LAMP primer combination and nucleic acid detection kit for simultaneously detecting influenza A virus and influenza B virus

Abstract

The invention discloses an LAMP (loop-mediated isothermal amplification) primer combination for simultaneously detecting influenza A and influenza B viruses and a nucleic acid detection kit, and relates to the technical field of virus detection, the LAMP primer combination for simultaneously detecting the influenza A and influenza B viruses comprises an LAMP primer designed based on an M gene of the influenza A viruses and an LAMP primer designed based on an NS gene of the influenza B viruses; the sequence of the specific primer probe for detecting the influenza A virus is SEQ No.1-7, and the sequence of the specific primer probe for detecting the influenza B virus is SEQ No.8-14. The kit can be used for detecting influenza A and B viruses, is high in detection sensitivity, strong in specificity and simple to operate, and has the following advantages by applying an LAMP probe method: interference of non-specific amplification is more effectively reduced; different fluorophores can be marked by specific probes of different pathogens, so that single-tube multiple detection is realized.

Description

Technical Field

[0001] This invention relates to the field of virus detection technology, specifically to a LAMP primer combination and nucleic acid detection kit for the simultaneous detection of influenza A and B viruses. Background Technology

[0002] Influenza viruses belong to the Orthomyxoviridae family and are single-stranded, negative-sense, segmented RNA viruses. Based on differences in nucleoproteins and matrix proteins, they can be classified into four types: A, B, C, and D (or A, B, C, and D). Influenza A viruses, based on the protein structure and gene specificity of hemagglutinin (HA) and neuraminidase (NA) on the viral surface, can be further divided into multiple subtypes; currently, 18 HA subtypes (H1-18) and 11 NA subtypes (N1-11) have been identified. Influenza B viruses are divided into the Victoria and Yamagata lineages. Currently, the viruses causing seasonal influenza outbreaks are the H1N1 and H3N2 subtypes of influenza A viruses and the Victoria and Yamagata lineages of influenza B viruses. The incubation period for influenza... The course of the illness is generally 1 to 7 days, mostly 2 to 4 days. It mainly begins with fever, headache, and muscle and joint pain, with body temperature reaching 39 to 40°C. Common symptoms include sore throat, dry cough, nasal congestion, and runny nose. Systemic symptoms such as chills, shivering, fatigue, and loss of appetite may also occur. Some patients may have mild or no symptoms. Influenza virus infection can exacerbate chronic underlying diseases. Children usually have higher fever levels than adults, and children with influenza B often experience more gastrointestinal symptoms such as nausea, vomiting, and diarrhea. Newborns may only present with lethargy, refusal to feed, and apnea. The clinical manifestations of the elderly may be atypical, often with no fever or low-grade fever, but significant cough, sputum production, wheezing, and chest pain. They may also present with anorexia and altered mental status. In cases without complications, the course of the disease is self-limiting, with fever gradually subsiding and systemic symptoms improving 3 to 5 days after onset, but cough and recovery of strength often take a longer time. Currently, the main detection methods for influenza viruses include virus isolation and culture, immunological detection, and molecular biological detection methods. Isolation and culture techniques: Pathogen isolation and culture was the gold standard for early pathogen detection. This method has high specificity and sensitivity, but the clinical testing time is too long, the process is cumbersome, and it is not suitable for large-scale testing. Immunological detection: Based on the antigen-antibody immune reaction, pathogens are detected at the protein level. This method has problems such as low detection sensitivity, specificity being greatly affected by environmental factors, a long detection window period, and inability to meet diagnostic and treatment needs. It is only suitable for initial screening and cannot be used as a basis for timely diagnosis. It also cannot identify different subtypes of the same type of pathogen. The most common molecular biological diagnostic method is polymerase chain reaction (PCR). PCR is currently the gold standard for the detection of most pathogens. However, the PCR instrument required for amplification is expensive, and the reaction time is long, generally requiring 1-2 hours or longer. In addition, most of these PCR test kits require low-temperature storage and transportation, which places high demands on the storage and transportation environment of the reagents. Moreover, the use of the reagents requires a high level of professional skills from the operators, which is not conducive to clinical promotion. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides a LAMP primer combination and nucleic acid detection kit for the simultaneous detection of influenza A and B viruses, thus solving the problems mentioned in the background section.

