LAMP primer set and kit for detection of influenza a virus

Abstract

The application relates to the field of molecular biology and discloses a LAMP primer group and a kit for detecting influenza A virus H1N1. The primer group comprises an outer primer pair, an inner primer pair and a loop primer pair; the outer primer pair comprises a forward outer primer with a nucleotide sequence as shown in SEQ ID NO. 1 and a reverse outer primer with a nucleotide sequence as shown in SEQ ID NO. 2; the inner primer pair comprises a forward inner primer with a nucleotide sequence as shown in SEQ ID NO. 3 and a reverse inner primer with a nucleotide sequence as shown in SEQ ID NO. 4; and the loop primer pair comprises a forward loop primer with a nucleotide sequence as shown in SEQ ID NO. 5 and a reverse loop primer with a nucleotide sequence as shown in SEQ ID NO. 6. The primer group has good thermal stability, is suitable for 70 DEG C high-temperature LAMP amplification, reduces non-specific binding, and has good sensitivity and specificity.

Description

Cross-references to related applications

[0001] This application claims priority to Chinese Patent Application No. 202511562311.1, filed on October 29, 2025, entitled "LAMP Primer Set and Reagent Kit for Detection of Influenza A Virus", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of molecular biology technology, specifically to a LAMP primer set and kit for detecting influenza A virus. Background Technology

[0003] Influenza A is the most common seasonal influenza, caused by the influenza A virus, which is currently the dominant influenza virus active globally. Influenza A viruses belong to the Orthomyxoviridae family and can be classified into several subtypes based on the antigenic characteristics and sequence features of their surface antigens HA (haemagglutinin) and NA (neuraminidase). These include 18 antigenic subtypes of HA (H1-H18) and 11 antigenic subtypes of NA (N1-N11). Influenza A viruses are highly mutated; the nucleotide sequences encoding HA and / or NA are prone to mutation, leading to changes in the antigenic epitopes of HA and / or NA. This causes the original specific immunity in the population to fail, resulting in the highly contagious, rapidly spreading, and easily widespread epidemic characteristics of influenza A. Although influenza is self-limiting, it can still pose a fatal threat to immunocompromised individuals (such as young children, pregnant women, and the elderly). Antiviral treatment administered within 24-48 hours of the onset of influenza can effectively combat the disease. Therefore, early, rapid, and accurate diagnosis of influenza A is crucial for timely intervention, treatment, and control of the disease's spread.

[0004] The clinical significance of influenza virus detection methods largely depends on their speed. Currently, clinically used methods for influenza virus detection include immunofluorescence assays, enzyme immunoassays, immunochromatography, reverse transcription-polymerase chain reaction (RT-PCR), and real-time quantitative PCR. These methods are either time-consuming or require specialized equipment, making widespread application difficult.

[0005] Loop-mediated isothermal amplification (LAMP) is a highly efficient, rapid, and specific nucleic acid amplification technique first proposed by Notomi et al. in 2000 (see "Notomi, T., et al. (2000). Loop-mediated isothermal amplification of DNA. NucleicAcids Research, 28(12), e63."). LAMP utilizes a DNA polymerase with strand displacement activity (such as Bst DNA polymerase) and a set of specifically designed primers to achieve exponential amplification of the target DNA sequence under isothermal conditions (typically 60-65°C). Unlike traditional polymerase chain reaction (PCR), LAMP does not require thermal cycling equipment and only requires a constant temperature heating device to complete the reaction. Therefore, it is particularly suitable for on-site testing or point-of-care diagnosis with limited resources (see the literature "Mori, Y., & Notomi, T. (2009). Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases. Journal of Infection and Chemotherapy, 15(2), 62-69"). Furthermore, due to its simple and rapid operation (usually completed within 30-60 minutes) and low equipment requirements, LAMP technology has been widely used in pathogen detection, food safety monitoring, environmental microbiology analysis, and genetic disease diagnosis (see the literature "Parida, M., Sannarangaiah, S., Dash, PK, Rao, PVL, & Morita, K. (2008). Loop-mediated isothermal amplification (LAMP): principle, features, and future prospects. Journal of Clinical Microbiology, 46(2), 610-614").

[0006] Currently, loop-mediated isothermal amplification (LAMP) technology is typically performed at a constant temperature of 60-65℃. The aerosols from the amplification products easily contaminate the environment, especially in primary laboratories where improper operation can lead to false positive results. Increasing the amplification incubation temperature can reduce aerosol contamination, and high-temperature amplification can effectively reduce primer dimer formation, improving detection specificity, making it particularly suitable for complex samples (such as blood or sputum containing inhibitors). However, the insufficient thermostability of enzymes and primer sets at high temperatures is the main bottleneck of current high-temperature LAMP technology. Existing LAMP primers for influenza A virus H1N1 exhibit insufficient thermostability and are prone to non-specific binding when amplified at high temperatures (e.g., 70℃); simultaneously, high temperatures reduce primer-template binding efficiency, significantly deteriorating the detection limit. Summary of the Invention

[0007] This invention provides a LAMP primer set for the detection of influenza A virus (H1N1) to solve the problems of insufficient primer thermostability, low template binding rate and low detection limit under high temperature amplification conditions.

