Primer group and kit for simultaneously detecting multiple respiratory pathogen nucleic acids based on twenty-six PCR (Polymerase Chain Reaction)

Abstract

The invention discloses a primer group and a kit for simultaneously detecting nucleic acids of various respiratory pathogens based on twenty-six PCR (Polymerase Chain Reaction). The kit comprises primer pool freeze-dried powder 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 nose / enterovirus (HRV / HEV), bordetella pertussis (BP), human coronavirus (HCoV), human bocavirus (HBoV) and chlamydia pneumoniae (CP), PCR (polymerase chain reaction) freeze-dried powder and a buffer solution. According to the kit, primer system optimization is carried out on various pathogens, brand new primers and probe pools are designed, clinical sensitivity and specificity can be achieved, and meanwhile, the kit is matched with a fully-closed full-automatic detection card box integrating sample treatment and amplification and is suitable for rapid detection of respiratory tract pathogens.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid detection, and particularly relates to a primer set and a kit for simultaneously detecting nucleic acids of multiple respiratory pathogens based on 26-plex PCR. Background Art

[0002] Respiratory tract infections are one of the most common diseases in the world. The incidence rate occupies an important position in the overall incidence rate of residents in various countries. Approximately 10% of residents suffer from respiratory tract infections every year. Respiratory tract infections are mainly caused by various respiratory viruses and some bacteria, mycoplasmas and chlamydias. Common viruses include influenza A virus (FluA), influenza B virus (FluB), respiratory syncytial virus (RSV), human parainfluenza virus (hPIV), Mycoplasma pneumoniae (MP), Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), human respiratory adenovirus (hAdV), human respiratory rhinovirus / enterovirus (HRV / HEV), Bordetella pertussis (BP), human coronavirus (HCoV), human bocavirus (HBoV), and Chlamydia pneumoniae (CP). More and more new pathogenic respiratory viruses have been discovered, which makes the diagnosis and treatment of clinically related diseases very difficult. There are a large number of respiratory viruses, and effectively identifying respiratory pathogens and clarifying the types of respiratory infection pathogens are of great significance for clinical diagnosis and treatment and epidemic prevention and control.

[0003] There are many detection methods for respiratory pathogens at home and abroad, including traditional isolation and culture methods, immunological detection techniques, and emerging molecular biology techniques. In recent years, nucleic acid-based molecular detection methods have become revolutionary technologies for detecting respiratory pathogens due to their rapidity, sensitivity, specificity, and time-saving characteristics. Fluorescent quantitative qPCR reactions can perform at most 4-5 multiplex detections simultaneously. However, this detection requires reading multiple different fluorescent agents at the same time, and the corresponding optical detection equipment is designed complexly and is expensive. It becomes powerless when more than 4-5 pathogens need to be detected. Summary of the Invention

[0004] Aiming at the technical problem in the prior art that multiplex qPCR detection requires reading signals of multiple different fluorescent agents, the present invention aims to provide a new primer set and a kit for simultaneously detecting nucleic acids of multiple respiratory pathogens.

[0005] The primer set and kit for simultaneous detection of multiple respiratory pathogen nucleic acids of the present invention can simultaneously detect 12 respiratory pathogens, solving the problems that it is difficult to simultaneously detect multiple viruses in the existing respiratory virus nucleic acid detection and it is difficult to effectively control the detection process. The present invention optimizes the primer system for detecting influenza A virus (FluA), influenza B virus (FluB), respiratory syncytial virus (RSV), human parainfluenza virus (hPIV), Mycoplasma pneumoniae (MP), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human respiratory adenovirus (HADV), human respiratory rhinovirus / enterovirus (HRV / HEV), Bordetella pertussis (BP), human coronavirus (HCoV), human bocavirus (HBoV) and Chlamydia pneumoniae (CP) by such multiplex PCR, and designs a new primer and quencher probe pool, which can sensitively and specifically meet the early detection of these 12 respiratory pathogens clinically.