[0004] To achieve the above objectives, the present invention is implemented through the following technical solution: a LAMP primer combination for simultaneous detection of influenza A and influenza B viruses, comprising a LAMP primer combination for simultaneous detection of influenza A and influenza B viruses: LAMP primers designed based on the conserved M gene of influenza A virus (which has ≥95% homology in all subtypes of influenza A virus and has high species specificity), and LAMP primers designed based on the conserved NS gene of influenza B virus (which has ≥98% homology in both lineages of influenza B virus and has no risk of cross-species reaction); The specific primer and probe sequences for detecting influenza A virus are SEQ No. 1-7, and the specific primer and probe sequences for detecting influenza B virus are SEQ No. 8-14. The primer-probe sequence with the name of SEQ No. 7 is FluA-P, and the primer-probe sequence with the name of SEQ No. 14 is FluB-P. FluA-P and FluB-P are dual-labeled probes.

[0005] A nucleic acid detection kit utilizes the aforementioned LAMP primer combination for simultaneous detection of influenza A and B viruses. The nucleic acid detection kit is used for the simultaneous detection of influenza A and B viruses and includes RT-LAMP probe lyophilized reagents and sample release agents to achieve simultaneous detection of influenza A and B viruses in a single reaction.

[0006] Furthermore, the RT-LAMP probe lyophilization reagent includes the LAMP primer combination, reaction buffer, dNTPs, enzyme mixture, and lyophilization protectant, wherein the lyophilization reagent is lyophilized microspheres prepared by mixing the components.

[0007] Furthermore, the sample release agent includes one of 10-150mM Tris-HCl buffer, 100-600mM NaCl, 1-100mM EDTA, 0.1-30% Triton X-100, 0.1-20% NP-40, and 0.01-10% SDS.

[0008] Furthermore, the reaction buffer comprises 10–200 mM Tris-HCl buffer and 1–20 mM... 10–200 mM ammonium sulfate, 10–200 mM potassium chloride, 0.01%–1% Tween 20, with a reaction buffer pH of 8.0–8.9.

[0009] Furthermore, the dNTPs include 0.2–1.8 mM dATP, 0.2–1.8 mM dGTP, 0.2–1.8 mM dCTP, and 0.2–1.8 mM dTTP.

[0010] Furthermore, the enzyme mixture includes BST DNA polymerase, reverse transcriptase, and ribonuclease H II (RNase H II).

[0011] Furthermore, the freeze-drying protectant includes raffinose, mannitol, BSA, and PEG8000, with mass fractions of 5%–20%, 0.5%–5%, 0.01%–5%, and 0.02%–10%, respectively.

[0012] Furthermore, the preparation method of the RT-LAMP probe lyophilization reagent is as follows: mix all components evenly, prepare microspheres at 25 μL / sphere, quickly freeze with liquid nitrogen and then transfer to a lyophilizer for lyophilization, or dispense into eight-tube lyophilization in situ.

[0013] Furthermore, the ribonuclease H II can specifically hydrolyze the phosphodiester bonds of RNA on the DNA / RNA hybrid strand, but does not hydrolyze single-stranded / double-stranded DNA or RNA. The mechanism of action of this enzyme is to break the phosphodiester bonds that connect RNA bases to DNA bases at the 5' direction, generating 3' hydroxyl groups and phosphate groups at the 5' end of ribonucleic acid.

[0014] This invention provides a LAMP primer combination and nucleic acid detection kit for the simultaneous detection of influenza A and B viruses, which has the following beneficial effects: 1. This LAMP primer combination and nucleic acid detection kit for the simultaneous detection of influenza A and B viruses can be used to detect both influenza A and B viruses with high sensitivity, strong specificity, and simple operation. Utilizing the LAMP probe method, it offers the following advantages compared to traditional LAMP dye methods, colorimetric methods, and pH indicator colorimetric methods: it more effectively reduces interference from non-specific amplification; and it enables multiplex detection in a single tube by labeling different fluorescent groups with specific probes for different pathogens.

[0015] 2. This LAMP primer combination and nucleic acid detection kit for the simultaneous detection of influenza A and B viruses includes lyophilized reagents for the RT-LAMP probe method for detecting influenza A and B viruses. These lyophilized reagents are convenient to use; simply rehydrate the sample with the sample release agent before testing, eliminating the need for solution preparation and reducing the risk of operational errors. The lyophilized reagents can be transported at room temperature, reducing transportation costs. The reaction can be completed in just 30 minutes at a constant temperature, without the need for complex temperature control procedures. Compared to conventional qPCR reactions, which require 1–2 hours to complete, this significantly shortens the detection time. Furthermore, when the pathogen detection concentration is high, amplification peaks can be observed within 15 minutes. Attached Figure Description

[0016] Figure 1 This is a diagram showing the positive detection results of the nucleic acid detection kit of the present invention across two channels; Figure 2 This diagram illustrates the in-situ lyophilization preparation of the RT-LAMP probe method lyophilization reagents for influenza A and B viruses according to the present invention. Figure 3 This diagram illustrates the lyophilization preparation of lyophilized reagent microspheres for influenza A and B viruses using the RT-LAMP probe method according to the present invention. Detailed Implementation

[0017] The embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention.

