Cathogen joint detection method based on micro-fluidic chip technology and application

Abstract

The invention belongs to the technical field of molecular biology, and particularly discloses a joint pathogen detection method based on a micro-fluidic chip technology and application of the joint pathogen detection method. The detection method is based on parallel control of multiple reaction cavities of a micro-fluidic chip, and specific primers and probe mediated PCR amplification and signal detection are combined; 20 pathogens, namely 13 common pathogens and 7 high-risk pathogens, of respiratory infectious diseases can be jointly detected. Compared with a conventional method, the method has the advantages that respiratory infectious diseases and high-risk pathogens can be synchronously screened, pathogen subtypes can be accurately recognized, and the method is adaptive to multi-scene rapid detection so as to fill the blank of a combined screening technology; meanwhile, the detection cost is reduced, the flux is improved, primary popularization is easy, clinical misdiagnosis and missed diagnosis are reduced, and support is provided for public health prevention and control.

Description

Technical Field

[0001] This invention relates to the field of molecular biology technology, specifically to a method and application for the joint detection of pathogens based on microfluidic chip technology. Background Technology

[0003] Highly pathogenic pathogens are characterized by their strong pathogenicity, high incidence of severe illness and mortality after infection, and frequent confusion of early symptoms with common infections. Failure to identify them promptly significantly increases the difficulty of prevention and control and raises public health risks. Highly pathogenic pathogens also pose a significant threat to human health. Currently, there are no vaccines or specific treatments for dengue fever, measles, and hemorrhagic fever with renal syndrome. Humans are generally susceptible to yellow fever virus, and its symptoms are difficult to distinguish from diseases such as malaria, typhoid fever, and dengue fever. Japanese encephalitis has a rapid onset and progression, with atypical early symptoms. Once respiratory failure occurs, treatment is challenging and the mortality and disability rates are high. Early diagnosis and aggressive, standardized treatment in the early stages of Japanese encephalitis can minimize mortality and disability rates.

[0004] For the aforementioned viruses and pathogens, the main detection techniques include virus isolation and culture, serological detection, and molecular biological detection. However, these methods have high requirements for experimental techniques and conditions; some observations require special training, others require specialized equipment, and they are time-consuming and have relatively low sensitivity, limiting their clinical application and promotion. Compared with traditional virus isolation methods, molecular biological detection methods are more sensitive and faster. With the rapid development of molecular biology technology, real-time quantitative PCR and LAMP technologies can be used for virus detection. However, these molecular biological methods need to be performed in the laboratory, limiting their application in rapid on-site detection; moreover, these methods all suffer from insufficient throughput and limited detection range. Although next-generation sequencing has significant sensitivity advantages and can simultaneously detect multiple samples, it is expensive, and the large amount of data generated during sequencing requires analysis by professional bioinformaticians, making it unsuitable for routine laboratory testing. Therefore, a rapid detection method that is fast, easy to operate, inexpensive, and has high sensitivity and specificity still needs further research and development.

[0005] While microfluidic chip technology has been gradually applied to pathogen detection due to its advantages such as low reagent consumption, fast analysis speed, and parallel detection capability, existing patented solutions still suffer from core shortcomings: narrow detection range, low typing accuracy, and poor adaptability to various scenarios. These limitations prevent it from filling the technological gap in "joint screening of respiratory and highly virulent pathogens" and also fail to meet the practical needs of precise clinical diagnosis and efficient public health control. Therefore, developing a microfluidic chip-based pathogen joint detection technology that comprehensively covers target sites, provides accurate typing, and is adaptable to multiple application scenarios has become an urgent technical challenge. Summary of the Invention

[0006] This invention aims to provide a method and application for the combined detection of pathogens based on microfluidic chip technology, to address the shortcomings of existing detection technologies in terms of detection speed, ease of operation, sensitivity, and specificity. To achieve the above objective, this invention provides the following technical solution:

[0007] A method for the combined detection of multiple pathogens based on a microfluidic chip includes the following primer pairs and probes:

[0008] (1) A specific primer pair and a probe with a nucleotide sequence as shown in SEQ ID NO.1-2 for detecting H1N1 virus nucleotide sequence; and the probe is labeled with a fluorescent reporter group ROX at the 5' end and a quencher group BHQ2 at the 3' end;

[0009] (2) A specific primer pair and a probe with a nucleotide sequence as shown in SEQ ID NO.4-5 for detecting MERS virus nucleotide sequences; and the probe is labeled with a fluorescent reporter group FAM at the 5' end and a quencher group BHQ1 at the 3' end;

[0010] (3) A specific primer pair and a probe with a nucleotide sequence as shown in SEQ ID NO.7-8 for detecting the N gene nucleotide sequence of the novel coronavirus (Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2); and the probe is labeled with a fluorescent reporter group FAM at the 5' end and a quencher group BHQ1 at the 3' end;

[0011] (4) A specific primer pair and a probe with a nucleotide sequence as shown in SEQ ID NO.10-11 for detecting the nucleotide sequence of the 1ab gene of the novel coronavirus (Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2); and the probe is labeled with a fluorescent reporter group ROX at the 5' end and a quencher group BHQ2 at the 3' end.

