Kit for single-hole detection of multiple respiratory pathogen nucleic acids and pre-use method thereof

Abstract

The invention provides a kit for single-hole detection of nucleic acids of various respiratory pathogens, and belongs to the technical field of pathogen detection, the kit comprises 2-5 primer combinations and a film-forming agent; the film-forming agent is polyethylene wax; each primer combination comprises a plurality of primer pairs, and the primer pairs are used for amplifying specific respiratory tract pathogen nucleic acid. According to the invention, the reaction is divided into two systems, the two systems are separated by a film-forming agent, amplification of the two reaction systems is carried out in the same reaction tube, after multiple amplification is completed, an isolating membrane is heated and melted, two groups of amplification products are mixed, and then the amplification products are detected. The multiplex RT-PCR kit which is good in detection specificity, high in sensitivity, good in repeatability and high in detection accuracy and can be used for simultaneously detecting various pathogenic bacteria is successfully constructed.

Description

Technical Field

[0001] The present invention belongs to the technical field of pathogen detection, and in particular relates to a kit for detecting nucleic acids of multiple respiratory pathogens with a single hole and a pre-use method thereof. Background Art

[0002] Respiratory tract infection is one of the most common clinical diseases, and its incidence rate ranks first among acute infectious diseases. It is generally caused by bacteria and viruses, of which about 90% are caused by respiratory viruses. These pathogen infections will cause similar clinical symptoms such as fever, cough, pneumonia, or no significant increase in white blood cells. Acute respiratory tract infections are often mixed infections or secondary infections of viruses and bacteria, or single or multiple viral infections. As the disease continues to develop, mixed infections are more common. This increases the difficulty of controlling the patient's condition and determining the pathogen. Therefore, early diagnosis, early treatment, and early isolation are the basis of prevention and treatment. Only after the pathogens that cause respiratory infections are clearly identified, can targeted treatment be carried out to improve efficacy, reduce the burden on patients, and effectively reduce the abuse of antibiotics.

[0003] At present, there are many methods for detecting respiratory pathogens, mainly including virus culture, serological detection and molecular biology detection. Among them, virus culture and serological detection are time-consuming, low in sensitivity, high in culture requirements and prone to false negatives. Commonly used molecular biology methods include ordinary PCR, nested PCR, fluorescent quantitative PCR and constant temperature amplification. For the detection of multiple respiratory pathogens, due to the limitation of the detection channel, it is generally composed of multiple tubes of reagents, which is not only cumbersome to operate, but also takes a long time to operate, and the throughput is also reduced. Therefore, multiple nucleic acid detection schemes have emerged, and currently there are mainly: solid phase / liquid phase chip method, dissolution curve analysis method, mass spectrometry, fragment analysis method, etc. The multiple PCR system contains multiple primers, probes or molecular beacons at the same time, and the reaction system is complex; and there may be incompatible amplification conditions between multiple targets, mutual inhibition of amplification sites, and mutual interference between multiple primers and probes. These factors will lead to a sharp increase in experimental difficulty, poor sensitivity and accuracy of detection, so how to simplify the complexity of the multiple PCR reaction system and ensure the performance of multi-target detection is the key to solving the application of multiple PCR detection. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a multiplex RT-PCR kit with good detection specificity, high sensitivity, good repeatability and high detection accuracy, which can be used to simultaneously detect multiple pathogens.

[0005] The invention provides a single-hole kit for detecting nucleic acids of multiple respiratory pathogens, comprising 2 to 5 primer combinations and a film-forming agent; the film-forming agent is polyethylene wax; each primer combination comprises a plurality of primer pairs, and the primer pairs are used to amplify specific respiratory pathogen nucleic acids.

[0006] Preferably, the polyethylene wax is prepared from a thermoplastic resin and paraffin wax, and the thermoplastic resin has a melting range of 95 to 107°C and a melting point of 96 to 100°C.

[0007] Preferably, the molecular weight of the polyethylene wax is 1000-3500, and the purity of the polyethylene wax is ≧95%.

[0008] Preferably, the respiratory pathogens detected include influenza A virus, influenza B virus, adenovirus, bocavirus, rhinovirus, parainfluenza virus, coronavirus, respiratory syncytial virus, metapneumovirus, Mycoplasma pneumoniae and Chlamydia.