[0018] like Figures 1-3 As shown, the present invention provides a technical solution: a LAMP primer combination for simultaneous detection of influenza A and influenza B viruses, comprising: LAMP primers designed based on the conserved M gene of influenza A virus (which has ≥95% homology in all subtypes of influenza A virus and has high species specificity), and LAMP primers designed based on the conserved NS gene of influenza B virus (which has ≥98% homology in both lineages of influenza B virus and has no risk of cross-species reaction); The specific primer and probe sequences for detecting influenza A virus are SEQ No. 1-7, and the specific primer and probe sequences for detecting influenza B virus are SEQ No. 8-14, as shown in Table 1: Table 1 The primer-probe sequence with the name FluA-P (SEQ No. 7) and the primer-probe sequence with the name FluB-P (SEQ No. 14) are both dual-labeled probes. FluA-P is labeled with a FAM fluorescent group at its 5' end and a BHQ1 quencher group at its 3' end. FluB-P is labeled with a VIC fluorescent group at its 5' end and a BHQ1 quencher group at its 3' end. The / rC / in the probe sequence is a ribonucleic acid (RNA) modification, which can enhance the specificity of probe binding to the target sequence and the sensitivity of enzyme digestion.

[0019] A nucleic acid detection kit, which utilizes the aforementioned LAMP primer combination for simultaneous detection of influenza A and B viruses, the nucleic acid detection kit for simultaneous detection of influenza A and B viruses, includes RT-LAMP probe lyophilized reagent and sample release agent, to achieve simultaneous detection of influenza A and B viruses in a single reaction; Preferably, the specific primer and probe concentrations for detecting influenza A virus are: FluA-F3: 0.2 μM, FluA-B3: 0.2 μM, FluA-FIP: 1.6 μM, FluA-BIP: 1.6 μM, FluA-LF: 0.8 μM, FluA-LB: 0.8 μM, and FluA-P: 0.2 μM. Preferably, the specific primer and probe concentrations for detecting influenza B virus are: FluB-F3: 0.2 μM, FluB-B3: 0.2 μM, FluB-FIP: 1.6 μM, FluB-BIP: 1.6 μM, FluB-LF: 0.8 μM, FluB-LB: 0.8 μM, and FluB-P: 0.2 μM. The RT-LAMP probe method lyophilization reagent includes the LAMP primer combination, reaction buffer, dNTPs, enzyme mixture, and lyophilization protectant. The lyophilization reagent is a 25 μL lyophilized microsphere prepared by mixing the components. The sample release agent includes at least one of 10-150mM Tris-HCl buffer, 100-600mM NaCl, 1-100mM EDTA, 0.1-30% Triton X-100, 0.1-20% NP-40, and 0.01-10% SDS (which can be used in combination according to sample type to enhance virus release efficiency); preferably, the sample release agent includes: 10mM Tris-HCl buffer, 100mM NaCl, 0.5% Triton X-100, and 0.1% NP-40, and the pH value of the sample release agent is 8.5; The reaction buffer includes 10–200 mM Tris-HCl buffer and 1–20 mM... The reaction buffer contains 10-200 mM ammonium sulfate, 10-200 mM potassium chloride, and 0.01%-1% Tween 20, with a pH of 8.0-8.9. This buffer system maintains the optimal ionic strength and pH environment for LAMP amplification, significantly improving enzyme activity stability. Preferably, the reaction buffer comprises: 20 mM Tris-HCl buffer, 8 mM... The reaction buffer consisted of 10 mM ammonium sulfate, 50 mM potassium chloride, and 0.1% Tween 20, with a pH of 8.5. The dNTPs include 0.2–1.8 mM dATP, 0.2–1.8 mM dGTP, 0.2–1.8 mM dCTP, and 0.2–1.8 mM dTTP. The equimolar ratio of these four dNTPs can avoid a decrease in amplification efficiency due to substrate concentration imbalance. Preferably, the dNTPs include: 1.4 mM dATP, 1.4 mM dGTP, 1.4 mM dCTP, and 1.4 mM dTTP. The enzyme mixture comprises: 0.1-1 U / μL BST DNA polymerase, 0.1-1 U / μL reverse transcriptase, and 0.04-100 mU / μL ribonuclease H II (RNase H II); preferably, the enzyme mixture comprises: 0.32 U / μL BST DNA polymerase, 0.2 U / μL reverse transcriptase, and 1 mU / μL ribonuclease H II (RNase H II). The freeze-drying protectant includes raffinose, mannitol, BSA, and PEG8000, with mass fractions of 5%–20%, 0.5%–5%, 0.01%–5%, and 0.02%–10%, respectively. This combination can significantly reduce enzyme activity loss during freeze-drying and reconstitution, ensuring that the enzyme activity retention rate is ≥90% after 6 months of storage at room temperature. Preferably, the freeze-drying protectant includes raffinose, mannitol, BSA, and PEG8000, with mass fractions of 5%, 2%, 0.1%, and 2%, respectively. The preparation method of RT-LAMP probe lyophilization reagent is as follows: Mix all components evenly, prepare microspheres at 25 μL / sphere, flash freeze in liquid nitrogen, transfer to a lyophilizer for lyophilization, and follow the lyophilization procedure as follows: pre-freezing stage (-40℃, 3h, vacuum 0Pa) → sublimation drying (-40℃ 12h, -30℃ 5h, -20℃ 2h, -10℃ 1h, all at 10Pa) → desorption drying (0℃ 1h, 10℃ 2h, 20℃ 5h, all at 10Pa); or dispense into eight-tube lyophilization in situ; Ribonuclease H II can specifically hydrolyze the phosphodiester bonds of RNA on DNA / RNA hybrid strands, but it does not hydrolyze single-stranded / double-stranded DNA or RNA. The mechanism of action of this enzyme is to break the phosphodiester bonds that connect RNA bases to DNA bases at the 5' direction, generating 3' hydroxyl groups and 5' phosphate groups at the 5' end of ribonucleic acid. This specific cleavage action can further reduce false positive signals caused by non-specific amplification.