[0012] (5) A specific primer pair and a probe with a nucleotide sequence as shown in SEQ ID NO.13-14 for detecting influenza A virus; and the probe is labeled with a fluorescent reporter group FAM at the 5' end and a quencher group BHQ1 at the 3' end;

[0013] (6) A specific primer pair and a probe with a nucleotide sequence as shown in SEQ ID NO.16-17 for detecting influenza B virus; and the probe is labeled with a fluorescent reporter group FAM at the 5' end and a quencher group BHQ1 at the 3' end;

[0014] (7) A specific primer pair and a probe with a nucleotide sequence as shown in SEQ ID NO.19-20 for detecting parainfluenza virus type 1 (PIV-1); and the probe is labeled with a fluorescent reporter group FAM at the 5' end and a quencher group MGB at the 3' end.

[0015] (8) A specific primer pair and a probe with a nucleotide sequence as shown in SEQ ID NO.22-23 for detecting parainfluenza virus type 2 (PIV-2); and the probe is labeled with a fluorescent reporter group FAM at the 5' end and a quencher group MGB at the 3' end.

[0016] (9) A specific primer pair and a probe with a nucleotide sequence as shown in SEQ ID NO.25-26 for detecting parainfluenza virus type 3 (PIV-3); and the probe is labeled with a fluorescent reporter group FAM at the 5' end and a quencher group BHQ1 at the 3' end;

[0017] (10) A specific primer pair and a probe with a nucleotide sequence as shown in SEQ ID NO.28-29 for detecting parainfluenza virus type 4 (PIV-4); and the probe is labeled with a fluorescent reporter group FAM at the 5' end and a quencher group MGB at the 3' end.

[0018] (11) A specific primer pair and a probe with a nucleotide sequence as shown in SEQ ID NO.31-32 for detecting respiratory syncytial virus type A (RSV-A); and the probe is labeled with a fluorescent reporter group ROX at the 5' end and a quencher group BHQ2 at the 3' end.

[0019] (12) A specific primer pair and a probe with a nucleotide sequence as shown in SEQ ID NO.34-35 for detecting respiratory syncytial virus type B (RSV-B); and the probe is labeled with a fluorescent reporter group FAM at the 5' end and a quencher group BHQ1 at the 3' end.

[0020] (13) A specific primer pair for detecting adenovirus (AdV) nucleotide sequences as shown in SEQ ID NO.37-38 and a probe with nucleotide sequences as shown in SEQ ID NO.39; and the probe is labeled with a fluorescent reporter group FAM at the 5' end and a quencher group MGB at the 3' end;

[0021] (14) A specific primer pair and a probe with a nucleotide sequence as shown in SEQ ID NO.40-41 for detecting human rhinovirus (HRV); and the probe is labeled with a fluorescent reporter group FAM at the 5' end and a quencher group BHQ1 at the 3' end.

[0022] (15) A specific primer pair and a probe with a nucleotide sequence as shown in SEQ ID NO. 43-44 for detecting Mycoplasma pneumoniae (MP); and the probe is labeled with a fluorescent reporter group FAM at the 5' end and a quencher group MGB at the 3' end.

[0023] (16) A specific primer pair and a probe with a nucleotide sequence as shown in SEQ ID NO.46-47 for detecting enterovirus 71 (EV71); and the probe is labeled with a fluorescent reporter group FAM at the 5' end and a quencher group BHQ1 at the 3' end.

[0024] (17) A specific primer pair and a probe with a nucleotide sequence as shown in SEQ ID NO.49-50 for detecting Coxsackievirus A16 (CV-A16) nucleotide sequence; and the probe is labeled with a fluorescent reporter group FAM at the 5' end and a quencher group BHQ1 at the 3' end;

[0025] (18) A specific primer pair and a probe with a nucleotide sequence as shown in SEQ ID NO.52-53 for detecting dengue virus (DENV); and the probe is labeled with a fluorescent reporter group FAM at the 5' end and a quencher group BHQ1 at the 3' end.