[0006] The primer set for simultaneous detection of nucleic acids of multiple respiratory pathogens of the present invention includes the following primer pairs: FluA-F forward primer, FluA-R reverse primer, FluB-F forward primer, FluB-R reverse primer, RSVA-F forward primer, RSVA-R reverse primer, RSVB-F forward primer, RSVB-R reverse primer, hPIV1-F forward primer, hPIV1-R reverse primer, hPIV2-F forward primer, hPIV2-R reverse primer, hPIV3-F forward primer, hPIV3-R reverse primer, hPIV4-F forward primer, hPIV4-R reverse primer, MP-F forward primer, MP-R reverse primer, SARS-CoV-2-ORF forward primer, SARS-CoV-2-ORF reverse primer, SARS-CoV-2-N forward primer, SARS-CoV-2-N reverse primer, SARS-CoV-2-E forward primer, SARS-CoV-2-E reverse primer, HADV-F forward primer, HADV-R1 reverse primer, HADV-R2 reverse primer, HADV-R3 reverse primer, HADV-R4 reverse primer, HRV / HEV-F forward primer, HRV / HEV-R reverse primer, HRV / HEV-R1 reverse primer, HRV / HEV-R2 reverse primer, HRV / HEV-R3 reverse primer, HRV / HEV-R4 reverse primer, BP-F forward primer, BP-R reverse primer, HCoV-OC43-F forward primer, HCoV-OC43-R reverse primer, HCoV-NL63-F forward primer, HCoV-NL63-R reverse primer, HCoV-HKU1-F forward primer, HCoV-HKU1-R reverse primer, HCoV-229E-F forward primer, HCoV-229E-R reverse primer, HBoV1-F forward primer, HBoV1-R reverse primer, CP-F forward primer and CP-R reverse primer.

[0007] Preferably, the primer set for simultaneous detection of multiple respiratory pathogen nucleic acids of the present invention further includes the following quenching probes: FluA-P quenching probe, FluB-P quenching probe, RSVA-P quenching probe, RSVB-P quenching probe, hPIV1-P quenching probe, hPIV2-P quenching probe, hPIV3-P quenching probe, hPIV4-P quenching probe, MP-P quenching probe, SARS-CoV-2-ORF quenching probe, SARS-CoV-2-N quenching probe, SARS-CoV-2-E quenching probe, HADV-P quenching probe, HRV / HEV-P quenching probe, BP-P quenching probe, HCoV-OC43-P quenching probe, HCoV-NL63-P quenching probe, HCoV-HKU1-P quenching probe, HCoV-229E-P quenching probe, HBoV1-P quenching probe, and CP-P quenching probe.

[0008] Preferably, the primer set for simultaneous detection of multiple respiratory pathogen nucleic acids of the present invention further includes the following primer set and quenching probe: RP-F forward primer, RP-R reverse primer, and RP-P quenching probe.

[0009] Preferably, the primer set for simultaneous detection of multiple respiratory pathogen nucleic acids of the present invention specifically includes the following primer pairs:

[0010] FluA-F forward primer as shown in SEQ ID NO:1, FluA-R reverse primer as shown in SEQ ID NO:2;

[0011] FluB-F forward primer as shown in SEQ ID NO:4, FluB-R reverse primer as shown in SEQ ID NO:5;

[0012] RSVA-F forward primer as shown in SEQ ID NO:7, RSVA-R reverse primer as shown in SEQ ID NO:8;

[0013] RSVB-F forward primer as shown in SEQ ID NO:10, RSVB-R reverse primer as shown in SEQ ID NO:11;

[0014] hPIV1-F forward primer as shown in SEQ ID NO:13, hPIV1-R reverse primer as shown in SEQ ID NO:14;

[0015] hPIV2-F forward primer as shown in SEQ ID NO:16, hPIV2-R reverse primer as shown in SEQ ID NO:17;

