Primer probe group and application thereof in detection of influenza A virus
By using primer probe sets and strand displacement amplification technology, the problems of low sensitivity, high false positive rate, and complex operation in influenza A virus detection have been solved, enabling efficient and specific simultaneous screening of H5, H7, and H9 subtypes, reducing costs and false positives.
Patent Information
- Application Number
- CN202511820513.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-04
- Publication Date
- 2026-02-17
AI Technical Summary
Existing technologies for detecting influenza A virus suffer from low sensitivity, high false positive rates, complex operation, and high costs. In particular, sequence differences between different subtypes can lead to false negatives, making it difficult to achieve universal screening with a single tube.
A primer and probe set, including F3, B3, FIP, BIP, LF, LB primers and quench-fluorescence complementary probes, was used in combination with strand displacement amplification technology to optimize conserved bases, achieving high coverage of H5, H7, and H9 subtypes, and avoiding false positives through a fluorescence signal activation strategy.
It enables simultaneous screening of H5, H7, and H9 subtypes, reduces reagent types and inventory pressure, maintains high amplification efficiency and specificity, avoids false negatives and false positives, achieves single-copy sensitivity, and is easy to interpret.
Smart Images
Figure CN121538352A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to methods for the determination or testing of enzymes, nucleic acids or microorganisms, and more particularly to a primer probe set and its application in the detection of influenza A virus. Background Technology
[0002] Influenza A virus is one of the most threatening respiratory pathogens to human and livestock health. Its genome is a segmented single-stranded negative-sense ribonucleic acid. Based on antigenic differences in hemagglutinin (HA) and neuraminidase (NA), 18 HA subtypes (H1-H18) and 11 NA subtypes (N1-N11) have been identified. Among them, subtypes H5, H7, and H9 not only cause serious diseases in poultry but have also crossed species barriers multiple times, infecting humans and causing outbreaks of highly pathogenic avian influenza (HPAI) or low pathogenic avian influenza (LPAI). These subtypes are listed by the World Health Organization as key zoonotic pathogens for monitoring.
[0003] Currently, the gold standard for laboratory testing of influenza A virus, both domestically and internationally, remains real-time fluorescent reverse transcription polymerase chain reaction (qRT-PCR). This method has high sensitivity and specificity, but it relies on expensive thermal cyclers, specialized laboratories, and high-level protective measures. The amplification time typically requires 60-120 minutes, making it difficult to conduct in the field, at the grassroots level, or in resource-scarce areas.
[0004] Loop-mediated isothermal amplification (LAMP) in isothermal amplification technology can complete 10 LAMP cycles in 15-40 minutes at a constant temperature of 60-65℃. 9 Rapid on-site amplification, requiring only a simple isothermal device, has become a research hotspot for on-site rapid detection. However, the end product of traditional LAMP is magnesium pyrophosphate turbidity or a visible fluorescent dye (such as SYBR Green I or calcein), which can only be interpreted under open conditions. The dye binds to any double-stranded DNA, making it highly susceptible to false positives due to aerosol contamination or primer dimers. Furthermore, it cannot distinguish between specific and non-specific amplification, and the sensitivity is usually only 10^-10. 1 copy.
[0005] To improve the specificity of LAMP, strategies such as RNase H-based chimeric RNA-DNA probes, molecular beacons, and CRISPR-Cas post-processing probes have emerged in recent years. However, RNase H is sensitive to ionic strength and temperature, resulting in significant baseline drift, and the sensitivity remains at around 10. 1 Copying is problematic; structural probes are expensive to synthesize and easily interfered with by high-concentration primers in the LAMP system; CRISPR-Cas and other methods require additional enzymes and equipment, which deviates from the original intention of LAMP to be simple and low-cost.
[0006] On the other hand, significant intraspecific sequence differences exist within the H5, H7, and H9 subtypes. Taking the M2 gene as an example, representative strains of H5N1, H7N9, and H9N2 exhibit 5%-15% base mutations in the F1c region. Using conventional single-sequence primers / probes can easily lead to decreased amplification efficiency or even failure due to single-base mismatches, resulting in false negatives. Existing technologies typically design multiple sets of primers and probes for different subtypes, resulting in high reagent costs, cumbersome operations, and complex inventory management, making it difficult to achieve universal screening with a single tube.
