A nucleic acid detection kit for simultaneous detection of nine respiratory pathogens based on a common qPCR detection platform

Abstract

The present invention discloses a nucleic acid detection kit for simultaneously detecting 9 respiratory pathogens based on a common qPCR detection platform, comprising the following non-mixing: reaction solution A, reaction solution B, and reaction solution C. Reaction solution A includes forward and reverse primers and fluorescent probes for influenza A virus, influenza B virus, and respiratory syncytial virus; reaction solution B includes forward and reverse primers and fluorescent probes for human parainfluenza virus, Mycoplasma pneumoniae, and the new coronavirus; reaction solution C includes forward and reverse primers and fluorescent probes for human respiratory adenovirus, human respiratory rhinovirus / enterovirus, and Bordetella pertussis. The fluorescent groups of the fluorescent probes used for different pathogens in the same reaction solution are different. The nucleic acid detection kit for simultaneously detecting 9 respiratory pathogens based on a common qPCR detection platform of the present invention has high specificity and high sensitivity, and is suitable for rapid detection of respiratory pathogens.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid detection, and specifically relates to a nucleic acid detection kit for simultaneously detecting nine respiratory pathogens based on a common qPCR detection platform. Background Art

[0002] Respiratory infections are among the most common illnesses worldwide. Their incidence rates contribute significantly to the overall morbidity of populations worldwide. Approximately 10% of the population suffers from respiratory infections each year. Respiratory infections are primarily caused by various respiratory viruses and some bacteria, mycoplasmas, and chlamydia. Common viruses include influenza A virus (FluA), influenza B virus (FluB), respiratory syncytial virus (RSV), human parainfluenza virus (hPIV), Mycoplasma pneumoniae (MP), novel coronavirus (SARS-CoV-2), human respiratory adenovirus (hAdV), human respiratory rhinovirus / enterovirus (HRV / HEV), and Bordetella pertussis (BP). The increasing number of new pathogenic respiratory viruses being discovered complicates the diagnosis and treatment of clinically relevant diseases. Given the diverse nature of respiratory viruses, effectively identifying respiratory pathogens and clarifying the specific pathogens responsible for respiratory infections are crucial for clinical diagnosis, treatment, and epidemic prevention and control.

[0003] Numerous methods exist for detecting respiratory pathogens both domestically and internationally, including traditional isolation and culture methods, immunological detection techniques, and emerging molecular biology technologies. In recent years, nucleic acid-based molecular detection methods, characterized by their rapidity, sensitivity, specificity, and time-saving nature, have become a revolutionary technology for detecting respiratory pathogens. Conventional fluorescence quantitative qPCR (qPCR) detection platforms can only perform four to five multiplex assays simultaneously. Therefore, how to simultaneously detect a wider range of pathogens and improve detection efficiency using conventional qPCR platforms remains a pressing technical challenge. Summary of the Invention

[0004] This invention addresses the technical problem that existing fluorescent quantitative qPCR detection platforms have difficulty detecting more than four or five pathogens. The purpose is to provide a nucleic acid detection kit for the simultaneous detection of nine respiratory pathogens based on a common qPCR detection platform. The kit of the present invention is capable of simultaneously detecting nine respiratory pathogens on a common qPCR detection platform. This is achieved by setting up three independent, non-mixing reaction solutions. In each reaction solution, fluorescent probes targeting different pathogens are equipped with different fluorescent groups for differentiation, thereby achieving the goal of simultaneous detection of nine respiratory pathogens.

[0005] The primer set and kit for simultaneous detection of nucleic acids of multiple respiratory pathogens of the present invention can simultaneously detect 9 respiratory pathogens, solving the problem that existing respiratory virus nucleic acid detection is difficult to detect multiple viruses at the same time and difficult to effectively control the detection process. The present invention optimizes the primer system for this type of multiple PCR to detect influenza A virus (FluA), influenza B virus (FluB), respiratory syncytial virus (RSV), human parainfluenza virus (hPIV), Mycoplasma pneumoniae (MP), new coronavirus (SARS-CoV-2), human respiratory adenovirus (HADV), human respiratory rhinovirus / enterovirus (HRV / HEV) and Bordetella pertussis (BP), and designs a new primer and fluorescent probe pool, which can achieve clinically sensitive and specific early detection of these 9 respiratory pathogens.

[0006] The nucleic acid detection kit for simultaneously detecting nine respiratory pathogens based on a common qPCR detection platform of the present invention comprises the following mixed solutions that do not mix with each other: reaction solution A, reaction solution B, and reaction solution C;

[0007] In the present invention, the term "mutually immiscible" means that reaction liquid A, reaction liquid B, and reaction liquid C do not mix with each other and are separate and independent; the "mixed liquid" in the "mutually immiscible mixed liquid" refers to reaction liquid A, or reaction liquid B, or reaction liquid C, and these three substances themselves are in the form of a mixed liquid; if the kit also includes a positive quality control product and a negative quality control product, then the "mixed liquid" can also refer to the positive quality control product and the negative quality control product.

[0008] The reaction solution A includes a reaction premix, a FluA-F forward primer, a FluA-R reverse primer, a FluA-P fluorescent probe, a FluB-F forward primer, a FluB-R reverse primer, a FluB-P fluorescent probe, an RSVA-F forward primer, an RSVA-R reverse primer, an RSVA-P fluorescent probe, an RSVB-F forward primer, an RSVB-R reverse primer, and an RSVB-P fluorescent probe;

[0009] The reaction solution B includes a reaction premix, an hPIV1-F upstream primer, an hPIV1-R downstream primer, an hPIV1-P fluorescent probe, an hPIV2-F forward primer, an hPIV2-R reverse primer, an hPIV2-P fluorescent probe, an hPIV3-F forward primer, an hPIV3-R reverse primer, an hPIV3-P fluorescent probe, an hPIV4-F forward primer, an hPIV4-R reverse primer, an hPIV4-P fluorescent probe, an MP-F forward primer, an MP-R reverse primer, and a forward primer, MP-P fluorescent probe, SARS-CoV-2-ORF forward primer, SARS-CoV-2-ORF reverse primer, SARS-CoV-2-ORF fluorescent probe, SARS-CoV-2-N forward primer, SARS-CoV-2-N reverse primer, SARS-CoV-2-N fluorescent probe, SARS-CoV-2-E forward primer, SARS-CoV-2-E reverse primer, SARS-CoV-2-E fluorescent probe;

[0010] The reaction solution C includes a reaction premix, an HADV-F upstream primer, an HADV-R1 downstream primer, an HADV-R2 downstream primer, an HADV-R3 downstream primer, an HADV-R4 downstream primer, an HADV-P fluorescent probe, an HRV / HEV-F forward primer, an HRV / HEV-R1 reverse primer, an HRV / HEV-R2 reverse primer, an HRV / HEV-R3 reverse primer, an HRV / HEV-R4 reverse primer, an HRV / HEV-P fluorescent probe, a BP-F forward primer, a BP-R reverse primer, and a BP-P fluorescent probe.

[0011] In the present invention,

[0012] The reaction solution A may also include RP-F upstream primer, RP-R downstream primer and RP-P fluorescent probe as internal reference controls;

[0013] The reaction solution B may also include RP-F upstream primer, RP-R downstream primer and RP-P fluorescent probe as internal reference controls;

[0014] The reaction solution C may further include an RP-F upstream primer, an RP-R downstream primer, and an RP-P fluorescent probe as internal reference controls.