[0016] The hPIV3-F forward primer shown in SEQ ID NO:19 and the hPIV3-R reverse primer shown in SEQ ID NO:20;

[0017] The hPIV4-F forward primer shown in SEQ ID NO:22 and the hPIV4-R reverse primer shown in SEQ ID NO:23;

[0018] The MP-F forward primer shown in SEQ ID NO:25 and the MP-R reverse primer shown in SEQ ID NO:26;

[0019] The SARS-CoV-2-ORF-F forward primer shown in SEQ ID NO:28 and the SARS-CoV-2-ORF-R reverse primer shown in SEQ ID NO:29;

[0020] The SARS-CoV-2-N-F forward primer shown in SEQ ID NO:31 and the SARS-CoV-2-N-R reverse primer shown in SEQ ID NO:32;

[0021] The SARS-CoV-2-E-F forward primer shown in SEQ ID NO:34 and the SARS-CoV-2-E-R reverse primer shown in SEQ ID NO:35;

[0022] The HADV-F forward primer shown in SEQ ID NO:37, the HADV-R1 reverse primer shown in SEQ ID NO:38, the HADV-R2 reverse primer shown in SEQ ID NO:39, the HADV-R3 reverse primer shown in SEQ ID NO:40, and the HADV-R4 reverse primer shown in SEQ ID NO:41;

[0023] The HRV / HEV-F forward primer shown in SEQ ID NO:43, the HRV / HEV-R1 reverse primer shown in SEQ ID NO:44; the HRV / HEV-R2 reverse primer shown in SEQ ID NO:45; the HRV / HEV-R3 reverse primer shown in SEQ ID NO:46; the HRV / HEV-R4 reverse primer shown in SEQ ID NO:47;

[0024] The BP-F forward primer shown in SEQ ID NO:49 and the BP-R reverse primer shown in SEQ ID NO:50;

[0025] The forward primer for HCoV-OC43-F as shown in SEQ ID NO:52, and the reverse primer for HCoV-OC43-R as shown in SEQ ID NO:53;

[0026] The forward primer for HCoV-NL63-F as shown in SEQ ID NO:55, and the reverse primer for HCoV-NL63-R as shown in SEQ ID NO:56;

[0027] The forward primer for HCoV-HKU1-F as shown in SEQ ID NO:58, and the reverse primer for HCoV-HKU1-R as shown in SEQ ID NO:59;

[0028] The forward primer for HCoV-229E-F as shown in SEQ ID NO:61, and the reverse primer for HCoV-229E-R as shown in SEQ ID NO:62;

[0029] The forward primer for HBoV1-F as shown in SEQ ID NO:64, and the reverse primer for HBoV1-R as shown in SEQ ID NO:65;

[0030] The forward primer for CP-F as shown in SEQ ID NO:67, and the reverse primer for CP-R as shown in SEQ ID NO:68.

[0031] Preferably, the primer set for simultaneous detection of multiple respiratory pathogen nucleic acids of the present invention further comprises:

[0032] The forward primer for RP-F as shown in SEQ ID NO:70, and the reverse primer for RP-R as shown in SEQ ID NO:71.

[0033] Preferably, the primer set for simultaneous detection of multiple respiratory pathogen nucleic acids of the present invention further comprises the following quenching probes:

[0034] The quenching probe FluA-P as shown in SEQ ID NO:3;

[0035] The quenching probe FluB-P as shown in SEQ ID NO:6;

[0036] The quenching probe RSVA-P as shown in SEQ ID NO:9;

[0037] The quenching probe RSVB-P as shown in SEQ ID NO:12;

[0038] The quenching probe hPIV1-P as shown in SEQ ID NO:15;

[0039] The hPIV2-P quenching probe as shown in SEQ ID NO:18;

[0040] The hPIV3-P quenching probe as shown in SEQ ID NO:21;

[0041] The hPIV4-P quenching probe as shown in SEQ ID NO:24;

