding reagent.

[0035] In some embodiments, the RNA binding reagent comprises one or more antisense oligonucleotides comprising a peptide backbone. In some embodiments, the one or more antisense oligonucleotides comprises an antisense oligonucleotide labelled with fluorescein amidite, an antisense oligonucleotide labelled with biotin, the antisense oligonucleotide labelled with digoxigenin, or any combination thereof.

[0036] In some embodiments, the control reagent comprises a bacterial protein. In some embodiments, the control reagent comprises streptavidin labelled with gold.

[0037] In some embodiments, the enhancement component comprises beads. In some embodiments, the enhancement component comprises cysteamine labelled with gold.

[0038] In some embodiments, whereby in operation, the sample preparation assembly is maintained at a temperature within a range of about 5°C to about 35°C.

[0039] In some embodiments, the lateral flow assay device comprises one or more test strips for displaying the one or more signals for determining if the virus is present in a sample and, if present, if the virus belongs to a first subtype.

[0040] In some embodiments, the one or more signals comprises one signal for detecting if the virus is present in the sample and, if the virus is present, for determining if the virus belongs to the first subtype. In some embodiments, the one or more signals comprises: a first signal for detecting if the virus is present in the sample; and a second signal for determining if the virus belongs to the first subtype. In some embodiments, the one or more signals further comprises one or more additional signals for determining if the virus belongs to one or more additional subtypes.

[0041] In some embodiments, the one or more test strips comprises a plurality of test strips. In some embodiments, the plurality of test strips comprises: a first test strip for providing a first signal of the one or more signals, the first signal for detecting if the virus is present in the sample; and a second test strip for providing a second signal of the one or more signals, the second signal for determining if the virus belongs to the first subtype. In some embodiments, the plurality of test strips comprises: a first test strip for providing a first signal of the one or more signals, the first signal for detecting if the virus is present in the sample and, if the virus is present, for determining if the virus belongs to the first subtype; and a second test strip for providing a second signal of the one or more signals, the second signal for determining if the virus belongs to a second subtype. In some embodiments, the plurality of lateral flow strips comprises one or more additional lateral flow strips for providing one or more additional signals, the one or more additional signals for determining if the virus belongs to one or more additional subtypes.

[0042] In some embodiments, the lateral flow assay device comprises one or more sample reception openings for receiving one or more aliquots of the mixture. In some embodiments, the lateral flow assay device comprises one or more control regions and one or more test regions for providing the one or more signals. In some embodiments, the one or more sample reception openings is fluidly connected to the one or more control regions and the one or more test regions.

[0043] In some embodiments, the one or more test strips comprises the one or more control regions and the one or more test regions.

[0044] In some embodiments, the one or more control regions comprises antibodies for binding the control reagent. In some embodiments, the one or more test regions comprises antibodies for binding an RNA complex comprising the one or more target RNA and the RNA binding reagent.

[0045] In some embodiments, the one or more signals comprises a viral signal for confirming that the virus is present within the sample, a control signal for confirming that the lateral flow assay device works properly, a subtype signal for determining whether the virus belongs to the first subtype, or any combination thereof. In some embodiments, the one or more control regions provides the control signal. In some embodiments, the one or more test regions provides one or both of the viral signal and the subtype signal.

[0046] In some embodiments, the sample comprises an animal sample. In some embodiments, the sample comprises a livestock sample. In some embodiments, the sample comprises an avian sample. In some embodiments, the sample comprises a poultry sample.

[0047] In some embodiments, the virus comprises an influenza virus. In some embodiments, the virus comprises an avian influenza virus.

[0048] In some embodiments, the antisense oligonucleotide comprising a peptide backbone binds specifically to RNA from an avian influenza virus. In some embodiments, the RNA from an avian influenza virus comprises RNA transcribed from a matrix gene, an H5 gene, an H7 gene or any combination thereof.

[0049] In some embodiments, the one or more target RNA of the virus is transcribed from a matrix gene, a hemagglutinin gene, a neuraminidase gene, or any combination thereof. In some embodiments, the one or more target RNA of the virus is transcribed from a matrix gene and a hemagglutinin gene. In some embodiments, the one or more target RNA of the virus is transcribed from one or both of an H5 gene and an H7 gene.