[0009] Preferably, when the kit comprises two primer combinations, the first primer combination comprises:

[0010] The primer pair for amplifying coronavirus OC43 is shown in SEQ ID NO.1 and SEQ ID NO.2; the primer pair for amplifying coronavirus 229E is shown in SEQ ID NO.3 and SEQ ID NO.4; the primer pair for amplifying coronavirus NL63 is shown in SEQ ID NO.5 and SEQ ID NO.6; the primer pair for amplifying seasonal influenza A virus H3N2 is shown in SEQ ID NO.7 and SEQ ID NO.8; the primer pair for amplifying boca virus is shown in SEQ ID NO.9 and SEQ ID NO.10; the primer pair for amplifying influenza A virus H7N9 is shown in SEQ ID NO.11 and SEQ ID NO.12; the primer pair for amplifying Mycoplasma pneumoniae is shown in SEQ ID NO.13 and SEQ ID NO.14; the primer pair for amplifying SARS-CoV is shown in SEQ ID NO.15 and SEQ ID NO.15; the primer pair for amplifying respiratory syncytial virus group B is shown in SEQ ID NO.17 and SEQ ID NO. NO.18; a primer pair for amplifying parainfluenza virus type 3, as shown in SEQ ID NO.19 and SEQ ID NO.20; a primer pair for amplifying rhinovirus type 2, as shown in SEQ ID NO.21 and SEQ ID NO.22; a primer pair for amplifying Chlamydia trachomatis, as shown in SEQ ID NO.23 and SEQ ID NO.24; a primer pair for amplifying Chlamydia pneumoniae, as shown in SEQ ID NO.25 and SEQ ID NO.26;

[0011] The second primer combination includes:

[0012] A primer pair for amplifying coronavirus HKU1 is shown in SEQ ID NO.27 and SEQ ID NO.28; a primer pair for amplifying influenza A virus H1N1 (2009) is shown in SEQ ID NO.29 and SEQ ID NO.30; a primer pair for amplifying influenza A virus H1N1 is shown in SEQ ID NO.31 and SEQ ID NO.32; a primer pair for amplifying influenza A virus H5N2 is shown in SEQ ID NO.33 and SEQ ID NO.34; a primer pair for amplifying influenza A virus H3N2 is shown in SEQ ID NO.35 and SEQ ID NO.36; a primer pair for amplifying metapneumovirus HMPV is shown in SEQ ID NO.37 and SEQ ID NO.38; a primer pair for amplifying respiratory syncytial virus group A is shown in SEQ ID NO.39 and SEQ ID NO.40; a primer pair for amplifying influenza B virus Yamagata strain is shown in SEQ ID NO.41 and SEQ ID NO. NO.42; a primer pair for amplifying influenza B virus Victoria strain, as shown in SEQ ID NO.43 and SEQ ID NO.44; a primer pair for amplifying parainfluenza virus type 1, as shown in SEQ ID NO.45 and SEQ ID NO.46; a primer pair for amplifying parainfluenza virus type 2, as shown in SEQ ID NO.47 and SEQ ID NO.48; a primer pair for amplifying parainfluenza virus type 4, as shown in SEQ ID NO.49 and SEQ ID NO.50; a primer pair for amplifying rhinovirus type 1, as shown in SEQ ID NO.51 and SEQ ID NO.52; a primer pair for amplifying adenovirus group B, as shown in SEQ ID NO.53 and SEQ ID NO.54; a primer pair for amplifying adenovirus group C, as shown in SEQ ID NO.55 and SEQ ID NO.56; a primer pair for amplifying adenovirus group E, as shown in SEQ ID NO.57 and SEQ ID NO.58.

[0013] Preferably, the kit further comprises a PCR reaction premix, a negative control substance and a positive control substance.

[0014] The present invention provides a pre-use method of the kit, comprising mixing a first primer combination, a PCR reaction premix, water and a nucleic acid extract of a sample to be detected to form a first reaction system, then adding a film-forming agent to the surface of the first reaction system to form an isolation layer, and finally mixing a second primer combination, a PCR reaction premix, water and a nucleic acid extract of the sample to be detected on top of the isolation layer to form a second reaction system.

[0015] The present invention also provides an amplification system for detecting nucleic acids of multiple respiratory pathogens by multiple RT-PCR using the kit, comprising the following components:

[0016]

[0017] Preferably, the final concentration of each primer in the first primer combination or the second primer combination in the reaction system is 50-250 nM.