[0007] Therefore, there is an urgent need for a primer-probe set and its application in detecting influenza A virus. Summary of the Invention
[0008] This invention aims to overcome the shortcomings of the prior art, and to this end, the following technical solution is adopted: A first aspect of the present invention is to provide a primer-probe set for detecting influenza A virus, wherein the influenza A virus comprises H5 subtype, H7 subtype, and H9 subtype; the primer-probe set includes: The F3 primer consists of the base sequence shown in SEQ ID NO: 1; Primer B3 consists of the base sequence shown in GCCTGCTCACTCGATCCA (SEQ ID NO: 2); The FIP primers consist of the base sequence shown in SEQ ID NO: 3; The BIP primers consist of the base sequence shown in SEQ ID NO: 4; The LF primers consist of the base sequence shown in SEQ ID NO: 5; The LB primers consist of the base sequence shown in SEQ ID NO: 6; A first probe, wherein the base sequence of the first probe is identical to that of the FIP primer or the BIP primer, and the 5' end of the first probe is labeled with either a quenching group or a fluorescent group; and The second probe has a base sequence that is the reverse complementary sequence of the 5' end of the first probe or a segment of the reverse complementary sequence of the 5' end of the first probe, and the 3' end of the second probe is marked with either the quenching group or the fluorescent group, wherein the absorption spectrum of the quenching group can completely cover the emission spectrum of the fluorescent group.
[0009] Preferably, the base sequence of the first probe is the same as that of the FIP primer, and the quenching group is labeled at the 5' end of the first probe; the base sequence of the second probe is a segment of the reverse complementary sequence at the 5' end of the first probe, and the fluorescent group is labeled at the 3' end of the second probe, and the absorption spectrum of the quenching group can completely cover the emission spectrum of the fluorescent group.
[0010] Furthermore, the second probe consists of the base sequence shown in CACAGCATCGGTCTCACAGRCA (SEQ ID NO: 7), and the 3' end of the second probe is labeled with the fluorescent group, and the absorption spectrum of the quenching group can completely cover the emission spectrum of the fluorescent group.
[0011] Preferably, the quenching group is BHQ-1 and the fluorescent group is 6-FAM.
[0012] Furthermore, the first probe consists of the base sequence shown in BHQ1-TGYCTGTGAGACCGATGCTGTGACAGAAGTGGCTTTTGGCC (SEQ ID NO: 3), and the second probe consists of the base sequence shown in CACAGCATCGGTCTCACAGRCA-6-FAM (SEQ ID NO: 7).
[0013] Preferably, in the presence of a molar concentration meter, the primer-probe set comprises: Four copies of F3 primers; Four copies of B3 primers; 16 FIP primers; 16 BIP primers; 8 sets of LF primers; 8 LB primers; Five copies of the first probe; and Five samples of the second probe were used.
[0014] A second aspect of the present invention is to provide a kit for detecting influenza A virus, wherein the influenza A virus comprises H5 subtype, H7 subtype and H9 subtype; the kit includes: the primer and probe set as described above.
[0015] Preferably, it also includes: strand displacement DNA polymerase Bst 3.0, Moloney murine leukemia virus reverse transcriptase, RNase inhibitor, buffer suitable for LAMP reaction, dNTPs, and magnesium ions.
[0016] A third aspect of the present invention is to provide a method for detecting influenza A virus for non-diagnostic purposes, wherein the influenza A virus comprises H5 subtype, H7 subtype, and H9 subtype; the detection method includes the following steps: S1. Provide the sample to be tested; S2. Mix the sample to be tested with the components in the reagent kit as described above to form a reaction system; S3. The amplification reaction is carried out under isothermal conditions; S4. Real-time detection of fluorescence signals in the reaction system.
[0017] A fourth aspect of the invention is to provide the use of the primer-probe set as described above or the kit as described above in the preparation of products for detecting influenza A virus, which consists of H5 subtype, H7 subtype and H9 subtype.
[0018] Preferably, the influenza A virus consists of the H5N1 subtype, the H7N9 subtype, and the H9N2 subtype.
