A microfluidic chip and detection method for nucleic acid detection of respiratory pathogens

Abstract

This invention relates to a microfluidic chip for nucleic acid detection of respiratory pathogens, comprising multiple independent chambers, with a chamber number ≥3; at least one chamber contains primers and probes for detecting GAPDH and at least one chamber contains primers and probes for detecting Bacillus atrophus DNA; the remaining chambers contain primer sets and probes for detecting one or more of the following pathogen genes: Novel Coronavirus N gene, Novel Coronavirus 1ab gene, Coronavirus 229E, Coronavirus OC43, Coronavirus NL63, Coronavirus HKU1, adenovirus (universal), Respiratory Syncytial Virus A and B, Human Metapneumovirus, Rhinovirus, Influenza A virus, Influenza B virus, Human Parainfluenza Virus 1, Human Parainfluenza Virus 2, Human Parainfluenza Virus 3, Human Parainfluenza Virus 4, and Mycoplasma pneumoniae. This invention enables simultaneous detection of multiple respiratory pathogens, is simple and rapid to operate, and reduces the chance of contamination.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid amplification technology, specifically relating to a microfluidic chip and detection method for nucleic acid detection of respiratory pathogens. Background Technology

[0002] Common acute respiratory infections include acute upper respiratory tract infection, acute tracheobronchopneumonia, and bronchiectasis, among others, with inconsistent clinical manifestations. A wide variety of pathogens can cause respiratory infections, such as viruses, bacteria, mycoplasma, chlamydia, and Legionella; moreover, a single pathogen can cause multiple clinical manifestations, and the same clinical manifestation can be caused by multiple pathogens. In clinical practice, it is often difficult to target the specific pathogen for treatment, easily leading to inappropriate treatment, prolonged fever in some patients, even worsening of the condition, or overuse of antibiotics. Therefore, multivariate detection of pathogens causing respiratory infections is of great significance for timely clinical diagnosis and epidemic reporting.

[0003] Approximately 70%-80% of acute upper respiratory tract infections are caused by viruses. These mainly include influenza viruses (A, B, and C), parainfluenza viruses, respiratory syncytial virus (RSV), adenoviruses, rhinoviruses, Coxsackieviruses, measles viruses, and rubella viruses. Bacterial infections can occur directly or subsequently after viral infections, with hemolytic streptococci being the most common, followed by Haemophilus influenzae, Streptococcus pneumoniae, and Staphylococcus aureus. Gram-negative bacilli are occasionally seen. The main manifestations of these infections are rhinitis, pharyngitis, or tonsillitis. 80% of upper respiratory tract diseases and some lower respiratory tract diseases are caused by pathogens other than bacteria. Atypical pathogens, including respiratory viruses, are the most common, such as Legionella pneumophila, Mycoplasma pneumoniae, Chlamydia pneumoniae, adenoviruses, RSV, influenza viruses, and parainfluenza viruses. The etiology is not only complex but also often involves multiple pathogens. Accurate etiological analysis is not only the basis for diagnosis but also the foundation for rationally selecting treatment plans.

[0004] Currently, commonly used methods for detecting respiratory pathogens include: pathogen isolation and culture, tissue and cell culture, serology, direct detection methods (including electron microscopy), indirect and direct immunofluorescence antibody methods (IFA / DFA), enzyme immunoassay, and nucleic acid amplification. Pathogen isolation and culture, along with tissue and cell culture, is often considered the gold standard, but it suffers from drawbacks such as complex operation, long culture time, high technical difficulty, and low positive rate. While electron microscopy directly detects pathogen particles, its positive rate is not high, and the detection time is long, making it unsuitable for rapid clinical diagnosis. Nucleic acid detection utilizes hybridization or polymerase chain reaction (PCR) techniques, offering high sensitivity and specificity, and enabling the detection of minute quantities. However, hybridization PCR involves amplification followed by hybridization, requiring the opening of tubes to remove PCR products. This exposes high concentrations of products to air, easily leading to cross-contamination between samples.