[0026] (19) A specific primer pair and a probe with a nucleotide sequence as shown in SEQ ID NO.55-56 for detecting Hantaan virus (HTNV); and the probe is labeled with a fluorescent reporter group FAM at the 5' end and a quencher group BHQ1 at the 3' end.

[0027] (20) A specific primer pair and a probe with a nucleotide sequence as shown in SEQ ID NO.58-59 for detecting yellow fever virus (YFV); and the probe is labeled with a fluorescent reporter group FAM at the 5' end and a quencher group MGB at the 3' end.

[0028] (21) A specific primer pair and a probe with a nucleotide sequence as shown in SEQ ID NO. 61-62 for detecting Chikungunya virus (CHIKV); and the probe is labeled with a fluorescent reporter group ROX at the 5' end and a quencher group BHQ2 at the 3' end.

[0029] (22) A specific primer pair and a probe with a nucleotide sequence as shown in SEQ ID NO.64-65 for detecting measles virus (MV); and the probe is labeled with a fluorescent reporter group ROX at the 5' end and a quencher group BHQ2 at the 3' end.

[0030] (23) A specific primer pair and a probe with a nucleotide sequence as shown in SEQ ID NO.67-68 for detecting Russian spring-summer encephalitis virus (RSSEV); and the probe is labeled with a fluorescent reporter group ROX at the 5' end and a quencher group BHQ2 at the 3' end.

[0031] (24) A specific primer pair and a probe with a nucleotide sequence as shown in SEQ ID NO.70-71 for detecting Japanese Encephalitis Virus (JEV), wherein the 5' end of the probe is labeled with a fluorescent reporter group FAM and the 3' end is labeled with a quencher group MGB.

[0032] This invention provides a method for joint detection of pathogens based on microfluidic chip technology, comprising the following steps:

[0033] (1) Extract DNA from the pathogen to be tested;

[0034] (2) Inject the DNA of the pathogen to be tested extracted in step (1) into the sample detection well of the microfluidic chip, and use the DNA of the pathogen to be tested as a template to perform PCR amplification using the primers described in claim 1 to obtain the amplification result of the pathogen to be tested.

[0035] (3) Determine the type of pathogen to be tested based on the amplification results.

[0036] Furthermore, the PCR amplification reaction system consists of: 10 μL ExTaq HS, 1 μL upstream primer, 1 μL downstream primer, 1 μL probe, and 6 μL sterile water.

[0037] Furthermore, the PCR amplification reaction conditions are: 95℃ pre-denaturation for 30 s; 95℃ for 5 s, 60℃ for 30 s, for 40 cycles.

[0038] Furthermore, the microfluidic chip has one negative control well, one amplification control well, and at least 10 sample detection wells.

[0039] Furthermore, the sample detection well has 14 wells.

[0040] Furthermore, the fabrication steps of the microfluidic chip include:

[0041] (1) Drying: Place in a vacuum drying oven and dry continuously at 0.15 MPa and -55℃ for 4 hours. Then, raise the temperature to 15℃ at a rate of 10℃ every 1 hour, and then restore to normal temperature and pressure.

[0042] (2) Hot bonding: Using a flat-plate hot-press bonding device, the sealing film that matches the chip substrate is aligned with the dried chip, and the sealing film is quickly hot-pressed and bonded at 260℃ for 5 s.

[0043] (3) Cutting: Remove excess sealing film from the edges by laser cutting to obtain the microfluidic chip;

[0044] This invention also provides an application of a pathogen co-detection method based on microfluidic chip technology in the field of pathogen detection.

[0045] The present invention also provides a pathogen co-detection method based on microfluidic chip technology, which can jointly detect 20 pathogens, including 13 common pathogens and 7 highly virulent pathogens of respiratory infectious diseases.

[0046] The beneficial effects of this technical solution are:

[0047] 1. Expand testing coverage and fill the gap in combined screening technologies.

[0048] This method, relying on the parallel detection capabilities of microfluidic chips, can simultaneously design detection targets for respiratory infectious disease pathogens (such as influenza virus, novel H1N1 influenza A virus, MERS virus, etc.) and highly virulent pathogens (such as dengue virus, yellow fever virus, Japanese encephalitis virus, chikungunya virus, etc.). It breaks through the limitation of existing microfluidic detection technologies that focus on a single field, and realizes the joint screening of two types of high-threat pathogens. It effectively fills the technical gap in "coordinated detection of respiratory and highly virulent pathogens" and provides more comprehensive etiological data support for public health prevention and control.