[0018] The present invention also provides an amplification program corresponding to the amplification system, and the amplification program is as follows:

[0019]

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention analyzes the genome sequence of respiratory pathogens, selects appropriate target gene fragments, and designs multiple groups of different PCR primers to construct a multiplex PCR kit that can be used to simultaneously detect the above pathogens. However, when detecting the detection effect of the primers, it is found that the primers themselves, as well as the dimers between primers, cross-interference and other factors will affect the accuracy and sensitivity of the multiplex PCR detection system. For example, the detection specificity of individual primers is poor; after combining different primers together, the detection effect is significantly reduced compared to when they are used alone. In order to solve this problem, the present invention divides the reaction into two systems, separates them with a film-forming agent, and amplifies the two reaction systems in the same reaction tube. After the multiple amplification is completed, the isolation membrane is heated and melted to mix the two groups of amplification products, and then the amplification products are detected. The present invention successfully constructs a multiplex RT-PCR kit that can be used to simultaneously detect multiple pathogens with good detection specificity, high sensitivity, good repeatability and high detection accuracy.

[0022] The kit provided by the present invention is suitable for screening clinical cases of acute respiratory infection. A single test can achieve simultaneous screening of 29 possible infection types, greatly reducing the test cost and saving test time. It is suitable for routine clinical testing and emergency testing in response to public health emergencies.

[0023] The results of the examples of the present invention show that the kit provided by the present invention has multiple detection capabilities and can realize the detection of complex infection samples; at the same time, it can realize low-concentration sample detection, and the sensitivity can reach 10 copies / reaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 A schematic diagram of a single-tube amplification during use of the kit provided by the present invention;

[0025] Figure 2The specificity test results in Experimental Example 1 are OC43, 229E, HKU1, and NL63;

[0026] Figure 3 The specific test results in Experimental Example 1 are SeasonalH3N2, H1N1(2009), hBov, H1N1;

[0027] Figure 4 The specific test results in Experimental Example 1 are H5N2, H3N2, H7N9, and MP;

[0028] Figure 5 The specific test result SARS-CoV in Experimental Example 1;

[0029] Figure 6 The specificity test results in Experimental Example 1 are RSVA, RSVB, Yamagata, and Victoria;

[0030] Figure 7 The specific test results in Experimental Example 1 are HMPV, CT, Cpn, and HPIV1;

[0031] Figure 8 The specific test results in Experimental Example 1 are HPIV2, HPIV3, HPIV4, and HRV1;

[0032] Fig. 9 The specific test results HRV2, HADVB, HADVC, and HADVE in Experimental Example 1;

[0033] Fig.10 The sensitivity test results in Experimental Example 2;

[0034] Fig.11 The test results of using H1N1 and H5N2 samples as test objects and using the kit provided by the present invention to perform tests according to the test method, system and procedure described in Example 1;

[0035] Fig.12 The HPIV2 sample was used as the detection object, and the detection results were obtained by using the kit provided by the present invention according to the detection method, system and procedure described in Example 1. DETAILED DESCRIPTION

[0036] The invention provides a single-hole kit for detecting nucleic acids of multiple respiratory pathogens, comprising 2 to 5 primer combinations and a film-forming agent; the film-forming agent is polyethylene wax; each primer combination comprises a plurality of primer pairs, and the primer pairs are used to amplify specific respiratory pathogen nucleic acids.

[0037] In the present invention, the polyethylene wax is prepared from a thermoplastic resin and paraffin wax, the melting range of the thermoplastic resin is 95-107°C, and the melting point is 96°C-100°C; the molecular weight of the polyethylene wax is 1000-3500, and the purity of the polyethylene wax is ≧95%. In the present invention, the film-forming agent is used to separate different reaction systems in a single tube. The film-forming agent of the present invention is added to the surface layer of the first reaction system and solidifies to form a colorless and transparent isolation film, thereby separating different reaction systems and realizing the amplification of more than two reaction systems simultaneously in a single tube. In the present invention, the film-forming agent is purchased from Jiangsu Tianwen New Material Technology Co., Ltd.

