Kit for detecting twelve pathogens of human respiratory tract

Abstract

The invention discloses a kit for detecting twelve pathogens in the human respiratory tract, and relates to the technical field of kits, the kit comprises a reagent strip, a first hole site chamber arranged on the reagent strip, a nucleic acid extraction suction head arranged above the first hole site chamber, a second hole site chamber arranged on the reagent strip on one side of the first hole site chamber, and a third hole site chamber arranged on the reagent strip on the other side of the second hole site chamber, a chip reagent canning suction head is arranged above the second hole site cavity, a third hole site cavity is formed in the reagent strip on the side, away from the first hole site cavity, of the second hole site cavity, and a UV light curing adhesive dispensing suction head is arranged above the third hole site cavity; a reagent washing liquid storage assembly is arranged on the side, away from the second hole position cavity, of the third hole position cavity, the reagent washing liquid storage assembly comprises a plurality of storage units and is used for storing different types of reagent washing liquid, and a limiting groove is formed in the portion, on one side of the reagent washing liquid storage assembly, of the reagent strip; and an amplification chamber assembly is arranged in the limiting groove. The kit is high in use flexibility and strong in convenience.

Description

Technical Field

[0001] The present invention relates to the technical field of test kits, in particular to a test kit for detecting twelve pathogens of the human respiratory tract. Background Art

[0002] Respiratory infections are complex, with pathogens including bacteria, viruses, mycoplasmas, chlamydia, and fungi, with over 80% being viral. These infections can occur in all seasons and at any age, and are transmitted through virus-laden droplets, aerosols, or contaminated utensils. Respiratory infections are the leading cause of death among young children (<5 years old) worldwide, killing approximately 5 million children annually. Common pathogens that can cause respiratory infections in clinical practice include influenza A virus (FluA), influenza B virus (FluB), respiratory syncytial virus (RSV), adenovirus (Adv), parainfluenza virus (PIV), metapneumovirus (MPV), bocavirus (HBoV), rhinovirus (HRV), coronavirus (Cov), Mycoplasma pneumoniae (MP), Chlamydia pneumoniae (CP), and the novel coronavirus. These twelve diseases are extremely difficult to differentiate based solely on clinical symptoms, and mixed infections with multiple pathogens are common in clinical practice. Therefore, developing an efficient, sensitive, and cost-effective detection method is crucial for the differential diagnosis, prevention, and control of respiratory diseases.

[0003] The key steps in molecular biological testing of samples include nucleic acid extraction and purification, nucleic acid amplification, and nucleic acid detection. The quality and speed of nucleic acid extraction and purification are crucial for accurate and rapid detection of samples. Currently, a variety of methods are available for nucleic acid extraction and purification, including PC extraction, high-salt precipitation, centrifugal columns, and biomagnetic beads. These methods require operator intervention, making them cumbersome and prone to human contamination, leading to false positives. Poor sample extraction quality can also affect amplification results, leading to false negatives.

[0004] The invention relates to genetic nucleic acid testing and nucleic acid extraction and amplification testing. Currently, these methods are commonly performed manually through processes such as lysis and binding, magnetic bead extraction, and purification. However, manual operation varies from person to person, and the precision and accuracy of the operation are generally slow, labor costs are high, and test data may exhibit subtle discrepancies. Furthermore, nucleic acid transfer and subsequent PCR amplification are required. Furthermore, there is no multiplex RT-PCR method that can simultaneously detect twelve respiratory pathogens: FluA, FluB, RSV, Adv, PIV, hMPV, HBoV, HRV, Cov, MPn, CPn, and SC2.

[0005] Although the nucleic acid extraction instrument has improved and guaranteed the speed and accuracy of sample lysis, magnetic bead extraction, and purification, its shortcomings are also obvious. A nucleic acid extraction kit must be used by eight people before it can be used for extraction, so there are problems with limitations and flexibility, and the cost of manufacturing nucleic acid extraction kits is high. Therefore, a product is needed that can meet the needs of a single-person nucleic acid lysis, extraction, and purification method, can perform twelve human respiratory pathogen tests, and can be tested on demand. Summary of the Invention