[0049] 2. Improve the accuracy of subtyping detection to support precision clinical diagnosis and treatment.

[0050] To address the shortcomings of existing technologies in terms of "fuzzy subtyping," this method optimizes the design and immobilization process of DNA primers and probes on the chip surface. This allows for precise identification of pathogens requiring further subtyping (such as different subtypes of influenza virus, respiratory syncytial virus A / B, etc.), clearly distinguishing specific subtypes and variation characteristics. This avoids diagnostic ambiguity caused by broad-based testing, providing clinicians with accurate etiological subtyping information, assisting doctors in developing targeted treatment plans (such as adjusting medication for different influenza subtypes), reducing misdiagnosis and missed diagnosis, and lowering the incidence of severe illness, mortality, and disability.

[0051] 3. Enhance scene adaptability to meet the needs of rapid response in multiple scenarios.

[0052] This method inherits the core advantages of microfluidic chips—low reagent consumption, fast analysis speed, and simple operation—eliminating the need for complex laboratory equipment and professional bioinformatics analysts. Detection can be completed with simple operation, and the detection cycle is shortened by more than 50% compared to traditional molecular biology methods. This characteristic makes it suitable for scenarios requiring rapid response, such as fever clinics, port quarantine, and primary healthcare institutions. It can quickly produce test results, effectively solving the problems of low detection efficiency and insufficient scenario adaptability of existing technologies. This buys crucial time for early pathogen identification and intervention, reducing the risk of disease transmission.

[0053] 4. Strengthen the effectiveness of joint prevention and control efforts and reduce the burden on public health.

[0054] This method, through "early and accurate identification + multi-pathogen joint screening," can quickly distinguish between common respiratory infections and highly infectious diseases, as well as single and mixed infections, providing clear guidance for clinical diagnosis and treatment. At the same time, it provides public health departments with real-time pathogen epidemic data, supporting the precise adjustment of prevention and control strategies. It can effectively reduce cross-infection caused by missed or misdiagnosed cases, reduce the risk of disease spread, and alleviate the medical burden and economic losses of patients' families and society from both the diagnosis and treatment and prevention and control ends, thus safeguarding human health and public health security. Attached Figure Description

[0055] Figure 1 This is a schematic diagram of the structure of the chip based on the microfluidic technology proposed in this invention;

[0056] Figure 2 The melting curves of pathogen plasmid standards using specific primers and probes were used to validate the RT-PCR reaction system; where A is the melting curve of dengue virus, B is the melting curve of Hantavirus, C is the melting curve of chikungunya virus, and D is the melting curve of Japanese encephalitis virus.

[0057] Figure 3 Evaluation of the positive sample concordance rate of microfluidic chips - Example 1;

[0058] Figure 4 Evaluation of the positive sample concordance rate of microfluidic chips - Example 2;

[0059] Figure 5 Evaluation of the concordance rate of negative samples for microfluidic chips - Example 1;

[0060] Figure 6 Example 2: Evaluation of the concordance rate of negative samples for microfluidic chips;

[0061] Figure 7 Example 1: Evaluation of Specificity for Microfluidic Chip Sample Detection;

[0062] Figure 8 Example 2: Evaluation of the specificity of microfluidic chip sample detection. Detailed Implementation

[0063] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0064] Example 1: Optimization of nucleic acid amplification system using designed primers and probes

[0065] I. Analysis, Comparison, and Design:

[0066] The whole genome sequences of 20 pathogens were downloaded and analyzed from the Genebank database. Primers that integrate multiple subtypes of viruses were designed, and nucleic acid amplification systems were optimized using synthetic standard plasmids of 20 pathogens.

[0067] Table 1 Optimization scheme for real-time fluorescence quantitative nucleic acid amplification system

[0068]

[0069] II. Extraction of Pathogen Nucleic Acid

[0070] Pathogen nucleic acid was extracted using the whole blood DNA / RNA rapid extraction kit from Tiangen Biotech, following the instructions in the manual.

[0071] III. Amplification Reaction Procedure

[0072] Remove the RT-PCR premix (TAKARA RR391A), primers, and probes from the refrigerator, dissolve them thoroughly, vortex to mix, and then prepare different reaction mixtures according to the different reaction system schemes in Table 1.

[0073] After preparing the mixture, vortex to mix thoroughly and place on an ice box. Prepare sample reaction tubes, add 18 μL of the reaction mixture to each tube, then add 2 μL of the sample to be tested. Cap or cover the tubes and centrifuge briefly to prevent liquid from adhering to the walls and air bubbles from forming. The reaction conditions for sample testing are: 95℃ pre-denaturation for 30 s; 95℃ for 5 s, 60℃ for 30 s, for 40 cycles.