[0008] In a first aspect, the present invention provides a LAMP primer set for the detection of influenza A virus (H1N1), the primer set comprising an outer primer pair, an inner primer pair, and a loop primer pair; the outer primer pair comprising a forward outer primer with a nucleotide sequence as shown in SEQ ID NO. 1 and a reverse outer primer with a nucleotide sequence as shown in SEQ ID NO. 2; the inner primer pair comprising a forward inner primer with a nucleotide sequence as shown in SEQ ID NO. 3 and a reverse inner primer with a nucleotide sequence as shown in SEQ ID NO. 4; and the loop primer pair comprising a forward loop primer with a nucleotide sequence as shown in SEQ ID NO. 5 and a reverse loop primer with a nucleotide sequence as shown in SEQ ID NO. 6.

[0009] Secondly, the present invention also provides a LAMP high-temperature amplification buffer, wherein the components of the high-temperature amplification buffer include MgSO4, KCl and (NH4)2SO4; the concentration of MgSO4 in the high-temperature amplification buffer is 4.4 mM; the concentration of KCl in the high-temperature amplification buffer is 48 mM; and the concentration of (NH4)2SO4 in the high-temperature amplification buffer is 44 mM.

[0010] In one optional embodiment, the high-temperature amplification buffer further comprises dNTPs and / or fluorescent dyes; the concentration of dNTPs in the high-temperature amplification buffer is 0.48 mM; and the mass percentage concentration of the fluorescent dye in the high-temperature amplification buffer is 8%.

[0011] In one optional embodiment, the high-temperature amplification buffer further comprises dNTPs and / or fluorescent dyes; the concentration of dNTPs in the amplification buffer is 0.48 mM; and the mass percentage concentration of the fluorescent dye in the amplification buffer is 8%, corresponding to 4 μM.

[0012] In one optional embodiment, the high-temperature amplification buffer further comprises a solvent; the solvent is a 20 mM Tris-HCl buffer at pH 8.8.

[0013] Thirdly, the present invention also provides a kit for detecting influenza A virus (H1N1), the kit comprising the above-mentioned primer set and high-temperature amplification buffer.

[0014] In one optional embodiment, the kit further includes a high-temperature mixed enzyme working solution; the high-temperature mixed enzyme working solution comprises a high-temperature Bst enzyme, a high-temperature RT enzyme, and a high-temperature enzyme diluent.

[0015] In one alternative embodiment, the high-temperature Bst enzyme comprises high-temperature Bst enzyme-093.

[0016] In one alternative embodiment, the high-temperature enzyme diluent comprises Tween-20, BSA, glycerol, and CMIT / MIT.

[0017] Fourthly, the present invention also provides a method for detecting influenza A virus (H1N1), the method being for purposes other than disease diagnosis and treatment, the method comprising: using the above-described kit to detect a sample to be tested.

[0018] In one optional embodiment, the detection method includes: extracting RNA from the sample to be tested to obtain the nucleic acid to be tested; mixing the nucleic acid to be tested, the primer set, the high-temperature amplification buffer, and the high-temperature mixed enzyme working solution to obtain a LAMP reaction system; performing LAMP amplification on the LAMP reaction system using a constant-temperature fluorescence detector; and determining whether influenza A virus H1N1 exists in the sample to be tested based on the real-time fluorescence signal obtained after LAMP amplification.

[0019] In one optional implementation, the LAMP amplification conditions in the detection method are: constant temperature incubation at 68~72℃ for 30~60 min.

[0020] In one optional implementation, the LAMP amplification conditions in the detection method are: incubation at a constant temperature of 70°C for 30-60 minutes.

[0021] In one optional embodiment, the isothermal fluorescence detector includes at least one of real-time quantitative PCR, a dedicated isothermal fluorescence LAMP instrument, and a multifunctional microplate fluorescence detector.

[0022] Fifthly, the present invention also provides the application of the above-mentioned primer set or the above-mentioned high-temperature amplification buffer or the above-mentioned kit or the above-mentioned detection method in the detection of influenza A virus (H1N1), wherein the application is not for the purpose of disease diagnosis and treatment.

[0023] The technical solution of this invention has the following advantages: 1. This invention provides a LAMP primer set for the detection of influenza A virus (H1N1), comprising an outer primer pair, an inner primer pair, and a loop primer pair; the outer primer pair comprises a forward outer primer with the nucleotide sequence shown in SEQ ID NO.1 and a reverse outer primer with the nucleotide sequence shown in SEQ ID NO.2; the inner primer pair comprises a forward inner primer with the nucleotide sequence shown in SEQ ID NO.3 and a reverse inner primer with the nucleotide sequence shown in SEQ ID NO.4; the loop primer pair comprises a forward loop primer with the nucleotide sequence shown in SEQ ID NO.5 and a reverse loop primer with the nucleotide sequence shown in SEQ ID NO.6. The primer set provided by this invention exhibits good thermostability, is suitable for LAMP amplification detection systems at 70°C, reduces non-specific binding, and has good detection sensitivity and specificity. The detection limit of the primer set of this invention reaches 50 C / R, and there is no cross-reactivity with H3N2, influenza B virus (FluB), and human parainfluenza virus type 1 (HPIV1). The primer set of this invention provides support for the clinical diagnosis, epidemiological monitoring, and quality control of vaccine development for H1N1 influenza virus.

[0024] 2. This invention also provides a high-temperature amplification buffer for LAMP, comprising MgSO4, KCl, and (NH4)2SO4; the concentration of MgSO4 in the amplification buffer is 4.4 mM; the concentration of KCl in the amplification buffer is 48 mM; and the concentration of (NH4)2SO4 in the amplification buffer is 44 mM. This invention's high-temperature amplification buffer, based on existing technologies, removes components such as betaine and Tween-20, facilitating subsequent experimental operations with high-temperature reagents, such as lyophilization. Optimization of ion concentrations improves enzyme reactivity, making it more conducive to HA gene amplification. This invention's high-temperature amplification buffer is compatible with lyophilization processes, suitable for rapid on-site detection needs, and can be widely applied in clinical diagnosis, epidemiological monitoring, and food safety testing, providing key technical support for the standardization and industrialization of high-temperature LAMP technology.