[0019] Compared with the prior art, the beneficial effects of this utility model are reflected in: The primer and probe set of this invention achieves high coverage of H5, H7, and H9 subtypes by introducing degenerate bases optimized through conservation analysis at key sites. This allows for simultaneous screening without subtype-specific detection, significantly reducing reagent types and inventory pressure, while maintaining excellent amplification efficiency and specificity, avoiding false negatives due to sequence differences. Furthermore, the strategy of coupling quenching-fluorescence complementary probes with strand displacement amplification ensures that the fluorescence signal is activated only when the target sequence is present, resulting in extremely low background that can be clearly interpreted by the naked eye or instruments, completely avoiding false positives caused by contamination or non-specific amplification in traditional dye methods. Attached Figure Description
[0020] Figure 1 This is the real-time fluorescence curve for detecting the nucleic acid of influenza A virus H5N1 pathogen in Example 2; Figure 2 This is the real-time fluorescence curve for detecting the nucleic acid of the H7N9 influenza A virus pathogen in Example 2; Figure 3 This is the real-time fluorescence curve for detecting the nucleic acid of the H9N2 influenza A virus pathogen in Example 2; Figure 4 The real-time fluorescence curve for detecting the nucleic acid of influenza A virus H5N1 pathogen in Comparative Example 1; Figure 5 The real-time fluorescence curve for detecting the nucleic acid of influenza A virus H5N1 pathogen in Comparative Example 2; Figure 6 This is a heatmap showing the detection sensitivity of each primer-probe set for detecting the nucleic acid of each influenza A virus pathogen in Comparative Example 3. Detailed Implementation
[0021] The specific embodiments of the present invention will be described in detail below.
[0022] Unless otherwise defined, the technical or scientific terms used in the claims and description shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.
[0023] The word "comprising" or similar terms used in the specification and claims of this patent application mean that the objects preceding "comprising" include the objects listed after "comprising" or their equivalents, and do not exclude other objects. Example
[0024] The complete gene sequence of the M2 strain, a representative of H5N1: ATGAGTCTTCTAACCGAGGTCGAAACGTACGTTCTCTCTATCGTCCCGTCAGGCCCCCTCAAAGCCGAGATCGCGCAGAGACTTGAGGATGTCTTTGCAGGAAAGAACACCGATCTCGAGGCTCTCATGGAATGGCTAAAGACAAGACCAATCCTGTCACCTCTGACTAAAGGGATTTTAGGATTTGTGTTCACGCTCACCGTGCCCAGTGAGCGAGGACTGCAGCGTAGACGCTTTGTCCAGAATGCCTTAAATGGAAATGGAGATCCAAACAATATGGATAGGGCAGTTAAGCTATACAAGAAGCTGAAAAGAGAAATAACATTCCATGGGGCTAAGGAGGTCGCACTCAGCTACTCAACCGGTGCACTTGCCAGTTGTATGGGTCTCATATACAACAGGATGGGAACGGTGACCACAGAAGTGGCTTTTGGCCTAGTGTGTGCCACTTGTGAGCAGATTGCAGATTCACAGCATCGGTCTCACAGACAGATGGCAACTACCACCAACCCACTAATCAGGCATGAGAACAGAATGGTGCTGGCCAGCACTACAGCTAAGGCTATGGAGCAGATGGCTGGATCGAGTGAGCAGGCAGCGGAAGCCATGGAGGTTGCTAGTCAGGCTAGGCAGATGGTGCAGGCAATGAGGACAATTGGGACTCATCCTAGCTCCAGTGCCGGTCTGAAAGATAATCTTCTTGAAAATTTGCAGGCCTACCAAAAACGAATGGGAGTGCAAATGCAGCGATTCAAGTGATCCTCTTGTTGTTGCCGCAAGTATCATTGGGATACTGCACTTGATATTGTGGATTCTTGATCGTCTTTTCTTCAAATGCATTTATCGTCGCCTTAAATACGGTTTGAAAAGAGGGCCTTCTACGGAAGGGGTACCTGAGTCTATGAGGGAAGAGTATCGGCAGGAACAGCAGAGTGCTGTGGATGTTGACGATGGTCATTTTGTCAACATAGAGCTGGAGTAA(SEQ ID NO:8); Full gene sequence of H7N9 representative strain M2: ATGAGTCTTCTAACCGAGGTCGAAACGTACGTTCTCTCTATCATTCCATCAGGCCCCCTCAAAGCCGAGATCGCACAGAGACTTGAGGATGTTTTTGCAGGGAAGAACGCAGATCTCGAGGCTCTCATGGAGTGGATAAAGACAAGACCAATCCTGTCACCTCTGACTAAGGGGATTTTAGGGTTTGTGTTCACGCTCACCGTGCCCAGTGAGCGAGGACTGCAGCGTAGACGGTTTGTCCAAAACGCCCTAAATGGGAATGGAGACCCAAACAACATGGACAAGGCGGTTAAATTATACAAGAAACTGAAGAGGGAAATGACATTTCATGGAGCAAAGGAAGTTGCACTCAGTTACTCAACTGGTGCGCTTGCCAGCTGCATGGGTCTCATATACAACAGAATGGGGACTGTGACCGCAGAAGGGGCTCTTGGACTAGTATGTGCCACTTGTGAGCAGATTGCTGACGCACAACATCGGTCCCACAGGCAGATGGCGACTACTACTAACCCACTAATTAGGCATGAGAATAGAATGGTACTAGCCAGCACTACGGCTAAGGCTATGGAGCAGATGGCTGGATCAAGTGAACAGGCAGCGGAAGCCATGGAAGTTGCAAGTCAGGCTAGGCAAATGGTGCAGGCTATGAGAACAGTTGGGACTCACCCTAACTCCAGTACAGGTCTAAAAGATGATCTTATTGAAAATTTGCAGGCCTACCAGAACCGGATGGGAGTGCAACTGCAGCGGTTCAAGTGAGCCTCTAGTCGTTGCAGCTAACATTATTGGGATATTGCACTTGATATTGTGGATTCTTGATCGTCTTTTCTTCAAATGCATTTATCGTCGTTTTAAATACGGTTTGAAAAGAGGGCCTTCTACGGAAGGAATGCCTGAGTCTATGAGGGAAGAATATCGGCAGGAACAGCAGAATGCTGTGGATGTTGACGATGGTCATTTTGTCAACATAGAGCTGAAGTAAAAA(SEQ ID NO:9);as well as; Full gene sequence of H9N2 representative strain M2: ATGAGTCTTCTAACCGAGGTCGAAACGTACGTTCTCTCTATCATCCCATCAGGCCCCCTCAAAGCCGAGATCGCGCAGAGACTTGAGGATGTTTTTGCAGGGAAGAACACAGATCTTGAGGCTCTCATGGAATGGCTAAAGACAAGACCAATCCTGTCACCTCTGACTAAGGGGATTTTAGGGTTTGTGTTCACGCTCACCGTGCCCAGTGAGCGAGGACTGCAGCGTAGACGATTTGTCCAAAATGCCCTAAATGGGAATGGAGACCCAAACAACATGGACAGGGCAGTTAAACTATACAAGAAGCTGAAGAGGGAAATGACATTCCATGGAGCAAAGGAAGTTGCACTCAGTTACTCAACTGGTGCGCTTGCCAGTTGCATGGGTCTCATATACAACCGGATGGGAACAGTGACCACAGAAGTGGCTCTTGGCCTAGTATGTGCCACTTGTGAACAGATTGCTGATGCCCAACATCGGTCCCACAGGCAGATGGCGACTACCACCAACCCACTAATCAGGCATGAGAACAGAATGGTACTAGCCAGCACTACGGCTAAGGCCATGGAGCAGATGGCTGGATCAAGTGAGCAGGCAGCAGAAGCCATGGAAGTCGCAAGTCAGGCTAGGCAAATGGTGCAGGCTATGAGGACAATTGGGACTCACCCTAGTTCCAGTGCAGGTCTAAAAGATGATCTTATTGAAAATTTGCAGGCTTACCAGAAACGGATGGGAGTGCAAATGCAGAGATTCAAGTGATCCTCTCGTTGTTGCAGCAAGTATCATTGGGATATTGCACTTGATATTGTGGATTCTTGATCGTCTTTTCTTCAAATGCATTTATCGTCGCTTTAAATACGGTTTGAAAAGAGGGCCTTCTACGGAAGGAGTGCCTGAGTCTATGAGGGAAGAGTATCGGCAGGAACAGCAGAATGCTGTGGATGTTGACGATGGTCATTTTGTCAACATAGAGCTGGAGTAA(SEQ ID NO:10); Using the target gene sequence, a primer-probe set for detecting influenza A virus was constructed, comprising: The F3 primer consists of the base sequence shown in SEQ ID NO: 1; Primer B3 consists of the base sequence shown in GCCTGCTCACTCGATCCA (SEQ ID NO: 2); The FIP primers consist of the base sequence shown in SEQ ID NO: 3; The BIP primers consist of the base sequence shown in SEQ ID NO: 4; The LF primers consist of the base sequence shown in SEQ ID NO: 5; The LB primers consist of the base sequence shown in SEQ ID NO: 6; The first probe has the same base sequence as the FIP primer, and the 5' end of the first probe is marked with a quenching group BHQ-1, which is composed of the base sequence shown in BHQ1-TGYCTGTGAGACCGATGCTGTGACAGAAGTGGCTTTTGGCC (SEQ ID NO: 3). The second probe has a base sequence that is a segment of the reverse complementary sequence at the 5' end of the first probe, and the 3' end of the second probe is labeled with a fluorescent group 6-FAM, which is composed of the base sequence shown in CACAGCATCGGTCTCACAGRCA-6-FAM (SEQ ID NO: 7); the absorption spectrum of the quencher group BHQ-1 can completely cover the emission spectrum of the fluorescent group 6-FAM.