[0074] IV. Melting Curve Analysis

[0075] The melting curves of the constructed pathogen standard plasmids reacting with self-designed specific primers and probes meet the experimental requirements of RT-PCR, such as... Figure 2 Experiments were conducted to verify the different schemes mentioned above, and scheme v1 was found to produce the best detection results. Therefore, scheme v1 was selected as the primer and probe system used in the chip.

[0076] Example 2: Fabrication and Application of Microfluidic Chips

[0077] I. Fabrication of Microfluidic Chips

[0078] The fabrication of the microfluidic chip includes the following steps: (1) Drying: Placed in a vacuum drying oven, dried continuously at 0.15 MPa and -55℃ for 4 h, then heated to 15℃ at a rate of 10℃ every 1 h, and then restored to room temperature and pressure; (2) Thermal bonding: Using a flat-plate thermal bonding device, the sealing film matching the chip substrate is aligned with the dried chip, and the sealing film is rapidly thermally bonded at 260℃ and 5 s; (3) Cutting: Excess sealing film at the edges is removed by laser cutting to obtain the microfluidic chip. The microfluidic chip has 1 negative quality control well, 1 amplification quality control well, and 14 sample detection wells (wells 3 to 16), such as Figure 1 As shown.

[0079] The prepared microfluidic chip also includes pre-packaged reagents required for detection: (1) negative control wells (well 1): blank wells; (2) amplification control wells (well 2): ​​pre-loaded with 10 μL of ExTaqHS, 1 μL of GAPDH-F and 1 μL of GAPDH-R; (3) sample detection wells (wells 3 to 16): wells 3 to 12 (10 wells in total) are currently used detection wells, each well pre-loaded with 2 specific primers for 2 target pathogens, 1 corresponding probe and RT-PCR premix (specifically the v1 scheme reaction system screened in Example 1: 10 μL of ExTaqHS, 1 μL of upstream primer, 1 μL of downstream primer, 1 μL of probe and 6 μL of sterile water); wells 13 to 16 (4 wells in total) are unused reserved wells, currently pre-loaded with the same basic RT-PCR premix as the detection wells (without specific primers and probes), used to supplement corresponding reagents when expanding detection targets in the future, without affecting the normal reaction of the detection wells already used in this example.

[0080] II. Detection Methods

[0081] Inject 10 μL of extracted pathogen DNA per well into the sample detection well of the microfluidic chip, and place the chip in the microfluidic chip detector for reaction. The reaction conditions for sample detection are: 95℃ pre-denaturation for 30 s; 95℃ for 5 s, 60℃ for 30 s, for 40 cycles. After detection, analyze the results.

[0082] Example 3: Evaluation of the detection sensitivity of microfluidic chips

[0083] The microfluidic chip and detection method prepared in Example 2 were used to detect 20 pathogens according to gradient concentration (copies are the number of copies). The results are detailed in Tables 2-25.

[0084] Table 2. Detection results of H1N1 plasmids at different concentrations

[0085]

[0086] The linear correlation coefficient of this experiment was 0.9992, the lowest detection limit was 12 copies / μL, and the results for H1N1 and H2O were negative.

[0087] Table 3. Detection results of MERS plasmids at different concentrations

[0088]

[0089] The linear correlation coefficient of this experiment was 0.9952, the lowest detection limit was 1.2 copies / μL, and the H2O result was negative.

[0090] Table 4. Results of SARS-CoV-2 plasmid N gene detection at different concentrations.

[0091]

[0092] The linear correlation coefficient of this experiment was 0.9996, the lowest detection limit was 1.2 copies / μL, and the H2O result was negative.

[0093] Table 5. Results of SARS-CoV-2 plasmid 1ab gene detection at different concentrations.

[0094]

[0095] The linear correlation coefficient of this experiment was 0.9991, the lowest detection limit was 1.2 copies / μL, and the H2O result was negative.

[0096]

[0097] Table 6. Detection results of Influenza A virus plasmids at different concentrations.

[0098] The linear correlation coefficient of this experiment was 0.9948, the lowest detection limit was 1.2 copies / μL, and the H2O result was negative.

[0099] Table 7. Detection results of Influenza B virus plasmids at different concentrations.

[0100]

[0101] The linear correlation coefficient of this experiment was 0.9988, the lowest detection limit was 1.2 copies / μL, and the H2O result was negative.

