70817-02 ASSAYS, KITS AND METHODS FOR DETECTION OF INFLUENZA PRIORITY [0001] This application is related to and claims the priority benefit of U.S. Provisional Patent Application No. 63/676,999 filed July 30, 2024. The contents of the aforementioned application is hereby incorporated by reference in its entirety into this disclosure. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH [0002] This invention was made with government support under: (1) Grant No. UL1TR002529 awarded by the National Institutes of Health, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award, and (2) Grant No. FF-NIA20-0000000087 awarded by the Foundation for Food and Agriculture Research. The United States Government has certain rights in the invention. SEQUENCE LISTINGS [0003] The sequences herein are also provided in computer readable form in a Sequence Listing XML file (file entitled “70817-02SequenceListing_F30JUL2025”; file size = 88,010 bytes; date created July 30, 2025) filed herewith and herein incorporated by reference. The information recorded in computer readable form is identical to the written Sequence Listing provided herein, pursuant to 37 C.F.R. § 1.821(f). BACKGROUND [0004] Outbreaks of highly pathogenic avian influenza (HPAI) have resulted in substantial economic losses to the global poultry industry and continue to pose a serious and growing threat to public health. Traditionally considered an avian disease, HPAI has increasingly demonstrated expanded host tropism, with documented infections in seals, polar bears, goats, dairy cattle, and humans. As of Fall 2023, HPAI viruses have been reported on every continent except Australia, highlighting the virus’s unprecedented geographic and host range expansion. Badara, Wildlife under threat as avian influenza reaches Antarctica, WOAH – World Organisation for Animal Health (2024). [0005] The socioeconomic impact of recent outbreaks is profound. For example, an H5N1 HPAI outbreak in Indiana in 2023 caused over $100 million in poultry industry losses and triggered broad international trade restrictions. USDA Animal and Plant Health Inspection Service, USDA confirms highly pathogenic Avian influenza in a commercial turkey flock in Dubois County, Indiana, USDA APHIS (2022). Similarly, the 2022 U.S. outbreak led to the culling of
70817-02 approximately 40 million birds, with total economic costs estimated between $2.5 to $3 billion USD. Farahat et al., The resurgence of Avian influenza and human infection: A brief outlook, New Microbes New Infections 53: 101122 (2023). On a global scale, HPAI outbreaks affected 67 countries in 2022 alone, leading to the loss of more than 131 million domestic poultry. Of particular concern is the first documented multi-state outbreak of H5N1 in dairy cattle in the United States, beginning in March 2024, underscoring the potential for cross-species transmission and agricultural disruption beyond avian species. USDA Animal and Plant Health Inspection Service, Highly pathogenic Avian influenza (HPAI) detections in livestock, USDA APHIS (2024); American Veterinary Medical Association, Avian influenza virus type A (H5N1) in U.S. dairy cattle, AVMA (2024); US Centers for Disease Control and Prevention, Current H5N1 bird flu situation in diary cows, USCDC (2024). These developments underscore the urgent need for robust and scalable diagnostic tools to detect avian influenza viruses (AIVs), particularly the H5 subtype, in both avian and non-avian hosts. Peiris et al., Avian influenza virus (H5N1): a threat to human health, Clinical Microbiology Reviews 20: 243-267 (2007); Kotey et al., Phylogenetic and genetic characterization of influenza A H9N2 viruses isolated from backyard poultry in selected farms in Ghana, Veterinary Medicine & Science 8: 1570-1577 (2022). [0006] Influenza viruses are members of the Orthomyxoviridae family and are classified into four types – A, B, C, and D – based on their nucleoprotein and matrix protein antigens. Influenza A viruses, the primary agents of avian influenza, are further subtyped by their hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins, of which 18 HA and 11 NA subtypes have been identified to date. Notably, 16 HA and 9 NA subtypes have been found in avian reservoirs, establishing birds as the primary source of influenza A virus genetic diversity. Alexander, A review of avian influenza in different bird species, Veterinary Microbiology 74: 3-13 (2000); US Centers for Disease Control, Influenza type A viruses, USCDC (2024); Fan et al., Structures of influenza A virus RNA polymerase offer insight into viral genome replication, Nature 573: 287- 290 (2019). [0007] Avian influenza viruses (AIVs) are broadly classified as low pathogenic (LPAIV) or highly pathogenic (HPAIV) based on their virulence in chickens. Kiss et al., Molecular characterization of highly pathogenic H5N1 avian influenza viruses isolated in Sweden in 2006, Virology J 5: 113 (2005); Capua et al., Monitoring for highly pathogenic avian influenza in wild birds in Italy, Veterinary Record 147: 640 (2000); Capua et al., Mortality of Muscovy ducks (Cairina moschata) and domestic geese (Anser anser var. domestica) associated with natural infection with a highly pathogenic avian influenza virus of H7N1 subtype, Avian pathology: J W.V.P.A 30: 179-183 (2001). H5 and H7 subtypes are of particular concern, as these can undergo mutation and recombination events that convert LPAIV strains into HPAIV strains, often resulting
70817-02 in severe outbreaks with high mortality rates in poultry, especially in galliform species (e.g., chickens and turkeys). Alexander & Brown, History of highly pathogenic avian influenza, Revue scientifique et technique (International Office of Epizootics) 28: 19-38 (2009). [0008] Traditional diagnostic methods for AIV detection, including reverse transcription polymerase chain reaction (RT-PCR) and real-time quantitative RT-PCR (RT-qPCR), are highly sensitive and specific, but they require expensive instrumentation, skilled personnel, and centralized laboratory facilities, making them impractical for large-scale surveillance or field- based diagnostics, particularly in resource-limited or remote settings. Indeed, conventional diagnostics cannot be performed in the field and, thus, require additional time and money to ship the samples to laboratories for processing. As such, laboratory-based tests require long processing times, especially when a large number of samples are submitted for testing. This testing delay, or bottleneck between testing and diagnosis, can result in long wait times for poultry production facility operators or veterinarians and loss of valuable livestock. [0009] To address these limitations, loop-mediated isothermal amplification (LAMP) has emerged as a potentially promising alternative. LAMP is a rapid, highly sensitive, and cost- effective nucleic acid amplification method that operates at a constant temperature, eliminating the need for thermocyclers. LAMP assays are typically resistant to inhibitors, and results can be obtained using simple equipment, such as a heat block or water bath, making the technology well- suited for point-of-care and field deployment. [0010] The RT-LAMP technique incorporates reverse transcriptase (RT) to enable detection of RNA viruses, including AIVs. Notomi et al., Loop-mediated isothermal amplification of DNA, Nucleic Acids Research 28: E63 (2000). RT-LAMP assays have been successfully developed for numerous RNA viruses, including SARS-CoV-2, West Nile virus, and Dengue virus, among others. Kitagawa et al., Evaluation of rapid diagnosis and novel coronavirus disease (COVID-19) using loop-mediated isothermal amplification, J Clinical Virology: The Official Publication of the Pan American Soc’y for Clinical Virology 129: 10446 (2020); Parida et al., Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of West Nile virus, J Clinical Microbiology 42: 257-263 (2004); Lau et al., Colorimetric detection of Dengue by single tube reverse-transcription-loop-mediated isothermal amplification, PLoS ONE 10: e0138694 (2015); Soliman & El-Matbouli, Reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of viral hemorrhagic septicaemia virus (VHS), Veterinary Microbiology 114: 205-213 (2006); Sano et al., Development of an RT-LAMP assay for rapid detection of SFTS virus, Viruses 13: 693 (2021); Fukuta et al., Detection of Japanese yam mosaic virus by RT-LAMP, Archives Virology 148: 1713-1720 (2003).
70817-02 [0011] While LAMP does show promise as an effective field diagnostic tool, a major limitation of using LAMP (or RT-LAMP) as a mainstream assay for pathogen screening is the occurrence of false positives – either due to poor reagent handling or carryover contamination from previous experiments. Additionally, the accuracy of LAMP is heavily dependent on the primers used and, prior to this disclosure, optimal primer sets had yet to be identified. Indeed, designing LAMP and RT-LAMP primers has proven challenging. [0012] In view of the rapid spread and growing host range of HPAI viruses – especially H5N1 – there remains a critical unmet need for field-deployable, sensitive, cost-effective, and specific diagnostic assays that enable early detection, rapid response, and containment of emerging outbreaks across avian and mammalian species. BRIEF SUMMARY [0013] Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assays are provided. In at least one embodiment, such assays can comprise at least one RT-LAMP primer set that targets a ribonucleic acid (RNA) fragment of a targeted influenza virus. In certain embodiments, the RT-LAMP assay comprises at least one RT-LAMP primer set that targets an RNA fragment of an influenza virus, wherein the assay allows for single-step identification of the presence or absence of the influenza virus in a sample. The at least one RT-LAMP primer set can comprise one or more of primer sets A-L in Table 2, or comprise primers having a nucleotide sequence identical to, or having at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, each primer sequence of the corresponding primer set A-L in Table 2. Each of the least one RT-LAMP primer set can comprise 4 to 6 primers. [0014] The targeted influenza virus can be an influenza A virus and, optionally, of hemagglutinin (HA) subtype H5 or HA subtype H7. The targeted influenza virus can be an avian influenza virus (AIV) and, optionally, highly pathogenic avian influenza (HPAI). The targeted influenza virus can be of a neuraminidase (NA) subtype N1. The targeted influenza virus can be selected from the group consisting of H5N1, H5N2, H7N3, and H7N9. [0015] In certain embodiments the assay processes and provides a visual result in 60 minutes or less. The visual result can be indicative of the presence or absence of the influenza virus in the sample. The visual result can be a color-coded or colorimetric result. [0016] The sample can be an oropharyngeal sample, a blood sample, a mammary secretion, a nasal sample, or an environmental water sample. The sample can be obtained from a subject that is a mammal or a bird. The subject can be avian, bovine, human, or swine. The subject can be housed in a high-density animal facility selected from the group consisting of a poultry production facility, a swine production facility, a bovine production facility, a zoological facility or other
70817-02 captive wildlife facility, and any other intensive animal-housing environment suitable for the maintenance or exhibition of animals in captivity. [0017] In certain embodiments, the assay comprises a pH- or magnesium-based indicator. Each of the RT-LAMP primer sets can have a limit of detection (LOD) of at least 500 copies/reaction. Each of the one or more RT-LAMP primer sets can be coupled with a colorimetric reagent. In certain embodiments, the colorimetric reagent is pH sensitive or magnesium sensitive. The colorimetric reagent can be phenol red. The assay can further comprise a fluorescent indicator. [0018] In certain embodiments, each RT-LAMP primer set is at least about 98% specific to the targeted RNA fragment. [0019] In at least one exemplary embodiment, the assay is a paper-based RT-LAMP assay. For example, the assay can be paper-based and comprise at least one reaction strip affixed to a backing and, optionally, one or more spacers positioned between each reaction strip. In such embodiments, each reaction strip can be loaded with, coated with, embedded in, or otherwise associated with at least one RT-LAMP primer set and a master mix. Additionally, the paper-based assay can further comprise a control strip affixed to the backing, wherein one or more spacers are optionally positioned between the control strip and each reaction strip. [0020] Methods for identifying the presence or absence of an influenza virus in a sample are also provided. Such methods can comprise providing any of the assays described herein, obtaining a sample from a subject; combining the sample and the at least one RT-LAMP primer set; heating the combination to initiate amplification of the targeted RNA fragment; and detecting a visual result in the heated combination indicative of the presence or absence of the targeted influenza virus in the sample. The visual result can be, in certain embodiments, provided in 60 minutes or less of initiating the heating step. The sample can be an unprocessed biofluid of the subject. The sample can be an unprocessed environmental water sample, an unprocessed oropharyngeal sample (i.e., an oropharyngeal swab), an unprocessed cloacal swab, an unprocessed nasal sample (i.e., a nasal swab), an unprocessed mammary secretion sample, or an unprocessed blood sample. [0021] In certain embodiments, detecting a visual result further comprises one or more of: measuring a relative clarity of the heated combination using a turbidimeter; and analyzing colorimetric data in the visual result using one or more of a fluorescent reader, an ultraviolet light reader, or camera. In certain embodiments, if the visual result is indicative of the presence of the targeted influenza virus, the method further comprises treating the subject for an influenza viral infection. In certain embodiments, the at least one RT-LAMP primer set is coupled with a colorimetric reagent that is pH sensitive or magnesium sensitive. In certain embodiments, the colorimetric agent is phenol red. In certain embodiments, treating the subject comprises isolating