[0006] In order to overcome the shortcomings of the existing technology, the purpose of the present invention is to provide a kit for detecting twelve pathogens of the human respiratory tract, which solves the problem of insufficient flexibility in the use of traditional equipment by setting up a reagent washing liquid storage component and an amplification chamber component.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A test kit for detecting twelve pathogens of the human respiratory tract comprises a reagent strip, and further comprising: a first hole position chamber arranged on the reagent strip, a nucleic acid extraction tip arranged above the first hole position chamber, a second hole position chamber arranged on the reagent strip on one side of the first hole position chamber, a chip reagent canned tip arranged above the second hole position chamber, a third hole position chamber arranged on the reagent strip on a side of the second hole position chamber away from the first hole position chamber, a UV light curing glue dispensing tip arranged above the third hole position chamber, the nucleic acid extraction tip, the chip reagent canned tip and the UV light curing glue dispensing tip having different volumes, a reagent washing liquid storage assembly arranged on the reagent strip on a side of the third hole position chamber away from the second hole position chamber, the reagent washing liquid storage assembly comprising a plurality of storage units and storing different types of reagent washing liquids, a limiting groove being provided on the reagent strip on one side of the reagent washing liquid storage assembly, and an amplification chamber assembly being arranged in the limiting groove.

[0009] As a preferred technical solution of the present invention, the reagent washing storage component includes a fourth hole position chamber arranged on the reagent strip, a fifth hole position chamber is arranged on one side of the fourth hole position chamber, and a sixth hole position chamber is arranged on the side of the fifth hole position chamber away from the fourth hole position chamber. Different actual washing liquids are stored in the fourth hole position chamber, the fifth hole position chamber and the sixth hole position chamber respectively.

[0010] As a preferred technical solution of the present invention, a seventh hole position chamber is provided on the reagent strip on the side of the sixth hole position chamber away from the fifth hole position chamber, and magnetic beads are provided in the seventh hole position chamber. An eighth hole position chamber is provided on the reagent strip on the side of the seventh hole position chamber away from the sixth hole position chamber, and reagent eluent is provided in the eighth hole position chamber. A ninth hole position chamber is provided on the reagent strip on the side of the eighth hole position chamber away from the seventh hole position chamber, and UV light curing glue is stored in the ninth hole position chamber.

[0011] As a preferred technical solution of the present invention, the amplification chamber assembly includes a connecting frame, which is arranged in correspondence with the limiting groove, and the bottom of the connecting frame is respectively fixedly connected to the first chip PCR chamber, the second chip PCR chamber, the third chip PCR chamber and the fourth chip PCR chamber.

[0012] As a preferred technical solution of the present invention, a first sample addition port, a second sample addition port, a third sample addition port and a fourth sample addition port are respectively arranged above the first chip PCR chamber, the second chip PCR chamber, the third chip PCR chamber and the fourth chip PCR chamber.

[0013] This invention offers the following advantages: High production capacity: Through the "one mold, eight cavities" injection mold design, eight test kits can be produced in three minutes, reaching a daily production capacity of 1,280 test kits, significantly improving production efficiency. Low cost: Each test kit costs less than RMB 0.4 and is sold for approximately RMB 0.8, significantly reducing manufacturing and marketing costs.

[0014] Mold Durability: Made of high-quality stainless steel, it can be repeatedly injected over 250,000 times, with a precision error controlled within 0.05mm-0.08mm, ensuring long-term stability. Automated Process: Through fully automatic canning and hot-pressing film sealing technology, the operation speed is 10-15 times that of manual operation, reducing manual intervention and avoiding operational errors. Integrated Design: The reagent strips are pre-loaded with reagents and tips required for nucleic acid extraction, washing, amplification, etc., eliminating the need for manual preparation and simplifying the testing process. On-Demand Testing: Supports single-person testing, flexibly adapting to clinical point-of-care testing needs, breaking through the limitations of traditional multiple-person testing systems.

[0015] Multiple sealing technology: Paraffin oil and paraffin rings are used for double sealing to effectively prevent aerosol contamination and reduce the risk of false positives / false negatives. Optimized primer probe design: Specific primer probes are designed for 12 pathogens and conserved regions of human reference genes, combined with fluorescent labels (FAM, HEX, ROX, CY5) and non-fluorescent quenching groups (BHQ1 / BHQ2) to ensure detection sensitivity and specificity. Lyophilized system: The reagents are stored in the form of lyophilized pellets with high stability, easy transportation and long-term storage, and stable performance after reconstitution. Simultaneous detection of 12 pathogens: By optimizing the multiple RT-PCR reaction conditions, a single test can simultaneously distinguish 12 respiratory pathogens including influenza A virus (FluA), influenza B virus (FluB), respiratory syncytial virus (RSV), adenovirus (Adv), and new coronavirus (SC2).