[0020] Example 1: Kit preparation; The RT-LAMP probe mixture was prepared according to the following formulation: 20 mM Tris-HCl buffer (pH 8.5), 8 mM MgSO4, 10 mM ammonium sulfate, 50 mM potassium chloride, 0.1% Tween 20, 1.4 mM dNTPs mixture, 0.32 U / μL BST DNA polymerase, 0.2 U / μL reverse transcriptase, 1 mU / μL RNase H II, 5% raffinose, 2% mannitol, 0.1% BSA, 2% PEG8000, and LAMP primers and probes (concentrations added according to preferred values); The mixture was dropped into 25 μL / particle to form microspheres, which were then rapidly frozen in liquid nitrogen and transferred to a freeze dryer for freeze drying to obtain freeze-dried microspheres. Prepare the sample release solution: 10mM Tris-HCl buffer, 100mM NaCl, 0.5% Triton X-100, 0.1% NP-40, and adjust the pH to 8.5; The lyophilized microspheres and sample release agent were assembled into a complete kit and stored in a sealed container. Example 2: Sample detection; Sample processing: Take samples such as pharyngeal swabs and nasal swabs, add sample release agent, vortex mix well, and let stand at room temperature for 5 minutes to obtain the sample solution to be tested; Reaction system construction: Place one lyophilized microsphere in a reaction tube, add 25 μL of the sample solution to be tested, and mix gently; Detection: Place the reaction tube into the fluorescence quantitative detector, set the constant temperature to 65℃, react for 30 min, and simultaneously set the FAM channel (excitation wavelength 494nm, emission wavelength 518nm) to detect influenza A virus and the VIC channel (excitation wavelength 535nm, emission wavelength 556nm) to detect influenza B virus. The instrument collects fluorescence signals once every 1 min. Result interpretation: If a specific amplification peak appears in a certain channel, it is determined to be positive for the corresponding virus; if no amplification peak appears, it is negative. Example 3: Performance verification; Specificity verification: The samples were tested with influenza A H1N1 and H3N2 subtypes, influenza B Victoria and Yamagata lineages, and control samples such as respiratory syncytial virus and adenovirus. Only the corresponding channels of influenza A and B samples showed amplification peaks, while the control samples showed no amplification. Sensitivity verification: Serial dilutions of influenza A and B virus nucleic acids (10¹–10⁻⁶) were performed. 6 The kit can detect viral nucleic acid at 10¹ copies / μL. Stability verification: After 6 months of storage at room temperature, the detection performance of the kit did not decrease significantly; after 3 freeze-thaw cycles, the sensitivity and specificity remained stable. Based on the above description, the present invention can be used to simultaneously detect influenza A and B viruses with high detection sensitivity, strong specificity, and simple operation. Compared with traditional LAMP dye method, colorimetric method, and pH indicator colorimetric method, it has the following advantages: more effectively reduces interference from non-specific amplification; and can achieve single-tube multiplex detection by labeling different fluorescent groups with specific probes of different pathogens. Furthermore, the RT-LAMP probe lyophilized reagents for detecting influenza A and B viruses in the nucleic acid detection kit are in lyophilized form, which is convenient to use. Simply rehydrate the sample with the sample release agent before testing, eliminating the need for solution preparation and reducing the risk of operational errors. The lyophilized reagents can be transported at room temperature, thereby reducing transportation costs. The reaction can be completed in just 30 minutes at a constant temperature, without the need for complicated temperature change procedures. Compared to conventional qPCR reactions, which require 1 to 2 hours to complete the detection, this greatly shortens the detection time. Moreover, when the pathogen detection concentration is high, the amplification peak can be observed within 15 minutes.