70817-02 the subject and/or administering a therapeutically effective amount of an antiviral medication to the subject. [0022] Kits are also provided. In certain embodiments, such a kit comprises one or more assays described herein, at least one swab for obtaining a sample; and a heating element to initiate amplification of the targeted RNA fragment when the at least one RT-LAMP primer set of the assay and the sample are combined. The kit can further comprise a fluorescent indicator and a fluorescent reader, an ultraviolet light reader, or a camera to provide colorimetric result data indicative of the presence or absence of the targeted influenza virus in the sample. The kit can further comprise a sealable container with a transport media therein. In certain embodiments, the kit is portable. In certain embodiments, the kit is capable of use in a non-laboratory setting. The heating element can be a water bath. The heating element can be a hair dryer or other air-based heating implement. The at least one RT-LAMP primer set can be coupled with a colorimetric reagent that is pH sensitive or magnesium sensitive. [0023] In certain embodiments, the kit further comprises a dropper and/or a sealable container for receiving a paper-based assay. [0024] Uses of the one or more RT-LAMP assays hereof in detecting the presence or absence of a targeted influenza virus in a sample are also provided. [0025] Still further, methods of detecting influenza in a sample are provided. In certain embodiments, such a method comprises amplifying a targeted RNA fragment of an influenza virus in the sample using: (1) at least one RT-LAMP primer set comprising one or more primer sets A- L in Table 2, or primers having a nucleotide sequence identical to, or having at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, each primer sequence of the corresponding primer set A-L in Table 2 (e.g., by performing RT-LAMP methods as described herein); and/or at least one primer or probe sequence identical to, or having at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, one or more of SEQ ID NOS: 76-78 (e.g., by performing a one-step RT-qPCR assay as described herein). BRIEF DESCRIPTION OF THE DRAWINGS [0026] The disclosed embodiments and other features, advantages, and disclosures contained herein, and the matter of attaining them, will become apparent and the present disclosure will be better understood by reference to the following description of various exemplary embodiments of the present disclosure taken in conjunction with the accompanying drawings, figures, and tables, wherein: [0027] The disclosed embodiments and other features, advantages, and disclosures contained herein, and the matter of attaining them, will become apparent and the present disclosure will be
70817-02 better understood by reference to the following description of various exemplary embodiments of the present disclosure taken in conjunction with the accompanying drawings, figures, and tables, wherein: [0028] FIG. 1 shows data from the screening of reverse-transcription loop-mediated isothermal amplification (RT-LAMP) primer sets for H5 subtype avian influenza virus (AIV) using a fluorescent RT-LAMP assay performed with the NEB® WarmStart® DNA/RNA LAMP Kit. The screening was conducted to evaluate amplification performance and specificity across multiple candidate primer sets. [0029] FIGS. 2A-2D show data from a limit of detection (LOD) experiment using the various selected primer sets in a fluorescent H5 subtype AIV qLAMP assay format. The assay was conducted using serial dilutions of in vitro transcribed (IVT) RNA template at concentrations of 18,900, 1,890, 500, 250, 125, 50, 25, 5, and 1 copies per reaction to evaluate analytical sensitivity and establish the LOD of the assay for H5 subtype AIV detection. FIG.2A shows LOD data from primer set HPAIV.HA.5, FIG.2B shows LOD data from primer set HPAIV.HA.4, FIG.2C shows LOD data from primer set HPAIV.HA.6, and FIG. 2D shows LOD data from primer set HPAIV.HA.8. [0030] FIG. 3A shows a photograph of a colorimetric liquid RT-LAMP assay using the HPAIV.HA.5 primer set from a LOD experiment. The colorimetric liquid RT-LAMP assay was performed to test different concentrations of synthetic RNA (1,890, 500, 250, 125, 50, 25, 5, and 1 copies/reaction), with y denoting a yellow color of the reaction, and unmarked wells being pink. [0031] FIG 3B shows a photograph of a colorimetric liquid RT-LAMP assay using the HPAIV.HA.4 primer set from a LOD experiment. The colorimetric liquid RT-LAMP assay was performed to test different concentrations of synthetic RNA (1,890, 500, 250, 125, 50, 25, 5, and 1 copies/reaction), with Y denoting a yellow color of the reaction, and unmarked wells being pink. [0032] FIGS. 4A and 4B show data from a cross-reactivity test of HPAIV.HA.5 against various avian and bovine pathogens. FIG. 4A shows the cross-reactivity data of HPAIV.HA.5 against various avian pathogens including Newcastle Disease Virus (NDV) (non-virulent), Infectious Bronchitis Virus (IBV), Avian Infectious Laryngotracheitis Virus (ILT), Mycoplasma gallisepticum MG), Ornithobacterium rhinotracheale (ORT), and Avibacterium (Av) paragallinarum (Infectious Coryza). FIG. 4B shows the cross-reactivity data of HPAIV.HA.5 against various bovine pathogens including Bovine Rhinitis A Virus (BRAV) , bovine rhinitis B virus (BRBV), Bovine Coronavirus (BCV), Influenza D Virus (IDV), Bovine Adenovirus type 3 (BAV-3), Bovine Herpesvirus type 1 (BHV-1), Bovine Viral Diarrhea Virus type 1 (BVDV-1), Bovine Respiratory Syncytial Virus (BRSV), Bovine Adenovirus type 7 (BAV-7), and Bovine Parainfluenza Virus type 3 (BPI3V).
70817-02 [0033] FIG.5 is a schematic and colorimetric evaluation of a paper-based device hereof, with A) displaying a step-by-step workflow diagram for use of the paper-based device in diagnosing a subject with an influenza infection (the non-primer control is indicated by the control zone); B) showing a schematic representation of at least one embodiment of the paper device; and C) showing a colorimetric readout obtained from negative and positive tests using the paper-based device hereof. Pink (or negative results) are labeled “P” and Yellow (or positive results) are labeled “Y”. [0034] FIG. 6 shows data from a paper-based RT-LAMP LOD study using the HPAIV.HA.5 primer set, which was performed to test different concentrations of synthetic RNA. Samples marked “Y” are yellow and unmarked samples are red/pink. [0035] FIGS.7A-7F show data from comparison of detection methods for HPAIV in spiked and non-spiked oropharyngeal swabs collected from poultry. Three detection methods were used – the paper RT-LAMP assay hereof, quantitative polymerase chain reaction (qPCR) as per WHO protocol, and the qPCR methodology described herein. FIG. 7A shows a photograph of a paper RT-LAMP hereof, spiked with 1,000 copies of HPAIV, and FIGS.7B and 7C show quantitative data related to the fluorescent intensity measured from the 30 spiked samples by qPCR, as per the WHO protocol and via the protocol described herein, respectively. FIG.7D shows a photograph of a paper RT-LAMP hereof, which is not spiked. FIGS.7E and 7F show quantitative data related to the fluorescent intensity measured from the unspiked samples by qPCR, as per the WHO protocol and via the protocol described herein, respectively. [0036] As such, an overview of the features, functions and/or configurations of the components depicted in the various figures will now be presented. It should be appreciated that not all of the features of the components of the figures are necessarily described and some of these non- discussed features (as well as discussed features) are inherent from the figures themselves. Other non-discussed features may be inherent in component geometry and/or configuration. Furthermore, wherever feasible and convenient, like reference numerals are used in the figures and the description to refer to the same or like parts or steps. The figures are in a simplified form and not to precise scale. DETAILED DESCRIPTION [0037] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to the embodiments illustrated in the drawings, tables, and figures and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of this disclosure is thereby intended.
70817-02 [0038] The present disclosure includes various assays, kits, and methods to target and/or detect and/or treat the presence or absence of influenza viruses, such as to diagnose and treat influenza and, in particular, avian influenza. These assays (and methods of treatment using such assays) can be portable, disposable, and capable of providing fast and accurate results in the field without the need for a laboratory and other complex equipment. The terms “treat,” “treating,” “treated,” or “treatment” (with respect to a disease or condition) is an approach for obtaining beneficial or desired results including and preferably clinical results and includes, but is not limited to, one or more of the following: improving a condition associated with a disease, curing a disease, lessening severity of a disease, delaying progression of a disease, alleviating one or more symptoms associated with a disease, increasing the quality of life of one suffering from a disease, prolonging survival and/or prophylactic or preventative treatment. [0039] Additionally the assays presented herein provide rapid and accurate results (as compared to conventionally available assays and other methodologies). Perhaps more specifically, the novel primer sets of the assays, kits, and methods hereof increase testing accuracy (e.g., at or about 99% or 100% analytical sensitivity and at or about 89% or more analytical specificity) and decrease testing time to at or about 60 minutes, or less than 60 minutes, thus providing fast and accurate results. These sensitive screening models – whether employed alone or in combination with traditional techniques – are crucial for containing the spread of the virus and safeguarding poultry (and other livestock and human) populations. Indeed, the assays hereof can be used as point-of- need tests (PONT) for on-site sample testing without requiring transportation of the samples to a laboratory setting. This can be especially beneficial in remote areas with limited laboratory facilities. [0040] Assays & Primer Sets [0041] In at least one embodiment, a portable assay or method using the same comprises a reverse- transcription loop-mediated isothermal amplification (RT-LAMP) assay that utilizes novel primers (e.g. primer sets, including, without limitation primer sets comprising SEQ ID NOS: 1- 72 as set forth in Table 2 below). Also disclosed herein are detection methods using RT-LAMP assays that is capable of specifically detecting the presence of one or more influenza viruses and/or influenza virus hemagglutinin (HA) subtypes, such as H5N1, H7N1, or other subtypes, in a biological sample selected from the group consisting of pharyngeal, oropharyngeal, nasal, blood, mammary secretion, or other biological specimens, including environmental water samples obtained from a location where the subject mammal drinks, thereby providing a diagnosis of influenza infection. Accordingly, the assays, kits and methods hereof can be used to rapidly and accurately diagnose influenza infection in the field such that treatment, where desired, can be administered.
70817-02 [0042] RT-LAMP uses 4-6 primers that can recognize 6-8 distinct regions of target ribonucleic acid (RNA) for a highly specific amplification reaction. [0043] In RT-LAMP, a reverse transcriptase first synthesizes complementary DNA (cDNA) from the viral RNA target sequence. A strand-displacing DNA polymerase then initiates nucleic acid synthesis, and two specifically designed primers generate self-complementary “loop” structures that promote successive rounds of amplification. Extension occurs on these loop regions, with additional primers annealing to the newly formed structures to drive rapid, exponential amplification. The resulting DNA products are typically long concatamers (>20^kb) composed of numerous tandem repeats of the relatively short (80–250^bp) target sequence, separated by single-stranded loop regions. Although such concatameric products are generally unsuitable for most downstream molecular manipulations, the degree of target amplification achievable is sufficiently high to enable sensitive detection by multiple modalities, including colorimetric, fluorescent, turbidity-based, or other signal-generating readouts. [0044] Real-time fluorescence detection using intercalators or probes, lateral flow, and agarose gel detection, for example, are all directly compatible with RT-LAMP reactions. Instrumentation for RT-LAMP typically requires consistent heating to the desired reaction temperature and, where desired, real-time fluorescence for quantitative measurements. Optimized settings for running RT- LAMP assays on isothermal instruments are known in the art, and the assay can be performed using the techniques described in detail in at least Notomi et al. (2000), supra; Nagamine et al., “Accelerated reaction by loop-mediated isothermal amplification using loop primers,” Molecular & Cellular Probes 16(3): 223-229 (2002), both of which are incorporated herein by reference in their entireties. [0045] In certain instances, LAMP can be so prolific that the products and byproducts of these reactions can be visualized by the naked eye. For example, magnesium pyrophosphate produced during the reaction can be observed as a white precipitate or added indicators (e.g., calcein or hydroxynaphthol blue) can be used to signal a positive reaction or an indicative pH change. [0046] In certain embodiments, the RT-LAMP assay can be coupled with a colorimetric reagent that is sensitive to magnesium or pH and allows for visualization of the result with the naked eye and/or quantification using a camera. Such colorimetric reagents, for example, can include a phenol red. In certain embodiments, the RT-LAMP assays hereof coupled with a colorimetric reagent has a limit of detection (LOD) of 500 copies of RNA per reaction (25 copies/µL), with an analytical specificity of 100%, and/or an analytical sensitivity in the range of 60% - 100%. In an exemplary embodiment, the primers described herein are coupled with a composition comprising phenol red, such as, for example and without limitation, Warmstart® LAMP 2 x Master Mix.