[0025] 3. This invention also provides a kit for detecting influenza A virus (H1N1), comprising the aforementioned primer set and a high-temperature amplification buffer. The primer set used in the kit exhibits good thermostability, is suitable for LAMP amplification detection systems at 70°C, reduces non-specific binding, and provides good detection sensitivity and specificity. The high-temperature amplification buffer in the kit has optimized ion concentration, resulting in higher enzyme reactivity and compatibility with lyophilization processes, making it suitable for rapid on-site detection. Therefore, the kit of this invention provides an innovative solution for the rapid diagnosis of influenza A virus. Attached Figure Description

[0026] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0027] Figure 1 This is a primer set amplification performance verification - blank group.

[0028] Figure 2 This is the primer set amplification performance verification - experimental group.

[0029] Figure 3 This is a primer set thermal stability verification - blank group.

[0030] Figure 4 This is the primer set thermal stability verification - experimental group.

[0031] Figure 5 This is a validation of the performance of potassium chloride concentration optimization - blank group.

[0032] Figure 6 This is the experimental group for verifying the performance of potassium chloride concentration optimization.

[0033] Figure 7 This is a validation of the performance of magnesium sulfate concentration optimization - blank group.

[0034] Figure 8 This is the experimental group for verifying the performance of magnesium sulfate concentration optimization.

[0035] Figure 9 This is a blank group for performance verification of ammonium sulfate concentration optimization.

[0036] Figure 10 This is the experimental group for performance verification of ammonium sulfate concentration optimization.

[0037] Figure 11 This is a performance verification of the high-temperature buffer solution.

[0038] Figure 12 The primer set is a sensitivity test; (A) Primer set of Example 1; (B) Primer set of Comparative Example 1-1.

[0039] Figure 13 The detection rate test of primer sets; (A) Primer set of Example 1; (B) Primer set of Comparative Example 1-1.

[0040] Figure 14 This is a specific detection of the primer set in Example 1. Detailed Implementation

[0041] The following embodiments are provided to better understand the present invention, but the following embodiments do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.

[0042] Unless otherwise specified, all experimental steps or conditions in the examples were performed according to conventional experimental procedures and conditions in the art. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0043] Information on reagent materials used in the embodiments of this invention: Enzyme-free water was purchased from Sangon Biotech (Shanghai) Co., Ltd.; the RT-LAMP Dye Assay Kit (UDGplus) (catalog number 13762ES60) was purchased from Yisheng Biotechnology Co., Ltd.; the fluorescent dye SYBR-16 was purchased from Thermo Fisher Scientific, catalog number S7578; for details on the high-temperature Bst enzyme-093, high-temperature RT enzyme, and high-temperature enzyme diluent, please refer to the literature "FADS-Based Directed Evolution of a Robust Bst DNA Polymerase Adapting High-Temperature Loop-Mediated Isothermal Amplification (HT-LAMP)"; the high-temperature enzyme diluent consisted of: 8% (g / 100 mL) Tween-20, 5% (g / 100 mL) BSA, 4% (v / v) glycerol, and 2% (g / 100 mL) Tween-20. CMIT / MIT (isothiazolinone bactericide and preservative, composed of CMIT (5-chloro-2-methyl-4-isothiazolin-3-one) and MIT (2-methyl-4-isothiazolin-3-one) in a mass ratio of 3:1, with enzyme-free water as the solvent) in mL.

[0044] The H1N1 RNA template was purchased from Twist Bioscience, and the primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0045] Example 1: A LAMP primer set for the detection of influenza A virus (H1N1) This embodiment provides a LAMP primer set for the detection of influenza A virus (H1N1). The primer set consists of an outer primer pair, an inner primer pair, and a loop primer pair. The outer primer pair consists of a forward outer primer HA-F3 with a nucleotide sequence as shown in SEQ ID NO.1 and a reverse outer primer HA-B3 with a nucleotide sequence as shown in SEQ ID NO.2. The inner primer pair consists of a forward inner primer HA-FIP with a nucleotide sequence as shown in SEQ ID NO.3 and a reverse inner primer HA-BIP with a nucleotide sequence as shown in SEQ ID NO.4. The loop primer pair consists of a forward loop primer HA-LF with a nucleotide sequence as shown in SEQ ID NO.5 and a reverse loop primer HA-LB with a nucleotide sequence as shown in SEQ ID NO.6. The target sequence of the primer set is the influenza A virus H1N1 HA gene with accession number NC_026433.1.

[0046] Table 1 Primer sequences

[0047] Example 2: A LAMP high-temperature amplification buffer This embodiment provides a high-temperature amplification buffer for LAMP, which is a 20 mM Tris-HCl buffer (pH 8.8) containing 4.4 mM MgSO4, 48 mM KCl, 44 mM (NH4)2SO4, 0.48 mM dNTPs and 2 μL of the fluorescent dye SYBR-16 (corresponding to 4 μM).