[0042] The MP-P quenching probe as shown in SEQ ID NO:27;

[0043] The SARS-CoV-2-ORF-P quenching probe as shown in SEQ ID NO:30;

[0044] The SARS-CoV-2-N-P quenching probe as shown in SEQ ID NO:33;

[0045] The SARS-CoV-2-E-P quenching probe as shown in SEQ ID NO:36;

[0046] The HADV-P quenching probe as shown in SEQ ID NO:42;

[0047] The HRV / HEV-P quenching probe as shown in SEQ ID NO:48;

[0048] The BP-P quenching probe as shown in SEQ ID NO:51;

[0049] The HCoV-OC43-P quenching probe as shown in SEQ ID NO:54;

[0050] The HCoV-NL63-P quenching probe as shown in SEQ ID NO:57;

[0051] The HCoV-HKU1-P quenching probe as shown in SEQ ID NO:60;

[0052] The HCoV-229E-P quenching probe as shown in SEQ ID NO:63;

[0053] The HBoV1-P quenching probe as shown in SEQ ID NO:66;

[0054] The CP-P quenching probe as shown in SEQ ID NO:69.

[0055] Preferably, the primer set for simultaneous detection of nucleic acids of multiple respiratory pathogens of the present invention further includes the following quenching probe: the RP-P quenching probe as shown in SEQ ID NO:72.

[0056] Kit for simultaneous detection of nucleic acids of multiple respiratory pathogens of the present invention, the kit comprising: the primer set and reaction premix of the present invention.

[0057] Preferably, in the kit for simultaneous detection of nucleic acids of multiple respiratory pathogens of the present invention, the reaction premix comprises an enzyme mixture, a nucleotide mixture, divalent magnesium ions and a buffer solution.

[0058] The enzyme mixture comprises: a thermostable DNA polymerase at 0.01 - 1 IU / µL, preferably 0.01 - 0.1 IU / µL, a reverse transcriptase at 100 - 400 U / µL, and an RNase inhibitor at 30 - 50 U / µL; the nucleotide mixture is deoxyribonucleoside triphosphate with a concentration of 10 - 500 µmol / L, preferably 100 - 200 µmol / L; the divalent magnesium ions are selected from magnesium chloride or magnesium sulfate with a concentration of 1 - 4 mmol / L, preferably 2.5 - 3.5 mmol / L; the buffer solution is a Tris buffer solution with pH = 8.0; the concentrations of each primer and each quenching probe are 10 - 500 nmol / L, preferably 100 - 200 nmol / L.

[0059] The kit further comprises an internal reference, and the internal reference is MS2 phage.

[0060] Preferably, the DNA sequences of the plasmid pseudovirus standards are respectively as shown in SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93 and SEQ ID NO:94.

[0061] The kit further comprises a microarray qPCR detection cassette, and each sub-detection area in the microarray qPCR detection cassette is respectively fixed with a fluorescent nucleic acid hybridized with a corresponding quenching probe.

[0062] The specific primers and quenching probes of the present invention are shown in Table 1

[0063] Table 1 Each primer and quenching probe

[0064]

[0065]

[0066] The sequence of the plasmid pseudovirus DNA standard product is shown in Table 2:

[0067]

[0068]

[0069]

[0070]

[0071] The kit for simultaneous detection of nucleic acids of multiple respiratory pathogens of the present invention is stored in the dark at room temperature.