[0050] According to another aspect of this disclosure, there is provided a kit for detecting an infectious agent, the kit comprising: a system of the present disclosure; and instructions for using the system of the present disclosure for detecting the infectious agent.BRIEF DESCRIPTION OF THE DRAWINGS

[0051] For a more complete understanding of the disclosure, reference is made to the following description and accompanying drawings, in which:

[0052] FIG. 1 is a diagrammatic top view of an exemplary viral detection and identification system comprising a lateral flow assay device and a sample preparation assembly, according to some embodiments of this disclosure;

[0053] FIG. 2 is a diagrammatic top view of an exemplary lateral flow assay device of the system shown in FIG. 1, the lateral flow assay device comprising a plurality of test strips;

[0054] FIG. 3 is a diagrammatic perspective view of an exemplary test strip of the system shown in FIG. 1;

[0055] FIG. 4 is a flowchart showing the steps of an exemplary method for detecting and identifying a virus using a lateral flow assay, according to some embodiments of this disclosure; and

[0056] FIG. 5 is a flowchart showing further steps of the exemplary method shown in FIG. 4.DETAILED DESCRIPTION

[0057] The present disclosure relates to methods, systems, and kits for detecting and identifying a virus using a lateral flow assay. The methods, systems, and kits comprise improved designs and features.

[0058] The methods and systems disclosed herein comprise detection of one or more target RNA of a virus to detect if the virus is present in the sample and, if the virus is present, determining if the virus belongs to a subtype. The methods and systems disclosed herein may be configured to identify whether a virus present in a sample belongs to a particular subtype of the virus or one of several particular subtypes of the virus.

[0059] As used herein, the term “subtype” or “viral subtype” is intended to refer to a distinct variation within a species of virus. The distinct variation may be determined or classified based on any number of factors or considerations, including but not limited to, viral species, phenotype, genotype, mode of infection, surface antigens (and reactions from antigens thereto), mode of reproduction, mode of transmission, host reservoir, and the like. Without limitation, “subtype” or “viral subtype” may comprise the meaning of the terms “serotype”, “serovar”, “strain”, “variant”, “variation”, “serogroup”, “serocomplex”, the like, or any combination thereof. In some embodiments, the term “subtype” or “viral subtype” may be used interchangeably with the term “serotype” or “serovar”.

[0060] Without being bound by any particular theory, use of RNA as opposed to peptides or protein as a target viral marker may allow for earlier detection of a virus. Transcription of RNA occurs before translation of peptides and proteins, and thus RNA may be more abundant at earlier points of the viral life cycle relative to the abundance of viral peptides and proteins. Moreover, target viral peptides and proteins for detection and identification are often outside antigens which may not be as numerous or abundant as viral RNA. Furthermore, targeting specific RNA sequences or molecules may facilitate identification of any number of factors or characteristics of a virus beyond its subtype including, but not limited to, pathogenicity, lethality, and transmissibility.

[0061] The methods and systems disclosed herein also facilitate rapid and accurate detection and identification of a virus while being cost-efficient. In some embodiments, a sample may be processedand analyzed within 20 minutes or less. Samples do not require pre-processing as would be the case for other methods and systems, such as those requiring PCR. Furthermore, the methods and systems disclosed herein may be performed or used without any heating or cooling steps. In some embodiments, the methods and systems disclosed herein are configured to be stored and / or to work at room temperature.

[0062] Turning now to FIG. 1, a viral detection system in some embodiments is shown and is generally identified using reference numeral 100, which may be used for processing and analyzing a sample to determine and identify the presence and subtype of viruses in the sample.

[0063] The viral detection system 100 may be used for home-based testing for disease diagnosis and prognosis. However, those skilled in the art will appreciate that the viral detection system 100 may be used in other suitable places such as health centers, clinics, hospitals, and the like.

[0064] As shown in FIG. 1, the viral detection system 100 in these embodiments comprises a sample preparation assembly 110 for processing a sample and a lateral flow assay device 105 (also denoted a “lateral flow device”) for analyzing the sample to determine and identify the presence and subtype of viruses in the sample.

[0065] As used herein, the term “sample preparation assembly” refers to any instruments, devices, compounds, materials, or apparatuses, which are suitable to prepare a sample for detection and identification of a virus. The sample preparation assembly 110 in these embodiments comprises a vessel 115, a reagent 120, and a sample collector 125. The vessel 115 may be any receptacle having a lid or a cap including, for example, an Eppendorf™ tube, a Falcon™ tube, a conical tube, a test tube, or the like. In some embodiments, the vessel 115 comprises a plurality of vessels. Without limitation, this plurality of vessels may facilitate testing of a plurality of samples, identification of multiple viruses,identification of multiple subtypes, or a combination thereof. In some embodiments, the vessel 115 comprises a plurality of vessels for identifying a plurality of subtypes.