[0016] Clinically validated for high accuracy: 84 clinical sample test results were fully consistent with Sanger sequencing results, confirming the reliability of the detection method. Reduced reagent waste: The single-dose design avoids reagent waste associated with batch testing, reducing laboratory operating costs. Energy-saving and environmentally friendly: Automated processes reduce energy consumption, and the freeze-drying process reduces reliance on cold chain transportation, in line with green production concepts. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The figure shows a reagent strip schematically illustrating the overall structure of a test kit for detecting twelve pathogens of the human respiratory tract.

[0018] Figure 2 The front cross-sectional structure of a reagent strip used to detect twelve pathogens in the human respiratory tract is shown.

[0019] In the figure: 1. Reagent strip; 2. First hole chamber; 3. Nucleic acid extraction pipette tip; 4. Second hole chamber; 5. Chip reagent canned pipette tip; 6. Third hole chamber; 7. UV light curing glue dispensing pipette tip; 8. Fourth hole chamber; 9. Fifth hole chamber; 10. Sixth hole chamber; 11. Seventh hole chamber; 12. Eighth hole chamber; 13. Ninth hole chamber; 14. Limiting groove; 15. Connecting frame; 16. First chip PCR chamber; 17. Second chip PCR chamber; 18. Third chip PCR chamber; 19. Fourth chip PCR chamber; 20. First sample port; 21. Second sample port; 22. Third sample port; 23. Fourth sample port. DETAILED DESCRIPTION

[0020] The preferred embodiments of the present invention are described below in conjunction with the attached test strips. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.

[0021] Example 1, please refer to Figure 1-2A test kit for detecting twelve pathogens of the human respiratory tract comprises a reagent strip 1, and further comprises: a first hole position chamber 2 provided on the reagent strip 1, a nucleic acid extraction pipette tip 3 provided above the first hole position chamber 2, a second hole position chamber 4 provided on the reagent strip 1 on one side of the first hole position chamber 2, a chip reagent canned pipette tip 5 provided above the second hole position chamber 4, a third hole position chamber 6 provided on the reagent strip 1 on the side of the second hole position chamber 4 away from the first hole position chamber 2, a UV light curing glue dispensing pipette tip provided above the third hole position chamber 6. Head 7, the nucleic acid extraction tip 3, chip reagent canned tip 5 and UV light curing glue dispensing tip 7 have different volumes, the third hole position chamber 6 is provided with a reagent washing liquid storage component on the side of the reagent strip 1 away from the second hole position chamber 4, the reagent washing liquid storage component includes a plurality of storage units and stores different types of reagent washing liquids, a limiting groove 14 is provided on the reagent strip 1 on one side of the reagent washing liquid storage component, and an amplification chamber component is provided in the limiting groove 14; the reagent strip 1 is processed and formed by mold opening and injection molding;

[0022] This kit is used to detect primer-probe combinations and amplification reaction solutions for twelve respiratory pathogens and human endogenous internal controls; a special 1000-μl pipette tip is placed in the first well chamber on reagent strip 1; a special 100-μl pipette tip is placed in the second well chamber; and a special 100-μl pipette tip is placed in the third well chamber.

[0023] The reagent washing storage component includes a fourth hole position chamber 8 arranged on the reagent strip 1, a fifth hole position chamber 9 is arranged on one side of the fourth hole position chamber 8, and a sixth hole position chamber 10 is arranged on the side of the fifth hole position chamber 9 away from the fourth hole position chamber 8. Different actual washing liquids are stored in the fourth hole position chamber 8, the fifth hole position chamber 9 and the sixth hole position chamber 10 respectively.

[0024] A seventh hole position chamber 11 is provided on the reagent strip 1 on the side of the sixth hole position chamber 10 away from the fifth hole position chamber 9, and magnetic beads are provided in the seventh hole position chamber 11. An eighth hole position chamber 12 is provided on the reagent strip 1 on the side of the seventh hole position chamber 11 away from the sixth hole position chamber 10, and reagent eluent is provided in the eighth hole position chamber 12. A ninth hole position chamber 13 is provided on the reagent strip 1 on the side of the eighth hole position chamber 12 away from the seventh hole position chamber 11, and UV light curing glue is stored in the ninth hole position chamber 13.

[0025] The amplification chamber assembly includes a connecting frame 15, which is arranged in correspondence with the limiting groove 14. The bottom of the connecting frame 15 is respectively fixedly connected to the first chip PCR chamber 16, the second chip PCR chamber 17, the third chip PCR chamber 18 and the fourth chip PCR chamber 19.

[0026] A first sample addition port 20, a second sample addition port 21, a third sample addition port 22 and a fourth sample addition port 23 are respectively arranged above the first chip PCR chamber 16, the second chip PCR chamber 17, the third chip PCR chamber 18 and the fourth chip PCR chamber 19.