[0015] In the present invention, the primer pairs and fluorescent probes are specifically as follows:

[0016] A FluA-F forward primer as shown in SEQ ID NO: 1, a FluA-R reverse primer as shown in SEQ ID NO: 2; a FluA-P fluorescent probe as shown in SEQ ID NO: 3;

[0017] FluB-F forward primer as shown in SEQ ID NO:4, FluB-R reverse primer as shown in SEQ ID NO:5; FluB-P fluorescent probe as shown in SEQ ID NO:6;

[0018] RSV A-F forward primer as shown in SEQ ID NO: 7, RSV A-R reverse primer as shown in SEQ ID NO: 8; RSV A-P fluorescent probe as shown in SEQ ID NO: 9;

[0019] RSVB-F forward primer as shown in SEQ ID NO: 10, RSVB-R reverse primer as shown in SEQ ID NO: 11; RSVB-P fluorescent probe as shown in SEQ ID NO: 12;

[0020] hPIV1-F forward primer as shown in SEQ ID NO: 13, hPIV1-R reverse primer as shown in SEQ ID NO: 14; hPIV1-P fluorescent probe as shown in SEQ ID NO: 15;

[0021] hPIV2-F forward primer as shown in SEQ ID NO: 16, hPIV2-R reverse primer as shown in SEQ ID NO: 17; hPIV2-P fluorescent probe as shown in SEQ ID NO: 18;

[0022] hPIV3-F forward primer as shown in SEQ ID NO: 19, hPIV3-R reverse primer as shown in SEQ ID NO: 20; hPIV3-P fluorescent probe as shown in SEQ ID NO: 21;

[0023] hPIV4-F forward primer as shown in SEQ ID NO: 22, hPIV4-R reverse primer as shown in SEQ ID NO: 23; hPIV4-P fluorescent probe as shown in SEQ ID NO: 24;

[0024] The MP-F forward primer is shown in SEQ ID NO: 25, the MP-R reverse primer is shown in SEQ ID NO: 26; the MP-P fluorescent probe is shown in SEQ ID NO: 27;

[0025] A SARS-CoV-2-ORF-F forward primer as shown in SEQ ID NO:28, a SARS-CoV-2-ORF-R reverse primer as shown in SEQ ID NO:29; and a SARS-CoV-2-ORF-P fluorescent probe as shown in SEQ ID NO:30;

[0026] The SARS-CoV-2-NF forward primer as shown in SEQ ID NO:31, the SARS-CoV-2-NR reverse primer as shown in SEQ ID NO:32; the SARS-CoV-2-NP fluorescent probe as shown in SEQ ID NO:33;

[0027] A SARS-CoV-2-EF forward primer as shown in SEQ ID NO:34, a SARS-CoV-2-ER reverse primer as shown in SEQ ID NO:35; a SARS-CoV-2-EP fluorescent probe as shown in SEQ ID NO:36;

[0028] A HADV-F forward primer as shown in SEQ ID NO: 37, a HADV-R1 reverse primer as shown in SEQ ID NO: 38, a HADV-R2 reverse primer as shown in SEQ ID NO: 39, a HADV-R3 reverse primer as shown in SEQ ID NO: 40, and a HADV-R4 reverse primer as shown in SEQ ID NO: 41; and a HADV-P fluorescent probe as shown in SEQ ID NO: 42;

[0029] An HRV / HEV-F forward primer as set forth in SEQ ID NO:43, an HRV / HEV-R1 reverse primer as set forth in SEQ ID NO:44; an HRV / HEV-R2 reverse primer as set forth in SEQ ID NO:45; an HRV / HEV-R3 reverse primer as set forth in SEQ ID NO:46; an HRV / HEV-R4 reverse primer as set forth in SEQ ID NO:47; and an HRV / HEV-P fluorescent probe as set forth in SEQ ID NO:48;

[0030] BP-F forward primer as shown in SEQ ID NO:49, BP-R reverse primer as shown in SEQ ID NO:50; BP-P fluorescent probe as shown in SEQ ID NO:51;

[0031] The RP-F forward primer is shown in SEQ ID NO: 52, the RP-R reverse primer is shown in SEQ ID NO: 53; and the RP-P fluorescent probe is shown in SEQ ID NO: 54.

[0032] In the present invention,

[0033] In reaction solution A, the fluorescent groups of the four fluorescent probes, influenza A virus, influenza B virus, respiratory syncytial virus, and human conserved gene RP fragment, are different;

[0034] In reaction solution B, the fluorescent groups of the four fluorescent probes for human parainfluenza virus, Mycoplasma pneumoniae, novel coronavirus, and human conserved gene RP fragment are different;

[0035] In reaction solution C, the fluorescent groups of the four fluorescent probes, namely human respiratory adenovirus, human respiratory rhinovirus / enterovirus, Bordetella pertussis and human conserved gene RP fragment, are different.

[0036] However, the fluorescent groups in reaction solution A, reaction solution B, and reaction solution C can be the same.

[0037] In the present invention, an example is:

[0038] In reaction solution A, the fluorescent groups of the four fluorescent probes, influenza A virus, influenza B virus, respiratory syncytial virus, and human conserved gene RP fragment, are each selected from FAM, VIC, CY5, and ROX, and the fluorescent groups of the four fluorescent probes are different;

[0039] In reaction solution B, the fluorescent groups of the four fluorescent probes for human parainfluenza virus, Mycoplasma pneumoniae, novel coronavirus, and human conserved gene RP fragment are each selected from FAM, VIC, CY5, and ROX, and the fluorescent groups of each fluorescent probe are different;

[0040] In reaction solution C, the fluorescent groups of the four fluorescent probes, human respiratory adenovirus, human respiratory rhinovirus / enterovirus, Bordetella pertussis and human conserved gene RP fragment, are each selected from FAM, VIC, CY5 and ROX, and the fluorescent groups of each fluorescent probe are different.

[0041] In the present invention, specifically

[0042] In reaction solution A, the fluorescent group connected to the FluA fluorescent probe is labeled with FAM, the fluorescent group connected to the FluB fluorescent probe is labeled with VIC, and the fluorescent group connected to the RSVA and RSVB fluorescent probes are both labeled with CY5;

[0043] In reaction solution B, the fluorescent group labeled with hPIV1, hPIV2, hPIV3, and hPIV4 fluorescent probes is FAM, the fluorescent group labeled with MP fluorescent probe is VIC, and the fluorescent group labeled with SARS-CoV-2-ORF, SARS-CoV-2-N, and SARS-CoV-2-E fluorescent probes is CY5;

[0044] In reaction solution C, the fluorescent group connected to the fluorescent probe of HADV is labeled with FAM, the fluorescent group connected to the fluorescent probe of HRV / HEV is labeled with VIC, and the fluorescent group connected to the fluorescent probe of BP is labeled with CY5;

[0045] In reaction solution A, reaction solution B, and reaction solution C, the fluorescent group connected to the fluorescent probe of the human conserved gene RP fragment is labeled with ROX.

[0046] During the qPCR reaction, if the sample to be tested contains FluA, the labeled FAM probe in tube A containing reaction solution A will produce a fluorescent signal; if the sample to be tested contains FluB, the labeled VIC probe in tube A will produce a fluorescent signal; if the sample to be tested contains RSV, the labeled CY5 probe in tube A will produce a fluorescent signal;

[0047] If the sample to be tested contains hPIV, the labeled FAM probe in tube B containing reaction solution B will produce a fluorescent signal; if the sample to be tested contains MP, the labeled VIC probe in tube B will produce a fluorescent signal; if the sample to be tested contains SARS-CoV-2, the labeled CY5 probe in tube B will produce a fluorescent signal;

[0048] If the sample to be tested contains HADV, the labeled FAM probe in tube C containing reaction solution C will produce a fluorescent signal; if the sample to be tested contains HRV / HEV, the labeled VIC probe in tube C will produce a fluorescent signal; if the sample to be tested contains BP, the labeled CY5 probe in tube C will produce a fluorescent signal.