[0066] The sample preparation assembly 110 may also comprise a dispenser (not shown) for loading a processed sample (i.e., the mixture) onto the lateral flow device 1 10. The dispenser may be a separate instrument or tool such as, for example, a dropper, a pipette, or the like. Those skilled in the art will appreciate that the vessel 115 may also contain the dispenser as, for example, a dropper attached to or forming the lid, a spigot, a spout, or the like.

[0067] As used herein, the term “sample” refers to a biological material from any organism that may be either infected by or carrying a virus. The sample may, for example, be fecal material or any bodily fluid such as saliva, mucous, sputum, blood, serum, plasma, or urine. In some embodiments, the sample includes saliva or oral samples from poultry. The sample collector 125 may be a needle, a swabbing tool, a plunger, a spatula, or the like.

[0068] The reagent 120 comprises a viral transport medium, a binding component, an enhancement component, or any combination thereof. As used herein, the term “viral transport medium” refers to any solution or liquid that facilitates preservation of viruses by preventing viral degradation. In some embodiments, the viral transport medium comprises one or more components for facilitating lysis of viral cells to allow release of RNA from an interior of the viral cells into the viral transport medium.

[0069] The binding reagent may comprise a control reagent and an RNA binding reagent. As used herein, the term “control reagent” refers to a compound or material that, when provided to the lateral flow device 110, allows for a quality check that the methods and systems disclosed herein are working properly. The control reagent may comprise compounds that bind to one or more components of the lateral flow device 110 to visualize a control signal. Without limitation, the control reagent may be acompound or material that interacts with the lateral flow device 110 to produce fluorescent compounds, luminescent compounds, coloured compounds, reporter enzymes, or any combination thereof. In some embodiments, the control reagent comprises streptavidin-gold.

[0070] As used herein, the term “RNA binding reagent” refers to any compound or material that, when provided to viral RNA, interacts to form an RNA complex. The RNA binding reagent may comprise antisense oligonucleotides configured to interact with certain sequences or molecules of viral RNA. Without being bound by any particular theory, these antisense oligonucleotides may allow for increased selectivity and sensitivity for detecting and identifying viruses, particularly by binding with specific RNA sequences or molecules only present in certain viruses and / or certain viral subtypes.

[0071] Those skilled in the art will appreciate that these antisense oligonucleotides may be configured to bind with RNA from conserved regions of any type or subtype of virus to detect and identify said virus. Without limitation, the targeted virus may include adenoviruses, coronaviruses, noroviruses, influenza viruses, papillomaviruses, enteroviruses, flaviviruses, orthopoxviruses, hepatitis viruses, and the like. A person skilled in the art will also understand that these examples of targeted viruses are associated with particular viral families (e.g., adenovirus with the family Adenoviridae). In some embodiments, the targeted virus may comprise a particular genus or species of these viral families. In some embodiments, the targeted virus comprises a genus or a species of influenza. In some embodiments, the targeted virus comprises avian influenza virus.

[0072] In some embodiments, the RNA binding reagent comprises one or more antisense oligonucleotides configured to bind RNA from an avian influenza virus. In some embodiments, the RNA binding reagent comprises one or more antisense oligonucleotides configured to bind RNA transcribed from a matrix gene and / or a hemagglutinin gene. In some embodiments, the one or moreantisense oligonucleotides are configured to bind RNA transcribed from a matrix gene, an H5 gene, and an H7 gene of an avian influenza virus.

[0073] As used herein, the term “matrix gene” is intended to refer to a gene sequence that is conserved across influenza A virus (as known in the prior art). As used herein, the term “hemagglutinin gene” is intended to refer to a gene sequence that encodes for the viral hemagglutinin surface glycoprotein. As used herein, the term “neuraminidase” gene is intended to refer to a gene sequence that encodes for the viral neuraminidase enzyme. A person skilled in the art will appreciate that there are many mutations and variations of the hemagglutinin and the neuraminidase genes.

[0074] For example, and without limitation, the antisense oligonucleotides binding specifically to RNA transcribed from a matrix gene may have the sequence listings of SEQ ID NOs. 1 to 8 as set out in Table 1 below:Table 1. Exemplary Antisense Oligonucleotide Sequence Listings Targeting Matrix Gene of Influenza A Virus

[0075] In some embodiments, the antisense oligonucleotides binding specifically to RNA transcribed from a matrix gene comprise any two of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8. In some embodiments, the antisense oligonucleotides binding specifically to RNA transcribed from a matrix gene comprise: SEQ ID NO. 1 and SEQ ID NO. 2; SEQ ID NO. 3 and SEQ ID NO. 4; SEQ ID NO. 5 and SEQ ID NO. 6; or SEQ ID NO. 7 and SEQ ID NO. 8.