70817-02 [0047] Warmstart® LAMP 2 x Master Mix, which contains phenol red, is characterized by its transition from pink to yellow as the RT-LAMP reaction occurs and the pH decreases. Positive and negative results using the primers hereof for detection of an influenza virus and the pink and yellow color distinction can be observed in FIGS.3A, 3B, and 6. [0048] In certain embodiments, a paper-based device is provided that can detect influenza virus present in a biological sample. In certain embodiments, the paper-based device can detect influenza virus present in a biological sample, such as H5 subtype AIV, at a minimum of 500 copies per reaction. The paper-based device can be a microfluidic paper-based analytical device (µPAD) that is user-friendly, portable, and customizable. [0049] As noted above, although some methods exist for detecting influenza virus infection, conventional approaches have not enabled accurate detection of influenza viruses in a field setting. The colorimetric RT-LAMP assays for an influenza virus provided herein however, are accurate and sensitive. [0050] The colorimetric RT-LAMP assays offer at least six advantages: (1) they can be conducted on the farm/in the field using a simple consumer-grade water bath; (2) they can provide a visual readout and, thus, allow for analysis with the naked eye; (3) they provide a response within 60 minutes; (4) they do not require sample processing (e.g., extraction of nucleic acids); (5) they can detect an influenza virus (e.g., a highly pathogenic avian influenza (HPAI) virus) with a high degree of accuracy (such as 96% accuracy or 100% accuracy); and (6) they can utilize a simple swab for sampling. This approach has the potential to significantly enhance influenza virus detection (such as, for example, influenza A virus detection) and contribute to the control and prevention of influenza outbreaks (such as, for example, AIV outbreaks). [0051] RT-LAMP is well-suited for point-of-care and field diagnostics using all manner of sample types. Further, the RT-LAMP reaction is robust and tolerant of inhibitors, allowing for crude sample prep and minimal nucleic acid purification, if desired. [0052] In at least one illustrative RT-LAMP assay hereof, the assay is for single-step identification of a targeted infectious agent in a sample. The RT-LAMP assay comprises at least one novel LAMP primer set that targets an RNA of the targeted infectious agent in a sample, wherein the targeted infectious agent is an influenza virus. The assay not only allows for detection of an influenza virus within a sample, but also the distinction thereof from other pathogens in a single step. Further, the RT-LAMP assays hereof can achieve at least a sensitivity and specificity of about 100%, which is a significant increase of sensitivity as compared to existing RT-LAMP models, and offer approximately ten times the detection capability of conventional reverse transcription polymerase chain reaction (RT-PCR) methods. In certain embodiments, the novel
70817-02 RT-LAMP primer sets are also designed to enhance amplification speed of the assay such that the RT-LAMP assays hereof are more rapid than conventional LAMP or RT-LAMP assays. [0053] In at least one embodiment, the RT-LAMP primer set comprises F3, B3, FIP, BIP primers. Additionally, the RT-LAMP primer set can also comprise loop primers (e.g., labelled as LF (loop forward) and/or LB (loop backward)). [0054] The influenza virus to be detected by the RT-LAMP assays hereof can be any influenza virus. In certain embodiments, the influenza virus is a strain of influenza A virus. In certain embodiments, the influenza virus is an AIV and, optionally, a highly pathogenic AIV (HPAI). In certain embodiments, the influenza A virus is HA subtype H5 or HA subtype H7. In certain embodiments, the influenza virus is of neuraminidase (NA) subtype N1. In certain embodiments, the influenza virus can be H5N1, H5N2, H7N3, or H7N9. [0055] In at least one embodiment, treatment and detection methods using a RT-LAMP assay and primers, can specifically detect the presence of an influenza virus (such as, for example, influenza A virus) in 60 minutes or less (e.g., in about 60 minutes or less, in about 60-55 minutes or less, in about 55-50 minutes or less, or in about 50-45 minutes or less). [0056] The targeted nucleic acid for each pathogen is, preferably, an RNA segment or region having little to no sequence homology with non-target influenza viruses or other pathogens. While certain such RNA targets have been previously reported, the present investigators have newly identified additional conserved RNA regions and have designed the primers listed in Tables^2 and^3 to be specific for these newly discovered RNA sequences. Targeting such regions using the RT-LAMP assays described herein enables rapid and specific detection of influenza virus infections within a flock, herd, or other susceptible population. Early identification allows for timely intervention, including removal or isolation of infected subjects and/or initiation of treatment, thereby reducing the risk of an outbreak and improving overall disease control and biosecurity management. [0057] In certain embodiments, the influenza virus is an AIV and, optionally, a highly pathogenic AIV (HPAI). AIVs, in particular, present a significant threat to the global poultry industry, causing substantial mortality and economic damage. These viruses can infect various bird species, raising concerns about animal-to-animal transmission and potential fatalities. Reperant et al., ADME Adaptive pathways of zoonotic influenza viruses: from exposure to establishment in humans, Vaccine 30(30): 4419-4434 (2012); Nakauchi et al., Development of a reverse transcription loop- mediated isothermal amplification assay for the rapid diagnosis of avian influenza A (H7N9) virus infection, J Virological Methods 204: 101-104 (2014); Padzil et al., Loop-mediated isothermal amplification (LAMP) as a promising point-of-care diagnostic strategy in avian virus research, Animals: An Open Access J from MDPI 12(1): 76 (2021). The recent HPAI H5N1 outbreak in
70817-02 U.S. dairy cattle highlights the virus’s ability to jump from wild birds to domestic animals and humans, underscoring the risk of a future influenza pandemic. Burrough et al., Highly pathogenic avian influenza A (H5N1) clade 2.3.4.4b virus infection in domestic dairy cattle and cats, Emerging Infectious Diseases 30(7): 1335-1343 (2024); U.S. Centers for Disease Control & Prevention, Highly pathogenic avian influenza A (H5N1) virus infection reported in a person in the U.S., CDC Media Release (April 1, 2024); Garg, Outbreak of highly pathogenic avian influenza A (H5N1) viruses in U.S. dairy cattle and detection of two human cases – United States, MMWR Morbidity and Mortality Weekly Report 73(21): 501-505 (2024). [0058] Accordingly, while the specific examples described herein utilize avian influenza virus as a representative example, that such viruses are zoonotic and can cross species. To the extent the viral RNA is substantially conserved with respect to the primer targets hereof, the present assays, methods, and kits can be used in non-avian species for early and/or rapid detection – including with avian and mammalian subjects such as, and without limitation, avian, bovine, humans, and swine. As such, the samples tested by such assays, methods and kits can be obtained from a subject that is a mammal or a bird. In certain embodiments, the subject is selected from the group consisting of an avian species, a bovine species, a human, and a swine species. In certain embodiments, the subject is housed in a high-density animal facility selected from the group consisting of a poultry production facility (e.g., a commercial poultry production facility including, without limitation, a broiler grow-out house, a turkey finishing house, or a layer facility), a swine production facility (e.g., a commercial swine production facility including, without limitation, a grow-finish barn, a finishing barn, or a wean-to-finish facility), a bovine production facility (e.g., a commercial bovine production facility including, without limitation, a feedlot, dairy barn, or calf-raising facility), a zoological facility or other captive wildlife facility, and any other intensive animal-housing environment suitable for the maintenance or exhibition of animals in captivity. [0059] Each RT-LAMP primer set of the assay is designed to target and amplify the targeted RNA from a targeted influenza virus, while maintaining little to no amplification of other pathogens or negative samples. Each RT-LAMP primer set can include 4 to 6 DNA primers (however the number of primers used can be modified, as desired). [0060] In other embodiments, the RT-LAMP primer set(s) hereof each comprise a RT-LAMP primer set listed in Tables 2 and 3. In certain embodiments, the RT-LAMP primer set(s) comprise one or more primers of any of SEQ ID NOS.1-72 and 76-78. [0061] It will be appreciated that the primers listed in Tables 2, 3, and 5 are designed to not only exhibit a high degree of specificity for the targeted RNA fragment, but also to enhance amplification speed. Indeed, the data provided herein supports that the primer sets provided herein
70817-02 render the RT-LAMP assays hereof more rapid and more sensitive than conventional RT-LAMP assays. [0062] Any number of RT-LAMP primer sets can be used in the same assay; for example, and without limitation, an assay can comprise one or more of the following: a RT-LAMP primer set comprising SEQ ID NOS: 31-26, a RT-LAMP primer set comprising SEQ ID NOS: 25-30, a RT- LAMP primer set comprising SEQ ID NOS: 43-48; a RT-LAMP primer set comprising SEQ ID NOS: 19-24, a RT-LAMP primer set comprising SEQ ID NOS: 37-42, a RT-LAMP primer set comprising SEQ ID NOS: 49-54, a RT-LAMP primer set comprising SEQ ID NOS: 55-60, a LAMP primer set comprising SEQ ID NOS: 13-18, a RT-LAMP primer set comprising SEQ ID NOS: 7-12, a LAMP primer set comprising SEQ ID NOS: 1-6, a RT-LAMP primer set comprising SEQ ID NOS: 61-66, and/or a RT-LAMP primer set comprising SEQ ID NOS: 67-72. In another embodiment, for example, a first RT-LAMP primer set can comprise SEQ ID NOS: 31-26, a second RT-LAMP primer set can comprise SEQ ID NOS: 25-30, and/or a third RT-LAMP primer set can comprise SEQ ID NOS: 43-48. [0063] In at least one embodiment, the assays hereof comprise one or more of the RT-LAMP primer sets described herein. In certain embodiments, such assays comprise one or more of the RT-LAMP primer sets that comprise one or more primers of any of SEQ ID NOS.1-72. In certain embodiments, such assays comprise one or more of the RT-LAMP primer sets that comprise one or more primers having a nucleotide sequence identical to, or having at or about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any of SEQ ID NOS.1-72. [0064] The term “identity” with respect to a reference to an amino acid sequence is defined as the percentage of nucleic acid residues in a candidate sequence that are identical with the residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity and not considering any conservative substitutions as part of the sequence identity. Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the product by 100 to achieve a percentage. Thus, two copies of exactly the same sequence have 100% identity, whereas two sequences that have nucleic acid deletions, insertions, or substitutions relative to one another have a lower degree of identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill of the art, for instance, using publicly available computer software. For example, determination of percent identity or similarity between sequences can be done, for example, by using the GAP program (Genetics Computer Group, software; now available via Accelrys online), and alignments can be done using, for example, the ClustalW algorithm (VNTI software, InforMax Inc.). Further, a sequence database can be searched using the nucleic acid sequence of interest. Algorithms for database searching are typically based on the BLAST
70817-02 software (Altschul et al., 1990), but those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. [0065] In certain embodiments, the assay comprises one or more of the RT-LAMP primer sets A- L in Table 2. In certain embodiments, the assay comprises one or more of the RT-LAMP primer sets A-L in Table 2, wherein each of the primers has a nucleotide sequence identical to, or having at or about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, each of their associated SEQ ID NOS. For example and without limitation, the assay can comprise the RT-LAMP primer sets of primer set A, wherein each of the primers has a nucleotide sequence identical to, or having at or about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, each of SEQ ID NOS: 1-6. The assay can comprise the RT-LAMP primer sets of primer set B, wherein each of the primers has a nucleotide sequence identical to, or having at or about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, SEQ ID NOS: 7-12. The assay can comprise the RT-LAMP primer sets of primer set C, wherein each of the primers has a nucleotide sequence identical to, or having at or about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, SEQ ID NOS: 13-18. The assay can comprise the RT-LAMP primer sets of primer set D, wherein each of the primers has a nucleotide sequence identical to, or having at or about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, SEQ ID NOS: 19-24. The assay can comprise the RT-LAMP primer sets of primer set E, wherein each of the primers has a nucleotide sequence identical to, or having at or about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, SEQ ID NOS: 25- 30. The assay can comprise the RT-LAMP primer sets of primer set F, wherein each of the primers has a nucleotide sequence identical to, or having at or about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, SEQ ID NOS: 31-36. The assay can comprise the RT- LAMP primer sets of primer set G, wherein each of the primers has a nucleotide sequence identical to, or having at or about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, SEQ ID NOS: 37-42. The assay can comprise the RT-LAMP primer sets of primer set H, wherein each of the primers has a nucleotide sequence identical to, or having at or about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, SEQ ID NOS: 43-48. The assay can comprise the RT-LAMP primer sets of primer set I, wherein each of the primers has a nucleotide sequence identical to, or having at or about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, SEQ ID NOS: 49-54. The assay can comprise the RT-LAMP primer sets of primer set J, wherein each of the primers has a nucleotide sequence identical to, or having at or about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, SEQ ID NOS: 55-60. The assay can comprise the RT-LAMP primer sets of primer set K, wherein
70817-02 each of the primers has a nucleotide sequence identical to, or having at or about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, SEQ ID NOS: 61-66. The assay can comprise the RT-LAMP primer sets of primer set L, wherein each of the primers has a nucleotide sequence identical to, or having at or about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, SEQ ID NOS: 67-72. The inclusion of primers with the above sequence identity thresholds is intended to encompass functionally equivalent variants, such as those incorporating degenerate bases, conservative substitutions, or sequence modifications that do not substantially affect primer specificity or efficiency in the disclosed RT-LAMP assays. [0066] In certain embodiments, each RT-LAMP primer set of the assay is at least about 98% specific to the targeted RNA fragment of the influenza virus. [0067] The results of the RT-LAMP assays hereof can, in some embodiments, be seen with the naked eye. While conventional versions of RT-LAMP assays require SYBR Green staining for signal detection (which necessitates opening the tube after thermal incubation) the RT-LAMP assays hereof can be performed with a turbidimeter (e.g., a Loopamp real-time turbidimeter) to detect a positive signal. A turbidimeter measures the relative clarity of the sample and does not require opening the tube, which reduces the risk of environmental diffusion and cross- contamination during gene amplification. [0068] In certain embodiments, magnesium pyrophosphate produced during the reaction can be observed as a white precipitate or added indicators (e.g., calcein, magnesium-based indicators, or hydroxynaphthol blue) can be used to signal a positive reaction or an indicative pH change. [0069] In certain embodiments, the RT-LAMP assays hereof can be coupled with or include indicators (e.g., colorimetric reagents or indicators) to allow for visual inspection of assay results without opening the reaction tube. Such assay results can provide a visual result that corresponds to the presence or absence of the targeted influenza virus in the sample. In some cases, the visual result is color-coded and/or colorimetric, and in other cases the result can be a letter, number, word, symbol, lines, or other representation indicative of the presence or absence of the targeted influenza virus. [0070] Fluorescence can also be employed to facilitate signal detection. In at least one embodiment, the RT-LAMP assays hereof further comprise fluorescent dye in the reagents mix for assay or a fluorescent tag coupled with the primers themselves. Fluorescent data/intensities can thereafter be collected (using thermocyclers or a fluorometer, for example) and analyzed. In the above non-limiting example where a loop primer is directed to a unique RNA fragment associated with AIV, a particular fluorescent indicator can be coupled with such primer so that visualization of the fluorescence of that particular fluorescent indicator is indicative of the sample being positive for the AIV.