[0048] Example 3: A kit for detecting influenza A virus (H1N1) This embodiment provides a kit for detecting influenza A virus (H1N1), which consists of a high-temperature mixed enzyme working solution, a 10× mixed primer working solution (containing the primer set of Example 1), a 4× high-temperature amplification buffer (i.e., a 4-fold concentration of the high-temperature amplification buffer of Example 2), and a negative control group. The high-temperature mixed enzyme working solution comprises high-temperature Bst enzyme-093, high-temperature RT enzyme, and high-temperature enzyme diluent; the concentration of high-temperature Bst enzyme-093 in the high-temperature mixed enzyme working solution is 50 U / μL, and the concentration of high-temperature RT enzyme is 20 U / μL; the preparation method of the high-temperature mixed enzyme working solution is as follows: high-temperature Bst enzyme-093 and high-temperature RT enzyme are diluted and dissolved to concentrations of 50 U / μL and 20 U / μL, respectively, using the high-temperature enzyme diluent. The 10× mixed primer working solution is composed of HA-F3, HA-B3, HA-FIP, HA-BIP, HA-LF, HA-LB and enzyme-free water; the concentrations of HA-F3, HA-B3, HA-FIP, HA-BIP, HA-LF and HA-LB in the 10× mixed primer working solution are 2, 2, 16, 16, 4 and 4 μM, respectively; the preparation method of the 10× mixed primer working solution is as follows: HA-F3 (100 μM, 2 μL), HA-B3 (100 μM, 2 μL), HA-FIP (100 μM, 16 μL), HA-BIP (100 μM, 16 μL), HA-LF (100 μM, 4 μL), HA-LB (100 μM, 4 μL) and enzyme-free water (56 μL) are mixed; The negative control was enzyme-free water.

[0049] Example 4: A method for detecting influenza A virus (H1N1) This embodiment provides a method for detecting influenza A virus (H1N1). The method uses the kit from Example 3, and the detection process includes the following steps: 1. Extract RNA from the sample to be tested to obtain the nucleic acid to be tested; 2. The reagent kit of Example 3 was used to detect the nucleic acid to be tested. The specific operation was as follows: 25 μL of LAMP amplification system was prepared. The specific reagent volumes were: 6.25 μL of 4× high temperature amplification buffer, 2.5 μL of 10× mixed primer working solution, 1 μL of high temperature mixed enzyme working solution, 2 μL of nucleic acid template, and 13.25 μL of enzyme-free water.

[0050] 3. After the system is prepared, use a real-time fluorescence PCR instrument (ABI Q1 real-time fluorescence PCR instrument) to perform high-temperature LAMP amplification at 70℃. Based on the real-time fluorescence signal obtained, determine whether H1N1 can be detected. The conditions for high-temperature LAMP amplification at 70℃ were as follows: incubation at 37℃ for 2-5 min, incubation at 70℃ for 30-60 min, inactivation at 85℃ for 5 min, and melting curve analysis program (95℃, 15s; 60℃, 1 min; 95℃, 1s). The negative control was enzyme-free water.

[0051] Comparative Example 1-1: A LAMP primer set for the detection of influenza A virus (H1N1) This comparative example provides a LAMP primer set for the detection of influenza A virus (H1N1), and the primer set sequences are shown in SEQ ID NO.7~11 (the primer set sequences are shown in Table 2).

[0052] Table 2 Primer sequences

[0053] Comparative Examples 1-2: A LAMP primer set for the detection of influenza A virus (H1N1) This comparative example provides a LAMP primer set for the detection of influenza A virus (H1N1), and the primer set sequences are shown in SEQ ID NO.12~17 (the primer set sequences are shown in Table 3).

[0054] Table 3 Primer sequences

[0055] Comparative Examples 2-1 to 2-7: A LAMP High-Temperature Amplification Buffer This comparative example provides a high-temperature LAMP amplification buffer. Based on Example 2, the concentrations of KCl, MgSO4, and (NH4)2SO4 were adjusted, as detailed below: Comparative Example 2-1: Based on Example 2, the concentrations of KCl, MgSO4 and (NH4)2SO4 were adjusted to 50 mM, 4 mM and 40 mM, respectively; Comparative Examples 2-2 to 2-3: Based on Example 2, the KCl concentration was adjusted to 51 mM and 54 mM, respectively; Comparative Examples 2-4 to 2-5: Based on Example 2, the MgSO4 concentration was adjusted to 4.2 mM and 4.6 mM, respectively; Comparative Examples 2-6 to 2-7: Based on Example 2, the concentration of (NH4)2SO4 was adjusted to 38 mM and 41 mM, respectively.

[0056] Comparative Example 3-1: A kit for detecting influenza A virus (H1N1) This comparative example provides a kit for detecting influenza A virus (H1N1). The kit is based on Example 3, except that the primer sets are replaced with the primer sets of Comparative Example 1-1. The kit consists of a high-temperature mixed enzyme working solution, a 10× mixed primer working solution (containing the primer sets of Comparative Example 1-1), a 4× high-temperature amplification buffer (i.e., 4 times the concentration of the high-temperature amplification buffer of Example 2), and a negative control group. The 10× mixed primer working solution is composed of HA-F3-2, HA-B3-2, HA-FIP-2, HA-BIP-2, HA-LB-2, and enzyme-free water. The concentrations of HA-F3-2, HA-B3-2, HA-FIP-2, HA-BIP-2, and HA-LB-2 in the 10× mixed primer working solution are 2, 2, 16, 16, and 4 μM, respectively. The 10× mixed primer working solution is prepared by mixing HA-F3-2 (100 μM, 2 μL), HA-B3-2 (100 μM, 2 μL), HA-FIP-2 (100 μM, 16 μL), HA-BIP-2 (100 μM, 16 μL), HA-LB-2 (100 μM, 4 μL), and enzyme-free water (60 μL).