[0049] In addition, if the reactions in tubes A, B, and C all contain internal reference genes, the labeled ROX probe should produce a fluorescent signal to detect instrument failure, reagent factors, polymerase activity factors, etc.

[0050] In the present invention, the reaction premix solution includes an enzyme mixture, a nucleotide mixture, divalent magnesium ions and a buffer solution.

[0051] In the present invention, the enzyme mixture comprises: 0.01-1 IU / μL of heat-resistant DNA polymerase, 100-400 U / μL of reverse transcriptase, and 30-50 U / μL of RNase inhibitor; the nucleotide mixture is deoxyribonucleotide triphosphates with a concentration of 10-500 μmol / L; the divalent magnesium ion is selected from magnesium chloride or magnesium sulfate with a concentration of 1-4 mmol / L; the buffer solution is Tris buffer with a pH of 8.0; and the concentrations of each primer and each fluorescent probe are respectively 10-500 nmol / L.

[0052] Preferably, the enzyme mixture comprises: 0.01-0.1 IU / μL of heat-resistant DNA polymerase, 100-400 U / μL of reverse transcriptase, and 30-50 U / μL of RNase inhibitor; the nucleotide mixture is deoxyribonucleotide triphosphates with a concentration of 100-200 μmol / L; divalent magnesium ions are selected from magnesium chloride or magnesium sulfate with a concentration of 2.5-3.5 mmol / L; the buffer solution is Tris buffer with a pH of 8.0; and the concentrations of each primer and each fluorescent probe are respectively 100-200 nmol / L.

[0053] In the present invention, the kit may further include a positive quality control product and a negative quality control product.

[0054] In the present invention, the positive quality control product is a plasmid pseudovirus standard product.

[0055] In the present invention, the negative quality control product is sterilized diethyl pyrocarbonate treated water.

[0056] In the present invention, the DNA sequences of the plasmid pseudovirus standards are shown as SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69 and SEQ ID NO:70, respectively.

[0057] The specific primers and fluorescent probes of the present invention are shown in Table 1.

[0058] Table 1 Primers and fluorescent probes

[0059]

[0060]

[0061]

[0062] The sequences of plasmid pseudoviral DNA standards are shown in Table 2:

[0063]

[0064]

[0065]

[0066]

[0067] The nucleic acid detection kit for simultaneously detecting nine respiratory pathogens of the present invention is stored at room temperature in the dark.

[0068] The nucleic acid detection kit for simultaneous detection of nine respiratory pathogens of the present invention can simultaneously and specifically and sensitively detect nine respiratory pathogens. The nucleic acid detection kit for simultaneous detection of nine respiratory pathogens includes amplification primers and fluorescent probe combinations for nine respiratory pathogens. Among them, the amplification reaction of the nine respiratory pathogens contains multiple fluorescent probes, primers, enzyme mixtures, nucleotide mixtures, divalent magnesium ions, buffer solutions, etc. with different pathogen targets. The presence of influenza A virus (FluA), influenza B virus (FluB), respiratory syncytial virus (RSV), human parainfluenza virus (hPIV), Mycoplasma pneumoniae (MP), new coronavirus (SARS-CoV-2), human respiratory adenovirus (HADV), human respiratory rhinovirus / enterovirus (HRV / HEV) and Bordetella pertussis (BP), including the conserved gene RP fragment of humans, can be detected in the same reaction system; the kit also contains positive quality control products and negative quality control products, thereby improving the accuracy of PCR detection. The nucleic acid detection kit for simultaneous detection of nine respiratory pathogens of the present invention maintains high specificity while significantly improving the sensitivity of detection, and is suitable for rapid detection of respiratory pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 Figure 2 is the result of FluA detection;

[0070] Figure 2 Figure 1 is the result of FluB detection;

[0071] Figure 3 This is the RSV test result diagram;

[0072] Figure 4 This is the hPIV test result diagram;

[0073] Figure 5 This is the MP test result diagram;

[0074] Figure 6 This is a graph of SARS-CoV-2 test results;

[0075] Figure 7 This is the hAdV detection result diagram;

[0076] Figure 8 This is the HRV / HEV test result diagram;

[0077] Figure 9 This is the BP test result diagram;

[0078] Figure 10 This is a graph showing the results of cross-reaction detection. DETAILED DESCRIPTION

[0079] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention is further described below with reference to specific examples.

[0080] Example 1

[0081] The nucleic acid detection kit for simultaneous detection of 9 respiratory pathogens includes: reaction solution A, reaction solution B, reaction solution C, positive quality control and negative quality control;

[0082] Reaction solution A includes reaction premix, FluA-F forward primer, FluA-R reverse primer, FluA-P fluorescent probe, FluB-F forward primer, FluB-R reverse primer, FluB-P fluorescent probe, RSVA-F forward primer, RSVA-R reverse primer, RSVA-P fluorescent probe, RSVB-F forward primer, RSVB-R reverse primer, RSVB-P fluorescent probe, RP-F upstream primer, RP-R downstream primer and RP-P fluorescent probe;

[0083] Reaction solution B includes reaction premix, hPIV1-F upstream primer, hPIV1-R downstream primer, hPIV1-P fluorescent probe, hPIV2-F forward primer, hPIV2-R reverse primer, hPIV2-P fluorescent probe, hPIV3-F forward primer, hPIV3-R reverse primer, hPIV3-P fluorescent probe, hPIV4-F forward primer, hPIV4-R reverse primer, hPIV4-P fluorescent probe, MP-F forward primer, MP-R reverse primer, MP-P fluorescent probe, S ARS-CoV-2-ORF forward primer, SARS-CoV-2-ORF reverse primer, SARS-CoV-2-ORF fluorescent probe, SARS-CoV-2-N forward primer, SARS-CoV-2-N reverse primer, SARS-CoV-2-N fluorescent probe, SARS-CoV-2-E forward primer, SARS-CoV-2-E reverse primer, SARS-CoV-2-E fluorescent probe, RP-F upstream primer, RP-R downstream primer, and RP-P fluorescent probe;

[0084] Reaction solution C includes reaction premix, HADV-F upstream primer, HADV-R1 downstream primer, HADV-R2 downstream primer, HADV-R3 downstream primer, HADV-R4 downstream primer, HADV-P fluorescent probe, HRV / HEV-F forward primer, HRV / HEV-R1 reverse primer, HRV / HEV-R2 reverse primer, HRV / HEV-R3 reverse primer, HRV / HEV-R4 reverse primer, HRV / HEV-P fluorescent probe, BP-F forward primer, BP-R reverse primer, BP-P fluorescent probe, RP-F upstream primer, RP-R downstream primer and RP-P fluorescent probe.

[0085] The forward and reverse primers and fluorescent probe sequences are shown in SEQ ID NO: 1 to SEQ ID NO: 54, and the concentrations are 200 nmol / L respectively.

[0086] The reaction premix includes: a heat-resistant DNA polymerase at a concentration of 0.05 IU / μL, a reverse transcriptase at 125 IU / μL, an RNase inhibitor at 40 U / μL, a divalent magnesium ion at a concentration of 3 mmol / L, a deoxyribonucleotide triphosphate at a concentration of 200 μmol / L, and a buffer solution of Tris buffer at pH 8.0.

[0087] The positive quality control product is a plasmid pseudovirus standard product, and the sequences of the pseudovirus standard products are shown in SEQ ID NO: 55 to SEQ ID NO: 70 respectively.

[0088] The negative control material is sterilized diethyl pyrocarbonate treated water.