70817-02 [0071] In certain embodiments, the colorimetric agent is pH sensitive (e.g., phenol red). While specific embodiments and examples are provided herein, it will be appreciated that any colorimetric reagent sensitive to pH or magnesium can be employed. [0072] Where multiple primer sets are used in the same assay, indicators can be used to easily identify visual results related to the targeted influenza virus(es) or other pathogens present in the sample. In at least one embodiment, for example, the first primer set can be labeled (at their 5'- ends, for example) with a stable, fluorescent material of a first intensity, the second primer set can be labeled with a stable, fluorescent material of a second intensity, and the third primer set can be labeled with a stable, fluorescent material of a third intensity using methods commonly known in the relevant arts. When one or more of the primer sets anneal to a complementary target amplicon (i.e. the RNA fragment of the targeted influenza virus), the 5'→3' exonucleolytic activity of DNA polymerase cleaves the label from the primer. This cleavage releases the fluorescent label, producing an increased fluorescence signal characteristic of the fluorescent material associated with the primer set that hybridized to the target. In such embodiments, measurement of the resulting fluorescence intensity can identify which primer set(s) bound to the target sequence present in the sample. While fluorescent indicators are described above, it will be appreciated that any type of indicators can be used with the novel assays of the present disclosure, including other indicators now known or hereinafter developed. [0073] Additionally, certain embodiments of the RT-LAMP assays can optionally utilize a fluorescent reader, an ultraviolet light reader, and/or a camera for signal detection and/or the display of assay results (e.g., where indicators are used). In these embodiments, the visual results may be color-metric and/or digitally provided, such as, for example, through a wireless device, laptop computer, or cell phone and may utilize WiFi, Bluetooth, or cellular data. [0074] The RT-LAMP assays (and primer sets thereof) can detect the targeted influenza RNA fragments in various sample types and, in certain embodiments, does not require that such samples be processed prior to running the assay. The sample can be an unprocessed biological sample collected from the subject. For example, a sample can comprise a simple environmental water sample, an unprocessed oropharyngeal sample (e.g., obtained via a pharyngeal swab), an unprocessed cloacal swab, an unprocessed nasal sample (e.g., obtained via a nasal swab), an unprocessed mammary secretion sample (e.g., obtained from expressed milk), or an unprocessed blood sample. The ability to use unprocessed samples is advantageous for several reasons, at least one of which being that the assay translates easily to field use due to the ease of incubation. In certain embodiments, the samples, once collected, can be housed in a tube or vial containing a transport medium suitable for the collection, transport and/or handling of the specimen. For example, and without limitation, the transport medium can be liquid amies transport media.
70817-02 [0075] In certain embodiments, the assays can be prepared as paper-based assays. Paper-based RT-LAMP assays are a promising diagnostic tool for detecting influenza (e.g., AIVs). One of their main advantages is their simplicity and ease of use, as they do not require sophisticated laboratory infrastructure or trained personnel. This makes them suitable for resource-limited settings. Moreover, the paper-based RT-LAMP assays hereof are highly specific and sensitive, as demonstrated herein in connection with the RT-LAMP assay for detecting H5 subtype AIV, which showed a LOD of 500 copies/reaction, indicating its high diagnostic potential for AIV detection. [0076] In certain embodiments, the assay is a microfluidic paper-based analytical device (µPAD). Referring to subpart B of FIG. 5, the paper-based assay 500 can comprise, for example, at least one reaction strip 502 affixed to a backing 506 (for example, by double-sided tape, an adhesive 507, or via any other appropriate means). The backing 506 can be any suitable backing to provide support to the reaction strip 502. In certain embodiments, the backing 506 is a polyester backing. [0077] The assay 500 can further comprise a control strip 504 (not shown) affixed to the backing 506. In certain embodiments, the control strip 504 is separate, and positioned apart, from each reaction strip 502. In certain embodiments, the control strip 504 is in fluid communication with one or more reaction strips 502. The control strip 504 can be used as a reference and/or assay performance control to confirm that the assay has been properly executed, that the reagents are functional, and/or that the sample has adequately migrated along the strip as is known in the art. In certain embodiments, the control strip 504 can include one or more control reagents, such as primers unrelated to the target sequence(s), labeled oligonucleotides, immobilized dyes, and/or other indicator materials that produce a detectable signal independent of the presence or absence of the targeted influenza virus. [0078] The paper-based assay 500 can, optionally, further comprise one or more spacers 508 positioned between each reaction strip 502 to, for example, prevent crosstalk among the strips. A spacer 508 can also be positioned in between a reaction strip 502 and a control strip 504 if so desired. A spacer 502 can be composed of any appropriate material such as, for example, polystyrene. [0079] Each reaction strip 502 can loaded with, coated with, embedded in, or otherwise associated with at least one RT-LAMP primer set and a master mix. The master mix can be any mix described herein (e.g., in the Examples). In certain embodiments, the master mix comprises WarmStart® RTx reverse transcriptase, Bst 2.0 WArmStart® DNA polymerase, Antarctic thermolabile UDG, and a dNTP mixture. The master mix can further comprise an indicator such as colorimetric reagent (e.g., phenol red). The master mix can further comprise a fluorescent indicator. The master mix can further comprise one or more salts, such as a magnesium sulfate (MgSO4) and/or potassium chloride (KCl), in amounts effective to facilitate primer annealing and polymerase
70817-02 activity. The master mix can further comprise one or more additives, such as non-ionic detergents (e.g., Tween®^20), sugars or sugar alcohols (e.g., trehalose), proteins (e.g., bovine serum albumin (BSA)), and/or osmolytes or betaine-type compounds (e.g., betaine), in amounts effective to stabilize assay components, enhance enzyme activity, and/or improve amplification efficiency. [0080] In addition to their current diagnostic capabilities, paper-based RT-LAMP assays have promising prospects. One such prospect is the development of multiplex RT-LAMP assays, which can simultaneously detect multiple target analytes. This includes the multiplex RT-LAMP assay, which can offer quick, highly sensitive, inexpensive, and robust diagnostics for detecting multiple AIVs. Additionally, these paper-based RT-LAMP assays can be integrated with digital technologies, such as smartphone applications, for real-time monitoring and analysis. This integration can lead to the creation of affordable PONTs usable in both clinical and non-clinical settings, such as field surveillance of AIV in wild bird populations. The paper-based assay 500 format can offer at least three distinct advantages: (1) the user only needs to handle the sample and does not need to mix various reaction reagents (buffers, master mix, primer sets, etc.); (2) paper-based assays have the potential to be multiplexed by fabricating them in a side-by-side strip; and (3) when fabricated using roll-to-roll manufacturing, the multiplexed paper-based assays 500 can be scaled up to millions to billions of tests per year (if needed). Davidson et al., A paper-based colorimetric molecular test for SARS-CoV-2 in saliva, Biosensors & Bioelectronics X 9: 100076 (2021). [0081] Kits [0082] Kits for testing one or more samples are also provided. Such diagnostic kits can be configured for field use such as, for example, in a poultry production facility (e.g., a broiler house, a layer house, turkey barns, and other intensive poultry farming structures); a feed lot, dairy farm, or other type of cattle operation; a commercial swine production facility (e.g., a grow-finish barn, finishing barn, or wean-to-finish facility); and/or on-site at a zoological or other captive wildlife facility. Accordingly, the kits can be portable and capable of use in a non-laboratory setting. [0083] Still further, the design of the paper-based assays 500 allow for primer sets to be regularly updated in view of new viral outbreaks and for the use of highly specific primers to minimize false positives. Furthermore, assay performance can be enhanced by employing straightforward techniques like heat or chemical treatment, as well as sample dilution. [0084] In at least one embodiment, the diagnostic kits hereof comprise a molecular diagnostic assay comprising one or more of the RT-LAMP primer sets described herein. In certain embodiments, such assays comprise one or more of the RT-LAMP primer sets that comprise one or more primers of any of SEQ ID NOS.1-72. In certain embodiments, such assays comprise one or more of the RT-LAMP primer sets that comprise one or more primers having a nucleotide
70817-02 sequence identical to, or having at or about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any of SEQ ID NOS.1-72. [0085] In certain embodiments, the assay of the kit comprises one or more of the RT-LAMP primer sets A-L in Table 2. In certain embodiments, the assay comprises one or more of the RT- LAMP primer sets A-L in Table 2, wherein each of the primers has a nucleotide sequence identical to, or having at or about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, each of their associated SEQ ID NOS. For example and without limitation, the assay can comprise the RT-LAMP primer sets of primer set A, wherein each of the primers has a nucleotide sequence identical to, or having at or about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, each of SEQ ID NOS: 1-6. The assay can comprise the RT-LAMP primer sets of primer set B, wherein each of the primers has a nucleotide sequence identical to, or having at or about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, SEQ ID NOS: 7-12. The assay can comprise the RT-LAMP primer sets of primer set C, wherein each of the primers has a nucleotide sequence identical to, or having at or about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, SEQ ID NOS: 13-18. The assay can comprise the RT-LAMP primer sets of primer set D, wherein each of the primers has a nucleotide sequence identical to, or having at or about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, SEQ ID NOS: 19-24. The assay can comprise the RT-LAMP primer sets of primer set E, wherein each of the primers has a nucleotide sequence identical to, or having at or about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, SEQ ID NOS: 25-30. The assay can comprise the RT-LAMP primer sets of primer set F, wherein each of the primers has a nucleotide sequence identical to, or having at or about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, SEQ ID NOS: 31-36. The assay can comprise the RT-LAMP primer sets of primer set G, wherein each of the primers has a nucleotide sequence identical to, or having at or about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, SEQ ID NOS: 37-42. The assay can comprise the RT-LAMP primer sets of primer set H, wherein each of the primers has a nucleotide sequence identical to, or having at or about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, SEQ ID NOS: 43-48. The assay can comprise the RT-LAMP primer sets of primer set I, wherein each of the primers has a nucleotide sequence identical to, or having at or about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, SEQ ID NOS: 49-54. The assay can comprise the RT-LAMP primer sets of primer set J, wherein each of the primers has a nucleotide sequence identical to, or having at or about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, SEQ ID NOS: 55-60. The assay can comprise the RT-LAMP primer sets of primer set K, wherein each of the primers has a nucleotide sequence identical to, or having at