[0076] For example, and without limitation, the antisense oligonucleotides binding specifically to RNA transcribed from an H5 gene may have the sequence listings of SEQ ID NOs. 9 to 14 as set out in Table 2 below:Table 2. Exemplary Antisense Oligonucleotide Sequence Listings Targeting H5 Gene of Avian Influenza Virus

[0077] In some embodiments, the antisense oligonucleotides binding specifically to RNA transcribed from an H5 gene comprise any two of SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, and SEQ ID NO. 14. In some embodiments, the antisenseoligonucleotides binding specifically to RNA transcribed from an H5 gene comprise: SEQ ID NO. 9 and SEQ ID NO. 10; SEQ ID NO. 11 and SEQ ID NO. 12; or SEQ ID NO. 13 and SEQ ID NO. 14.

[0078] For example, and without limitation, the antisense oligonucleotides binding specifically to RNA transcribed from an H7 gene may have the sequence listings of SEQ ID NOs. 15 to 24 as set out in Table 3 below:Table 3. Exemplary Antisense Oligonucleotide Sequence Listings Targeting H7 Gene of Avian Influenza Virus

[0079] In some embodiments, the antisense oligonucleotides binding specifically to RNA transcribed from an H7 gene comprise any two of SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, and SEQ ID NO. 24. In some embodiments, the antisense oligonucleotides binding specifically to RNA transcribed from an H7 gene comprise: SEQ ID NO. 15 and SEQ ID NO. 16; SEQ ID NO. 17and SEQ ID NO. 18; SEQ ID NO. 19 and SEQ ID NO. 20; SEQ ID NO. 21 and SEQ ID NO. 22; or SEQ ID NO. 23 and SEQ ID NO. 24.

[0080] In some embodiments, the RNA binding reagent binds the one or more target RNA to form an RNA complex. In some embodiments, the RNA complex has a sandwich structure formed by the binding of the target RNA on either side by two antisense oligonucleotides. The RNA binding reagent may have labels to visualize a viral signal and / or a subtype signal when the RNA complex binds to one or more components of the lateral flow device 110. The labels may, for example, include fluorescent compounds, luminescent compounds, coloured compounds, reporter enzymes, or any combination thereof. In some embodiments, the labels comprise biotin, fluorescein amidite (FAM), and dioxigenin (DIG). In addition to binding the one or more components of the lateral flow device 110, the control reagent binds the RNA complex to visualize the one or more signals 145 of the lateral flow device 110, specifically the viral signal and / or the subtype signal.

[0081] In some embodiments, the RNA binding reagent comprises two labels for both binding one or more components of the lateral flow device 110 and for binding the control reagent. The RNA complex may, for example, comprise an antisense oligonucleotide labelled with FAM or DIG bound within the target RNA and an antisense oligonucleotide labelled with biotin, wherein the FAM or DIG binds one or more components of the lateral flow device 110 while the biotin binds the control reagent to provide the one or more signals 145.

[0082] In some embodiments, the one or more antisense oligonucleotides comprises a peptide backbone. Without being bound to any particular theory, a peptide backbone allows for improved stability and increased shelf-life as compared to other types of backbones.

[0083] As used herein, the term “enhancement component” refers to any compound or material that is suitable for augmenting and / or amplifying visualization of the one or more signals 145 providedon the lateral flow device 110. In some embodiments, the enhancement component comprises signal augmentation beads. In some embodiments, the signal augmentation beads comprise cysteamine labelled with gold. Without being bound by any particular theory, the size of the signal augmentation beads may contribute to the visualization of the one or more signals 145. In some embodiments, the signal augmentation beads comprise a size between about 3 nm to about 300 nm, between about 4 nm to about 250 nm, between about 5 nm to about 200 nm, or between about 6 nm to about 150 nm. In some embodiments, the signal augmentation beads comprise a size of about 5 nm, about 10 nm, about 15 nm, about 20 nm, about 40 nm, about 60 nm, about 80 nm, or about 150 nm.

[0084] In some embodiments, the enhancement component binds the control reagent to provide visualization of the one or more signals 145 of the lateral flow device 110. Without being bound by any particular theory, positively charged cysteamine binds to negatively charged streptavidin to provide the augmentation / amplification.