[0005] Therefore, developing a respiratory pathogen detection scheme that can simultaneously detect multiple pathogens, is simple and fast to operate, and can effectively prevent cross-contamination is of great significance for solving the problems of existing technologies. Summary of the Invention

[0006] (a) Technical problems to be solved

[0007] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a microfluidic chip for nucleic acid detection of respiratory pathogens, which can simultaneously detect multiple pathogens and has the advantages of simple operation, rapid operation, and closed-system amplification to effectively prevent cross-contamination. The present invention also relates to a method for detecting respiratory pathogens using the microfluidic chip.

[0008] (II) Technical Solution

[0009] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0010] In a first aspect, the present invention provides a microfluidic chip for nucleic acid detection of respiratory pathogens, comprising multiple independent chambers, each chamber being connected to a sample injection port and an exhaust port; the number of chambers is ≥3;

[0011] At least one of the multiple chambers contains primers and probes for detecting GAPDH, and at least one chamber contains primers and probes for detecting Bacillus atrophus DNA.

[0012] The remaining chambers are each independently coated with upstream and downstream primers and probes for detecting one or more of the following pathogen genes: novel coronavirus N gene, novel coronavirus 1ab gene, coronavirus 229E, coronavirus OC43, coronavirus NL63, coronavirus HKU1, adenovirus universal, respiratory syncytial virus A, respiratory syncytial virus B, human metapneumovirus, rhinovirus, influenza A virus, influenza B virus, human parainfluenza virus 1, human parainfluenza virus 2, human parainfluenza virus 3, human parainfluenza virus 4, and mycoplasma pneumoniae.

[0013] According to a preferred embodiment of the present invention, the upstream and downstream primers for detecting GAPDH are shown in SEQ ID NO: 55 and SEQ ID NO: 56, and the probe is shown in SEQ ID NO: 57; the upstream and downstream primers for detecting Bacillus atrophus DNA are shown in SEQ ID NO: 58 and SEQ ID NO: 59, and the probe is shown in SEQ ID NO: 60.

[0014] According to a preferred embodiment of the present invention, upstream and downstream primers for detecting influenza A virus are shown as SEQ ID NO:1 and SEQ ID NO:2, and probes are shown as SEQ ID NO:3; upstream and downstream primers for detecting influenza B virus are shown as SEQ ID NO:4 and SEQ ID NO:5, and probes are shown as SEQ ID NO:6.

[0015] The upstream and downstream primers for detecting human parainfluenza virus type 1 are shown in SEQ ID NO:7 and SEQ ID NO:8, and the probe is shown in SEQ ID NO:9;

[0016] The upstream and downstream primers for detecting human parainfluenza virus type 2 are shown in SEQ ID NO:10 and SEQ ID NO:11, and the probe is shown in SEQ ID NO:12;

[0017] The upstream and downstream primers for detecting human parainfluenza virus type 3 are shown in SEQ ID NO:13 and SEQ ID NO:14, and the probe is shown in SEQ ID NO:15;

[0018] The upstream and downstream primers for detecting human parainfluenza virus type 4 are shown in SEQ ID NO:16 and SEQ ID NO:17, and the probe is shown in SEQ ID NO:18;

[0019] The upstream and downstream primers for detecting adenovirus universally are shown in SEQ ID NO:19 and SEQ ID NO:20, and the probe is shown in SEQ ID NO:21;

[0020] The upstream and downstream primers used to detect coronavirus 229E are shown in SEQ ID NO:22 and SEQ ID NO:23, and the probe is shown in SEQ ID NO:24;

[0021] The upstream and downstream primers for detecting coronavirus NL63 are shown in SEQ ID NO:25 and SEQ ID NO:26, and the probe is shown in SEQ ID NO:27;

[0022] The upstream and downstream primers used to detect coronavirus HKU1 are shown in SEQ ID NO:28 and SEQ ID NO:29, and the probe is shown in SEQ ID NO:30;

[0023] The upstream and downstream primers for coronavirus OC43 are shown in SEQ ID NO:31 and SEQ ID NO:32, and the probe is shown in SEQ ID NO:33;

[0024] The upstream and downstream primers for detecting respiratory syncytial virus type A are shown in SEQ ID NO:34 and SEQ ID NO:35, and the probe is shown in SEQ ID NO:36.