[0025] Those skilled in the art will understand that the coding sequence of the M2 ion channel protein has a homology of more than 97% among different NA subtypes of influenza A virus, and its transmembrane region and N-terminal sequence have almost no deletions or insertions in the N1-N9 range, with only a few nonsense mutations or conserved amino acid substitutions. Therefore, the primer and probe set can be used to detect influenza A virus of subtypes H5, H7, and H9.
[0026] 0.4 μmol L -1 The F3 primer, 0.4 μmol L-1 The B3 primer, 1.6 μmol L -1 The FIP primers, 1.6 μmol L -1 The BIP primers, 0.8 μmol L -1 The LF primer, 0.8 μmol L -1 The LB primers, 0.5 μmol L -1 The first probe, 0.5 μmol L -1 The second probe, 8U of strand displacement DNA polymerase Bst 3.0, 200U of Moloney murine leukemia virus reverse transcriptase (M-MuLV RT), 40U of RNase inhibitor, and 20 mmol L -1 Tris-HCl, 1 mmol L -1 dNTPs, 8 mmol / L -1 MgSO4, 10 mmol L -1 (NH4)2SO4, 50 mmol L -1 The kit for detecting influenza A virus is prepared by adding KCl and 0.1 vol% Tween-20 to a final volume of 25 μL with ribonucleic acid-free water.
[0027] The reaction system was kept at 63℃ for 45 min, and the fluorescence signal of the FAM channel was collected every 30 s. Positive samples should show a visible fluorescence curve jump, and the signal should continue to increase over time, while negative samples should always maintain a low baseline with no visible rise. Example
[0028] The nucleic acids of influenza A virus H5N1 pathogen, influenza A virus H7N9 pathogen, and influenza A virus H9N2 pathogen were serially diluted to 10⁻⁶. 5 Up to 10 0 For each copy / μL sample, take 2.5μL of the sample as a template and perform detection according to the detection method in Example 1; The results are as follows Figure 1-3 As shown, influenza A virus subtypes H5, H7, and H9 can all be detected in 10... 0 The stable detection at the copy / μL level, with no signal increase in the negative (blank, i.e., sample without copy template) control group, indicates that the sensitivity of the primer-probe set reaches the single-copy level.
[0029] Additionally, the nucleic acids of the following pathogens were diluted to 10⁻¹⁰: H1N1 influenza A virus, H3N2 influenza A virus, influenza B virus, respiratory syncytial virus, human adenovirus ADV-4, novel coronavirus, Mycoplasma pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Legionella pneumophila, Streptococcus pneumoniae, Streptococcus pyogenes, and Acinetobacter baumannii. 5 For each copy / μL sample, take 2.5μL of the sample as a template and perform detection according to the detection method in Example 1; The results showed that all non-target pathogens were negative, indicating that the primer-probe set had good specificity and no cross-reaction with common clinical interfering agents.
[0030] The difference between this comparative example and Example 1 is that the first and second probes are replaced with 5×SYBR Green I. Taking the nucleic acid of the H5N1 influenza A virus pathogen as an example, it is still serially diluted to 10. 5 Up to 10 0 For each copy / μL sample, take 2.5μL of the sample as a template and perform the detection using the same method; The results are as follows Figure 4 As shown, the negative (blank) control group showed a signal detection, indicating a false positive.