[0085] Once a sample is procured using the sample collector 125, it is provided to the vessel 115 alongside the reagent 120 to incubate and form a mixture. An aliquot or the entirety of the mixture may then be loaded onto the lateral flow device 1 10 for analyzing the sample to detect and identify any virus present in the sample.

[0086] The lateral flow device 110 in these embodiments comprises a housing 130, a sample reception opening 135, and a test strip 140. The housing 130 may be any structure to hold the sample reception opening 135 and the test strip 140. In some embodiments, the housing 130 is a plastic casing. The housing 130 may have any type or number of markers or text to denote placement of the one or more signals 145. In some embodiments, the housing 130 comprises markers and / or text to denote placement of the control signal, the viral signal, and the subtype signal. In some embodiments, the housing comprises quantification markers, which may be used as a standard to which the intensity ofthe one or more signals 145 generated. In some embodiments, the housing 130 comprises one or more QR codes for providing instructions on use, for providing information on interpreting results, for storing information regarding the test strip 140, or the like.

[0087] The sample reception opening 135 is fluidly connected to the test strip 140 wherein a mixture provided to the sample reception opening 135 flows, through capillary action, through the test strip 140. The sample reception opening 135 may comprise the same material or a different material as the test strip 140.

[0088] The test strip 140 in these embodiments comprises one or more reactive regions 150 for providing the one or more signals 145. In some embodiments, the one or more reactive regions 150 comprises a control region and a test region. The control region and the test region may be positioned at any point along the test strip 140. The control region and the test region comprise antibodies for specifically binding to different components of the mixture. In some embodiments, the control region comprises antibodies that specifically bind to the control reagent for providing the control signal. In some embodiments, the control region comprises antibodies that specifically bind to streptavidin. In some embodiments, the test region comprises antibodies that specifically bind to the RNA complex. In some embodiments, the test region comprises antibodies that specifically bind to FAM or DIG.

[0089] In some embodiments, the test strip 140 comprises a plurality of test regions as well as the control region. In some embodiments, the test strip 140 comprises a first test region for providing a viral signal and a second test region for providing a subtype signal. For example, the first test region may comprise antibodies that specifically bind to FAM and the second test region may comprise antibodies that specifically bind to DIG. In some embodiments, the first test region is configured for detecting and identifying the matrix gene and the second test region is configured for detecting the hemagglutinin gene. Without being bound by any particular theory, the first test region for detectingand identifying the matrix gene can minimize the number of false-negative outcomes. In some embodiments, the viral signal comprises a signal for detecting the matrix gene and the subtype signal comprises a signal for detecting the hemagglutinin gene. In some embodiments, the subtype signal comprises a signal for detecting the H5 gene and / or the H7 gene.

[0090] Those skilled in the art will appreciate that various alternative embodiments are readily available. For example, and without limitation, the test strip 140 shown in FIG. 1 has three reactive regions 150 that may be a control region and two test regions. In some embodiments, the two test regions are configured for detecting the matrix gene and the hemagglutinin gene. In some embodiments, the hemagglutinin gene comprises the H5 gene or the H5 gene. However, a test strip 140 may not have a control region for providing a control signal, but rather, may only comprise a test region for providing a viral signal and / or a subtype signal. In some embodiments, a test strip 140 may comprise more than three reactive regions 150 providing for a control region and more than three test regions and thereby, a control signals and more than three viral signals and / or subtype signals. For example, and without limitation, the three test regions may be for providing a viral signal for detecting the matrix gene, a first subtype signal for detecting the H5 gene, and a second subtype signal for detecting the H7 gene.

[0091] Turning now to FIG. 2, another exemplary lateral flow device 110 of the viral detection system 100 is shown. The lateral flow device 110 comprises a housing 205, a plurality of sample reception openings (210, 230), and a plurality of test strips (215, 235). The plurality of test strips (215, 235) each comprise one or more reactive regions (220, 240) which in turn provide for one or more signals (225, 245). In some embodiments, the lateral flow device 110 comprises two test strips (215, 235) for detecting and identifying two different viruses and / or two different viral subtypes. While both test strips (215, 235) may comprise control regions that are substantially the same, the test regionsmay comprise different antibodies for binding different labels contained in two different reagents 120. In this example, the two test strips (215, 235) may require separate sample preparation assemblies 105 that comprise different reagents 120 configured for detecting and identifying different viruses and / or different viral subtypes.