[0057] Comparative Example 3-2: A kit for detecting influenza A virus (H1N1) This comparative example provides a kit for detecting influenza A virus (H1N1). The kit is based on Example 3, except that the primer sets are replaced with the primer sets of Comparative Examples 1-2. The kit consists of a high-temperature mixed enzyme working solution, a 10× mixed primer working solution (containing the primer sets of Comparative Examples 1-2), a 4× high-temperature amplification buffer (i.e., 4 times the concentration of the high-temperature amplification buffer of Example 2), and a negative control group. The 10× mixed primer working solution is composed of HA-F3-3, HA-B3-3, HA-FIP-3, HA-BIP-3, HA-LF-3, HA-LB-3, and enzyme-free water. The concentrations of HA-F3-3, HA-B3-3, HA-FIP-3, HA-BIP-3, HA-LF-3, and HA-LB-3 in the 10× mixed primer working solution are 2, 2, 16, 16, 4, and 4 μM, respectively. The preparation method of the 10× mixed primer working solution is as follows: HA-F3-3 (100 μM, 2 μL), HA-B3-3 (100 μM, 2 μL), HA-FIP-3 (100 μM, 16 μL), HA-BIP-3 (100 μM, 16 μL), HA-LF-3 (100 μM, 4 μL), and HA-LB-3 (100 μM, 4 μL) are mixed. Mix 56 μL of enzyme-free water with 5 μL of enzyme-free water.

[0058] Comparative Examples 3-3 to 3-9: A kit for detecting influenza A virus (H1N1) This comparative example provides a kit for detecting influenza A virus (H1N1). The kit is based on Example 3, except that the amplification buffer is replaced with the high-temperature amplification buffer of Comparative Examples 2-1 to 2-7.

[0059] Comparative Examples 4-1 to 4-9: A LAMP Detection Method for Influenza A Virus (H1N1) This comparative example provides a LAMP detection method for influenza A virus (H1N1). The method is based on Example 4, in which the kit of Example 3 is replaced with the kits of Comparative Examples 3-1 to 3-9 respectively.

[0060] Experiment Example 1: Primer set performance test In this experiment, H1N1 RNA was used as the test sample to perform performance tests on the primer sets in Example 1, Comparative Example 1-1, and Comparative Example 1-2. The specific process is as follows: 1. Prepare H1N1 RNA standards (purchased from Twist Bioscience), with an initial concentration of 10. 6 The H1N1 RNA template was diluted to 50 copies / μL with enzyme-free water.

[0061] 2. The RNA template was detected using the one-step RT-LAMP Dye Assay Kit (UDG plus) produced by Yisheng Biotechnology Co., Ltd. The specific operation was as follows: a 25 μL LAMP amplification system was prepared, with the following reagent volumes: 6 μL Bst Buffer, 2.5 μL 10× mixed primer working solution, 1 μL Bst Enzyme Mix (UDG plus), 2 μL RNA template, and 13.5 μL enzyme-free water.

[0062] The 10× mixed primer working solution corresponding to the primer set in Example 1 is as shown in Example 3; The 10× mixed primer working solution corresponding to the primer set of Comparative Example 1-1 is shown in Comparative Example 3-1; The 10× mixed primer working solutions corresponding to the primer sets of Comparative Examples 1-2 are shown in Comparative Example 3-2.

[0063] 3. After the system is configured, LAMP amplification is performed using a real-time fluorescence PCR instrument (ABI Q1 real-time fluorescence PCR instrument). Based on the real-time fluorescence signal obtained, it is determined whether H1N1 can be detected. The amplification conditions for LAMP testing were: incubation at 37°C for 3 min, incubation at 65°C for 45 min, inactivation at 85°C for 5 min, and melting curve analysis program (95°C, 15 s; 60°C, 1 min; 95°C, 1 s). The negative control (i.e., blank control) was enzyme-free water.

[0064] 4. Performance verification and evaluation criteria: Primer sets with a positive time of less than 10 minutes for positive samples (experimental group) and no obvious peak in blank group are considered to have good amplification effect.

[0065] 5. Experimental Results: Using Yisheng's commercial reagents, the primer sets of Example 1, Comparative Example 1-1, and Comparative Example 1-2 were tested at 65°C and a template concentration of 100 C / R (i.e., when the RNA concentration was 50 copies / μL, 2 μL was added to obtain 100 copies per reaction, abbreviated as 100 C / R) to determine whether they could amplify normally. The results are as follows: Figure 1 and Figure 2 As shown.

[0066] Figure 1 and Figure 2 The results showed that all three primer sets could amplify normally at 65℃, and the blank set did not produce a peak, indicating good amplification specificity. At a template concentration of 100 C / R, all three primer sets could amplify normally, with only slight differences in positive time. The average positive time for the Example 1 primer set was 6 min; the average positive time for the Comparative Example 1-1 primer set was 9 min; and the average positive time for the Comparative Example 1-2 primer set was 15 min. Under the same template and incubation temperature, the positive time for the Example 1 primer set was the shortest, followed by the Comparative Example 1-1 primer set, and the shortest for the Comparative Example 1-2 primer set.