[0102] Table 8. Detection results of PIV-1 plasmids at different concentrations

[0103]

[0104] The linear correlation coefficient of this experiment was 0.9998, the lowest detection limit was 12 copies / μL, and the results for PIV-1 and H2O were negative.

[0105] Table 9. Detection results of PIV-2 plasmids at different concentrations

[0106]

[0107] The linear correlation coefficient of this experiment was 0.9978, the lowest detection limit was 12 copies / μL, and the results for PIV-2 1 and H2O were negative.

[0108] Table 10 Detection results of PIV-3 plasmids at different concentrations

[0109]

[0110] The linear correlation coefficient of this experiment was 0.9991, the lowest detection limit was 12 copies / μL, and the results for PIV-31 and H2O were negative.

[0111] Table 11 Detection results of PIV-4 plasmids at different concentrations

[0112]

[0113] The linear correlation coefficient of this experiment was 0.9976, the lowest detection limit was 12 copies / μL, and the results for PIV-4 1 and H2O were negative.

[0114] Table 12 Detection results of RSV-A plasmids at different concentrations

[0115]

[0116] The linear correlation coefficient of this experiment was 0.9990, the lowest detection limit was 1.2 copies / μL, and the H2O result was negative.

[0117] Table 13 Detection results of RSV-B plasmids at different concentrations

[0118]

[0119] The linear correlation coefficient of this experiment was 0.9969, the lowest detection limit was 1.2 copies / μL, and the H2O result was negative.

[0120] Table 14 Detection results of different concentrations of AdV virus plasmids

[0121]

[0122] The linear correlation coefficient of this experiment was 0.9969, the lowest detection limit was 1.2 copies / μL, and the H2O result was negative.

[0123] Table 15 Detection results of HRV viral plasmids at different concentrations

[0124]

[0125] The linear correlation coefficient of this experiment was 0.9992, the lowest detection limit was 1.2 copies / μL, and the H2O result was negative.

[0126] Table 16 Detection results of MP plasmids at different concentrations

[0127]

[0128] The linear correlation coefficient of this experiment was 0.9856, the lowest detection limit was 12 copies / μL, and the results for MP 1 and H2O were negative.

[0129] Table 17 Detection results of EV 71 plasmids at different concentrations

[0130]

[0131] The linear correlation coefficient of this experiment was 0.9978, the lowest detection limit was 12 copies / μL, and the results for EV 71-1 and H2O were negative.

[0132] Table 18 Detection results of CV-A16 virus plasmids at different concentrations

[0133]

[0134] The linear correlation coefficient of this experiment was 0.9996, the lowest detection limit was 12 copies / μL, and the results for CV-A16-1 and H2O were negative.

[0135] Table 19 Detection results of DNEV plasmids at different concentrations

[0136]

[0137] The linear correlation coefficient of this experiment was 0.9979, the lowest detection limit was 12 copies / μL, and the results for DNEV1 and H2O were negative.

[0138] Table 20 Detection results of HTNV plasmids at different concentrations

[0139]

[0140] The linear correlation coefficient of this experiment was 0.9984, the lowest detection limit was 4.44 copies / μL, and the H2O result was negative.

[0141] Table 21 Detection results of YFV virus plasmids at different concentrations

[0142]

[0143] The linear correlation coefficient of this experiment was 0.9997, the lowest detection limit was 4.11 copies / μL, and the H2O result was negative.

[0144] Table 22 Detection results of CHIKV plasmids at different concentrations

[0145]

[0146] The linear correlation coefficient of this experiment was 0.9993, the lowest detection limit was 12 copies / μL, and the results for CHIKV 1 and H2O were negative.

[0147] Table 23 Detection results of MV plasmids at different concentrations

[0148]

[0149] The linear correlation coefficient of this experiment was 0.9975, the lowest detection limit was 12 copies / μL, and the results for MV 1 and H2O were negative.

[0150] Table 24 Detection results of RSSEV plasmids at different concentrations

[0151]

[0152] The linear correlation coefficient of this experiment was 0.9975, the lowest detection limit was 12 copies / μL, and the results for RSSEV 1 and H2O were negative.

[0153] Table 25 Detection results of JEV plasmids at different concentrations

[0154]

[0155] The linear correlation coefficient of this experiment was 0.9964, the lowest detection limit was 12 copies / μL, and the results for JEV1 and H2O were negative.

[0156] The results showed that the lowest detection limit for each pathogen on the chip was 1.2 copies / μL for MERS, SARS-CoV-2 (N, 1ab), Influenza A, Influenza B, RSV-A, RSV-B, AdV, and HRV; 12 copies / μL for H1N1, PIV-1, PIV-2, PIV-3, PIV-4, MP, EV 71, CV-A16, DNEV, CHIKV, MV, RSSEV, and JEV; 4.44 copies / μL for HTNV; and 4.11 copies / μL for YFV.