[0089] In the same reaction system, three tubes A, B, and C were used to detect the presence of influenza A virus (FluA), influenza B virus (FluB), respiratory syncytial virus (RSV), human parainfluenza virus (hPIV), Mycoplasma pneumoniae (MP), novel coronavirus (SARS-CoV-2), human respiratory adenovirus (HADV), human respiratory rhinovirus / enterovirus (HRV / HEV), and Bordetella pertussis (BP). Tube A contained FluA and FluB labeled with FAM and VIC, respectively, while both RSVA and RSVB were labeled with Tube A is labeled with CY5; Tube B is labeled with FAM for hPIV1 / hPIV2 / hPIV3 and hPIV4, VIC for MP, and CY5 for SARS-CoV-2-ORF / SARS-CoV-2-N and SARS-CoV-2-E; Tube C is labeled with FAM, VIC, and CY5 for HADV, HRV / HEV, and BP, respectively. Reaction solution A is placed in tube A, reaction solution B in tube B, and reaction solution C in tube C. Each tube contains an internal control: a fragment of the conserved human gene RP, labeled with ROX. This internal control can indicate false negatives, preventing misinterpretation of test results due to inhibitors in the sample or operational errors, thereby improving the accuracy of PCR testing.

[0090] The test is performed on a common qPCR detection platform. The steps are as follows:

[0091] Step S1: Pretreatment: Take out the positive control and negative control respectively, thaw them at room temperature, shake them thoroughly and centrifuge them at 3000 rpm / min for 20 s.

[0092] Step S2, nucleic acid extraction: nucleic acid extraction was performed simultaneously on the positive nasopharyngeal swab samples to be tested (as shown in Table 5) and the positive and negative controls. Specifically, nucleic acid extraction was performed using the QIAGEN MinElute PCR Purification Kit. The operation was referred to the QIAGEN MinElute PCR Purification Kit instruction manual.

[0093] Step S3, sample addition: take out the pre-aliquoted lyophilized eight-tube strip from the kit, add 5 μL each of the nucleic acid extracted in step 2, the positive control nucleic acid, and the negative control nucleic acid, cap the tube tightly, shake evenly, and centrifuge briefly at 3000 rpm / min for 20 s;

[0094] Step S4: placing the PCR reaction tube obtained in step S4 in a fluorescent quantitative PCR instrument for amplification and fluorescence signal detection according to the parameters in Table 3;

[0095] Table 3 Amplification detection cycle parameters of fluorescence quantitative qPCR instrument

[0096]

[0097] Step S5: Determine the sample detection result according to the judgment criteria in Table 4.

[0098] Table 4 Judgment criteria

[0099]

[0100] Influenza A virus (FluA), influenza B virus (FluB), respiratory syncytial virus (RSV), human parainfluenza virus (hPIV), Mycoplasma pneumoniae (MP), human respiratory adenovirus (HADV), human respiratory rhinovirus / enterovirus (HRV / HEV), Bordetella pertussis (BP), and a negative control (sterilized diethyl pyrocarbonate-treated water) were tested and analyzed. The positive and negative controls met the requirements, demonstrating the validity of the assay. FluA, FluB, RSV, hPIV, MP, HADV, HRV / HEV, and BP were all detected on the corresponding targets. No amplification was observed in samples containing other viruses or in the negative control, demonstrating the good specificity of the assay kit and method (results shown in Table 4).

[0101] Table 4 Sample test list

[0102]

[0103] Example 2 Sensitivity Detection

[0104] 8×10 8dilution series to 8×10 4 copy / mL, 8×10 3 copy / mL, 8×10 2 The negative sample was a nasopharyngeal swab sample collected from a healthy volunteer. The diluted template and negative sample were amplified using the kit described in Example 1. The amplification conditions were the same as those in Example 1. The results of the 13 target detection sensitivities were shown in Table 5. As can be seen from Table 6, when the viral template was 8×10 2 When the sample concentration is 8×10 3 The pathogen detection results of copy / mL are as follows Figures 1 to 9 When the kit of the present invention is used, there is no cross-reaction between primer pairs, and the sensitivity is as high as that of conventional qPCR.

[0105] Table 6 Detection sensitivity of 9 respiratory virus nucleic acids using this kit

[0106]

[0107]

[0108] Example 3 Specificity Detection

[0109] Some inactivated common pathogens of human infection were selected, including Chlamydia pneumoniae, Mycobacterium tuberculosis, Streptococcus pneumoniae, Staphylococcus aureus, Escherichia coli, Candida albicans (positive standard of national drug standard substance for influenza A and B), and FluA pseudovirus, with a concentration of 1×10 4 The specificity test was performed as shown in Example 1. The results showed that except for the positive result of FluA, the results of other samples were negative, which proved that the present invention had no cross reaction with other pathogen nucleic acids in this example (such as Figure 10 ).

[0110] Table 5 List of specific reaction samples

[0111]