[0031] The difference between this comparative example and Example 1 is as follows: The F3 primer consists of the base sequence shown in CAAAGCTCTATCAAAACCCAA (SEQ ID NO: 11); Primer B3 consists of the base sequence shown in TGCATATTCTGGAGCAATGA (SEQ ID NO: 12); The FIP primers consist of the base sequence shown in SEQ ID NO: 13; The BIP primers consist of the base sequence shown in SEQ ID NO: 14; Furthermore, no LF or LB primers were used, and 240 mU of RNase H II and 0.2 μmol L were employed. -1 The chimeric probe replaces the first and second probes; The chimeric probe consists of the base sequence shown in SEQ ID NO: 15, namely 6-FAM-CTACTAGACCrCAAAGTAAACG-BHQ1. Taking the nucleic acid of the H5N1 influenza A virus pathogen as an example, it is still serially diluted to 10. 5 Up to 10 0 For each copy / μL sample, take 2.5μL of the sample as a template and perform the detection using the same method; The results are as follows Figure 5 As shown, the negative (blank) control group showed a signal detection, indicating a false positive.
[0032] The difference between this comparative example and Example 1 is as follows: The primer and probe set for detecting the H5 subtype of influenza A virus includes: The F3 primer consists of the base sequence shown in AGGATGGGAACGGTGAC (SEQ ID NO: 16); Primer B3 consists of the base sequence shown in GCCTGCTCACTCGATCCA (SEQ ID NO: 17); The FIP primers consist of the base sequence shown in TGTCTGTGAGACCGATGCTGTGACAGAAGTGGCTTTTGGCC (SEQ ID NO: 18); The BIP primers consist of the base sequence shown in SEQ ID NO: 19; The LF primers consist of the base sequence shown in SEQ ID NO: 20; The LB primers consist of the base sequence shown in SEQ ID NO: 21; The first probe consists of the base sequence shown in BHQ1-TGTCTGTGAGACCGATGCTGTGACAGAAGTGGCTTTTGGCC (SEQ ID NO: 22); The second probe consists of the base sequence shown in CACAGCATCGGTCTCACAGACA-6-FAM (SEQ ID NO: 23); The primer and probe set for detecting the H7 subtype of influenza A virus includes: The F3 primer consists of the base sequence shown in SEQ ID NO: 24; Primer B3 consists of the base sequence shown in GCCTGCTCACTCGATCCA (SEQ ID NO: 25); The FIP primers consist of the base sequence shown in TGCCTGTGGGACCGATGTTGTGACAGAAGTGGCTTTTGGCC (SEQ ID NO: 26); The BIP primers consist of the base sequence shown in SEQ ID NO: 27, namely ACTACTAACCCACTAATTAGGCTCTGCTCCATAGCCTTAGCT. The LF primers consist of the base sequence shown in SEQ ID NO: 28; The LB primers consist of the base sequence shown in SEQ ID NO: 29; The first probe consists of the base sequence shown in BHQ1-TGCCTGTGGGACCGATGTTGTGACAGAAGTGGCTTTTGGCC (SEQ ID NO: 30); The second probe consists of the base sequence shown in CACAACATCGGTCCCACAGGCA-6-FAM (SEQ ID NO: 31); The primer and probe set for detecting the H9 subtype of influenza A virus includes: The F3 primer consists of the base sequence shown in CGGATGGGAACAGTGAC (SEQ ID NO: 32); Primer B3 consists of the base sequence shown in GCCTGCTCACTCGATCCA (SEQ ID NO: 33); The FIP primers consist of the base sequence shown in TGCCTGTGGGACCGATGTTGGGACAGAAGTGGCTTTTGGCC (SEQ ID NO: 34); The BIP primers consist of the base sequence shown in SEQ ID NO: 35; The LF primers consist of the base sequence shown in SEQ ID NO: 36; The LB primers consist of the base sequence shown in SEQ ID NO: 37; The first probe consists of the base sequence shown in BHQ1-TGCCTGTGGGACCGATGTTGGGACAGAAGTGGCTTTTGGCC (SEQ ID NO: 38); The second probe consists of the base sequence shown in CCCAACATCGGTCCCACAGGCA-6-FAM (SEQ ID NO: 39); The nucleic acids of influenza A virus H5N1 pathogen, influenza A virus H7N9 pathogen, and influenza A virus H9N2 pathogen were serially diluted to 10⁻⁶. 4 Up to 10 0 For each copy / μL sample, take 2.5μL of the sample as a template and perform detection according to the detection method in Example 1; The results are as follows Figure 6 As shown, each single-subtype primer can efficiently amplify the corresponding subtype template, with a sensitivity on the order of single copies, while the detection limit for other subtype templates is on the order of 10. 3 -10 4 The number of copies / μL differs by 3-4 orders of magnitude; however, the primer-probe set of Example 1 can detect single copies of each subtype template.