[0038] In the present invention, the respiratory pathogens detected preferably include influenza A virus, influenza B virus, adenovirus, bocavirus, rhinovirus, parainfluenza virus, coronavirus, respiratory syncytial virus, metapneumovirus, Mycoplasma pneumoniae and Chlamydia; further preferably, the 29 pathogens shown in Table 1 are included. In the present invention, all forward primers in the first primer combination are preferably labeled with the same fluorescent marker, and all forward primers in the second primer combination are preferably labeled with a fluorescent marker different from the first primer combination. In the specific implementation process, all forward primers in the first primer combination are preferably labeled with a FAM fluorescent marker, and all forward primers in the second primer combination are preferably labeled with a HEX fluorescent marker. The primer sequences and groupings of the present invention are shown in Table 1, and "Group 1" in Table 1 represents the first primer combination, and "Group 2" represents the second primer combination.

[0039] Table 1 Amplification primer sequences

[0040]

[0041]

[0042]

[0043] In the present invention, the kit further comprises a PCR reaction premix, a negative control and a positive control. In the present invention, the PCR reaction premix is ​​preferably an RT-PCR Master Mix, comprising a reverse transcriptase, a hot start DNA polymerase, dNTPs, Mg 2+ ; Preferably purchased from Nanjing Novozyme Biotechnology Co., Ltd. In the present invention, the negative control is preferably RNase-free ddH2O, and the positive control is preferably a recombinant plasmid containing a target gene of a pathogen.

[0044] The present invention provides a pre-use method of the kit, comprising mixing a first primer combination, a PCR reaction premix, water and a nucleic acid extract of a sample to be detected to form a first reaction system, then adding a film-forming agent to the surface of the first reaction system to form an isolation layer, and finally mixing a second primer combination, a PCR reaction premix, water and a nucleic acid extract of the sample to be detected on top of the isolation layer to form a second reaction system.

[0045] The present invention also provides an amplification system for using the kit to perform multiple RT-PCR to detect nucleic acids of multiple respiratory pathogens. The amplification system is preferably as shown in Table 2.

[0046] Table 2 Amplification system for multiplex RT-PCR detection

[0047]

[0048] In the present invention, the final concentration of each primer in the first primer combination or the second primer combination in the respective reaction systems is preferably 50 to 250 nM.

[0049] The present invention also provides an amplification program corresponding to the amplification system, and the amplification program is shown in Table 3.

[0050] Table 3 Amplification procedures for multiplex RT-PCR assays

[0051]

[0052] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0053] Example 1

[0054] A single-well kit for detecting nucleic acids of multiple respiratory pathogens, the composition of which is shown in Table 4.

[0055] The two primer combinations are shown in Table 1 , film-forming agent, PCR reaction premix, and positive control.

[0056] Table 4 Kit Composition

[0057]

[0058] Detection method:

[0059] 1. Sample collection

[0060] Collect a throat swab, nasal swab or sputum sample from the patient and place the sample in a collection tube. Use a nucleic acid extraction kit to extract nucleic acid.

[0061] 2. Reagent Preparation

[0062] Thaw the components in the kit at room temperature until they are completely dissolved and mix well. Centrifuge the thawed components in a microcentrifuge for 10 seconds to concentrate the liquid at the bottom of the tube.

[0063] 3. Preparation of reaction system

[0064] 1) Thaw RT-PCR Master Mix, PrimerMix and positive control completely at room temperature.

[0065] Prepare the first reaction system and the second reaction system except the template according to Table 5. After adding each component, vortex for 10 seconds and centrifuge for 10 seconds.

[0066] Table 5 RT-PCR reaction system (single reaction)

[0067] Component name Standard system addition amount (μL) Component name Standard system addition amount (μL) The first primer combination 2 The second primer combination 2 Nuclease-free purified water 2 Nuclease-free purified water 2 RT-PCRMasterMix 5 RT-PCRMasterMix 5 template 1 template 1 RT-PCR reaction solution 1 total 10 Total RT-PCR reaction solution 2 10

[0068] 4) Add the film-forming agent to the PCR plate with the RT-PCR reaction solution 1 in sequence. The film-forming agent floats on the surface of the reaction solution 1 and solidifies to form a colorless and transparent isolation film. Then add the RT-PCR reaction solution 2 and mix the solution thoroughly.

[0069] 5) Place the PCR plate in the reaction tank of the PCR instrument and run the PCR program. The PCR amplification program is shown in Table 3.