[0021] The embodiments of the present invention are given for illustrative and descriptive purposes only, and are not intended to be exhaustive or to limit the invention to the forms disclosed. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described in order to better illustrate the principles and practical application of the invention, and to enable those skilled in the art to understand the invention and to design various embodiments with various modifications suitable for a particular purpose.

Claims

1. A LAMP primer combination for simultaneous detection of influenza A and B viruses, characterized in that: Including LAMP primer combinations for simultaneous detection of influenza A and B viruses: LAMP primers designed based on the M gene of influenza A virus and LAMP primers designed based on the NS gene of influenza B virus; The specific primer and probe sequences for detecting influenza A virus are SEQ No. 1-7, and the specific primer and probe sequences for detecting influenza B virus are SEQ No. 8-14; The primer-probe sequence with the name of SEQ No. 7 is FluA-P, and the primer-probe sequence with the name of SEQ No. 14 is FluB-P. FluA-P and FluB-P are dual-labeled probes.

2. A nucleic acid detection kit, which utilizes the LAMP primer combination for simultaneous detection of influenza A and B viruses as described in claim 1, characterized in that: The nucleic acid detection kit is used to simultaneously detect influenza A and B viruses, including RT-LAMP probe lyophilized reagents and sample release agents, to achieve simultaneous detection of influenza A and B viruses in a single reaction.

3. The nucleic acid detection kit according to claim 2, characterized in that: The RT-LAMP probe method lyophilization reagent includes the LAMP primer combination, reaction buffer, dNTPs, enzyme mixture, and lyophilization protectant as described in claim 1. The lyophilization reagent is lyophilized microspheres prepared by mixing the components.

4. A nucleic acid detection kit according to claim 2, characterized in that: The sample release agent includes one of the following: 10-150mM Tris-HCl buffer, 100-600mM NaCl, 1-100mM EDTA, 0.1-30% Triton X-100, 0.1-20% NP-40, and 0.01-10% SDS.

5. A nucleic acid detection kit according to claim 3, characterized in that: The reaction buffer includes 10–200 mM Tris-HCl buffer and 1–20 mM... The reaction buffer contains 10–200 mM ammonium sulfate, 10–200 mM potassium chloride, and 0.01%–1% Tween 20. The pH of the reaction buffer is 8.0–8.

9.

6. A nucleic acid detection kit according to claim 3, characterized in that: The dNTPs include 0.2–1.8 mM dATP, 0.2–1.8 mM dGTP, 0.2–1.8 mM dCTP, and 0.2–1.8 mM dTTP.

7. A nucleic acid detection kit according to claim 3, characterized in that: The enzyme mixture includes BST DNA polymerase, reverse transcriptase, and ribonuclease H II (RNase H II).

8. A nucleic acid detection kit according to claim 3, characterized in that: The freeze-drying protectant includes raffinose, mannitol, BSA, and PEG8000, with mass fractions of 5%–20%, 0.5%–5%, 0.01%–5%, and 0.02%–10%, respectively.

9. A nucleic acid detection kit according to claim 3, characterized in that: The preparation method of the RT-LAMP probe lyophilization reagent is as follows: mix all components evenly, prepare small balls at 25 μL / ball, quickly freeze with liquid nitrogen and then transfer to a lyophilizer for lyophilization, or dispense into eight-tube lyophilization in situ.

10. A nucleic acid detection kit according to claim 7, characterized in that: The ribonuclease H II can specifically hydrolyze the phosphodiester bonds of RNA on the DNA / RNA hybrid strand, but does not hydrolyze single-stranded / double-stranded DNA or RNA. The mechanism of action of this enzyme is to break the phosphodiester bonds that connect RNA bases to DNA bases at the 5' direction, generating 3' hydroxyl groups and phosphate groups at the 5' end of ribonucleic acid.

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