[0072] The kit for simultaneous detection of nucleic acids of multiple respiratory pathogens of the present invention can specifically and sensitively detect 12 respiratory pathogens simultaneously. The kit for simultaneous detection of nucleic acids of multiple respiratory pathogens includes a combination of amplification primers and quenching probes for 12 respiratory pathogens. Among them, the amplification reactions of 12 respiratory pathogens contain multiple quenching probes, primers, enzyme mixtures, nucleotide mixtures, divalent magnesium ions, buffer solutions, etc. for different pathogen targets, and can detect whether there are influenza A virus (FluA), influenza B virus (FluB), respiratory syncytial virus (RSV), human parainfluenza virus (hPIV), Mycoplasma pneumoniae (MP), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human respiratory adenovirus (HADV), human respiratory rhinovirus / enterovirus (HRV / HEV), Bordetella pertussis (BP), human coronavirus (HCoV), human bocavirus (HBoV) and Chlamydia pneumoniae (CP) in the same reaction system. The cartridge is provided with an internal reference control, including the RP fragment of the conserved gene of humans; the internal reference control can show the occurrence of false negatives, avoiding the occurrence of false negative interpretation results caused by inhibitors in the sample or operation errors, thereby improving the accuracy of PCR detection. The kit for simultaneous detection of nucleic acids of multiple respiratory pathogens of the present invention maintains high specificity while significantly improving the detection sensitivity, and at the same time cooperates with a fully automated detection cartridge integrating fully enclosed sample processing and amplification, which is suitable for the rapid detection of respiratory pathogens.

[0073] The positive and progressive effects of the present invention are as follows: The kit of the present invention further includes a microarray qPCR detection cassette. In the microarray qPCR detection cassette, fluorescent nucleic acids hybridized with corresponding quenching probes are respectively fixed in each sub-detection area. Only a certain kind of pathogen's fluorescent nucleic acid is fixed in each sub-detection area, so as to distinguish the pathogens in the test sample. The present invention also optimizes the primer system for multiplex PCR to detect multiple respiratory pathogens, and designs a brand-new primer and probe pool, which can achieve rapid detection of multiple respiratory pathogens clinically with sensitivity and specificity. The PCR rapid detection kit for detecting multiple respiratory pathogens of the present invention can specifically and sensitively detect the targets of 12 multiple respiratory pathogens with 12 pathogen-specific combined primers.

[0074] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the drawings to fully understand the purpose, features and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 It is a detection result diagram of FluA; Figure 2 It is a detection result diagram of FluB; Figure 3 It is a detection result diagram of RSV; Figure 4 It is a detection result diagram of hPIV; Figure 5 It is a detection result diagram of MP; Figure 6 It is a detection result diagram of SARS-CoV-2; Figure 7 It is a detection result diagram of HADV; Figure 8 It is a detection result diagram of HRV / HEV; Figure 9 It is a detection result diagram of BP; Figure 10 It is a detection result diagram of HCoV; Figure 11 It is a detection result diagram of HBoV; Figure 12 It is a detection result diagram of CP; Figure 13 It is a detection result diagram of simulated mixed positive sample 1; Figure 14 It is a detection result diagram of simulated mixed positive sample 2; Figure 15 It is a detection result diagram of simulated mixed positive sample 3; Figure 16 It is a detection result diagram of negative control product; Figure 17 This is a graph of cross-reaction detection results. Specific implementation manners

[0076] In order to make the technical means, creative features, achieved purposes and functions of the present invention easy to understand, the present invention will be further described below in conjunction with specific examples.

[0077] Example 1 The kit for detecting nucleic acids of multiple respiratory pathogens includes: each primer and each quenching probe shown in SEQ ID NO: 1 to SEQ ID NO: 72, with a concentration of 200 nmol / L respectively, the concentration of heat-resistant DNA polymerase is 0.05 IU / µL, the reverse transcriptase is 125 IU / µL, the RNA enzyme inhibitor is 40 U / µL, the concentration of divalent magnesium ions is 3 mmol / L, and the concentration of deoxyribonucleotide triphosphate is 200 µmol / L.

[0078] Detection is carried out on the qPCR detection platform disclosed in the multiple ligation probe microarray detection patent document described in patent document CN2019107011550 or other related patents. The step process is as follows: Step S1, sample addition: Take out the above-mentioned kit stored in the dark at room temperature, add 120 μL of each nasopharyngeal swab positive sample to be tested (specifically shown in Table 4) to the sample chamber, and tighten the tube cap; Step S2, according to the FDx-1000 or the corresponding instrument operation manual, run the PCR program to perform amplification and fluorescence signal detection.