[0033] In summary, the primer-probe set of this invention achieves high coverage of H5, H7, and H9 subtypes by introducing degenerate bases optimized through conservation analysis at key sites. This allows for simultaneous screening without subtype-specific detection, significantly reducing reagent types and inventory pressure, while maintaining excellent amplification efficiency and specificity, avoiding false negatives due to sequence differences. Furthermore, the strategy of quenching-fluorescence complementary probes coupled with strand displacement amplification ensures that the fluorescence signal is activated only when the target sequence is present, resulting in extremely low background that can be clearly interpreted by the naked eye or instruments, completely avoiding false positives caused by contamination or non-specific amplification in traditional dye methods.
[0034] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the description and illustrations of the present invention should be included within the protection scope of the present invention.
Claims
1. A primer probe set for detecting an influenza A virus, the influenza A virus consisting of an H5 subtype, an H7 subtype, and an H9 subtype; characterized in that, The primer probe set comprises: an F3 primer, the base sequence of which is shown in SEQ ID NO: 1; a B3 primer, the base sequence of which is shown in SEQ ID NO: 2; an FIP primer, the base sequence of which is shown in SEQ ID NO: 3; a BIP primer, the base sequence of which is shown in SEQ ID NO: 4; an LF primer, the base sequence of which is shown in SEQ ID NO: 5; an LB primer, the base sequence of which is shown in SEQ ID NO: 6; a first probe, the base sequence of which is the same as that of the FIP primer or the BIP primer, and the 5' end of which is labeled with one of a quencher group or a fluorescent group; and a second probe, the base sequence of which is the reverse complement of the 5' end of the first probe or a segment of the reverse complement of the 5' end of the first probe, and the 3' end of which is labeled with the other of the quencher group or the fluorescent group, the absorption spectrum of the quencher group being able to completely cover the emission spectrum of the fluorescent group.
2. The primer probe set of claim 1, wherein The base sequence of the first probe is the same as that of the FIP primer, and the 5' end of the first probe is labeled with the quencher group; the base sequence of the second probe is a segment of the reverse complement of the 5' end of the first probe, and the 3' end of the second probe is labeled with the fluorescent group, the absorption spectrum of the quencher group being able to completely cover the emission spectrum of the fluorescent group.
3. The primer probe set according to claim 1 or 2, characterized in that, The quencher group is BHQ-1, and the fluorescent group is 6-FAM.
4. The primer probe set of claim 1, wherein The primer probe set comprises, in terms of molar concentration: 4 parts of the F3 primer; 4 parts of the B3 primer; 16 parts of the FIP primer; 16 parts of the BIP primer; 8 parts of the LF primer; 8 parts of the LB primer; 5 parts of the first probe; and 5 parts of the second probe.
5. A kit for detecting influenza A virus, which is constituted of H5 subtype, H7 subtype, and H9 subtype, characterized by comprising: The kit comprises the primer probe set according to any one of claims 1-4.
6. The kit of claim 5, wherein Further comprising: a strand displacement DNA polymerase Bst 3.0, a Moloney murine leukemia virus reverse transcriptase, an RNase inhibitor, a buffer suitable for LAMP reaction, dNTPs, and magnesium ions.
7. A method for detecting an influenza A virus for non-diagnostic purposes, said influenza A virus consisting of H5 subtype, H7 subtype, and H9 subtype; characterized by, The steps of the detection method comprise: S1, providing a sample to be tested; S2, mixing the sample to be tested with the components in the kit according to any one of claims 5-6 to form a reaction system; S3, performing an amplification reaction under isothermal conditions; S4, detecting a fluorescent signal in the reaction system in real time.
8. Use of the primer probe set according to any one of claims 1-4 or the kit according to any one of claims 5-6 in the preparation of a product for detecting influenza A virus, the influenza A virus consisting of H5 subtype, H7 subtype, and H9 subtype.
9. The primer probe set according to any one of claims 1 to 4, or the kit according to any one of claims 5 to 6, or the detection method according to claim 7, or the use according to claim 8, characterized in that, The influenza A virus consists of H5N1 subtype, H7N9 subtype, and H9N2 subtype.