[0067] Experiment Example 2: High-Temperature Stability Test of Primer Sets This experiment tested the high-temperature stability of the primer sets from Example 1, Comparative Example 1-1, and Comparative Example 1-2. The specific process is as follows: 1. Prepare H1N1 RNA standards (purchased from Twist Bioscience), with an initial concentration of 10. 6 The H1N1 RNA template was diluted to 50 copies / μL with enzyme-free water. 2. Prepare a 25 μL LAMP amplification system. The specific reagent volumes are as follows: 6.25 μL of 4× high-temperature amplification buffer (as shown in Comparative Example 2-1), 2.5 μL of 10× mixed primer working solution, 1 μL of high-temperature mixed enzyme working solution (as shown in Example 3), 2 μL of RNA template, and 13.25 μL of enzyme-free water. The 10× mixed primer working solution corresponding to the primer set in Example 1 is as shown in Example 3; The 10× mixed primer working solution corresponding to the primer set of Comparative Example 1-1 is shown in Comparative Example 3-1; The 10× mixed primer working solutions corresponding to the primer sets of Comparative Examples 1-2 are shown in Comparative Example 3-2.

[0068] 3. After the system is prepared, use a real-time fluorescence PCR instrument (ABI Q1 real-time fluorescence PCR instrument) to perform high-temperature LAMP amplification at 70℃. Based on the real-time fluorescence signal obtained, determine whether H1N1 can be detected. The conditions for high-temperature LAMP amplification at 70℃ were: incubation at 37℃ for 3 min, incubation at 70℃ for 45 min, inactivation at 85℃ for 5 min, and melting curve analysis program (95℃, 15s; 60℃, 1 min; 95℃, 1s). The negative control (i.e., blank control) was enzyme-free water.

[0069] 4. Performance verification and evaluation criteria: Primer sets with a positive time of less than 10 minutes for positive samples (experimental group) and no obvious peak in blank group are considered to have good amplification effect.

[0070] 5. Experimental Results: Using the unoptimized high-temperature amplification buffer (as shown in Comparative Example 2-1), the primer sets of Example 1, Comparative Example 1-1, and Comparative Example 1-2 were tested at 70°C and a template concentration of 100 C / R to determine whether they could amplify normally. The results are as follows: Figure 3 and Figure 4 As shown.

[0071] Figure 3 and Figure 4 The results showed that all three primer sets amplified normally at 70℃. With increasing incubation temperature, the blank groups of Comparative Example 1-1 and Comparative Example 1-2 primer sets showed peaks at the end of amplification, indicating that these two primer sets were not stable enough at 70℃. The blank group of Example 1 primer set did not show any peaks. In the experimental group, when the template concentration was 100 C / R, all three primer sets amplified normally, but the positive time of all three primer sets shifted with increasing incubation temperature. The average positive time for Example 1 primer set was 12 min; the average positive time for Comparative Example 1 primer set was 15 min; and the average positive time for Comparative Example 1-2 primer set was 27 min. Under the same template concentration and incubation temperature, the positive time of Example 1 primer set was the shortest, followed by Comparative Example 1 primer set, and then Comparative Example 1-2 primer set. In summary, the results indicate that the high-temperature stability of Example 1 primer set is good; therefore, Example 1 primer set was used for subsequent tests.

[0072] Experiment Example 3: Verification of Amplification Performance in High-Temperature Buffer Experiment 1: The performance of the high-temperature amplification buffers of Examples 2 and Comparative Examples 2-1 to 2-7 was tested using the primer set of Example 1. The specific process is as follows: 1. Prepare H1N1 RNA standards (purchased from Twist Bioscience), with an initial concentration of 10. 6 The H1N1 RNA template was diluted to 50 copies / μL with enzyme-free water. 2. Prepare a 25 μL LAMP amplification system. The specific reagent volumes are as follows: 6.25 μL of 4× high-temperature amplification buffer (as shown in Example 2 and Comparative Examples 2-1 to 2-7, respectively), 2.5 μL of 10× mixed primer working solution (as shown in Example 3), 1 μL of high-temperature mixed enzyme working solution (as shown in Example 3), 2 μL of RNA template, and 13.25 μL of enzyme-free water.

[0073] 3. After the system is prepared, use a real-time fluorescence PCR instrument (ABI Q1 real-time fluorescence PCR instrument) to perform high-temperature LAMP amplification at 70℃. Based on the real-time fluorescence signal obtained, determine whether H1N1 can be detected. The conditions for high-temperature LAMP amplification at 70℃ were: incubation at 37℃ for 3 min, incubation at 70℃ for 45 min, inactivation at 85℃ for 5 min, and melting curve analysis program (95℃, 15s; 60℃, 1 min; 95℃, 1s). The negative control (i.e., blank control) was enzyme-free water.

[0074] 4. Performance verification and evaluation criteria: Primer sets with a positive time of less than 10 minutes for positive samples (experimental group) and no obvious peak in blank group are considered to have good amplification effect.

[0075] 5. Experimental Results: Using the primer set from Example 1, the amplification performance of the high-temperature amplification buffers from Example 2 and Comparative Examples 2-1 to 2-7 was tested at 70°C and a template concentration of 100 C / R. The results are as follows: Figures 5-10 As shown.

[0076] (1) Optimization of potassium chloride concentration: The concentration of potassium chloride affects the reaction efficiency and specificity in LAMP reagents. It stabilizes the activity of Bst DNA polymerase by adjusting the ionic strength and promotes primer-template binding. Too high a potassium ion concentration will inhibit enzyme activity, leading to amplification delay or failure; too low a concentration will reduce primer annealing efficiency and increase non-specific amplification. Therefore, optimizing the concentration of potassium chloride in the reagent is beneficial to improving reaction efficiency and specificity.