[0112] Conclusion: The PCR detection kit provided by this invention can simultaneously detect multiple respiratory pathogens and can stably detect low-copy viruses. The kit is fully enclosed and fully automated, has a wide range of uses, and has broad application prospects. 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Artificial Sequence <400> 9 atctctgcaa cattgacata ttcaaccc 28 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <400> 10 cctcacctca agtcagaac 19 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <400> 11 gacactggta taccaacctg 20 <210> 12 <211> 26 <212> DNA <213> Artificial Sequence <400> 12 ctgaggagtt ttaccaatcg acatgt 26 <210> 13 <211> twenty one <212> DNA <213> Artificial Sequence <400> 13 gatccatgct cctctactac c 21 <210> 14 <211> 19 <212> DNA <213> Artificial Sequence <400> 14 actgggagac attgaggtg 19 <210> 15 <211> 25 <212> DNA <213> Artificial Sequence <400> 15 ccagccgact gagacaaggg atgat 25 <210> 16 <211> twenty three <212> DNA <213> Artificial Sequence <400> 16 tctgcagcta tgagtaatca cat 23 <210> 17 <211> twenty one <212> DNA <213> Artificial Sequence <400> 17 catctggaat gactcggaat g 21 <210> 18 <211> 31 <212> DNA <213> Artificial Sequence <400> 18 atgcattcac cagaagccag catagataga g 31 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <400> 19 gatgccaaca aagttgctct 20 <210> 20 <211> twenty four <212> DNA <213> Artificial Sequence <400> 20 cgtacaattc acgaagatta ctct 24 <210> twenty one <211> 33 <212> DNA <213> Artificial Sequence <400> twenty one ctcctcaatg tcttccacta gataggagca taa 33 <210> twenty two <211> 20 <212> DNA <213> Artificial Sequence <400> twenty two cctggagtcc catcaaaagt 20 <210> twenty three <211> twenty two <212> DNA <213> Artificial Sequence <400> twenty three gttccagaca aaatgggtct tg 22 <210> twenty four <211> 38 <212> DNA <213> Artificial Sequence <400> twenty four cgtctcaaaa tttgttgatc aagacaatac aattacac 38 <210> 25 <211> 19 <212> DNA <213> Artificial Sequence <400> 25 agtcaacaaa ccacgtatg 19 <210> 26 <211> 18 <212> DNA <213> Artificial Sequence <400> 26 gtcgaggtcg tataaggc 18 <210> 27 <211> twenty four <212> DNA <213> Artificial Sequence <400> 27 gttacgccgg tatgacctcg ccgg 24 <210> 28 <211> twenty one <212> DNA <213> Artificial Sequence <400> 28 ccctgtgggt tttacactta a 21 <210> 29 <211> 19 <212> DNA <213> Artificial Sequence <400> 29 acgattgtgc atcagctga 19 <210> 30 <211> 28 <212> DNA <213> Artificial Sequence <400> 30 ccgtctgcgg tatgtggaaa ggttatgg 28 <210> 31 <211> twenty two <212> DNA <213> Artificial Sequence <400> 31 ggggaacttc tcctgctaga at 22 <210> 32 <211> twenty two <212> DNA <213> Artificial Sequence <400> 32 cagacatttt gctctcaagc tg 22 <210> 33 <211> 26 <212> DNA <213> Artificial Sequence <400> 33 cttgctttgc tgctgcttga cagatt 26 <210> 34 <211> 26 <212> DNA <213> Artificial Sequence <400> 34 acaggtacgt taatagttaa tagcgt 26 <210> 35 <211> twenty two <212> DNA <213> Artificial Sequence <400> 35 atattgcagc agtacgcaca ca 22 <210> 36 <211> 28 <212> DNA <213> Artificial Sequence <400> 36 ggtattcttg ctagttacac tagccatc 28 <210> 37 <211> twenty two <212> DNA <213> Artificial Sequence <400> 37 caacatgacc aaagactggt tc 22 <210> 38 <211> twenty two <212> DNA <213> Artificial Sequence <400> 38 gagtacatgc gatccttgta tc 22 <210> 39 <211> twenty two <212> DNA <213> Artificial Sequence <400> 39 gaatacatgc gatctttgta tc 22 <210> 40 <211> twenty two <212> DNA <213> Artificial Sequence <400> 40 gagtacatgc gatccttgta ac 22 <210> 41 <211> 18 <212> DNA <213> Artificial Sequence <400> 41 ggagtacatg cggtcctt 18 <210> 42 <211> 28 <212> DNA <213> Artificial Sequence <400> 42 ccaactacaa cattggctac cagggctt 28 <210> 43 <211> 17 <212> DNA <213> Artificial Sequence <400> 43 tcctccggcc cctgaat 17 <210> 44 <211> twenty two <212> DNA <213> Artificial Sequence <400> 44 caaattgtca ccataagcag cc 22 <210> 45 <211> twenty two <212> DNA <213> Artificial Sequence <400> 45 caaattgtca ccataagcag tc 22 <210> 46 <211> twenty three <212> DNA <213> Artificial Sequence <400> 46 tatatatatt gtcaccataa gca 23 <210> 47 <211> twenty two <212> DNA <213> Artificial Sequence <400> 47 catatatgct gtgaccataa ga 22 <210> 48 <211> 31 <212> DNA <213> Artificial Sequence <400> 48 cggaaccgac tactttgggt gtccgtgttt c 31 <210> 49 <211> 20 <212> DNA <213> Artificial Sequence <400> 49 atatgccgag cacggacaac 20 <210> 50 <211> 19 <212> DNA <213> Artificial Sequence <400> 50 gaatgcagcg gcaagcagg 19 <210> 51 <211> 26 <212> DNA <213> Artificial Sequence <400> 51 gatgcgatgg tatgcatggg gttcgc 26 <210> 52 <211> 19 <212> DNA <213> Artificial Sequence <400> 52 gacttcagca tggcggtgt 19 <210> 53 <211> 19 <212> DNA <213> Artificial Sequence <400> 53 gtccgcgcag agccttcag 19 <210> 54 <211> 27 <212> DNA <213> Artificial Sequence <400> 54 cagatttgga cctgcgagcg ggttctg 27 <210> 55 <211> 500 <212> DNA <213> Artificial Sequence <400> 55 ttgaagatgt atttgctgga aagaataccg atcttgaggc tctcatggag tggctaaaga 60 caagaccaat cctgtcacct ctgactaagg ggattttagg atttgtgttc acgctcaccg 120 tgcccagtga gcgaggactg cagcgtagac gctttgtcca aaatgccctt aatgggaatg 180 gggatccaaa taatatggac agagcagtca aactttatcg aaagcttaag agggagataa 240 cattccatgg agccaaagaa atagcactca gttattctgc tggtgcactt gccagttgta 300 tgggactcat atacaacagg atgggggctg tgaccaccga atcagcattt ggccttatat 360 gtgcaacctg tgaacagatt gccgactccc agcataagtc tcacaggcaa atggtaacaa 420 caaccaatcc attgataaga catgagaaca gaatggttct ggccagcact acagctaagg 480 ctatggagca aatggctgga 500 <210> 56 <211> 498 <212> DNA <213> Artificial Sequence <400> 56 gtaatgtatt gtccttgaga gtgttggtaa acggaacatt cctcaaacat cccaatggat 60 acaagtcctt atcaactctg catagattga atgcatatga ccagagtgga aggcttgttg 120 ctaaacttgt tgctactgat gatcttacag tggaggatga agaagatggc catcggatcc 180 tcaactcact cttcgagcgt cttaatgaag gacattcaaa gccaattcga gcagctgaaa 240 ctgcggtggg agtcttatcc caatttggtc aagagcaccg attatcacca gaagagggag 300 acaattagac tggtcacgga agaactttat cttttaagta aaagaattga tgataacata 360 ttgttccaca aaacagtaat agctaacagc tccataatag ctgacatggt tgtatcatta 420​​​ttgtgaattt aaaataaa 498 <210> 57 <211> 500 <212> DNA <213> Artificial Sequence <400> 57 tcttagcata tgtagtacaa ttaccactat atggtgtgat agatacacct tgttggaaat 60 tacacacatc ccctctatgt acaaccaaca caaaagaagg gtcaaacatc tgtttaacaa 120 gaactgacag aggatggtac tgtgacaatg caggatcagt atctttcttc ccacaagctg 180 aaacatgtaa agttcaatcg aatcgagtat tttgtgacac aatgaacagt ttaacattac 240 caagtgaagt aaatctctgc aatgttgaca tattcaatcc caaatatgat tgtaaaatta 300 tgacttcaaa aacagatgta agcagctccg ttatcacatc tctaggagcc attgtgtcat 360 gctatggcaa aactaaatgt acagcatcca ataaaaatcg tggaatcata aagacatttt 420 ctaacgggtg tgattatgta tcaaataaag gggtggacac tgtgtctgta ggtaacacat 480 tatattatgt aaataagcaa 500 <210> 58 <211> 480 <212> DNA <213> Artificial Sequence <400> 58 cacaaatacc cacagcatcc gagccctcca cattaaatcc taattaaaaa acctagtcac 60 atgcttagtt attcaaaaac tacatcttag cagagaaccg tgatctatca agcaagaaca 120 aaattaaacc tggggcaaat aaccatggag ttgctgatcc acaggtcaag tgcaatcttc 180 ctaactcttg ctgttaatgc attgtacctc acctcaagtc agaacataac tgaggagttt 240 taccaatcga catgtagtgc agttagcaga ggttatttta gtgctttaag aacaggttgg 300 tataccagtg tcataacaat agaattaagt aatataaaag aaaccaaatg caatggaact 360 gacactaaag taaaacttat aaaacaagaa ttagataagt ataagaatgc agtaacagaa 420 ttacagctac ttatgcaaaa cacgccagct gccaacaacc gggccagaag agaagcacca 480 <210> 59 <211> 480 <212> DNA <213> Artificial Sequence <400> 59 tctttgaaac acctaagcaa ggacctctat ctggcagcat atctgatctc acagaatcaa 60 