[0079] Table 3 Amplification detection cycle parameters of the fluorescence quantitative qPCR instrument

[0080] Step S3, judge the sample detection result according to the following judgment criteria.

[0081] If Ct ≤ 38, the sample is positive; if 38 < Ct < 40, it is a sensitivity marginal sample, and re-sampling is required for detection; if Ct ≥ 40, the sample is negative.

[0082] Simulated clinical positive samples of influenza A virus (FluA), influenza B virus (FluB), respiratory syncytial virus (RSV), human parainfluenza virus (hPIV), Mycoplasma pneumoniae (MP), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human respiratory adenovirus (HADV), human respiratory rhinovirus / enterovirus (HRV / HEV), Bordetella pertussis (BP), human coronavirus (HCoV), human bocavirus (HBoV), and Chlamydia pneumoniae (CP), as well as simulated clinical mixed positive sample 1 (containing FluA, FluB, RSV, hPIV), simulated clinical mixed positive sample 2 (containing MP, SARS-CoV-2, HADV, HRV / HEV), simulated clinical mixed positive sample 3 (containing BP, HCoV, HBoV, CP), and negative control (sterilized diethyl pyrocarbonate-treated water) were tested and the results were analyzed. The negative and positive controls met the requirements, indicating that the experiment was valid. FluA, FluB, RSV, hPIV, MP, HADV, SARS-CoV-2, HRV / HEV, BP, HCoV, HBoV, and CP were all detected on their respective targets, and the target pathogens could also be detected in the simulated clinical mixed positive samples (as shown in Figures 13 to 16 ). There was no amplification in the samples containing other viruses and the negative control, indicating that the detection kit and method had good specificity (the results are shown in Table 4).

[0083] Table 4 Sample Detection List

[0084] Example 2 Sensitivity Detection The standards of 19 targets (including subtypes of various pathogens) prepared in-house were serially diluted from 8×10 8 copy / mL to 1.5×10 5 copy / mL, 1.5×10 4 copy / mL, and 1.5×10 3 copy / mL to be used as templates. The negative samples were nasopharyngeal swab samples collected from healthy volunteers. The above-diluted templates and negative samples were amplified separately using the kit described in Example 1, and the amplification conditions were the same as those in Example 1. According to the result determination criteria in Example 1, the sensitivity results of the 19-target detection are shown in Table 5. As can be seen from Table 5, when the virus template was 1.5×10 3 copy / mL, the kit described in Example 1 could still use it as a template for amplification. Therefore, in the case of a low-concentration template, the kit of the present invention could still perform detection, thus having high sensitivity. The detection results of the pathogens with a sample concentration of 1.5×10 4 copy / mL are as shown in Figures 1 to 12As shown. When using the kit of the present invention, there is no cross-reaction between primer pairs, and it has the same high sensitivity as ordinary qPCR.

[0085] Table 5 Sensitivity results of detecting nucleic acids of 9 respiratory viruses with this kit

[0086] Example 3 Specific detection Select some inactivated common pathogens of human infection, including Chlamydia pneumoniae simulated clinical positive samples, Mycobacterium tuberculosis simulated clinical positive samples, Streptococcus pneumoniae simulated clinical positive samples, Staphylococcus aureus simulated clinical positive samples, Escherichia coli simulated clinical positive samples, Candida albicans simulated clinical positive samples (positive reference standards for national drug standards of influenza A and B), and FluA pseudovirus simulated clinical positive samples, with a concentration of 1×10 4 copies / μL, and perform specific detection. The detection process is as shown in Example 1. The results show that except for the FluA positive sample showing a positive result, the results of other samples are all negative, proving that there is no cross-reaction between the present invention and the nucleic acids of other pathogens in this example (such as Figure 17 ).