[0070] 4. Separation of samples by capillary electrophoresis

[0071] 1) The PCR amplification product was centrifuged for 10 seconds in a microcentrifuge, and the capillary electrophoresis sample was prepared according to Table 6. Before the first use of the kit, 2 μL Size 600 was added to 998 μL deionized formamide (HiDi) in a 1.5 ml centrifuge tube, mixed evenly, and stored at 4°C to avoid repeated freezing and thawing.

[0072] 2) Briefly centrifuge to remove air bubbles from the sample

[0073] 3) Capillary electrophoresis was performed using a gene analyzer such as Applied Biosystems SeqStudio, 3730 / 3730xl, etc., and the "Fragment" electrophoresis method was selected. For specific operation methods, please refer to the operating instructions of the gene analyzer. The test data were analyzed using GeneMapper analysis software (Applied Biosystems).

[0074] Table 6. PCR amplification program

[0075]

[0076] Experimental Example 1

[0077] Kit Detection Specificity Test in Example 1

[0078] Positive controls of influenza A virus (H7N9, H1N1, H3N2, H5N2), influenza A virus H1N1 (2009), seasonal H3N2 virus, influenza B virus (Victoria strain and Yamagata strain), adenovirus (group B, group C and group E), bocavirus, rhinovirus (type I and type 2), parainfluenza virus (type 1, type 2, type 3 and type 4), coronavirus (229E, OC43, NL63 and HKU1), respiratory syncytial virus (group A and group B), metapneumovirus, novel coronavirus, Mycoplasma pneumoniae and Chlamydia (Chlamydia trachomatis and Chlamydia pneumoniae) (purchased from Nanjing GenScript Biotechnology Co., Ltd.) were used for specificity experiments at a concentration of 1×10 4 The specific detection method, system and procedure are as described in Example 1, and the results are as follows Figure 2 to Figure 9 As shown, it is shown that the kit provided by the present invention has multiple detection capabilities and can realize the detection of complex infection samples.

[0079] Experimental Example 2

[0080] Example 1 Sensitivity test of the kit

[0081] The positive control (mixture of 29 plasmid DNAs) was diluted two-fold to a lower concentration. The nucleic acid concentration in the subsequent reaction was 0.2 ng / μL-100 ng / μL / reaction. The detection was performed according to the detection method, system and procedure described in Example 1. The detection effect of this kit when detecting low-concentration samples is as follows: Fig.10 As shown, the results confirm that the kit provided by the present invention can realize low-concentration sample detection, and the sensitivity can reach 0.2ng / μL / reaction.

[0082] Experimental Example 3

[0083] Kit sample detection in Example 1

[0084] H1N1, H5N2, and HPIV2 samples were used as test objects and tested according to the test method, system, and procedure described in Example 1. The test kit was effective in testing samples. Figure 11-12 As shown, H1N1, H5N2, and HPIV2 samples can all produce normal peaks within their detection range. The results confirm that the kit provided by the present invention can amplify samples normally, there is no interference between primers, and there is no specific amplification.

[0085] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A kit for detecting nucleic acids of multiple respiratory pathogens in a single well, characterized in that: It comprises 2 to 5 primer combinations and a film-forming agent; the film-forming agent is polyethylene wax; each primer combination comprises a plurality of primer pairs, and the primer pairs are used to amplify specific respiratory pathogen nucleic acids.

2. The kit according to claim 1, characterized in that The polyethylene wax is prepared from thermoplastic resin and paraffin wax. The thermoplastic resin has a melting range of 95 to 107°C and a melting point of 96 to 100°C.

3. The kit according to claim 1 or 2, characterized in that The molecular weight of the polyethylene wax is 1000-3500, and the purity of the polyethylene wax is ≥95%.

4. The kit according to claim 1, characterized in that Respiratory pathogens tested include influenza A virus, influenza B virus, adenovirus, bocavirus, rhinovirus, parainfluenza virus, coronavirus, respiratory syncytial virus, metapneumovirus, Mycoplasma pneumoniae, and Chlamydia.