ccagttattc aatctgtgga tccggatcct taccaattgg catagccaag tattacggca 120 cagatcaaga attattaaaa gcctgcattg acctcaaaat aactgtacga agaacagtta 180 gatctggaga aatgatagta tacatggtag attcgatcca tgctcctcta ctaccatggt 240 ccagccgact gagacaaggg atgatatata atgccaataa agtagctcta gcacctcaat 300 gtctcccagt cgacaaagac atcagattca gggttgtatt tgtcaatgga acatcactag 360 gtacaattac aattgctaag gtcccaaaaa ctcttgcaga tcttgcatta ccgaactcaa 420 tatcagtgaa tctgctggtt acacttaggg caggagtatc aacggaacaa aaaggaatcc 480 <210> 60 <211> 480 <212> DNA <213> Artificial Sequence <400> 60[[ID=2|1]] tgaaacaatc agacctacaa tcagagtatt tgtcatcaat aataatgatc ctattgtaag 60 atctagactt ttattcttta atctacgaat tattatgagt aacactgcaa gagagggaca 120 tagagctggt gctctcctca gtcttttatc actaccttct gcagctatga gtaatcacat 180 caaactagcc atgcattcac cagaagccag catagataga gtagaaataa cagggtttga 240 gaataattca ttccgagtta ttccagatgc tcgatcaact atgtccagag gagaagtgct 300 ggccttcgaa gcattagctg aggacattcc tgataccctt aatcaccaaa ctccatttgt 360 aaataatgat gtggaagatg acatatttga tgaaacagag aaattcttgg atgtttgcta 420 tagtgtactt atgcaggcat ggatagtaac atgcaagtgc atgactgctc ctgatcaacc 480 <210> 61 <211> 480 <212> DNA <213> Artificial Sequence <400> 61 acaaatacgg gagtgtaaat gatctcgaca gtgacccgag ttacaaagtt tgtggctctg 60 gatcattacc aatcggattg gctaaataca ctgggaatga ccaggaatta ttacaagctg 120 caaccaaact ggacatagaa gtgagaagaa cagtcaaagc gaaagagatg gttgtgtata 180 cagtacaaaa tataaaacca gaactatacc catggtctaa tagactaaga aaaggaatgc 240 tgttcgatgc caacaaagtt gctcttgctc ctcaatgtct tccactagat aggagcataa 300 aatttagagt aatcttcgtg aattgtacgg caattggatc aataaccttg ttcaaaattc 360 ctaagtcaat ggcatcatta tctctaccca acacaatatc aatcaatctg caggtacata 420 tcaaaacagg ggttcagact gattccaaag ggatagttca aattttgaat gagaagggcg 480 <210> 62 <211> 480 <212> DNA <213> Artificial Sequence <400> 62 caggattgag gggaaagttg acaaaattat gcttactcaa aatacaattc agcaaacaaa 60 gaatgacact caacaaatca aaggttcact tgccacaatt gagggcctaa tcacgacaat 120 gaaaataatg gatcctggag tcccatcaaa agtaagtctc aggagtttaa acaaagaatc 180 agaacaagtt cctataattg ttactggtaa cggagacgtc tcaaaatttg ttgatcaaga 240 caatacaatt acacttgact cattagcaag acccatatta tctggaacca aacaaaaaac 300 cgatgagaga cgagcaggtg ttcgtataga tgcacttaaa ataacagtct cagaaatgat 360 tcgggatcta tttggagact gtgataagag caaaaagctt cttgaatcaa taaatatggc 420 aactacagag caagacatca atctgatcaa aaccaatgcc cttagaagta tcacctaaat 480 <210> 63 <211> 480 <212> DNA <213> Artificial Sequence <400> 63 aatcaaataa gtgtgggaac agaaaatcag gctttcaaca caaaattttc gtttttctgc 60 tctcactaaa taaattgagt tgggttttaa aaaccgccaa aaatttttaa tttagttttt 120 gctactttct catccctccc cctcacgggt gaaaaccccg gggcgtgggc cttagtgcgc 180 gataacactg ccaagggcat cactgccggc agtggcagtc aacaaaccac gtatgatcct 240 gcgcgaaccg aggccacctt gaccaccacc acctttgcgc tgcgccggta tgacctcgcc 300 gggcgcgcct tatacgacct cgacttttcg aagttaaacc cacaaacgcc aacgcgtgat 360 gccaacggcc agatcacctt taaccccttt ggcggctttg gtttgagtgg cagtgcaccc 420 caacagtgaa acgaggtcaa aaacaaggtc cccgtcgagg tggcccaaga ccccaccgat 480 <210> 64 <211> 480 <212> DNA <213> Artificial Sequence <400> 64 tcgtgttgtc tgtactgccg ttgccacata gatcatccaa atcctaaagg attttgtgac 60 ttaaaaggta agtatgtaca aatacctaca acttgtgcta atgaccctgt gggttttaca 120 cttaaaaaca cagtctgtac cgtctgcggt atgtggaaag gttatggctg tagttgtgat 180 caactccgcg aacccatgct tcagtcagct gatgcacaat cgtttttaaa cgggtttgcg 240 gtgtgagtgc agcccgtctt acaccgtgcg gcacaggcac tagtactgat gtcgtataca 300 gggcttttga catctacaat gataaagtag ctggttttgc taaattccta aaaactaatt 360 gttgtcgctt ccaagaaaag gacgaagatg acaatttaat tgattcttac tttgtagtta 420 agagacacac tttctctaac taccaacatg aagaaacaat ttataattta cttagggatt 480 <210> 65 <211> 490 <212> DNA <213> Artificial Sequence <400> 65 cctgctaaca atgctgcaat cgtgctacaa cttcctcaag gaacaacatt gccaaaaggc 60 ttctacgcag aagggagcag aggcggcagt caagcctctt ctcgttcctc atcacgtagt 120 cgcaacagtt caagaaattc aactccaggc agcagtatgg gaacttctcc tgctagaatg 180 gctggcaatg gcggtgatgc tgctcttgct ttgctgctgc ttgacagatt gaaccagctt 240 gagagcaaaa tgtctggtaa aggccaacaa caacaaggcc aaactgtcac taagaaatct 300 gctgctgagg cttctaagaa gcctcggcaa aaacgtactg ccactaaagc atacaatgta 360 acacaagctt tcggcagacg tggtccagaa caaacccaag gaaattttgg ggaccaggaa 420 ctaatcagac aaggaactga ttacaaacat tggccgcaaa ttgcacaatt tgcccccagc 480 gcttcagcgt 490 <210> 66 <211> 490 <212> DNA <213> Artificial Sequence <400> 66 agcctgaaga acatgtccaa attcacacaa tcgacggttc atccggagtt gttaatccag 60 taatggaacc aatttatgat gaaccgacga cgactactag cgtgcctttg taagcacaag 120 ctgatgagta cgaacttatg tactcattcg tttcggaaga gacaggtacg ttaatagtta 180 atagcgtact tctttttctt gctttcgtgg tattcttgct agttacacta gccatcctta 240 ctgcgcttcg attgtgtgcg tactgctgca atattgttaa cgtgagtctt gtaaaacctt 300 ctttttacgt ttactctcgt gttaaaaatc tgaattcttc tagagttcct gatcttctgg 360 tctaaacgaa ctaaatatta tattagtttt tctgtttgga actttaattt tagccatggc 420 agattccaac ggtactatta ccgttgaaga gcttaaaaag ctccttgaac aatggaacct 480 agtaataggt 490 <210> 67 <211> 480 <212> DNA <213> Artificial Sequence <400> 67 ctttcaagaa ggtctccatc atgtttgact cctcagtcag ctggcctggc aatgacaggc 60 tgttgtctcc aaatgagttt gaaatcaagc gcactgtgga tggggaagga tacaatgtgg 120 cccaatgcaa catgaccaaa gactggttcc tggttcagat gcttgccaac tacaacattg 180 gctaccaggg cttttacatc cctgagggat acaaggatcg catgtactcc tttttcagaa 240 acttccagcc tatgagcagg caggtggttg atgaggttaa ttacactgac tacaaagccg 300 tcaccttacc atatcaacac aacaactctg gctttgtagg ataccttgcg cctactatga 360 gacaagggga accttaccca gccaattatc catacccgct catcggaact actgccgtta 420 aaagtgttac ccaaaaaaag ttcctgtgcg acaggaccat gtggcgcata ccgttctcca 480 <210> 68 <211> 480 <212> DNA <213> Artificial Sequence <400> 68 ggctaactac ttcgagaaac ctagtaacgc cattgaagtt gcagagtgtt tcgctcagca 60 ctccccccgt gtagatcagg tcgatgagtc accgcattcc ccacgggcga ccgtggcggt 120 ggctgcgttg gcggcctgcc tatggggtaa cccataggac gctctaatac ggacatggcg 180 tgaagagtct attgagctag ttagtagtcc tccggcccct gaatgcggtt aatcctaact 240 gcggagcaca cacccttaat ccaaagggca gtgtgtcgta acgggcaact ctgcagcgga 300 accgactact ttgggtgtcc gtgtttcttt ttattcttgt attggctgct tatggtgaca 360 attaaagaat tgttaccata tagctattgg attggccatc cagtgtcaaa cagagctatt 420 gtatatctct ttgttggatt cacacctctc actcttgaaa cgttacacac cctcaattac 480 <210> 69 <211> 480 <212> DNA <213> Artificial Sequence <400> 69 gtgtcccgcg tcggacaggc acgcccggac caggcttctg cccgacatga aggcggtcag 60 gcagaactcc tgattcttgc ccttcagttt caaggccagc tcctggacag tgaagttctt 120 gtacagatgg gtcggcaagc cggcgacgtc agccgggctg tatatgccga gcacggacaa 180 caggatggat gcgatggtat gcatggggtt cgccttcagg ggcaatcctg cttgccgctg 240 cattcgacca tccggatcag ttcgagcgcg ggcttgcctt cgaaagtgag ttgcttggga 300 cccagaaaca tgcgcagcgg ccgttgttgc atgaagacgg cgcgcaacat gacttccatg 360 ggactgctgc cgggacgctt gagatccttg ccgaagcaga acggcacgtg cgcgtccggg 420 ctgctggccg cggcgcccag tttggcggcg atgccgcaga ccagcatgcg cgtcggggtg 480 <210> 70 <211> 480 <212> DNA <213> Artificial Sequence <400> 70 cccagcatgc ctgaggggcg ggctcagcgg ctgcgcagac tggcgcgcgc ggacggtcat 60 gggacttcag catggcggtg tttgcagatt tggacctgcg agcgggttct gacctgaagg 120 ctctgcgcgg acttgtggag acagccgctc accttggcta ttcagttgtt gctatcaatc 180 atatcgttga ctttaaggaa aagaaacagg aaattgaaaa accagtagct gtttctgaac 240 tcttcacaac tttgccaatt gtacagggaa aatcaagacc attaaaatt ttaactagat 300 taacaattat tgtctcggat ccatctcact gcaatgtttt gagagcaact tcttcaaggg 360 cccggctcta tgatgttgtt gcagtttttc caaagacaga aaagcttttt catattgctt 420 gcacacattt agatgtggat ttagtctgca taactgtaac agagaaacta ccattttact 480