[0092] Those skilled in the art will appreciate that a lateral flow device 110 may comprise more than two test strips (215, 235). In some embodiments, the lateral flow device 110 comprises three test strips, four test strips, five test strips, or six test strips. In some embodiments, the lateral flow device 110 comprises more than six test strips. In some embodiments, the system 100 may be configured for detecting and identifying more than two viruses and / or two viral subtypes.

[0093] Turning now to FIG. 3, an exemplary test strip 300 of the viral detection system 100 is shown. The test strip 300 may be used in the lateral flow devices 110 and may be substantially similar or identical to any one or more of the test strips (140, 215, 235) shown in FIGs. 1 and 2. The test strip 300 may have any length, width, size, shape, or comprise any material suitable for use in the lateral flow devices 110. In some embodiments, the test strip 300 is about 30 mm long, about 40 mm long, about 50 mm long, about 60 mm long, about 70 mm long, about 80 mm long, or about 90 mm long. In some embodiments, the test strip 300 is about 2 mm wide, about 3 mm wide, about 4 mm wide, about 5 mm wide, or about 6 mm wide.

[0094] The test strip 300 may comprise an absorption pad 302, a membrane 304, a conjugation pad 306, a sample pad 308, and a backing pad 310. As used herein, the term “absorption pad” may be used interchangeably with the term “wicking pad”. Each of the absorption pad 302, the membrane 304, the conjugation pad 306, the sample pad 308, and the backing pad 310 may have any length, width, size, shape, material, or physical property suitable for use in the exemplary lateral flow devices and the exemplary viral detection systems of the present disclosure.

[0095] As shown in FIG. 3, the test strip 300 may have the following exemplary and non-limiting dimensions and properties. The absorption pad 302 may have a length of about 18 mm. The conjugation pad 306 may have a length of about 6 mm. The sample pad 308 may have a length of about 17 mm. The backing pad 310 may have a length of about 60 mm. The absorption pad 302, the conjugation pad 306, and the sample pad 308 may comprise the same material or different materials having differences in physical properties including, but not limited to, absorption, thickness, and filtration. For example, and without limitation, the absorption pad 302, the conjugation pad 306, and the sample pad 308 may each comprise paper with different grades of absorption and / or filtration. A person skilled in the art will appreciate that other configurations and dimensions of the absorption pad 302, the conjugation pad 306, the sample pad 308, and the backing pad 310 are possible and contemplated.

[0096] The membrane 304 may have a length of about 24 mm and may be composed of nitrocellulose. The membrane 304 comprises the control region and the test region(s), each of which contain antibodies. As set out above for the test strip 140 shown in FIG. 1, and without limitation, the antibodies in membrane 304 may comprise antibodies for providing the control signal, the viral signal, and the subtype signal by being configured to bind to any one or more of streptavidin, FAM, DIG, and / or the RNA complex (comprising target RNA corresponding to the matrix gene and / or the hemagglutinin gene). The antibodies in membrane 304 for providing the viral signal and / or the subtype signal may further comprise separate antibodies for detection of the RNA complex and capture of the RNA complex. Without being bound by any particular theory, the concentration, abundance, and distribution of the antibodies on the membrane 304 may affect the efficacy of the test strip 300 for detecting and identifying a virus. For example, and without limitation, the membrane 304 may comprise about 1 mg / ml of antibodies in the control region, about 20 pg / ml of detection antibodies in the test region, and about 1 mg / ml of capture antibodies in the control region. A person skilled in theart will appreciate that other concentrations, abundances, and distributions of antibodies on the membrane are possible and contemplated.

[0097] FIGs. 4 and 5 are flowcharts showing the steps of a method 400 for detecting and identifying a virus, according to one embodiment of this disclosure. The method 400 comprises a step of receiving 405 a sample in a vessel, a step of admixing 410 a reagent with the sample in the vessel to provide a mixture, a step of incubating 415 the mixture, a step of loading 420 the mixture onto a lateral flow assay device, and a step of interpreting 425 one or more signals of the lateral flow device to detect and identify the virus.

[0098] Step 405 may comprise a step of procuring 405A the sample with a sample collector and a step of placing 405B the sample collector containing the sample within the vessel. Step 405A may, for example, involve swabbing an organism or a certain body part thereof to receive a sample. Alternatively, step 405A may involve retrieving a deposited biological material from a ground or a surface. In some embodiments, step 405A requires swabbing an organism for a certain amount of time to ensure that a received sample retrieves enough viral units to meet a threshold value for detection and identification. In some embodiments, step 405A comprises taking an oral swab and / or a nasopharyngeal swab.