[0027] The fourth well of reagent strip 1 is for reagent wash solution 1. The fifth well of reagent strip 1 is for reagent wash solution 2 and magnetic beads. The sixth well of reagent strip 1 is for reagent wash solution 3. The seventh well of reagent strip 1 is for reagent eluent. The eighth well of reagent strip 1 is for paraffin oil. The ninth and tenth wells of reagent strip 1 are for nucleic acid PCR amplification reagents and the paraffin ring of the PCR connector.

[0028] The reagent strip 1 for detecting twelve pathogens of the human respiratory tract is simple to manufacture and can be produced by injection molding with a mold and 8 cavities, one mold every 3 minutes, 160 pcs per hour with 8 pcs per mold, and a daily production capacity of about 1,280 pcs according to a normal 8-hour work system. The cost is less than RMB 0.4 per pc based on the current market price of high-quality PP raw materials, and the sales price is calculated as about RMB 0.8 for manual and mechanical wear and tear on the site, which greatly reduces costs and improves production capacity.

[0029] Since the injection molding mold is made of high-quality stainless steel, its practical life is about 250,000 injection moldings. Therefore, the precision error can still be maintained within the controllable range of 0.05mm-0.08mm during the 250,000 injection molding processes.

[0030] The nucleic acid extraction PCR amplification reagent strip 1 is canned in a fully automatic canning manner and is sealed by a hot pressing method. Compared with the manual operation time, the experimental time can be about ten to fifteen times that of the manual operation time, and has the advantages of higher accuracy, faster speed, smaller detection error, etc.

[0031] The test kit for detecting twelve pathogens of the human respiratory tract is double-sealed by paraffin oil and paraffin rings, thereby greatly reducing and eliminating the threat and pollution of amplification product aerosols to the laboratory or testing environment.

[0032] The kit for detecting twelve human respiratory pathogens is designed with specific primers for the conserved regions of the twelve respiratory pathogens and human endogenous genes, and by optimizing the multiplex PCR reaction conditions, an RT-PCR method is established that can simultaneously detect and distinguish the above 12 viruses and internal controls.

[0033] Example 2, please refer to Figure 1-2The device is used to detect processing reagents for twelve human respiratory pathogens, and primer probe combinations and amplification reaction solutions are designed for detecting twelve respiratory pathogens including FluA, FluB, RSV, Adv, PIV, hMPV, HBoV, HRV, Cov, MPn, CPn, and SC2 as well as internal controls.

[0034] Detection primers for FluA virus: upstream primer as shown in the figure, downstream primer as shown in the figure and Figure 1-2 The probe shown, the Figure 1-2 The 5' end of the probe shown is connected to a fluorescent reporter group, and the 3' end is connected to a non-fluorescent quencher group. The specific base sequences of the primer and probe are as follows:

[0035] FluA virus detection upstream primer: 5'-AGACAAGACCAATCCTGTCAC-3' Figure 1-2 As shown;

[0036] FluA virus detection downstream primer: 5'-AAGCGTCTACGCTGCAGTC-3' Figure 1-2 As shown;

[0037] FluA virus detection probe: 5'FAM-TTCACGCTCACCGTGCCC-3'BHQ1 Figure 1-2 As shown;

[0038] Detection primers for FluB virus: Figure 1-2 The upstream primers shown, Figure 1-2 The downstream primers shown and Figure 1-2 The probe shown, the Figure 1-2 The 5' end of the probe shown is connected to a fluorescent reporter group, and the 3' end is connected to a non-fluorescent quencher group. The specific base sequences of the primer and probe are as follows:

[0039] FluB virus detection upstream primer: 5'-TGTTTGGAGACACAATTGCCT-3' Figure 1-2 As shown;

[0040] FluB virus detection downstream primer: 5'-AACCAACAGTGTAAYTTTTCTGC-3' Figure 1-2 As shown;

[0041] FluB virus detection probe: 5'HEX-TTCTGCTTTGCCTTCTCCATCT-3'BHQ1 Figure 1-2 As shown;

[0042] Detection primers for RSV virus: Figure 1-2 The upstream primers shown, Figure 1-2The downstream primers shown and Figure 1-2 The probe shown, the Figure 1-2 The 5' end of the probe shown is connected to a fluorescent reporter group, and the 3' end is connected to a non-fluorescent quencher group. The specific base sequences of the primer and probe are as follows:

[0043] RSV virus detection upstream primer: 5'-GGGCAAATATGGAAACAT-3' Figure 1-2 As shown;