[0077] The results are as follows Figure 5 and Figure 6As shown, when the potassium ion concentration in the reagent was reduced to 48 mM, the blank group did not show a peak; the positive time in the positive group was 3-5 minutes earlier than that in the control group, the detection rate in the positive group was higher than that in the control group, and the amplification curve showed an "S" shape. When the potassium ion concentration in the reagent was gradually increased, the blank group showed non-specific amplification; in the positive group, although the positive time was also gradually advanced, the rising portion of the amplification curve was too flat, with no plateau phase or a steep plateau phase within the specified incubation time, and the maximum fluorescence value was constantly changing. Therefore, the concentration of potassium chloride in the high-temperature buffer was set to 48 mM.

[0078] (2) Optimization of magnesium sulfate concentration: Magnesium sulfate is a key cofactor in the LAMP reaction, providing Mg²⁺. + Maintaining the activity of Bst DNA polymerase and the stability of primer annealing is crucial. The concentration of magnesium sulfate directly affects amplification efficiency: too low a concentration leads to enzyme inactivation or weak primer binding, inhibiting amplification; too high a concentration exacerbates non-specific binding, causing false positives. Therefore, dynamically optimizing the magnesium sulfate concentration in the reagent is beneficial for improving the speed and specificity of the LAMP reaction and avoiding interference from primer dimers or background amplification.

[0079] The results are as follows Figure 7 and Figure 8 As shown, when the magnesium ion concentration in the reagent increased to 4.6 mM, the blank group showed a peak in the last 10 minutes of incubation, and its melting curve gradually showed a clear single-peak trend. Compared with the control group, when the magnesium ion concentration in the reagent was 4.2 mM and 4.4 mM, the blank group did not show a clear peak, and the melting curve did not show a clear single peak. As the magnesium ion concentration increased, the positive time of the positive group also gradually increased, the shape of the fluorescence curve gradually showed an "S" shape, and the melting curve also showed a sharp peak. When the magnesium ion concentration in the system increased to 4.4 mM, the blank group only showed a slight change in fluorescence value at the end of incubation (the last 10 minutes), but the positive time of the positive group was 5 minutes earlier than that of the control group. Therefore, the concentration of magnesium sulfate in the high-temperature buffer was set to 4.4 mM.

[0080] (3) Optimization of ammonium sulfate concentration: Ammonium sulfate affects reaction kinetics in LAMP reagents by adjusting ionic strength. NH4+ in ammonium sulfate can stabilize Bst DNA polymerase activity and weaken the hydrogen bonds in the DNA double strand, promoting template unwinding and primer binding. Excessive concentration may inhibit enzyme function, leading to decreased amplification efficiency; excessively low concentration reduces template unwinding efficiency, causing non-specific amplification. Optimizing the ammonium sulfate concentration can better balance unwinding ability and enzyme stability, ensuring high sensitivity and low background, and adapting to the needs of complex sample detection.

[0081] The results are as follows Figure 9 and Figure 10As shown, gradually increasing the concentration of ammonium sulfate in the system had almost no effect on the amplification of the blank group. However, it had a more significant effect on the positive group. Specifically, as the concentration of ammonium sulfate increased, the positive time of the positive group gradually advanced. When the concentration of ammonium sulfate was 44 mM, the positive time was 4 minutes earlier than that of the control group, and the consistency between positive samples also increased. Therefore, the concentration of ammonium sulfate in the high-temperature buffer was set to 44 mM.

[0082] Experiment 2: Using the primer set of Example 1, the concentrations of potassium chloride, magnesium sulfate, and ammonium sulfate were optimized according to the conditions of Experiment 1 to obtain the high-temperature amplification buffer described in Example 2. Under the conditions of template (H1N1 RNA standard, purchased from Twist Bioscience) concentration of 100 C / R and 60 C / R, the amplification effect before and after buffer optimization (corresponding to Example 2-1 and Example 2) was compared. The specific method was the same as in Experiment 1.

[0083] The results are as follows Figure 11 As shown, when the H1N1 template concentration was 100 C / R and 60 C / R, the optimized buffer amplification time was reduced by about 5 minutes on average at both test concentrations; the detection rate of the samples was also improved; the amplification curve of the positive amplification group showed a more obvious "S"-shaped amplification curve; and the time required to reach the amplification plateau was shortened, indicating that the high-temperature amplification buffer described in Example 2 has a better amplification effect.

[0084] Experiment Example 4: Primer set sensitivity test This experiment tested the sensitivity of the primer sets from Example 1 and Comparative Example 1-1. The specific procedure is as follows: The H1N1 RNA template (purchased from Twist Bioscience, initial concentration 10) was prepared with enzyme-free water. 6 The nucleic acid was serially diluted (copies / μL) to 25 C / R, 50 C / R, 75 C / R, 100 C / R, 125 C / R, and 150 C / R, using copies / R as the unit of measurement. This diluted nucleic acid was then used as the test nucleic acid. The test nucleic acid was subjected to 70°C high-temperature LAMP amplification using the method described in Example 4. The detection results are shown in [Figure 4]. Figure 12 .

[0085] like Figure 12 As shown, the limit of detection for the primer set in Example 1 was 50 C / R; the limit of detection for the primer set in Comparative Example 1-1 was 50 C / R.