[0157] Example 4: Evaluation of positive and negative sample concordance rates of microfluidic chips

[0158] Twenty pathogen standard reference materials or pathogen nucleic acid positive and negative samples were randomly numbered, and experiments were conducted by researchers using a blinded method according to the method in Example 2 to evaluate the chip's detection performance. It was found that all pathogen standard reference materials or pathogen nucleic acid positive samples were successfully detected, while negative samples were not detected. The concordance rate for positive samples was 100%, and the concordance rate for negative samples was 100% (partial results are attached). Figure 3-6 ).

[0159] Example 5: Evaluation of the Specificity of Microfluidic Chip Detection

[0160] 1. After mixing the above 20 pathogen standard reference materials (diluted to the lowest detection limit), the experiment was conducted by the experimenter according to the method in Example 2 to evaluate the detection effect of the chip. It was found that all pathogen standard reference materials could be successfully detected (see results). Figure 7 ).

[0161] 2. The 20 pathogen standard reference materials (diluted to the lowest detection limit) were randomly numbered, and one pathogen standard reference material was randomly selected by the experimenter using a blind method to replace the nucleic acid negative sample. The samples were then mixed and given to another experimenter to perform the experiment according to the method in Example 2 to evaluate the detection specificity of the chip. It was found that all pathogens except the negative sample could be detected (results are shown in...). Figure 8 ).

[0162] The above descriptions are merely embodiments of the present invention, and common knowledge regarding specific technical solutions or characteristics is not elaborated upon here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for detecting multiple pathogens based on a microfluidic technology chip, characterized in that, The primers and probes include the following: (1) a specific primer pair for detecting the nucleotide sequence of H1N1 virus as shown in SEQ ID NO. 1-2 and a probe with a nucleotide sequence as shown in SEQ ID NO. 3; (2) a specific primer pair for detecting the nucleotide sequence of MERS virus as shown in SEQ ID NO. 4-5 and a probe with a nucleotide sequence as shown in SEQ ID NO. 6; (3) a specific primer pair for detecting the nucleotide sequence of the N gene of the novel coronavirus as shown in SEQ ID NO. 7-8 and a probe with a nucleotide sequence as shown in SEQ ID NO. 9; (4) a specific primer pair for detecting the nucleotide sequence of the 1ab gene of the novel coronavirus as shown in SEQ ID NO. 10-11 and a probe with a nucleotide sequence as shown in SEQ ID NO. 12; (5) a specific primer pair for detecting the nucleotide sequence of the influenza A virus as shown in SEQ ID NO. 13-14 and a probe with a nucleotide sequence as shown in SEQ ID NO. 15; (6) a specific primer pair for detecting the nucleotide sequence of the influenza B virus as shown in SEQ ID NO. 16-17 and a probe with a nucleotide sequence as shown in SEQ ID NO. 18; (7) a specific primer pair for detecting the nucleotide sequence of parainfluenza virus type 1 as shown in SEQ ID NO. 19-20 and a probe with a nucleotide sequence as shown in SEQ ID NO. 21; (8) a specific primer pair for detecting the nucleotide sequence of parainfluenza virus type 2 as shown in SEQ ID NO. 22-23 and a probe with a nucleotide sequence as shown in SEQ ID NO. 24; (9) a specific primer pair for detecting the nucleotide sequence of parainfluenza virus type 3 as shown in SEQ ID NO. 25-26 and a probe with a nucleotide sequence as shown in SEQ ID NO. 27; (10) a specific primer pair for detecting the nucleotide sequence of parainfluenza virus type 4 as shown in SEQ ID NO. 28-29 and a probe with a nucleotide sequence as shown in SEQ ID NO. 30; (11) a specific primer pair for detecting the nucleotide sequence of respiratory syncytial virus type A as shown in SEQ ID NO. 31-32 and a probe with a nucleotide sequence as shown in SEQ ID NO. 33; (12) a specific primer pair for detecting the nucleotide sequence of respiratory syncytial virus type B as shown in SEQ ID NO. 34-35 and a probe with a nucleotide sequence as shown in SEQ ID NO. 36; (13) a specific primer pair for detecting the nucleotide sequence of adenovirus as shown in SEQ ID NO. 37-38 and a probe with a nucleotide sequence as shown in SEQ ID NO. 39; (14) a specific primer pair for detecting the nucleotide sequence of human rhinovirus as shown in SEQ ID NO. 40-41 and a probe with a nucleotide sequence as shown in SEQ ID NO. 42; (15) a specific primer pair for detecting a nucleotide sequence of Mycoplasma pneumoniae as shown in SEQ ID NO. 43-44 and a probe with a nucleotide sequence as shown in SEQ ID NO. 45; (16) a specific primer pair for detecting a nucleotide sequence of Enterovirus 71 as shown in SEQ ID NO. 46-47 and a probe with a nucleotide sequence as shown in SEQ ID NO. 48; (17) a specific primer pair for detecting a nucleotide sequence of Coxsackievirus A 16 as shown in SEQ ID NO. 49-50 and a probe with a nucleotide sequence as shown in SEQ ID NO. 51; (18) a specific primer pair for detecting a nucleotide sequence of Dengue virus as shown in SEQ ID NO. 52-53 and a probe with a nucleotide sequence as shown in SEQ ID NO. 54; (19) a specific primer pair for detecting a nucleotide sequence of Hantavirus as shown in SEQ ID NO. 55-56 and a probe with a nucleotide sequence as shown in SEQ ID NO. 57; (20) a specific primer pair for detecting a nucleotide sequence of Yellow fever virus as shown in SEQ ID NO. 58-59 and a probe with a nucleotide sequence as shown in SEQ ID NO. 60; (21) a specific primer pair for detecting a nucleotide sequence of Chikungunya virus as shown in SEQ ID NO. 61-62 and a probe with a nucleotide sequence as shown in SEQ ID NO. 63; (22) a specific primer pair for detecting a nucleotide sequence of Measles virus as shown in SEQ ID NO. 64-65 and a probe with a nucleotide sequence as shown in SEQ ID NO. 66; (23) a specific primer pair for detecting a nucleotide sequence of Tick-borne encephalitis virus as shown in SEQ ID NO. 67-68 and a probe with a nucleotide sequence as shown in SEQ ID NO. 69; (24) a specific primer pair for detecting a nucleotide sequence of Japanese encephalitis virus as shown in SEQ ID NO. 70-71 and a probe with a nucleotide sequence as shown in SEQ ID NO.