[0044] RSV virus detection downstream primer: 5'-GCACCCATATTGTTAGTGATG-3' Figure 1-2 As shown;

[0045] RSV virus detection probe: 5'ROX-CTTCACGAAGGCTCCACATACACA-3'BHQ2 Figure 1-2 As shown;

[0046] Detection primers for Adv virus: Figure 1-2 The upstream primers shown, Figure 1 The downstream primers shown and Figure 2 The probe shown, the Figure 2 The 5' end of the probe shown is connected to a fluorescent reporter group, and the 3' end is connected to a non-fluorescent quencher group. The specific base sequences of the primer and probe are as follows:

[0047] Adv virus detection upstream primer: 5'-CTGATCGCTAGGGTATTTAWACC-3' Figure 1-2 As shown;

[0048] Adv virus detection downstream primer: 5'-GCGGAGGAGAAAACTCTACTCGC-3' Figure 1 As shown;

[0049] Adv virus detection probe: 5'FAM-CACTCAAGAGTGGCCTCTTG-3'BHQ1 Figure 1 As shown;

[0050] Detection primers for HRV virus: Figure 1-2 The upstream primer, downstream primer, and probe shown in FIG. 5 are connected to a fluorescent reporter group at the 5' end of the probe and a non-fluorescent quencher group at the 3' end. The specific base sequences of the primers and probe are as follows:

[0051] HRV virus detection upstream primer: 5'-GAAGCCAWIIWITIGACAIGGTGIGA-3' Figure 1-2 As shown;

[0052] HRV virus detection downstream primer: 5'-GAAACACGGACACCCAAAGTAGT-3' Figure 1-2 As shown;

[0053] HRV virus detection probe: 5'HEX-TCCTCCGGCCCCTGAATGYG-3'BHQ1 Figure 1-2 As shown;

[0054] Detection primers for CPn pathogens: Figure 1-2 The upstream primer, downstream primer and Figure 1-2 The probes shown are as described Figure 1-2 The 5' end of the probe shown is connected to a fluorescent reporter group, and the 3' end is connected to a non-fluorescent quencher group. The specific base sequences of the primer and probe are as follows:

[0055] CPn pathogen detection upstream primer: 5'-GCATCTAATATATATTAAAGAAGGGG-3' Figure 1-2 As shown;

[0056] CPn pathogen detection downstream primer: 5'-GGCTTTTACCCCACCAAC-3' Figure 1-2 As shown;

[0057] CPn pathogen detection probe: 5'ROX-CTGATATCGCATAAACTCTTCCTCAACC-3'BHQ2 Figure 1-2 As shown;

[0058] Detection primers for hMPV virus: Figure 1-2 The upstream primer, the downstream primer and Figure 1 The probe shown, the Figure 1 The 5' end of the probe shown is connected to a fluorescent reporter group, and the 3' end is connected to a non-fluorescent quencher group. The specific base sequences of the primer and probe are as follows:

[0059] hMPV virus detection upstream primer: 5'-GYAACATCCCACAAAACCAGAG-3' Figure 1-2 As shown;

[0060] hMPV virus detection downstream primer: 5'-AGTTTAGTGAATATTARAGCACCTACAC-3' Figure 1-2 As shown;

[0061] hMPV virus detection probe: 5'FAM-CCTTCAGCACCAGACACACCNAT-3'BHQ1 Figure 1 As shown;

[0062] Detection primers for hPIV virus: Figure 2 The upstream primer, downstream primer, and probe shown in FIG. 5 are connected to a fluorescent reporter group at the 5' end of the probe and a non-fluorescent quencher group at the 3' end. The specific base sequences of the primers and probe are as follows:

[0063] hPIV virus detection upstream primer: 5'-CCTTCATTATCAATTGGTGATGC-3' Figure 2 As shown;

[0064] hPIV virus detection downstream primer: 5'-CCTAATTGTAAAACCTGATATGACTTC-3' Figure 1-2 As shown;

[0065] hPIV virus detection probe: 5'HEX-TTCATCAAACTTAATCACTCAAGGATGTG-3'BHQ1 Figure 1-2 As shown;

[0066] Detection primers for MPn: Figure 1-2 The upstream primers shown are Figure 1-2 The downstream primer and probe shown are connected to a fluorescent reporter group at the 5' end and a non-fluorescent quencher group at the 3' end. The specific base sequences of the primer and probe are as follows:

[0067] MPn detection upstream primer: 5'-CCAAAACGTGTTCTTTCTCCAT-3' Figure 1-2 As shown;

[0068] MPn virus detection downstream primer: 5'-TTAGCCATTCTACTCAATCCCA-3' Figure 1-2 As shown;

[0069] MPn detection probe: 5'ROX-ATAAACTCAACAAACAAACTGG-3'BHQ2 Figure 1-2 As shown;

[0070] Detection primers for CoV virus: Figure 1-2 The upstream primer, downstream primer and Figure 1-2 The probe shown, the Figure 1-2 The 5' end of the probe shown is connected to a fluorescent reporter group, and the 3' end is connected to a non-fluorescent quencher group. The specific base sequences of the primer and probe are as follows:

[0071] CoV virus detection upstream primer: 5'-GTTGATTCAACCTTTGTCACAGAATA-3' Figure 1-2 As shown;

[0072] CoV virus detection downstream primer: 5'-GCATAATCAGAATTATAACACACAAC-3' Figure 1-2 As shown;

[0073] CoV virus detection probe: 5'FAM-AAGCATTTTAGTATGATGATTT-3'BHQ1 Figure 1-2 As shown;

[0074] Detection primers for SC2 virus: Figure 1-2 The upstream primers shown, Figure 1-2 The downstream primers shown and Figure 1-2 The probe shown, the Figure 1-2 The 5' end of the probe shown is connected to a fluorescent reporter group, and the 3' end is connected to a non-fluorescent quencher group. The specific base sequences of the primer and probe are as follows:

[0075] SC2 virus detection upstream primer: 5'-GTCACACCTTCGGGAACG-3' Figure 1-2 As shown; SC2 virus detection downstream primer: 5'-ATGACTTGATCTATGAAATTTGG-3' Figure 1-2 As shown; SC2 virus detection probe: 5'HEX-CTACACAGGTGCCATCAAATTGG-3'BHQ1 Figure 1-2 As shown;

[0076] Detection primers for hBoV virus: Figure 1-2 The upstream primers shown, Figure 1-2 The downstream primers shown and Figure 1-2 The probe shown, the Figure 1-2 The 5' end of the probe shown is connected to a fluorescent reporter group, and the 3' end is connected to a non-fluorescent quencher group. The specific base sequences of the primer and probe are as follows:

[0077] hBoV virus detection upstream primer: 5'-GATAAARTTCCAAACTCATTTCCT-3' Figure 1-2 As shown;

[0078] hBoV virus detection downstream primer: 5'-AGTGCAGWATCCGTTTTCGTG-3' Figure 1-2 As shown;

[0079] hBoV virus detection probe: 5'ROX-CCTTTGTCCTACWCATTCACAGGAC-3'BHQ2 Figure 1-2 As shown;

[0080] Upstream primer for human endogenous reference gene detection: 5'-TGAGGAGAAGTCTGCCGTT-3' Figure 1-2 As shown; downstream primer for human endogenous reference gene detection: 5'-TAACCTTGATACCAACCTGCC-3' Figure 1-2 As shown;

[0081] Human endogenous reference gene detection probe: 5'CY5-CTCACCACCAACTTCATCCAC-3'BHQ2 Figure 1-2 As shown; preferably, the Figure 1-2 The fluorescent reporter group connected to the 5' end of the probe shown is FAM, and the non-fluorescent quencher group connected to the 3' end is BHQ1;

[0082] described Figure 1-2 The fluorescent reporter group connected to the 5' end of the probe shown is HEX, and the non-fluorescent quencher group connected to the 3' end is BHQ1;

[0083] described Figure 1-2 The fluorescent reporter group connected to the 5' end of the probe shown is ROX, and the non-fluorescent quencher group connected to the 3' end is BHQ2.

[0084] described Figure 1-2 The fluorescent reporter group connected to the 5' end of the probe shown is CY5, and the non-fluorescent quencher group connected to the 3' end is BHQ2.

[0085] Example 3, as Figure 1-2 As shown, this embodiment provides a reagent and consumables for detecting twelve pathogens of the human respiratory tract, including: the nucleic acid extraction and amplification reagent strip 1 described in Example 1, the primers and probes for detecting twelve pathogens of the human respiratory tract described in Example 2, and excipients or auxiliary agents for detecting the primers and probes of the twelve pathogens of the human respiratory tract. Wherein, the excipients or auxiliary agents refer to conventional excipients or auxiliary agents other than primers and probes that enable the primer-probe reagents for detecting twelve pathogens of the human respiratory tract to be used normally under normal working environment conditions. For example, Tris buffer, Mg2+, ribonucleic acid monomers (dA / G / C / UTPs), DNA polymerase, reverse transcriptase, UDG enzyme (Uracil-DNA Glycocasylase, uracil-DNA glycosylase), etc. for PCR detection.