70817-02 or about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, SEQ ID NOS: 61-66. The assay can comprise the RT-LAMP primer sets of primer set L, wherein each of the primers has a nucleotide sequence identical to, or having at or about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, SEQ ID NOS: 67-72. The inclusion of primers with the above sequence identity thresholds is intended to encompass functionally equivalent variants, such as those incorporating degenerate bases, conservative substitutions, or sequence modifications that do not substantially affect primer specificity or efficiency in the disclosed RT-LAMP assays. [0086] In certain embodiments, each RT-LAMP primer set of the assay is at least about 98% specific to the targeted RNA fragment of the influenza virus. [0087] The kit can further comprise at least one swab for obtaining a sample from a subject (e.g., a bird) and/or a vial or other container for receiving the at least one swab after a sample is collected. In at least one exemplary embodiment, the container can be used as the incubation environment for the collected sample and one or more RT-LAMP primer sets (i.e. where the amplification reaction is performed on the collected sample). Accordingly, the container can contain a transport media or the like as is known in the art, and/or any additional reagents that are useful in facilitating the DNA amplification reaction and/or visualizing the results thereof. For example, in at least one embodiment, UDG/UTG can be added to the media within the container to degrade leftover amplicons present therein after amplification of the targeted RNA. [0088] In at least one embodiment, the container is sealable and is at least partially transparent such that visual results present within the container can be visualized without opening the container itself. In certain embodiments, the kit comprises a sealable container for receiving the paper-based assay 500. In such embodiments, the container is at least partially transparent such that the visual results from the reaction strip(s) 502 can be visualized without opening the container itself. In certain embodiments, the container is water-resistant or waterproof. [0089] The assay of each kit can further comprise an indicator associated with each RT-LAMP primer set. As described above, the RT-LAMP primer sets can be configured to include the indicator (e.g., a fluorescent indicator coupled with an end of each primer) or the indicator can be added to the media housed by the container. [0090] In certain embodiments, the indicator of each kit comprises a colorimetric reagent. For example, in certain embodiments, one or more of the RT-LAMP primer sets can be coupled with a colorimetric reagent that is pH sensitive or magnesium sensitive. In certain embodiments, the colorimetric agent is phenol red. [0091] The kit can further comprise a heating element to initiate amplification of the targeted RNA fragment when the at least one RT-LAMP primer set and the sample are combined, for
70817-02 example, in the container and/or on the reaction strip 502 of the paper-based assay 500. In certain embodiments, the heating element is a water bath. The kit can also, optionally, comprise a fluorescent reader, an ultraviolet light reader, or a camera to provide color metric result data indicative of the presence or absence of a targeted pathogen in the sample. [0092] The kit can further comprise a dropper. [0093] Methods of Use [0094] Methods for identifying the presence or absence of an influenza virus in a sample (and, if identified as present, treating the subject) are also provided. In at least one embodiment, a method for identifying the presence or absence of an influenza virus in a sample comprises: providing at least one RT-LAMP primer set hereof (e.g., that targets an RNA fragment of a targeted influenza virus); obtaining a sample from a subject; combining the sample and the at least one RT-LAMP primer set; heating the combination to initiate amplification of the targeted RNA fragment; and detecting a visual result in the heated combination indicative of the presence or absence of the targeted influenza virus in the sample. In certain embodiments, if the visual result indicates the presence of the targeted influenza virus in the sample, the method further comprises treating the subject for the targeted influenza virus and/or a viral infection. [0095] As used herein, a “subject” is a mammal or a bird, preferably an avian species, but it can also be a human or non-human animal (including, without limitation, a laboratory, an agricultural, a domestic, or a wild animal). Thus, the assays and methods described herein are applicable to both human and veterinary disease applications. In certain embodiments, subjects that can be addressed using the methods hereof include subjects identified or selected as having or being at risk for having an influenza infection (e.g., exposure confirmed through contact tracing or the like). Such identification and/or selection can be made by clinical or diagnostic evaluation. In certain embodiments, treating the subject can comprise isolating the subject (e.g., from a commercial animal production facility). [0096] Uses of the one or more RT-LAMP assays are also provided. Such uses are not dissimilar to the methods outlined herein and can encompass the same (or similar) steps. In certain embodiments, a use of the one or more RT-LAMP assays hereof is disclosed in detecting the presence or absence of a targeted influenza virus in a sample. The targeted influenza virus can be an avian influenza virus. The targeted influenza virus can be an influenza A virus. The targeted influenza virus can be any described herein including, without limitation, influenza A, AIV, and/or a HPAI. [0097] Methods are also provided for detecting influenza in a sample. Such a method can comprise amplifying a targeted RNA fragment of an influenza virus in the sample using: (1) at least one RT-LAMP primer set comprising one or more primer sets A-L in Table 2, or primers
70817-02 having a nucleotide sequence identical to, or having at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, each primer sequence of the corresponding primer set A-L in Table 2 (e.g., by performing RT-LAMP as described herein); and/or at least one primer or probe sequence identical to, or having at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, one or more of SEQ ID NOS: 76-78 (e.g., by performing a one-step RT-qPCR assay as described herein). [0098] In certain embodiments, treating the subject can comprise administering a therapeutically effective amount of an antiviral medication to the subject. In certain embodiments, treating the subject can comprise isolating the subject (e.g., from a commercial animal production facility) and administering a therapeutically effective amount of an antiviral medication to the subject. [0099] At least one RT-LAMP primer set can be any of the RT-LAMP primer sets described herein. In at least one embodiment, for example, the each of the RT-LAMP primer sets can comprises one or more of primer sets A-L in Table 2, and/or comprise primers having a nucleotide sequence identical to, or having at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, each primer sequence of the corresponding primer set A-L in Table 2. [00100] In at least one illustrative embodiment, the sample comprises an unprocessed biofluid of the subject. The sample can be an unprocessed environmental water sample, an unprocessed oropharyngeal sample, an unprocessed nasal sample, an unprocessed mammary secretion sample, or an unprocessed blood sample. In certain embodiments, the sample can be a bovine nasal swab sample. Notwithstanding the foregoing, any sample capable of providing a medium sufficient to detect the targeted influenza virus using the methods hereof can be used. [00101] The methods and uses hereof can be performed within a single container that need not be opened once the sample is placed therein. In at least one embodiment, the container includes (a) the desired RT-LAMP primer set(s), each targeting an RNA fragment of a targeted influenza virus; (b) a media to facilitate the storage and/or amplification reaction (e.g., water or liquid amies transport media), one or more indicators (either coupled with each primer set or added to the media), and/or (c) any additional reagents desired. Accordingly, in at least one embodiment, the sample is collected from the subject (e.g., a nasopharyngeal or nasal swab is used to collect a sample from a subject’s throat or nasal cavity) and placed within the container that already houses the assay and associated reagents. The container can then be sealed (e.g., via a screw cap or the like) and the container and its contents heated to initiate the amplification reaction. [00102] Where the assay comprises a paper-based assay 500, a sample is obtained from the subject and, in certain embodiments, suspended or diluted in a suitable solution to form a sample mixture. The sample or sample mixture is then applied to one or more reaction strips 502 of the
70817-02 assay 500 so as to rehydrate the reagents thereon. The assay 500 can thereafter be placed within a sealable container, the container sealed, and the container and its contents heated under conditions sufficient to initiate the amplification reaction. In other embodiments, the assay 500 itself can be heated directly, without placement in a separate container, to initiate the amplification reaction. [00103] Detecting the visual result produced by the method can be performed using any of the modalities described above. In certain embodiments, the visual results can be seen with the naked eye (without the use of additional instruments). In other embodiments, the assay further comprises one or more indicators associated with each set of loop primers such that detection of a particular indicator is indicative of the targeted influenza virus being present within the sample. Accordingly, the methods can additionally comprise using a turbidimeter to measure the relative clarity of the heated combination, and/or a fluorescent reader (e.g., a fluorometer), an ultraviolet light reader, or camera to analyze color metric data in the visual result. [00104] These RT-LAMP assay methods and uses are advantageous over conventional methods because accurate results are more quickly produced, and the assays hereof are more sensitive and more specific than conventional methods. In certain embodiments, the methods hereof can provide a visual result indicative of the presence or absence of the targeted pathogen(s) within 60 minutes of initiating the heating step (e.g., initiating reaction of the primers with the sample). Further, such reactions can be conducted between about 60-65 ºC, which is well outside of ambient field temperatures. Importantly, the primers sets of the present disclosure have at least a 97% accuracy (96% sensitivity and 98% specificity), at least a 98% accuracy, at least a 99% accuracy, or even 100% accuracy, which is a significant improvement over conventional primers. [00105] A previous limitation of RT-LAMP assays for influenza screening has been the occurrence of false positives either due to poor reagent handling or carryover contamination from previous experiments. With the present assay, methods, and kits hereof, this concern can be minimized by employing multiple spaces for reaction preparation, pre-aliquoting the required reagents in each container to reduce contamination losses, and/or adding UDG and dUTP to degrade leftover amplicons in incubation environments (i.e., the containers). [00106] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the chemical and biological arts. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the subject of the present application, the preferred methods and materials are described herein. Additionally, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise.
70817-02 [00107] When ranges are used herein, all combinations and sub-combinations of ranges and specific embodiments therein are intended to be included. [00108] Additionally, the term “about,” when referring to a number or a numerical value or range (including, for example, whole numbers, fractions, and percentages), means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error) and thus the numerical value or range can vary between 1% and 15% of the stated number or numerical range (e.g., +/- 5 % to 15% of the recited value) provided that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). The term “comprising” (and related terms such as “comprise” or “comprises” or “having” or “including”) is not intended to exclude that in other certain embodiments, for example, an embodiment of any compound, composition of matter, composition, method, or process, or the like, described herein, may “consist of” or “consist essentially of” the described features. The term “substantially” can allow for a degree of variability in a value or range, for example, within 90%, within 95%, or within 99% of a stated value or of a stated limit of a range. [00109] Additionally, in describing representative embodiments, a method and/or process may have been presented as a particular sequence of steps. To the extent that the method or process does not rely on the particular order of steps set forth herein, the method or process should not be limited to the particular sequence of steps described. As one of ordinary skill in the art would appreciate, other sequences of steps may be possible. Therefore, the particular order of the steps disclosed herein should not be construed as limitations on the claims. In addition, the claims directed to a method and/or process should not be limited to the performance of their steps in the order written, and one skilled in the art can readily appreciate that the sequences may be varied and still remain within the spirit and scope of the present disclosure. [00110] It is therefore intended that this description and the appended claims will encompass, all modifications and changes apparent to those of ordinary skill in the art based on this disclosure. ENUMERATED CLAUSES [00111] Clause 1. A reverse-transcription loop-mediated isothermal amplification (RT- LAMP) assay comprising at least one RT-LAMP primer set that targets a ribonucleic acid (RNA) fragment of an influenza virus; wherein the assay allows for single-step identification of the presence or absence of the influenza virus in a sample; and the at least one RT-LAMP primer set comprises one or more of primer sets A-L in Table 2, wherein each primer has a nucleotide sequence identical to, or having at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, the primer sequence of the corresponding primer set A-L in Table 2.