Claims

1. A nucleic acid detection kit for simultaneous detection of 9 respiratory pathogens based on a common qPCR detection platform, characterized in that Including mixed solutions that do not mix with each other: reaction solution A, reaction solution B and reaction solution C; The reaction solution A includes a reaction premix, a FluA-F forward primer, a FluA-R reverse primer, a FluA-P fluorescent probe, a FluB-F forward primer, a FluB-R reverse primer, a FluB-P fluorescent probe, an RSVA-F forward primer, an RSVA-R reverse primer, an RSVA-P fluorescent probe, an RSVB-F forward primer, an RSVB-R reverse primer, and an RSVB-P fluorescent probe; The reaction solution B includes a reaction premix, an hPIV1-F upstream primer, an hPIV1-R downstream primer, an hPIV1-P fluorescent probe, an hPIV2-F forward primer, an hPIV2-R reverse primer, an hPIV2-P fluorescent probe, an hPIV3-F forward primer, an hPIV3-R reverse primer, an hPIV3-P fluorescent probe, an hPIV4-F forward primer, an hPIV4-R reverse primer, an hPIV4-P fluorescent probe, an MP-F forward primer, an MP-R reverse primer, and a forward primer, MP-P fluorescent probe, SARS-CoV-2-ORF forward primer, SARS-CoV-2-ORF reverse primer, SARS-CoV-2-ORF fluorescent probe, SARS-CoV-2-N forward primer, SARS-CoV-2-N reverse primer, SARS-CoV-2-N fluorescent probe, SARS-CoV-2-E forward primer, SARS-CoV-2-E reverse primer and SARS-CoV-2-E fluorescent probe; The reaction solution C includes a reaction premix, a HADV-F upstream primer, a HADV-R1 downstream primer, a HADV-R2 downstream primer, a HADV-R3 downstream primer, a HADV-R4 downstream primer, a HADV-P fluorescent probe, an HRV / HEV-F forward primer, an HRV / HEV-R1 reverse primer, an HRV / HEV-R2 reverse primer, an HRV / HEV-R3 reverse primer, an HRV / HEV-R4 reverse primer, an HRV / HEV-P fluorescent probe, a BP-F forward primer, a BP-R reverse primer, and a BP-P fluorescent probe; The primer pairs and fluorescent probes are specifically as follows: FluA-F forward primer as shown in SEQ ID NO: 1, FluA-R reverse primer as shown in SEQ ID NO: 2; FluA-P fluorescent probe as shown in SEQ ID NO: 3; FluB-F forward primer as shown in SEQ ID NO: 4, FluB-R reverse primer as shown in SEQ ID NO: 5; FluB-P fluorescent probe as shown in SEQ ID NO: 6; RSV A-F forward primer as shown in SEQ ID NO: 7, RSV A-R reverse primer as shown in SEQ ID NO: 8; RSV A-P fluorescent probe as shown in SEQ ID NO: 9; RSVB-F forward primer as shown in SEQ ID NO: 10, RSVB-R reverse primer as shown in SEQ ID NO: 11; RSVB-P fluorescent probe as shown in SEQ ID NO: 12; hPIV1-F forward primer as shown in SEQ ID NO: 13, hPIV1-R reverse primer as shown in SEQ ID NO: 14; hPIV1-P fluorescent probe as shown in SEQ ID NO: 15; hPIV2-F forward primer as shown in SEQ ID NO: 16, hPIV2-R reverse primer as shown in SEQ ID NO: 17; hPIV2-P fluorescent probe as shown in SEQ ID NO: 18; hPIV3-F forward primer as shown in SEQ ID NO: 19, hPIV3-R reverse primer as shown in SEQ ID NO: 20; hPIV3-P fluorescent probe as shown in SEQ ID NO: 21; hPIV4-F forward primer as shown in SEQ ID NO: 22, hPIV4-R reverse primer as shown in SEQ ID NO: 23; hPIV4-P fluorescent probe as shown in SEQ ID NO: 24; The MP-F forward primer is shown in SEQ ID NO: 25, the MP-R reverse primer is shown in SEQ ID NO: 26; the MP-P fluorescent probe is shown in SEQ ID NO: 27; A SARS-CoV-2-ORF-F forward primer as shown in SEQ ID NO:28, a SARS-CoV-2-ORF-R reverse primer as shown in SEQ ID NO:29; and a SARS-CoV-2-ORF-P fluorescent probe as shown in SEQ ID NO:30; The SARS-CoV-2-NF forward primer as shown in SEQ ID NO:31, the SARS-CoV-2-NR reverse primer as shown in SEQ ID NO:32; the SARS-CoV-2-NP fluorescent probe as shown in SEQ ID NO:33; A SARS-CoV-2-EF forward primer as shown in SEQ ID NO:34, a SARS-CoV-2-ER reverse primer as shown in SEQ ID NO:35; a SARS-CoV-2-EP fluorescent probe as shown in SEQ ID NO:36; A HADV-F forward primer as shown in SEQ ID NO: 37, a HADV-R1 reverse primer as shown in SEQ ID NO: 38, a HADV-R2 reverse primer as shown in SEQ ID NO: 39, a HADV-R3 reverse primer as shown in SEQ ID NO: 40, and a HADV-R4 reverse primer as shown in SEQ ID NO: 41; and a HADV-P fluorescent probe as shown in SEQ ID NO: 42; An HRV / HEV-F forward primer as set forth in SEQ ID NO:43, an HRV / HEV-R1 reverse primer as set forth in SEQ ID NO:44; an HRV / HEV-R2 reverse primer as set forth in SEQ ID NO:45; an HRV / HEV-R3 reverse primer as set forth in SEQ ID NO:46; an HRV / HEV-R4 reverse primer as set forth in SEQ ID NO:47; and an HRV / HEV-P fluorescent probe as set forth in SEQ ID NO:48; The BP-F forward primer is shown in SEQ ID NO: 49, the BP-R reverse primer is shown in SEQ ID NO: 50; and the BP-P fluorescent probe is shown in SEQ ID NO:

51.

2. The nucleic acid detection kit for simultaneous detection of nine respiratory pathogens based on a common qPCR detection platform according to claim 1, characterized in that The reaction solution A also includes the RP-F forward primer shown in SEQ ID NO: 52, the RP-R reverse primer shown in SEQ ID NO: 53; and the RP-P fluorescent probe shown in SEQ ID NO: 54 as an internal control; The reaction solution B also includes the RP-F forward primer shown in SEQ ID NO: 52, the RP-R reverse primer shown in SEQ ID NO: 53; and the RP-P fluorescent probe shown in SEQ ID NO: 54 as an internal control; The reaction solution C also includes the RP-F forward primer shown in SEQ ID NO: 52, the RP-R reverse primer shown in SEQ ID NO: 53; and the RP-P fluorescent probe shown in SEQ ID NO: 54 as an internal reference control.

3. The nucleic acid detection kit for simultaneous detection of nine respiratory pathogens based on a common qPCR detection platform according to claim 1 or 2, characterized in that In reaction solution A, the fluorescent groups of the four fluorescent probes, influenza A virus, influenza B virus, respiratory syncytial virus, and human conserved gene RP fragment, are different; In reaction solution B, the fluorescent groups of the four fluorescent probes for human parainfluenza virus, Mycoplasma pneumoniae, novel coronavirus, and human conserved gene RP fragment are different; In reaction solution C, the fluorescent groups of the four fluorescent probes, namely human respiratory adenovirus, human respiratory rhinovirus / enterovirus, Bordetella pertussis and human conserved gene RP fragment, are different.

4. The nucleic acid detection kit for simultaneous detection of nine respiratory pathogens based on a common qPCR detection platform according to claim 3, characterized in that In reaction solution A, the fluorescent groups of the four fluorescent probes, influenza A virus, influenza B virus, respiratory syncytial virus, and human conserved gene RP fragment, are each selected from FAM, VIC, CY5, and ROX, and the fluorescent groups of the four fluorescent probes are different; In reaction solution B, the fluorescent groups of the four fluorescent probes for human parainfluenza virus, Mycoplasma pneumoniae, novel coronavirus, and human conserved gene RP fragment are each selected from FAM, VIC, CY5, and ROX, and the fluorescent groups of each fluorescent probe are different; In reaction solution C, the fluorescent groups of the four fluorescent probes, human respiratory adenovirus, human respiratory rhinovirus / enterovirus, Bordetella pertussis and human conserved gene RP fragment, are each selected from FAM, VIC, CY5 and ROX, and the fluorescent groups of each fluorescent probe are different.

5. The nucleic acid detection kit for simultaneous detection of nine respiratory pathogens based on a common qPCR detection platform according to claim 4, characterized in that In reaction solution A, the fluorescent group connected to the FluA fluorescent probe is labeled with FAM, the fluorescent group connected to the FluB fluorescent probe is labeled with VIC, and the fluorescent group connected to the RSVA and RSVB fluorescent probes are both labeled with CY5; In reaction solution B, the fluorescent group labeled with hPIV1, hPIV2, hPIV3, and hPIV4 fluorescent probes is FAM, the fluorescent group labeled with MP fluorescent probe is VIC, and the fluorescent group labeled with SARS-CoV-2-ORF, SARS-CoV-2-N, and SARS-CoV-2-E fluorescent probes is CY5; In reaction solution C, the fluorescent group connected to the fluorescent probe of HADV is labeled with FAM, the fluorescent group connected to the fluorescent probe of HRV / HEV is labeled with VIC, and the fluorescent group connected to the fluorescent probe of BP is labeled with CY5; In reaction solution A, reaction solution B, and reaction solution C, the fluorescent group connected to the fluorescent probe of the human conserved gene RP fragment is labeled with ROX.

6. The nucleic acid detection kit for simultaneous detection of nine respiratory pathogens based on a common qPCR detection platform according to claim 1, characterized in that The reaction premix solution includes an enzyme mixture, a nucleotide mixture, divalent magnesium ions and a buffer solution.

7. The nucleic acid detection kit for simultaneous detection of nine respiratory pathogens based on a common qPCR detection platform according to claim 6, characterized in that: The enzyme mixture comprises: 0.01-1 IU / μL of heat-resistant DNA polymerase, 100-400 U / μL of reverse transcriptase, and 30-50 U / μL of RNase inhibitor; the nucleotide mixture is deoxyribonucleotide triphosphate with a concentration of 10-500 μmol / L; the divalent magnesium ion is selected from magnesium chloride or magnesium sulfate with a concentration of 1-4 mmol / L; the buffer solution is Tris buffer with a pH of 8.0; and the concentrations of each primer and each fluorescent probe are respectively 10-500 nmol / L.

8. The nucleic acid detection kit for simultaneous detection of nine respiratory pathogens based on a common qPCR detection platform according to claim 7, characterized in that: The enzyme mixture comprises: 0.01-0.1 IU / μL of heat-resistant DNA polymerase, 100-400 U / μL of reverse transcriptase, and 30-50 U / μL of RNase inhibitor; the nucleotide mixture is deoxyribonucleotide triphosphate with a concentration of 100-200 μmol / L; the divalent magnesium ion is selected from magnesium chloride or magnesium sulfate with a concentration of 2.5-3.5 mmol / L; the buffer solution is Tris buffer with a pH of 8.0; and the concentrations of each primer and each fluorescent probe are respectively 100-200 nmol / L.

9. The nucleic acid detection kit for simultaneous detection of nine respiratory pathogens based on a common qPCR detection platform according to claim 1, characterized in that The kit also includes a positive quality control product and a negative quality control product.

10. The nucleic acid detection kit for simultaneous detection of nine respiratory pathogens based on a common qPCR detection platform according to claim 9, characterized in that The positive quality control product is a plasmid pseudovirus standard product.

11. The nucleic acid detection kit for simultaneous detection of nine respiratory pathogens based on a common qPCR detection platform according to claim 9, characterized in that The negative quality control product is sterilized diethyl pyrocarbonate treated water.

12. The nucleic acid detection kit for simultaneous detection of nine respiratory pathogens based on a common qPCR detection platform according to claim 10, characterized in that: The DNA sequences of the plasmid pseudovirus standards are shown in SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69 and SEQ ID NO: 70, respectively.

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