[0086] Example 4, as Figure 1-2 As shown, embodiment 4 of the present invention provides a kit for detecting twelve pathogens of the human respiratory tract, the nucleic acid extraction and amplification reagent strip 1 described in embodiment 1, the primers and probes for detecting twelve pathogens of the human respiratory tract described in embodiment 2, and excipients or auxiliary agents for the primers and probes for detecting twelve pathogens of the human respiratory tract.

[0087] The excipients or auxiliary agents are conventional excipients or auxiliary agents other than primers and probes that enable the primer-probe reagents for detecting twelve human respiratory pathogens to be used normally under normal working conditions. For example, Tris buffer, Mg2+, ribonucleic acid monomers (dA / G / C / UTPs), DNA polymerase, reverse transcriptase, UDG enzyme (Uracil-DNA Glycocasylase), etc. for PCR detection.

[0088] Furthermore, in the kit for detecting twelve pathogens of the human respiratory tract described in Example 4 of the present invention, the primers and probes, excipients or auxiliary agents are made using a freeze-drying system to facilitate the storage and transportation of the kit.

[0089] The freeze-drying system requires not only the excipients or adjuvants of primers and probes for detecting twelve human respiratory pathogens, but also freeze-drying protective agents, such as trehalose dihydrate, mannitol, PEG8000, raffinose, dextran, glycine, etc.

[0090] Specifically, the final concentrations of the components of the amplification reaction solution during the reaction are: Tris-HCl 5-15 mmol / L, KCl 35-75 nmol / L, Mg2+ 2-8 mmol / L, dNTPs 0.3-0.6 mmol / L, UDG enzyme 0.02-0.2 U / ul, reverse transcriptase 1-5 U / uL, and DNA polymerase 0.05-0.3 U / ul;

[0091] Specifically, the final concentrations of the lyoprotectant components during the reaction are: trehalose dihydrate 220-330 mmol / L, mannitol 220-330 mmol / L, PEG8000 1-5 mmol / L, raffinose 0.5%-1% w / v, dextran 1%-5% w / v, and glycine 0.1%-0.5% w / v. The amplification reaction solution and lyoprotectant can be mixed with a 0.02 to 0.8 μM primer-probe combination for detecting twelve human respiratory pathogens to form a lyophilizable amplification reaction system reagent for detecting twelve human respiratory pathogens.

[0092] The kit for detecting twelve pathogens of the human respiratory tract described in Example 4 of the present invention is as follows Figure 1-2 Shown:

[0093]

[0094]

[0095]

[0096] Table 1 Amplification reaction system

[0097] (2) Amplification conditions

[0098] Through experimental testing, the denaturation / annealing time, denaturation / annealing temperature, number of cycles and other conditions were optimized, and the final PCR amplification conditions were determined as shown in Table 2 below.

[0099]

[0100] (3) System freeze-drying

[0101] The freeze-dried system has advantages such as good storage stability and convenient transportation. In the embodiment of the present invention, the amplification reaction solution reagent, lyoprotectant, and primer probe reagent are freeze-dried into freeze-dried pellets. When the freeze-dried pellets are used, they are all reconstituted with a nucleic acid elution solution.

[0102] Example 5, as Figure 1-2 As shown, the detection of respiratory samples

[0103] 1. 84 clinical samples were tested using the kit of the present invention.

[0104] 2. 84 respiratory clinical samples were subjected to nucleic acid extraction using a silica gel column method, and conserved fragments were amplified and subjected to Sanger sequencing. The test results were completely consistent with the test results of the present invention, as shown in Table 1. This shows that the method of the present invention has high accuracy in detecting twelve pathogens in the human respiratory tract.

[0105]

[0106] The standard parts used in the present invention can all be purchased commercially, and special-shaped parts can be customized according to the description in the specification and the attached test strips. The specific connection methods of each part are all conventional means such as bolts, rivets, welding, etc. that are mature in the prior art. The machinery, parts and equipment are all conventional models in the prior art. In addition, the circuit connections adopt conventional connection methods in the prior art and will not be described in detail here. Any content not described in detail in this specification belongs to the prior art known to professionals in this field.

[0107] In the description of the present invention, the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, a feature specified as "first" or "second" may explicitly or implicitly include one or more of such features. "Multiple" means two or more, unless otherwise specifically defined.