[0099] Step 410 may comprise a step of lysing 410A cells of the sample with a viral transport medium for releasing RNA from an interior of the cells of the sample, a step of binding 410B the one or more target RNA with a binding component to form an RNA complex, and a step of adding 410C an enhancement component to the sample for increasing a visibility of the one or more signals of the lateral flow assay device. In some embodiments, the reagent comprises the viral transport medium, the binding component, and the enhancement component as one mixture. In some embodiments, the viraltransport medium, the binding component, and the enhancement component are all added separately to the vessel.

[0100] At step 415, incubation of the mixture may occur at about room temperature or at a temperature higher than about room temperature. In some embodiments, incubation of the mixture may occur at a temperature within a range of about 0°C to about 40°C, about 2°C to about 38°C, about 4°C to about 36°C, about 6°C to about 34°C, or about 8°C to about 32°C. In some embodiments, incubation of the mixture occurs at a temperature within a range of about 5 °C to about 35 °C. In some embodiments, incubation of the mixture may occur at a temperature within a range of about 40°C to about 120°C, about 50°C to about 110°C, about 60°C to about 100°C, or about 70°C to about 90°C. In some embodiments, incubation of the mixture may occur at a temperature within about 60°C to about 100°C.

[0101] In some embodiments, step 415 may also occur after one or both of steps 410B and 410C. For example, after step 410B, the admixed sample, viral transport medium, and binding component may be incubated for about 5 minutes, after which, the enhancement component may be added and the mixture incubated for a further about 10 minutes.

[0102] At step 425 (425A, 425C, 425E), the presence of any viral signal, any subtype signal, and any control signal is determined. This can be done by visual inspection by human eyes or by a reader (e.g., any computing device with a camera). In some embodiments, the reader can convey data to a receiving instrument or apparatus (e.g., another computing device) via Wi-Fi™, Bluetooth™, the like, or any combination thereof. If the control signal is present, the lateral flow assay device worked properly (425B). If the viral signal is present, the virus is present in the sample (425D). If the subtype signal is present, the virus is positively identified as that subtype (425E).

[0103] Those skilled in the art will appreciate that such various embodiments and / or features thereof may be customized and / or combined as needed or desired. Moreover, although embodiments have been described above with reference to the accompanying drawings, those of skill in the art will appreciate that variations and modifications may be made without departing from the scope thereof as defined by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method of detecting and identifying a virus, the method comprising the steps of: receiving a sample in a vessel; admixing a reagent with the sample in the vessel to provide a mixture; incubating the mixture; and loading the mixture onto a lateral flow assay device for detecting one or more target RNA of the virus; and interpreting one or more signals of the lateral flow assay device, the one or more signals for detecting if the virus is present in the sample and, if the virus is present, for determining if the virus belongs to a first subtype.

2. The method of claim 1, wherein the step of receiving the sample in the vessel comprises: procuring the sample with a sample collector; and placing the sample collector containing the sample within the vessel.

3. The method of claim 2, wherein the step of procuring the sample with the sample collector comprises procuring an oral sample with a swabbing tool.

4. The method of any one of claims 1 to 3, wherein the step of admixing the reagent with the sample comprises two or more steps of: lysing cells of the sample with a viral transport medium for releasing RNA from an interior of the cells of the sample; binding the one or more target RNA with a binding component to form an RNA complex; andadding an enhancement component to the sample for increasing a visibility of the one or more signals of the lateral flow assay device.

5. The method of claim 4, wherein the binding component comprises a control reagent and an RNA binding reagent.

6. The method of claim 5, wherein the step of binding the one or more target RNA with the binding component to form the RNA complex comprises binding the one or more target RNA with the RNA binding reagent to form the RNA complex.

7. The method of claim 5 or 6, wherein the RNA binding reagent comprises one or more antisense oligonucleotides comprising a peptide backbone.

8. The method of any one of claims 5 to 7, wherein the control reagent comprises streptavidin labelled with gold.

9. The method of any one of claims 4 to 8, wherein the enhancement component comprises cysteamine labelled with gold.

10. The method of any one of claims 1 to 9, wherein one or both of the step of admixing a reagent with the sample and the step of incubating the mixture occur at a temperature within a range of about 5°C to about 35°C or about 60°C to about 100°C.