70817-02 [00112] Clause 2. The assay of clause 1, wherein the influenza virus is an influenza A virus. [00113] Clause 3. The assay of clause 1 or clause 2, wherein the influenza virus is avian influenza and, optionally, highly pathogenic avian influenza (HPAI). [00114] Clause 4. The assay of clause 2, wherein the influenza A virus is of hemagglutinin (HA) subtype H5 or HA subtype H7. [00115] Clause 5. The assay of any of the foregoing clauses, wherein the influenza virus is of neuraminidase (NA) subtype N1. [00116] Clause 6. The assay of any of the foregoing clauses, wherein the influenza virus is selected from the group consisting of H5N1, H5N2, H7N3, and H7N9. [00117] Clause 7. The assay of any of the foregoing clauses, wherein the assay processes and provides a visual result in 60 minutes or less, the visual result indicative of the presence or absence of the influenza virus in the sample. [00118] Clause 8. The assay of clause 7, wherein the visual result is a color-coded or colorimetric result. [00119] Clause 9. The assay of any of the foregoing clauses, wherein the sample is an oropharyngeal sample, a blood sample, a mammary secretion, a nasal sample, or an environmental water sample. [00120] Clause 10. The assay of any of the foregoing clauses, wherein the sample is obtained from a subject that is a mammal or a bird. [00121] Clause 11. The assay of any of the foregoing clauses, wherein the subject is avian, bovine, human, or swine. [00122] Clause 12. The assay of any of the foregoing clauses, wherein the subject is housed in a high-density animal facility selected from the group consisting of a poultry production facility, a swine production facility, a bovine production facility, a zoological facility or other captive wildlife facility, and any other intensive animal-housing environment suitable for the maintenance or exhibition of animals in captivity. [00123] Clause 13. The assay of any of the foregoing clauses, further comprising a pH- or magnesium-based indicator. [00124] Clause 14. The assay of any of the foregoing clauses, wherein each of the RT- LAMP primer sets has a limit of detection (LOD) of at least 500 copies/reaction. [00125] Clause 15. The assay of any of the foregoing clauses, wherein each of the one or more RT-LAMP primer sets is coupled with a colorimetric reagent. [00126] Clause 16. The assay of clause 15, wherein the colorimetric reagent is pH sensitive or magnesium sensitive.
70817-02 [00127] Clause 17. The assay of clause 15 or clause 16, wherein the colorimetric reagent is phenol red. [00128] Clause 18. The assay of any of the foregoing clauses, wherein each RT-LAMP primer set is at least about 98% specific to the targeted RNA fragment. [00129] Clause 19. The assay of any of the foregoing clauses, further comprising a fluorescent indicator. [00130] Clause 20. The assay of any of the foregoing clauses, wherein each of the least one RT-LAMP primer set comprises 4 to 6 primers. [00131] Clause 21. The assay of any of the foregoing clauses, wherein the assay is a paper- based RT-LAMP assay. [00132] Clause 22. The assay of any of the foregoing clauses, wherein the assay is paper- based and comprises: at least one reaction strip affixed to a backing; and optionally, one or more spacers positioned between each reaction strip; wherein each reaction strip is loaded with, coated with, embedded in, or otherwise associated with at least one RT-LAMP primer set and a master mix. [00133] Clause 23. The assay of clause 22, further comprising a control strip affixed to the backing, wherein one or more spacers are optionally positioned between the control strip and each reaction strip. [00134] Clause 24. A method for identification of the presence or absence of an influenza virus in a sample comprising: providing the assay of any one of clauses 1-23; obtaining a sample from a subject; combining the sample and the at least one RT-LAMP primer set; heating the combination to initiate amplification of the targeted RNA fragment; and detecting a visual result in the heated combination indicative of the presence or absence of the targeted influenza virus in the sample. [00135] Clause 25. The method of clause 24, wherein the visual result is provided in 60 minutes or less of initiating the heating step. [00136] Clause 26. The method of clause 24 or clause 25, wherein the sample is an unprocessed biofluid of the subject. [00137] Clause 27. The method of any one of clauses 24-26, wherein the sample is an unprocessed environmental water sample, an unprocessed oropharyngeal sample, an unprocessed nasal sample, an unprocessed mammary secretion sample, or an unprocessed blood sample. [00138] Clause 28. The method of any one of clauses 24-27, wherein detecting a visual result further comprises one or more of: measuring a relative clarity of the heated combination using a turbidimeter; and analyzing colorimetric data in the visual result using one or more of a fluorescent reader, an ultraviolet light reader, or camera.
70817-02 [00139] Clause 29. The method of any one of clauses 24-28, wherein if the visual result is indicative of the presence of the targeted influenza virus, the method further comprises treating the subject for an influenza viral infection. [00140] Clause 30. The method of any one of clauses 24-29, wherein the at least one RT- LAMP primer set is coupled with a colorimetric reagent that is pH sensitive or magnesium sensitive. [00141] Clause 31. The method of clause 30, wherein the colorimetric agent is phenol red. [00142] Clause 32. The method of clause 29, wherein treating the subject comprises isolating the subject and/or administering a therapeutically effective amount of an antiviral medication to the subject. [00143] Clause 33. A kit comprising: one or more assays of any one of clauses 1-23; at least one swab for obtaining a sample; and a heating element to initiate amplification of the targeted RNA fragment when the at least one RT-LAMP primer set of the assay and the sample are combined. [00144] Clause 34. The kit of clause 33, further comprising: a fluorescent indicator; and a fluorescent reader, an ultraviolet light reader, or a camera to provide colorimetric result data indicative of the presence or absence of the targeted influenza virus in the sample. [00145] Clause 35. The kit of clause 34, further comprising a sealable container with a transport media therein. [00146] Clause 36. The kit of any one of clauses 33-35, wherein the kit is portable and capable of use in a non-laboratory setting. [00147] Clause 37. The kit of any one of clauses 33-36, wherein the heating element is a water bath. [00148] Clause 38. The kit of any one of clauses 33-37, wherein the at least one RT-LAMP primer set is coupled with a colorimetric reagent that is pH sensitive or magnesium sensitive. [00149] Clause 39. The kit of any one of clauses 33-38, further comprising a dropper. [00150] Clause 40. The kit of any one of clauses 33-39, further comprising a sealable container for receiving the paper-based assay of clause 22 or clause 23. [00151] Clause 41. Use of the one or more RT-LAMP assays of any one of clauses 1-23 in detecting the presence or absence of a targeted influenza virus in a sample. [00152] Clause 42. The use of clause 41, wherein the targeted influenza virus is an avian influenza virus. [00153] Clause 43. The use of clause 41, wherein the targeted influenza virus is an influenza A virus.
70817-02 [00154] Clause 44. A method of detecting influenza in a sample, the method comprising amplifying a targeted RNA fragment of an influenza virus in the sample using at least one RT- LAMP primer set comprising one or more primer sets A-L in Table 2, or primers having a nucleotide sequence identical to, or having at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, each primer sequence of the corresponding primer set A-L in Table 2; and/or at least one primer or probe sequence identical to, or having at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, one or more of SEQ ID NOS: 76-78. [00155] Clause 45. The method of clause 44, wherein the influenza virus is an influenza A virus of hemagglutinin (HA) subtype H5 or HA subtype H7. EXAMPLES [00156] The following examples illustrate certain specific embodiments of the present disclosure and are not meant to limit the scope of the claimed invention in any way. EXAMPLE 1 Design of RT-LAMP Primers for Influenza Virus [00157] For avian influenza virus (AIV) detection, multiple studies have targeted conserved regions of the HA gene from H5 subtype viruses. For example, Hu et al. (2017) developed a digital LAMP (dLAMP) assay utilizing microfluidic droplet technology and demonstrated sensitive detection of 10 copies/μL of in vitro transcribed H5 RNA. Hu et al., Absolute quantification of H5-subtype avian influenza viruses using droplet digital loop-mediated isothermal amplification, Analytical Chemistry 89: 745-750 (2017). Table 1 summarizes previously reported LAMP-based assays for the detection of different subtypes of AIV, including the target gene, limit of detection (LOD), nucleic acid template type, and detection modality used. Detection readouts across the literature range from real-time fluorescence and turbidity to colorimetric changes, gel electrophoresis, lateral flow assays (LFA), and microarray integration. Detection methods included real-time fluorescent assays, turbidity assays, gel electrophoresis, digital LAMP (dLAMP), visual observation by colorimetry, integration with lateral flow assays (LFA), and microarrays. [00158] Table 1. Conventional LAMP assays far targeting different AIVs and their subtypes, including their target genes, LODs, type of template, and the detection method.
70817-02 Target Limit of Template Type of LAMP Assay Detection Gene/s Detection Type HA H9 10 IVT RNA A l l t h i e
[00159] To address the limitations of conventional diagnostic techniques, a reverse- transcription (RT)-LAMP method was developed that is capable of detecting various influenza viruses, including H5 subtype avian influenza virus (AIV), with a LOD as low as approximately 500 copies per reaction (based on a 25 µL sample). The RT-LAMP primers were designed using synthetic RNA corresponding to a genomic region that is highly conserved among influenza
70817-02 viruses, including those capable of zoonotic transmission. Sequence selection took into account broad representation from AIV isolates collected from diverse poultry populations across multiple geographic regions worldwide. [00160] The RT-LAMP primers were rationally designed to specifically amplify a selected hemagglutinin (HA) region of the viral genome. The target region was identified by comparative analysis of publicly available sequence data deposited in the Global Initiative on Sharing All Influenza Data (GISAID) database. Elbe & Buckland-Merrett, Data, disease and diplomacy: GISAID’s innovative contribution to global health, Global Challenges 1(1): 33-46 (2017). The selected HA region is conserved across genetically distinct H5N1 isolates and shares homology with HA regions from other influenza A subtypes, thereby providing a basis for cross-subtype detection. In particular, the HA region of AIV type A/H5N1 (GISAID Accession No. EPI1985974) from viral isolate A/turkey/Indiana/22-003707-003/2022 (|HA|4|WSS3056019) was selected as the template for synthetic gene construction. [00161] The H5 subtype HPAIV.HA gene of the selected highly pathogenic avian influenza virus (HPAIV) was synthesized and cloned into the pBluescript II KS (+) plasmid backbone (Genescript Biotech, USA), positioned downstream of a T7 promoter sequence and followed by a T7 terminator sequence to enable in vitro transcription of RNA templates for assay development and validation. The use of a conserved HA sequence in this context allows the resulting primers to be broadly applicable for the detection of diverse influenza viruses, including those with zoonotic potential, while maintaining high specificity for the intended targets. [00162] Restriction sites for SacI and NotI were engineered upstream and downstream of the HA gene insert, respectively. The recombinant plasmid carrying the target HA gene insert was then linearized with NotI-HF® (NEB R3189; Ipswich, MA, USA) in rCutSmart™ buffer for 15 minutes at the manufacturer-recommended temperature. The linearized plasmid DNA was purified using the Wizard® Clean-up System (Promega A7280; Madison, WI, USA) to obtain high-quality template DNA suitable for subsequent in vitro transcription. [00163] The purified, linearized DNA (including the T7 promoter) was then used as a template for in vitro transcription using HiScribe® T7 Quick High Yield RNA Synthesis Kit (NEB E2050; Ipswich, MA, USA) per the supplier's instructions. The in vitro-transcribed RNA was treated with 2 µl of DNase enzyme in the supplied DNase buffer for 30 minutes to remove any residual plasmid template DNA. Following DNase treatment, the RNA was purified using Monarch® RNA Clean-Up Kit (NEB T2030; Ipswich, MA, USA) according to the manufacturer’s protocol. RNA concentration and purity were determined using the NanoDrop™ 8000 spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, Delaware).