[0108] In the present invention, unless otherwise expressly specified or limited, the terms "mounted," "connected," "connect," "fixed," etc. should be understood broadly. For example, they may refer to fixed connection, detachable connection, or integration; mechanical connection or electrical connection; direct connection or indirect connection through an intermediate medium; internal communication between two components or interaction between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on specific circumstances.

[0109] In the present invention, unless otherwise expressly specified or limited, when a first feature is "above" or "below" a second feature, it may mean that the first and second features are in direct contact, or that the first and second features are in indirect contact through an intermediary. Furthermore, when a first feature is "above," "above," or "above" a second feature, it may mean that the first feature is directly above or diagonally above the second feature, or simply means that the first feature is at a higher level than the second feature. When a first feature is "below," "below," or "below" a second feature, it may mean that the first feature is directly below or diagonally below the second feature, or simply means that the first feature is at a lower level than the second feature.

[0110] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0111] The test strips attached to the embodiments disclosed in the present invention only involve structures related to the embodiments disclosed in the present invention. Other structures can refer to common designs. In the absence of conflicts, the same embodiment and different embodiments of the present invention can be combined with each other.

[0112] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A kit for detecting twelve pathogens of the human respiratory tract, comprising a reagent strip (1), characterized in that: Also includes: A first hole position chamber (2) is provided on the reagent strip (1), a nucleic acid extraction pipette tip (3) is provided above the first hole position chamber (2), a second hole position chamber (4) is provided on the reagent strip (1) on one side of the first hole position chamber (2), a chip reagent canned pipette tip (5) is provided above the second hole position chamber (4), a third hole position chamber (6) is provided on the reagent strip (1) on the side of the second hole position chamber (4) away from the first hole position chamber (2), a UV light curing glue dispensing pipette tip (7) is provided above the third hole position chamber (6), and a nucleic acid extraction pipette tip (3) is provided above the nucleic acid extraction pipette tip (3) of the reagent strip (1) on the one side of the second hole position chamber (4) away from the first hole position chamber (2). ), the volumes of the nucleic acid extraction pipette tip (3), the chip reagent canned pipette tip (5) and the UV light curing glue dispensing pipette tip (7) are different, a reagent washing liquid storage component is provided on the reagent strip (1) on the side of the third hole position chamber (6) away from the second hole position chamber (4), the reagent washing liquid storage component includes a plurality of storage units and stores different types of reagent washing liquids, a limiting groove (14) is provided on the reagent strip (1) on one side of the reagent washing liquid storage component, and an amplification chamber component is provided in the limiting groove (14).

2. A kit for detecting twelve pathogens of the human respiratory tract according to claim 1, characterized in that: The reagent washing storage component comprises a fourth hole position chamber (8) arranged on the reagent strip (1), a fifth hole position chamber (9) arranged on one side of the fourth hole position chamber (8), and a sixth hole position chamber (10) arranged on the side of the fifth hole position chamber (9) away from the fourth hole position chamber (8), wherein different actual washing solutions are stored in the fourth hole position chamber (8), the fifth hole position chamber (9) and the sixth hole position chamber (10).

3. A kit for detecting twelve pathogens of the human respiratory tract according to claim 2, characterized in that: A seventh hole position chamber (11) is provided on the reagent strip (1) on the side of the sixth hole position chamber (10) away from the fifth hole position chamber (9), and magnetic beads are provided in the seventh hole position chamber (11). An eighth hole position chamber (12) is provided on the reagent strip (1) on the side of the seventh hole position chamber (11) away from the sixth hole position chamber (10), and a reagent eluent is provided in the eighth hole position chamber (12). A ninth hole position chamber (13) is provided on the reagent strip (1) on the side of the eighth hole position chamber (12) away from the seventh hole position chamber (11), and UV light curing glue is stored in the ninth hole position chamber (13).

4. A kit for detecting twelve pathogens of the human respiratory tract according to claim 1, characterized in that: The amplification chamber assembly includes a connecting frame (15), the connecting frame (15) is arranged in correspondence with the limiting groove (14), and the bottom of the connecting frame (15) is fixedly connected to the first chip PCR chamber (16), the second chip PCR chamber (17), the third chip PCR chamber (18) and the fourth chip PCR chamber (19).

5. A kit for detecting twelve pathogens of the human respiratory tract according to claim 4, characterized in that: A first sample addition port (20), a second sample addition port (21), a third sample addition port (22) and a fourth sample addition port (23) are respectively provided above the first chip PCR chamber (16), the second chip PCR chamber (17), the third chip PCR chamber (18) and the fourth chip PCR chamber (19).

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