72.

2. A method for combined detection of pathogens based on microfluidic chip technology, characterized in that, comprising the following steps: (1) extracting DNA of a pathogen to be detected; (2) injecting the extracted DNA of the pathogen to be detected in step (1) into a sample detection hole of a microfluidic chip, using the DNA of the pathogen to be detected as a template, and performing PCR amplification using the primers of claim 1 to obtain an amplification result of the pathogen to be detected; (3) determining the type of the pathogen to be detected according to the amplification result.

3. The method according to claim 2, wherein the microfluidic chip technology-based pathogen combined detection method is characterized by, The reaction system of the PCR amplification is ExTaq HS 10 μL, upstream primer 1 μL, downstream primer 1 μL, probe 1 μL, and sterilized water 6 μL.

4. The method according to claim 2, wherein the microfluidic chip technology-based pathogen combined detection method is characterized by, The reaction conditions of the PCR amplification are pre-denaturation at 95℃ for 30 s, 95℃ for 5 s, 60℃ for 30 s, and 40 cycles.

5. The method according to claim 2, wherein the method is a method for detecting pathogens in combination based on microfluidic chip technology. The microfluidic chip is provided with one negative quality control hole, one amplification quality control hole, and at least 10 sample detection holes.

6. The method according to claim 5, wherein the method is a method for detecting pathogens in combination based on microfluidic chip technology. The sample detection holes are 14 holes.

7. The method according to claim 5, wherein the microfluidic chip technology-based pathogen combined detection method is characterized by, The preparation steps of the microfluidic chip comprise: (1) drying: placed in a vacuum drying oven, dried at 0.15 MPa, -55℃ for 4 h, then heated to 15℃ at a rate of 10℃ per interval of 1 h, and then returned to normal temperature and pressure; (2) thermal bonding: using a flat plate type hot press bonding equipment, the sealing film matched with the chip substrate is aligned with the dried chip, and then the sealing film is rapidly hot-pressed and bonded at 260℃ for 5 s; (3) cutting: the excess sealing film on the edge is removed by laser cutting, and the microfluidic chip is obtained.

8. The application of the method for detecting multiple pathogens based on microfluidic technology according to any one of claims 1-6 in the field of pathogen detection.

9. Use according to claim 7, characterized in that, The method can detect 20 kinds of pathogens, including 13 common pathogens of respiratory infectious diseases and 7 severe high-risk pathogens.

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