70817-02 [00164] Multiple sequence alignment of circulating H5 subtype AIV HA sequences from the National Center for Biotechnology Information (NCBI) database was performed using ClustalW (default parameters) to identify conserved regions within the target HA gene. These conserved sequences were used as the basis for primer design, ensuring broad strain coverage while minimizing the potential for cross-reactivity with non-target organisms. Based on this analysis, primers were designed using publicly available Primer Explorer V5 software (Eiken Chemical Co., Ltd., Tokyo, JP), with manual incorporation of degenerate nucleotide positions where sequence variability was observed. This design strategy enhances assay sensitivity and specificity for the detection of multiple circulating variants under standard reverse transcription polymerase chain reaction (RT-PCR) and fluorometric LAMP conditions. [00165] The specificity of all primer sets was evaluated using the BLASTN algorithm (Basic Local Alignment Search Tool, NCBI) against the nucleotide database to confirm target-specific binding and to assess potential cross-reactivity with non-target sequences. [00166] The resulting primer sets, listed in Table 2, were designated as HPAIV.HA.x, where HPAI refers to the target organism, HA refers to the target gene, and x is an arbitrary index that increases by one for each new primer set designed. [00167] All designed primer sets included loop primers (LF/LB) to increase reaction speed, and were synthesized and desalted by Invitrogen (Waltham, MA, USA). The qPCR primers and probes for the HPAIV strains listed in Table 2 were either from the World Health Organization (WHO)-recommended primer-probe set or newly designed using the PrimerQuest™ tool (Integrated DNA Technologies, Coralville, IA, USA (“IDT”)). All qPCR primers were synthesized by IDT in desalted form, whereas the probes were high-performance liquid chromatography (HPLC)-purified and configured as double-quenched probes to reduce background fluorescence and improve signal-to-noise ratio. [00168] Table 2. List of RT-LAMP primers designed for the HA target gene. Primer Primer Sequenc SEQ ID S t ID e (5'-3'） NO
70817-02 AATTCATCAGAGTGCCGGATTTTTCAGAG 10 HPAIV.HA.2_BIP GTCATTAGCTGGRTT HPAIVHA2LF A AT A TA A
70817-02 GTGTAGCTGGATGGCTCCTTTTTTCCGGC 46 HPAIV.HA.8_BIP ACTCTGATGAATT HPAIVHA LF A A A TT TT T
[ ] e prmers se n a e were en screene usng a uoromerc - AMP assay incorporating a double-stranded DNA-specific intercalating dye with spectral properties similar to SYTO9 (FIG.1). The dye was selected for its high fluorescence yield upon binding to double-stranded DNA, enabling real-time monitoring of RT-LAMP product accumulation throughout the reaction. The master mix for each reaction consisted of 2.5 µl of 10× HA LAMP primer mix, 12.5 µl of WarmStart® 2X LAMP Master Mix E1700 LAMP Kit (NEB, Ipswich, MA, USA), and 5 µl 5× LAMP fluorescent dye (diluted in nuclease-free water from 50X LAMP fluorescent dye provided with the kit) per reaction. From this master mix, 20 µl was dispensed evenly into each well of a 96-well plate, followed by 5 µl of either purified RNA template (at
70817-02 varying concentrations) or molecular-grade nuclease-free water for no-template control (NTC) reactions. The plate was capped using Versicap™ mat cap strips (Thermo Fisher Scientific Inc., Pittsburgh, PA, USA; Catalog No AB1820100) and placed in a qTower 3G Touch or qTower 3G real-time thermal cycler (Analytic Jena GmbH+Co. KG, Jena, Thuringia, Germany). Reactions were incubated at 65 °C with a ramp rate of 0.1 °C/second for 60 minutes. Real-time fluorescence was detected on the blue channel using settings for the FAM dye settings, with fluorescence measurements acquired every 60 seconds during the reactions. [00170] All assays were run in four technical replicates containing 5^µL of either 2.0^×^10³ copies/reaction or 105 copies/reaction of in vitro transcribed (IVT) synthetic RNA corresponding to the HA gene in the positive reactions, with the only other variable being the primer set used. For the NTC reactions, 5 µL of nuclease-free water was substituted for the synthetic HA gene IVT RNA. RT-LAMP reactions were carried out exactly as described above and fluorescence was measured continuously during amplification to assess primer performance. Only primer sets that yielded no amplification signal in the NTC reactions (indicating absence of false positives) and demonstrated robust amplification in the positive reactions were selected for further analysis. [00171] Primer sets were then scored according to a weighted schema previously described in Pascual-Garrigos et al., On-farm colorimetric detection of Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni in crude bovine nasal samples, Veterinary Research 52(1): 126 (2021). Briefly, primer sets were scored based upon the average reaction time, standard deviation of reaction time, average maximum fluorescent intensity, and standard deviation of maximum fluorescent intensity across all four replicates, as well as the number of false positives with weights of 20%, 15%, 5%, 5%, and 55% of the primer set score, respectively (The HPAIV.HA.5 RT-LAMP primer set (SEQ ID NOS: 25-30), in particular, showed high specificity for HPAIV.HA, excluding viruses from bovine and avian pathogens. [00172] ). [00173] Table 3. Performance characteristics of designed RT-LAMP primer sets for screening purposes to select the best-performing RT-LAMP primer sets for further downstream assays. Amplification Reaction Intensit Time False Positive Points Awarded ll .7
70817-02 (SEQ ID NOS: 31-36) HPAIV.HA.5 4 86989.87 7585.11 7 0 0 6 12 18 24 96.0 .0 .5 .3 .8 .6 .5 .1 .1 .0 .4
[00174] The reaction time for an individual reaction was defined as the time at which the second derivative of the time rate of change of the fluorescent intensity reached a maximum (i.e., roughly when the exponential phase first began). [00175] False positives were determined as any signal in an NTC reaction that displayed non-negligible fluorescent intensity increases over the course of the reaction and that increased above 10% of the maximum intensity of the instrument (approximately 140,000 RFU) in a negative reaction. False positives were penalized with increasing severity as the number of false positives increased. Primer sets were then sorted based on the total score. Primer sets scoring above 95 were selected and one additional primer set was selected if downstream screening failed. [00176] Under these criteria, primers HA.11 and HA.12 were deemed unsuitable for further analysis because they generated amplification signals in the NTC reactions, indicative of false- positive results. Although these primer sets also produced strong fluorescence signals and successfully amplified the positive control reactions, the occurrence of NTC amplification rendered them unreliable for diagnostic use.
70817-02 [00177] In contrast, the primer sets HPAIV.HA.1 – HPAIV.HA.10 exhibited no amplification in the NTC reactions, confirming the absence of false positive results, while producing strong fluorescence signals and consistently amplifying the positive controls. To compare performance, all primer sets were evaluated under identical conditions: final primer concentrations of 1.6 µM FIP/BIP, 0.2 µM F3/B3, 0.4 µM LoopF/LoopB, reaction mixture volume of 25 μL, incubation at 65 °C, and identical template input of 105 copies/reaction. Performance was assessed based on maximal fluorescence intensity, time to positive signal (TTP), and absence of false positives in the NTC reactions. Among these, the H5 subtype HPAIV.HA.4, H5 subtype HPAIV.HA.5, H5 subtype HPAIV.HA.6, and H5 subtype HPAIV.HA.7 primer sets demonstrated the highest amplification efficiency and most rapid detection, as evidenced by the scoring results summarized in Table 3. The HPAIV.HA.5 RT-LAMP primer set (SEQ ID NOS: 25-30), in particular, showed high specificity for HPAIV.HA, excluding viruses from bovine and avian pathogens. EXAMPLE 2 LAMP Limit of Detection Studies [00178] RT-LAMP & qLAMP Assays [00179] The limit of detection (LOD) of the RT-LAMP assay for the detection of H5 subtype AIV was evaluated using four selected primer sets (H5 subtype HPAIV.HA.4, H5 subtype HPAIV.HA.5, H5 subtype HPAIV.HA.6, and H5 subtype HPAIV.HA.8) in a fluorescent LOD study. Briefly, the LODs for each candidate primer set were determined using serial dilutions of in vitro-transcribed HA RNA (HA-IVT) in nuclease-free water. Dilution series were prepared corresponding to 1,980, 500, 250, 125, 50, 25, 5, and 1 copies/reaction in 25 µL total reaction volumes. RT-LAMP reactions were performed under the same assay conditions described above for primer screening and reaction plates were imaged at defined time points using a benchtop flatbed scanner (Epson Perfection V800 Photo Color, Epson America Inc., Long Beach, CA, USA) to document visible color changes in the reaction mixtures. The LOD was defined as the lowest RNA concentration at which all replicate reactions demonstrated a distinct, unambiguous color change. [00180] In parallel, the quantitative LAMP (qLAMP) assay was evaluated for analytic sensitivity by performing serial dilutions of the IVT-RNA template. The purpose was to determine the assay’s detection capability at low copy numbers and to establish the minimum number of target copies that could be reliably detected under the optimized qLAMP conditions. [00181] Across all primer sets tested, LOD values ranged from 250 to 18,900 copies per reaction. Specifically, the observed LODs for primer sets H5 subtype HPAIV.HA.4, 5, 6, and 8
70817-02 were 500, 500, 18,900, and 1,890 copies/reaction, respectively (FIG.2A-2D). Overall, the assay demonstrated a LOD of approximately 500 copies/reaction (25 µL), supporting its suitability for sensitive detection of H5 subtype AIV. This LOD is superior that achieved by conventional RT- PCR assays under comparable conditions, which typically range from 1,000 to 10,000 copies per reaction. Imai et al., Development of H5-RT-LAMP (loop-mediated isothermal amplification) system for rapid diagnosis of H5 avian influenza virus infection, Vaccine 24: 6679-6682 (2006); Poon et al., Detection of human influenza A viruses by loop-mediated isothermal amplification, J Clinical Microbiology 43: 427-430 (2005). Furthermore, the assay also demonstrated performance exceeding that reported for certain multiplex RT-qPCR assays. For example, Le^et^al. reported a detection limit of approximately 50^copies/µL (equating to about 1,000^copies per reaction). Le et al., Development of a multiplex RT-qPCR for the detection of different clades of avian influenza in poultry, Viruses 12: 100 (2020). In addition, RT-qPCR assays generally required specialized instrumentation, trained personnel, and controlled laboratory environments, and are therefore typically unsuitable for field deployment. [00182] The RT-LAMP assay described herein achieves a balance between sensitivity (limit of detection) and practical applicability, making it a valuable tool for the rapid identification of AIV, for example. Although the preferred operating temperature range for Bst DNA polymerase is approximately 60-65^°C, no additional reaction-temperature optimization was conducted because an intended use involves detection of multiple targets (e.g., multiple genes or pathogens) in a multiplexing format. Accordingly, a reaction temperature of 65^°C was selected (see methodologies described in Example 1), as it represents the optimal temperature for Bst DNA polymerase in paper-based RT-LAMP assays and provides conditions conducive to achieving maximum amplification efficiency across all intended targets. [00183] The two primer sets that demonstrated the lowest fluorescent LOD values (i.e., highest analytical sensitivity) in the previous LOD assays, as compared to all other primer sets evaluated – HPAIV.HA.4 and HPAIV.HA.5 – were subsequently subjected to further testing using the colorimetric liquid LAMP LOD determination method. This secondary evaluation was performed to confirm their sensitivity performance across different detection modalities and to assess their suitability for use in both fluorescence-based and colorimetric diagnostic formats. [00184] Colorimetric Liquid RT-LAMP Assay [00185] For the colorimetric liquid RT-LAMP assays, a pH-dependent master mix from New England BioLabs® Inc. (Ipswich, MA, USA) was used. This formulation incorporated the highly modified Bst 2.0 WarmStart® DNA Polymerase in a proprietary buffer containing magnesium sulfate (MgSO4) and phenol red, a visible pH-sensitive indicator dye. This DNA polymerase is a strand-shifting DNA polymerase I from Bacillus stearothermophilus (commonly