[0087] Table 6 List of samples for specific reaction

[0088] Conclusion: The PCR detection kit provided by the present invention that can simultaneously detect multiple respiratory pathogens can stably detect low-copy viruses. The kit has the characteristics of being fully enclosed and fully automatic, has a wide range of applications, and has broad application prospects.

Claims

1. A primer set for simultaneous detection of nucleic acids of multiple respiratory pathogens, characterized in that Specifically including the following primer pairs and quenching probes: FluA-F forward primer shown in SEQ ID NO:1, FluA-R reverse primer shown in SEQ ID NO:2, FluA-P quenching probe shown in SEQ ID NO:3; FluB-F forward primer shown in SEQ ID NO:4, FluB-R reverse primer shown in SEQ ID NO:5, FluB-P quenching probe shown in SEQ ID NO:6; RSVA-F forward primer shown in SEQ ID NO:7, RSVA-R reverse primer shown in SEQ ID NO:8, RSVA-P quenching probe shown in SEQ ID NO:9; RSVB-F forward primer shown in SEQ ID NO:10, RSVB-R reverse primer shown in SEQ ID NO:11, RSVB-P quenching probe shown in SEQ ID NO:12; hPIV1-F forward primer shown in SEQ ID NO:13, hPIV1-R reverse primer shown in SEQ ID NO:14, hPIV1-P quenching probe shown in SEQ ID NO:15; hPIV2-F forward primer shown in SEQ ID NO:16, hPIV2-R reverse primer shown in SEQ ID NO:17, hPIV2-P quenching probe shown in SEQ ID NO:18; hPIV3-F forward primer shown in SEQ ID NO:19, hPIV3-R reverse primer shown in SEQ ID NO:20, hPIV3-P quenching probe shown in SEQ ID NO:21; hPIV4-F forward primer shown in SEQ ID NO:22, hPIV4-R reverse primer shown in SEQ ID NO:23, hPIV4-P quenching probe shown in SEQ ID NO:24; MP-F forward primer shown in SEQ ID NO:25, MP-R reverse primer shown in SEQ ID NO:26, MP-P quenching probe shown in SEQ ID NO:27; SARS-CoV-2-ORF-F forward primer shown in SEQ ID NO:28, SARS-CoV-2-ORF-R reverse primer shown in SEQ ID NO:29, SARS-CoV-2-ORF-P quenching probe shown in SEQ ID NO:30; SARS-CoV-2-N-F forward primer shown in SEQ ID NO:31, SARS-CoV-2-N-R reverse primer shown in SEQ ID NO:32, SARS-CoV-2-N-P quenching probe shown in SEQ ID NO:33; The SARS-CoV-2-E-F forward primer shown in SEQ ID NO:34, the SARS-CoV-2-E-R reverse primer shown in SEQ ID NO:35, and the SARS-CoV-2-E-P quencher probe shown in SEQ ID NO:36; The HADV-F forward primer shown in SEQ ID NO:37, the HADV-R1 reverse primer shown in SEQ ID NO:38, the HADV-R2 reverse primer shown in SEQ ID NO:39, the HADV-R3 reverse primer shown in SEQ ID NO:40, the HADV-R4 reverse primer shown in SEQ ID NO:41, and the HADV-P quencher probe shown in SEQ ID NO:42; The HRV / HEV-F forward primer shown in SEQ ID NO:43, the HRV / HEV-R1 reverse primer shown in SEQ ID NO:44; the HRV / HEV-R2 reverse primer shown in SEQ ID NO:45; the HRV / HEV-R3 reverse primer shown in SEQ ID NO:46; the HRV / HEV-R4 reverse primer shown in SEQ ID NO:47, and the HRV / HEV-P quencher probe shown in SEQ ID NO:48; The BP-F forward primer shown in SEQ ID NO:49, the BP-R reverse primer shown in SEQ ID NO:50, and the BP-P quencher probe shown in SEQ ID NO:51; The HCoV-OC43-F forward primer shown in SEQ ID NO:52, the HCoV-OC43-R reverse primer shown in SEQ ID NO:53, and the HCoV-OC43-P quencher probe shown in SEQ ID NO:54; The HCoV-NL63-F forward primer shown in SEQ ID NO:55, the HCoV-NL63-R reverse primer shown in SEQ ID NO:56, and the HCoV-NL63-P quencher probe shown in SEQ ID NO:57; The HCoV-HKU1-F forward primer shown in SEQ ID NO:58, the HCoV-HKU1-R reverse primer shown in SEQ ID NO:59, and the HCoV-HKU1-P quencher probe shown in SEQ ID NO:60; The HCoV-229E-F forward primer shown in SEQ ID NO:61, the HCoV-229E-R reverse primer shown in SEQ ID NO:62, and the HCoV-229E-P quencher probe shown in SEQ ID NO:63; The HBoV1-F forward primer shown in SEQ ID NO:64, the HBoV1-R reverse primer shown in SEQ ID NO:65, and the HBoV1-P quencher probe shown in SEQ ID NO:66; The CP-F forward primer shown in SEQ ID NO:67, the CP-R reverse primer shown in SEQ ID NO:68, and the CP-P quenching probe shown in SEQ ID NO:

69.

2. The primer set for simultaneous detection of nucleic acids of multiple respiratory pathogens according to claim 1, wherein It further includes: The RP-F forward primer shown in SEQ ID NO:70, the RP-R reverse primer shown in SEQ ID NO:71, and the RP-P quenching probe shown in SEQ ID NO:

72.

3. A kit for simultaneously detecting nucleic acids of multiple respiratory pathogens, characterized in that The kit includes: the primer set according to any one of claims 1 to 2, and a reaction premix.

4. The kit for simultaneous detection of nucleic acids of multiple respiratory pathogens according to claim 3, characterized in that The reaction premix includes an enzyme mixture, a nucleotide mixture, divalent magnesium ions, and a buffer solution.

5. The kit for simultaneously detecting nucleic acids of multiple respiratory pathogens according to claim 4, characterized in that: The enzyme mixture includes: 0.01 - 1 IU / µL of thermostable DNA polymerase, 100 - 400 U / µL of reverse transcriptase, 30 - 50 U / µL of RNase inhibitor; the nucleotide mixture is deoxynucleoside triphosphate with a concentration of 10 - 500 µmol / L; the divalent magnesium ions are selected from magnesium chloride or magnesium sulfate with a concentration of 1 - 4 mmol / L; the buffer solution is Tris buffer with pH = 8.0; the concentrations of each primer and each quenching probe are 10 - 500 nmol / L respectively.

6. The kit for simultaneous detection of nucleic acids of multiple respiratory pathogens according to claim 5, wherein: The enzyme mixture includes: 0.01 - 0.1 IU / µL of thermostable DNA polymerase, 100 - 400 U / µL of reverse transcriptase, 30 - 50 U / µL of RNase inhibitor; the nucleotide mixture is deoxynucleoside triphosphate with a concentration of 100 - 200 µmol / L; the 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 pH = 8.0; the concentrations of each primer and each quenching probe are 100 - 200 nmol / L respectively.

7. The kit for simultaneous nucleic acid detection of multiple respiratory pathogens (26-plex PCR) according to claim 4, characterized in that The kit further includes an internal reference control, and the internal reference is MS2 phage.

8. The kit for simultaneous detection of nucleic acids of multiple respiratory pathogens according to claim 7, wherein, The DNA sequences of the plasmid pseudovirus standards are shown in SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, and SEQ ID NO:94 respectively.

9. The kit for simultaneously detecting nucleic acids of multiple respiratory pathogens according to claim 4, wherein The kit further includes a microarray qPCR detection cartridge, and each sub-detection area in the microarray qPCR detection cartridge is fixed with a fluorescent nucleic acid hybridized with the corresponding quenching probe.

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