11. The method of any one of claims 1 to 10, wherein the step of incubating the mixture comprises incubating the mixture at a temperature within a range of about 5°C to about 35°C or 60°C to about 100°C for an incubation time between about 3 minutes to about 10 minutes.

12. The method of any one of claims 1 to 11, wherein the lateral flow assay device comprises one or more control regions and one or more test regions for providing the one or more signals.

13. The method of claim 12, wherein the one or more control regions and the one or more test regions comprises antibodies for binding the mixture.

14. The method of claim 12 or 13 dependent from at least any one of claims 5 to 8, wherein the one or more control regions comprises antibodies for binding the control reagent.

15. The method of any one of claims 12 to 14 dependent at least from any one of claims 5 to 8, wherein the one or more test regions comprises antibodies for binding the RNA complex.

16. The method of any one of claims 12 to 15 dependent from at least any one of claims 5 to 8, wherein the step of adding the enhancement component to the sample comprises binding the enhancement component with one or both of the RNA complex and the one or more control regions.

17. The method of any one of claims 1 to 16, wherein the lateral flow assay device comprises one or more test strips for providing the one or more signals.

18. The method of claim 17, wherein the one or more signals comprises one signal for detecting if the virus is present in the sample and, if the virus is present, for determining if the virus belongs to the first subtype.

19. The method of claim 17, wherein the one or more signals comprises: a first signal for detecting if the virus is present in the sample and, if the virus is present, for determining if the virus belongs to the first subtype; and a second signal for determining if the virus belongs to a second subtype.

20. The method of claim 17, wherein the one or more signals comprises: a first signal for detecting if the virus is present in the sample; and a second signal for determining if the virus belongs to the first subtype.

21. The method of claim 19 or 20, wherein the one or more signals further comprises one or more additional signals for determining if the virus belongs to one or more additional subtypes.

22. The method of any one of claims 1 to 21, wherein the step of loading the mixture onto the lateral flow assay device comprises dispensing one or more aliquots of the mixture onto one or more sample reception openings of the lateral flow assay device.

23. The method of any one of claims 1 to 22, wherein the step of interpreting the one or more signals of the lateral flow assay device comprises: confirming that the lateral flow assay device works properly by identifying whether a control signal of the one or more signals is present;confirming that the virus is present within the sample by identifying whether a viral signal of the one or more signals is present; and determining whether the virus belongs to the first subtype by identifying whether a subtype signal of the one or more signals is present.

24. The method of claim 23 dependent from any one of claims 12 to 16, wherein the one or more control regions provides the control signal.

25. The method of claim 23 or 24 dependent from any one of claims 12 to 16, wherein the one or more test regions provides one or both of the viral signal and the subtype signal.

26. The method of any one of claims 1 to 25, wherein the sample comprises an animal sample.

27. The method of any one of claims 1 to 26, wherein the sample comprises an avian sample.

28. The method of any one of claims 1 to 27, wherein the virus comprises an influenza virus.

29. The method of any one of claims 1 to 28, wherein the virus comprises an avian influenza virus.

30. The method of any one of claims 1 to 43, wherein the one or more target RNA of the virus is transcribed from a matrix gene, a hemagglutinin gene, a neuraminidase gene, or any combination thereof.

31. The method of any one of claims 1 to 30, wherein the one or more target RNA of the virus is transcribed from a matrix gene, an H5 gene, an H7 gene or any combination thereof.

32. The method of claim 7, wherein the antisense oligonucleotide comprising a peptide binds specifically to RNA from an avian influenza virus.

33. The method of claim 32, wherein the RNA from an avian influenza virus comprises RNA transcribed from a matrix gene, an H5 gene, an H7 gene or any combination thereof.

34. A system for performing the method of any one of claims 1 to 33.

35. A system for detecting and identifying a virus, the system comprising: a sample preparation assembly for forming a mixture from a sample and a reagent; and a lateral flow assay device for detecting one or more target RNA of the virus within the mixture, wherein the lateral flow assay device displays one or more signals for determining if the virus is present in the sample and, if present, if the virus belongs to a first subtype.

36. The system of claim 35, wherein the sample comprises an avian sample.

37. The system of claim 35 or 36, wherein the virus comprises an influenza virus.

38. The system of any one of claims 35 to 37, wherein the one or more target RNA of the virus is transcribed from a matrix gene, an H5 gene, an H7 gene, or any combination thereof.

39. A kit for detecting an infectious agent, the kit comprising: the system of any one of claims 34 to 38; and instructions for using the system of any one of claims 34 to 38 for detecting the infectious agent.

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