5. The kit according to claim 1, characterized in that When the kit comprises two primer combinations, the first primer combination comprises: The primer pair for amplifying coronavirus OC43 is shown in SEQ ID NO.1 and SEQ ID NO.2; the primer pair for amplifying coronavirus 229E is shown in SEQ ID NO.3 and SEQ ID NO.4; the primer pair for amplifying coronavirus NL63 is shown in SEQ ID NO.5 and SEQ ID NO.6; the primer pair for amplifying seasonal influenza virus H3N2 is shown in SEQ ID NO.7 and SEQ ID NO.8; the primer pair for amplifying boca virus is shown in SEQ ID NO.9 and SEQ ID NO.10; the primer pair for amplifying influenza A virus H7N9 is shown in SEQ ID NO.11 and SEQ ID NO.12; the primer pair for amplifying Mycoplasma pneumoniae is shown in SEQ ID NO.13 and SEQ ID NO.14; the primer pair for amplifying SARS-CoV is shown in SEQ ID NO.15 and SEQ ID NO.15; the primer pair for amplifying respiratory syncytial virus group B is shown in SEQ ID NO.17 and SEQ ID NO.18; the primer pair for amplifying parainfluenza virus type 3 is shown in SEQ ID NO. ID NO.19 and SEQ ID NO.20; a primer pair for amplifying rhinovirus type 2, as shown in SEQ ID NO.21 and SEQ ID NO.22; a primer pair for amplifying Chlamydia trachomatis, as shown in SEQ ID NO.23 and SEQ ID NO.24; a primer pair for amplifying Chlamydia pneumoniae, as shown in SEQ ID NO.25 and SEQ ID NO.26; The second primer combination includes: The primer pair for amplifying coronavirus HKU1 is shown in SEQ ID NO.27 and SEQ ID NO.28; the primer pair for amplifying influenza A virus H1N1 (2009) is shown in SEQ ID NO.29 and SEQ ID NO.30; the primer pair for amplifying influenza A virus H1N1 is shown in SEQ ID NO.31 and SEQ ID NO.32; the primer pair for amplifying influenza A virus H5N2 is shown in SEQ ID NO.33 and SEQ ID NO.34; the primer pair for amplifying influenza A virus H3N2 is shown in SEQ ID NO.35 and SEQ ID NO.36; the primer pair for amplifying metapneumovirus HMPV is shown in SEQ ID NO.37 and SEQ ID NO.38; the primer pair for amplifying respiratory syncytial virus group A is shown in SEQ ID NO.39 and SEQ ID NO.40; the primer pair for amplifying influenza B virus Yamagata strain is shown in SEQ ID NO.41 and SEQ ID NO. NO.42; a primer pair for amplifying influenza B virus Victoria strain, as shown in SEQ ID NO.43 and SEQ ID NO.44; a primer pair for amplifying parainfluenza virus type 1, as shown in SEQ ID NO.45 and SEQ ID NO.46; a primer pair for amplifying parainfluenza virus type 2, as shown in SEQ ID NO.47 and SEQ ID NO.48; a primer pair for amplifying parainfluenza virus type 4, as shown in SEQ ID NO.49 and SEQ ID NO.50; a primer pair for amplifying rhinovirus type 1, as shown in SEQ ID NO.51 and SEQ ID NO.52; a primer pair for amplifying adenovirus group B, as shown in SEQ ID NO.53 and SEQ ID NO.54; a primer pair for amplifying adenovirus group C, as shown in SEQ ID NO.55 and SEQ ID NO.56; a primer pair for amplifying adenovirus group E, as shown in SEQ ID NO.57 and SEQ ID NO.

58.

6. The kit according to claim 1, characterized in that The kit also includes a PCR reaction premix, a negative control substance and a positive control substance.

7. The pre-use method of the kit according to claim 6, characterized in that: The first primer combination, PCR reaction premix, water and the nucleic acid extract of the sample to be tested are mixed to form a first reaction system, and then a film-forming agent is added to the surface of the first reaction system to form an isolation layer. Finally, the second primer combination, PCR reaction premix, water and the nucleic acid extract of the sample to be tested are mixed on the isolation layer to form a second reaction system.

8. An amplification system for multiple RT-PCR detection of nucleic acid of multiple respiratory pathogens using the kit according to claim 6, characterized in that: Includes the following components:

9. The amplification system according to claim 8, characterized in that: The final concentration of each primer in the first primer combination or the second primer combination in the reaction system is 50-250 nM.

10. The amplification program corresponding to the amplification system according to claim 8, characterized in that: The amplification procedure is as follows:

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