70817-02 referred to as Bst DNA polymerase or Bst 2.0), which has been genetically engineered to exhibit high catalytic activity under isothermal conditions without requiring a high-temperature denaturation step. The WarmStart® Colorimetric LAMP 2X Master Mix (DNA and RNA) (NEB, USA) also comprises reverse transcriptase (RTx) and enables rapid and highly reliable detection of both DNA (LAMP) and RNA (RT-LAMP). Furthermore, in the WarmStart® formulation (New England BioLabs, Ipswich, MA, USA), the enzyme is thermally activatable – remaining inactive at ambient or low temperatures, thereby preventing nonspecific amplification during reaction setup – and becomes catalytically active only after the reaction temperature reaches approximately 40 ºC. Accordingly, the mix enables visual detection of amplification products based on the pH shift generated during DNA synthesis, producing a distinct color change from pink to yellow that correlates with positive amplification events. More precisely, phenol red undergoes a distinct color change from pink to yellow when the pH of the reaction mixture decreases due to the release of a large number of protons during extensive nucleic acid synthesis catalyzed by Bst 2.0 WarmStart® DNA Polymerase. This pronounced color transition, coupled with the enzyme’s temperature- dependent activation (remaining inactive at lower temperatures and becoming active only upon heating) enhances the visibility and detectability of RT-LAMP results, making this master mix particularly advantageous for colorimetric endpoint analysis. [00186] The controlled activation minimized nonspecific background amplification and improved overall assay specificity in the RT-LAMP reactions. With this combination of enzyme and pH indicator, amplification was performed using a simple heat-maintaining device (e.g., in a water bath at 65 ºC), and the resulting color change in the amplicon was reliably detected by the naked eye within a short period of time, eliminating the need for complex instrumentation and/or a post-amplification processing step. [00187] Accordingly, the colorimetric RT-LAMP assay was performed in accordance with the manufacturer’s instructions for the WarmStart® Colorimetric LAMP 2X Master Mix Kit containing UDG (New England BioLabs, Ipswich, MA, USA; M1804), which included the pH-sensitive indicator dye previously described. The inclusion of Antarctic Thermolabile uracil-DNA glycosylase (UDG) and/or deoxyuridine triphosphate (dUTP) provided an additional safeguard against carryover contamination by degrading uracil-containing amplification products from previous reactions, thereby improving assay specificity and reducing false-positive results. The reactions consisted of 2.5 μL 10× primer mix (1.6 μM FIP and BIP primers, 0.4 μM each of LF and LB primers, 0.2 μM each of F3 and B3 primers), 12.5 μL WarmStart® Colorimetric LAMP 2X Master Mix, 5 μL nuclease-free water, and 5 μL template RNA or molecular biology water for NTCs in a total volume of 25 μL. All reactions were incubated in a qPCR cycler at 65 °C for 60
70817-02 minutes with the lid heated to 95 °C. Since the reaction was performed in a water bath at 65 ºC, all samples were incubated simultaneously for detection without needing a thermocycler. [00188] Both primer sets tested prompted a pink to yellow color change. The H5 subtype HPAIV.HA.5 primer set showed a LOD of 500 copies/reaction, while the other primer set (H5 subtype HPAIV.HA.4) exhibited a higher LOD than 1,890 copies/reaction (FIGS. 3A and 3B). These results indicate that the H5 subtype HPAIV.HA.5 primer set was the preferred primer set for H5 subtype AIV detection. EXAMPLE 3 Cross-Reactivity of HPAIV.HA.5 With Avian and Bovine Pathogens [00189] A comprehensive analytical specificity assessment of the selected HPAIV.HA.5 RT-LAMP primer set (SEQ ID NOS: 25-30) was conducted using genomic nucleic acid extracts from a broad panel of bovine and avian pathogens to evaluate potential cross-reactivity. The test panel included: bovine rhinitis A virus (BRAV; SD-1 strain, ATCC VR-668), bovine rhinits B virus (BRBV; EC-11 strain, ATCC VR-1806), bovine coronavirus (BCV; Mebus strain, BEI NR- 445), influenza D virus (IDV; D/bovine/660, provided by Dr. Benjamin Hause, South Dakota State University, USA), bovine adenovirus-3 (BAV-3; WBR-1 strain, ATCC VR-639), bovine herpesvirus-1 (BHV-1; Los Angeles strain; ATCC VR-188), bovine viral diarrhea virus-1 (BVDV-1; NADL strain, ATCC VR-534), bovine adenovirus-7 (BAV-7; Fukuroi strain, ATCC VR-768), bovine respiratory syncytial virus (BRSV; A 51908 strain, ATCC VR-1339), and bovine parainfluenza virus-3 (BPI3V; SF-4 strain, BEI NR-3234). In addition, six avian pathogens were also evaluated for cross-reactivity tests: Newcastle disease virus (NDV; non-virulent strain), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILT), Mycoplasma gallisepticum (MG), Ornithobacterium rhinotracheale (ORT), and Avibacterium paragallinarum (Av. Paragallinarum). Prior to this specificity analysis, all genomic extract materials were confirmed using qPCR, and 106 copies/reaction were used for input as templates. [00190] As shown in FIGS. 4A and 4B, for HPAIV.HA.5, fluorescence indicative of the qLAMP assay was exclusively observed in reactions that utilized HPAIV.HA.5 IVT templates. No fluorescence signals were detected in reactions containing genomic extracts from the ten other pathogens tested. These results support the assay’s ability to accurately identify HPAIV.HA.5, with no evidence of cross-reactivity with other bovine viral pathogens or the non-target avian pathogens. EXAMPLE 4
70817-02 Colorimetric Paper-Based RT-LAMP Assay [00191] The efficiency of a paper-based RT-LAMP assay using the same primer set H5 subtype HPAIV.HA.5 was also evaluated at varying template concentrations. A formulation previously described by the present inventors for an on-paper colorimetric assay for detecting SARS-CoV-2 was utilized, as described in Davidson et al. (2021), supra, and Wang et al., Fabrication of a paper-based colorimetric molecular test for SARS-CoV-2, MethodsX 8: 101586 (2021). [00192] Briefly, a paper-based RT-LAMP assay was constructed by assembling reading and reaction layers together, with spacers optionally positioned between to prevent crosstalk between the strips as shown in FIG.5, subpart B. The strips (or reading and reaction layers) were 5 mm by 5 mm of chromatography paper (Ahlstrom-Munksjo Grade Double White Opaque HIPS Polystyrene Litho Grade). A 3^mil optically clear MELINEX® polyester (PET) film (Tekra, MELINEX®^454) was used to fabricate the reading area and double-sided adhesive was applied to affix the reaction strips onto the MELINEX backing. The strips (or reading and reaction layers) were then separated by one or more 2.5 x 6 mm 20-mil polystyrene spacer(s) (Tekra Double White Opquae HIPS Polystyrene Litho Grade). [00193] Upon device assembly, 25 µL of master mix was added to each pad along with the appropriate primer set as described in Example 3. The paper-based RT-LAMP master mix included MgSO4 (2 mM), KCl (50 mM), WarmStart® RTx reverse transcriptase (0.3 U/µL), Antarctic thermolabile UDG (0.0004 U/µL), dUTP (0.14 mM), dNTP mixture (1.4 mM each dNTP), phenol red (0.25 mM), Tween® 20 (1% v/v), trehalose (10% w/v), BSA (500 µg/mL), and betaine (20 mM). [00194] The devices were then left for 1 hour to dry, after which 20 µL of either RNA-free water or a solution containing RNA was added to each individually for rehydration. The rehydrated devices were then placed in 2 x 2 inch 2 mil polypropylene bags (ULINE S-17954). The entire assay process, from preparing the cartridge to obtaining assay results, is presented in FIG.5, subpart A. [00195] To maintain a constant temperature of 149 ºF (or 65 ºC), a 12-quart EVERIE Sous Vide Container (Amazon B07GQWP85C) was filled with water and an Anova culinary AN500- US00 Sous Vide Precision Cooker (Amazon B07WQ4M5TS) was used for heating. Each appliance bag was securely attached to a standard clear film (617993, Office Depot, USA) and placed within a larger 1-gallon Savour Sous Vide Cooking Bag (Amazon B07NCSXMNN). After heating the water bath, the sealed and capped cooking bag was inserted. The samples were allowed to remain in the water for 60 minutes. Images of the pads were captured at 0 and 60 minutes with
70817-02 an Epson Perfection V800 Photo Scanner (Amazon B11B223201) configured to Pro Mode, 38- bit color image type, with a resolution of 600 dpi. [00196] The paper-based RT-LAMP assay's LOD was comparable to that of the colorimetric liquid LAMP assay, indicating its reliable detection ability with a low detection limit (500 copies/reaction; FIG.6). [00197] The results of RT-LAMP primer screening and detection limit testing demonstrate the optimized RT-LAMP assay's potential for detecting H5 subtype AIV, which could have significant implications for clinical diagnosis and epidemiological studies of H5 subtype AIV. The optimized RT-LAMP assay is positioned to be an essential diagnostic tool for the effective control of H5 subtype AIV through early detection. EXAMPLE 5 Evaluation of Paper RT-LAMP Assays on Contrived Samples [00198] The paper RT-LAMP assays of Example 4 were assembled, comprising 3*3 paper size and having dried 7.5 µL of master mix on the paper RT-LAMP. [00199] 1000 copies per reaction (2x the LOD in water) of HPAIV-IVT were spiked into resuspended 30 oropharyngeal swabs collected from healthy poultry for use in the assay, while another 30 samples were kept negative without spiking. Two assays were conducted – one with 7.5 µL of spiked samples added to the dried reaction pad (LAMP reagents on paper) of a paper LAMP assay and the other with .5 µL of non-spiked samples added to the dried reaction pad of another paper LAMP assay. The same samples were also tested with two qPCR assays for comparison and diagnostic assay evaluation. [00200] The paper RT-LAMP assays hereof successfully detected 1,000 copies of synthetic HPAIV-IVT spiked into resuspended nasal swab samples, even in the presence of inhibitors (FIGS.7A-7F). This confirmed 100% analytical sensitivity, analytical specificity, and accuracy at 1,000 copies/reaction (Table 4). When used on spiked oropharyngeal swabs, the paper-based assays hereof reliably identified HPAIV without false positives or negatives (comparable to both WHO_qPCR and the newly designed qPCR primers/probe described herein). This capability also eliminates the need for additional sample processing, making it particularly valuable for rapid and accurate HPAIV detection in resource-limited clinical settings. The high overall accuracy of 100% between the paper RT-LAMP and qPCR assays highlights its potential as an effective tool for diagnosing and monitoring HPAIV directly from oropharyngeal swabs. [00201] Table 4. Diagnostic performance of the paper RT-LAMP assay and qPCR assays (WHO or this study) in assessing spiked (30) and non-spiked (30) oropharyngeal swab samples obtained from poultry.
70817-02 qPCR (WHO or this study) Positive Negative
One-Step RT-qPCR Assay [00202] A one-step RT-qPCR assay was conducted utilizing the NEB Luna Universal One- Step RT-qPCR Kit (NEB #3006) and a qTower 3G Touch or qTower 3G real-time thermal cycler (Analytic Jena GmbH+Co. KG, Jena, Thuringia, Germany). The reaction mixture was prepared in accordance with the manufacturer’s instructions. Each 20 µL reaction contained the following components: 10 µL of Luna Universal One-Step Reaction Mix (2X), 1 µL of Luna WarmStart® RT Enzyme Mix (20X), 0.8 µL of Forward Primer (10 µM), 0.8 µL of Reverse Primer (10 µM), 0.4 µL of Probe (10 µM), 5 µL of RNA template/oropharyngeal swab sample, and 3 µL of nuclease-free water. The primers and probes used in this study are shown in Table 5. [00203] Table 5. qPCR Primers and Probes Targeting HPAIV-HA.5 Nucleotide sequence Primer/Probe References CGATCTAGAYGGGGTGAARCCTC H5HA-205- Sultankulova et al za d n
70817-02 /56-FAM/CG GAA ACC C/ZEN/A ATG HPAIV_H5-P This study TGC GAC GAATT/3IABkFQ/
ling conditions: Reverse transcription at 55 °C for 10 minutes, followed by initial denaturation at 95 °C for 10 seconds and annealing/extension at 60 °C for 45 seconds. [00205] Reactions were incubated at with a ramp rate of 0.1 °C/second for 60 minutes. Real-time fluorescence was detected on the blue channel using settings for the FAM dye settings, with fluorescence measurements acquired every 60 seconds during the reactions.