[0086] Experiment Example 5: Detection Rate Test of the Lowest Detection Limit This experiment tested the detection rate of the primer sets of Example 1 and Comparative Example 1-1 at the limit of detection. The specific process is as follows: The H1N1 RNA template (purchased from Twist Bioscience, initial concentration 10) was prepared with enzyme-free water. 6 RNA samples were diluted to the limit of detection (50 C / R) using copies / μL as the test nucleic acid. Twenty RNA sample groups were set up, and the test nucleic acid was subjected to 70°C high-temperature LAMP amplification using the method in Example 4. The detection results are shown in [Figure 4]. Figure 13 .

[0087] like Figure 13 As shown, the primer set of Example 1 detected 19 out of 20 RNA samples at 50 C / R, achieving a detection rate of 95%; and the blank group showed no peak, indicating good amplification efficiency and no non-specific amplification. The primer set of Comparative Example 1-1 detected 13 out of 20 RNA samples at 50 C / R, achieving a detection rate of 65%. The detection performance of the primer set of Comparative Example 1-1 at low concentrations was not as good as that of the primer set of Example 1. Therefore, the primer set of Example 1 was determined to be the optimal primer set.

[0088] Experiment Example 6: Primer set specificity detection This experimental example demonstrates the specificity detection of the primer set from Example 1. The specific procedure is as follows: RNA templates for H3N2 influenza virus, influenza B virus (FluB), and human parainfluenza virus type 1 (HPIV1) were prepared using enzyme-free water (all purchased from Twist Bioscience, original concentration 10). 6 The nucleic acid samples (copies / μL) were diluted to 50 C / R as test nucleic acids. LAMP amplification at 70°C was performed using the method described in Example 4. The specificity of the primer set from Example 1 was tested, and the results are shown in […]. Figure 14 .

[0089] like Figure 14 As shown, the primer set of Example 1 could not be amplified under the templates of H3N2 influenza virus, influenza B virus, and human parainfluenza virus type 1, but only under the template of H1N1, indicating that the primer set of Example 1 has good specificity.

[0090] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A LAMP primer set for detecting influenza A virus, characterized in that, The primer set includes an outer primer pair, an inner primer pair, and a loop primer pair; the outer primer pair includes a forward outer primer with a nucleotide sequence as shown in SEQ ID NO.1 and a reverse outer primer with a nucleotide sequence as shown in SEQ ID NO.2; the inner primer pair includes a forward inner primer with a nucleotide sequence as shown in SEQ ID NO.3 and a reverse inner primer with a nucleotide sequence as shown in SEQ ID NO.4; the loop primer pair includes a forward loop primer with a nucleotide sequence as shown in SEQ ID NO.5 and a reverse loop primer with a nucleotide sequence as shown in SEQ ID NO.

6.

2. A LAMP high-temperature amplification buffer, characterized in that, The high-temperature amplification buffer comprises MgSO4, KCl and (NH4)2SO4; the concentration of MgSO4 in the high-temperature amplification buffer is 4.4 mM; the concentration of KCl in the high-temperature amplification buffer is 48 mM; and the concentration of (NH4)2SO4 in the high-temperature amplification buffer is 44 mM.

3. The high-temperature amplification buffer according to claim 2, characterized in that, The high-temperature amplification buffer also contains dNTPs and / or fluorescent dyes; the concentration of dNTPs in the high-temperature amplification buffer is 0.48 mM; and the mass percentage concentration of the fluorescent dye in the high-temperature amplification buffer is 8%.

4. The high-temperature amplification buffer according to claim 2, characterized in that, The high-temperature amplification buffer also includes a solvent; the solvent is a 20 mM Tris-HCl buffer with a pH of 8.

8.

5. A kit for detecting influenza A virus, characterized in that, The kit includes the primer set as described in claim 1 and the high-temperature amplification buffer as described in any one of claims 2 to 4.

6. The reagent kit according to claim 5, characterized in that, The kit also includes a high-temperature mixed enzyme working solution; the components of the high-temperature mixed enzyme working solution include high-temperature Bst enzyme, high-temperature RT enzyme, and high-temperature enzyme dilution solution; Optionally, the high-temperature Bst enzyme includes high-temperature Bst enzyme-093.

7. The reagent kit according to claim 6, characterized in that, The components of the high-temperature enzyme diluent include Tween-20, BSA, glycerol, and CMIT / MIT.

8. A method for detecting influenza A virus H1N1, wherein the method is not for the purpose of disease diagnosis and treatment, characterized in that, The method includes: testing the sample to be tested using the kit according to any one of claims 5 to 7.

9. The detection method according to claim 8, characterized in that, The detection method includes: extracting RNA from the sample to be tested to obtain the nucleic acid to be tested; mixing the nucleic acid to be tested, the primer set according to claim 1, the high-temperature amplification buffer according to any one of claims 2 to 4, and the high-temperature mixed enzyme working solution to obtain a LAMP reaction system; performing LAMP amplification on the LAMP reaction system using a constant-temperature fluorescence detector; and determining whether influenza A virus H1N1 exists in the sample to be tested based on the real-time fluorescence signal obtained after LAMP amplification. Optionally, in the detection method, the LAMP amplification conditions are: incubation at a constant temperature of 68~72℃ for 30~60 min; Optionally, the isothermal fluorescence detector includes at least one of real-time quantitative PCR, a dedicated isothermal fluorescence LAMP instrument, and a multifunctional microplate fluorescence detector.

10. The application of the primer set of claim 1, the high-temperature amplification buffer of any one of claims 2-4, the kit of claims 5-7, or the detection method of claim 8 or 9 in the detection of influenza A virus, wherein the application is not for the purpose of disease diagnosis and treatment.

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