[0025] The upstream and downstream primers for detecting respiratory syncytial virus type B are shown in SEQ ID NO:37 and SEQ ID NO:38, and the probe is shown in SEQ ID NO:39.

[0026] The upstream and downstream primers used for detecting Mycoplasma pneumoniae are shown in SEQ ID NO:40 and SEQ ID NO:41, and the probe is shown in SEQ ID NO:42;

[0027] The upstream and downstream primers used to detect human metapneumovirus are shown in SEQ ID NO:43 and SEQ ID NO:44, and the probe is shown in SEQ ID NO:45.

[0028] The upstream and downstream primers used for detecting rhinovirus are shown in SEQ ID NO:46 and SEQ ID NO:47, and the probe is shown in SEQ ID NO:48;

[0029] The upstream and downstream primers used to detect the N gene of the novel coronavirus are shown in SEQ ID NO:49 and SEQ ID NO:50, and the probe is shown in SEQ ID NO:51.

[0030] The upstream and downstream primers used to detect the novel coronavirus 1ab gene are shown in SEQ ID NO:52 and SEQ ID NO:53, and the probe is shown in SEQ ID NO:54.

[0031] According to a preferred embodiment of the present invention, at least one of the plurality of chambers is blank and not coated with any detection reagent.

[0032] According to a preferred embodiment of the present invention, during the process of coating each chamber with primers and probes, 1 μL of the primer set and probe mixture is applied to the corresponding chamber using a pipette, dried, and then the chamber is sealed with a membrane; wherein, the total concentration of the primer set and probe mixture is 0.4-1 μmol / L, preferably 0.5 μmol / L.

[0033] According to a preferred embodiment of the present invention, the number of chambers is ≥20.

[0034] According to a preferred embodiment of the present invention, the microfluidic chip includes a detection plate body, which is a sheet-like plate body including a first side and a second side. A plurality of chamber holes, branch sample inlet channels communicating with a first end of the chamber holes, an exhaust channel communicating with a second end, exhaust holes communicating with each exhaust channel, a main channel communicating with each branch sample inlet channel, and a sample injection port located at the starting end of the main channel are recessed on the first side. The chamber holes are through-hole structures; the branch sample inlet channels, exhaust channels, and exhaust holes are non-through-hole structures.

[0035] The opening side of the vent is covered with a hydrophobic and breathable membrane. A first sealing sheet is laminated to the first side of the detection strip body to block the branch sample inlet channel, the vent channel, and the first side of the chamber hole. A second sealing sheet is laminated to the second side of the detection strip body to block the second side of the chamber hole, so that the chamber hole becomes a sealed chamber.

[0036] The microfluidic chip also includes a third sealing sheet and a cover plate for sealing the vent and injection holes after injection is completed.

[0037] According to a preferred embodiment of the present invention, the horizontal cross-section of the chamber is crescent-shaped, elliptical, or spindle-shaped, and the width of each chamber gradually narrows from the middle to both ends. Each chamber has a convex arc surface; the convex arc surface faces the outer peripheral edge of the detection plate body. With this structure, when an optical instrument is used to detect the probe signal within the chamber from the outer peripheral edge, the convex arc surface acts as a focusing lens, thereby facilitating the acquisition of a higher optical signal.

[0038] According to a preferred embodiment of the present invention, the aspect ratio of the chamber is between 3:1.4 and 2, and the aspect ratio is between 3:1 and 1.5. Preferably, the length of the chamber is 2.95-3.1 mm, the width is 1.5-1.7 mm, and the thickness (depth) is 1.2-1.4 mm.

[0039] According to a preferred embodiment of the present invention, the volume of the chamber is 5-10 μL.

[0040] Secondly, the present invention provides a method for nucleic acid detection of respiratory pathogens, wherein the method uses the aforementioned microfluidic chip for nucleic acid detection, and the detection steps are as follows:

[0041] S1. Sample processing and template extraction to obtain the template to be tested;

[0042] S2. Prepare RT-PCR amplification reagents and mix them with the template to be tested to make a liquid amplification system reagent;

[0043] S3. Inject the liquid amplification system reagent into the injection port of the microfluidic chip to fill each chamber with the liquid amplification system reagent, and then seal the injection port and the vent port.

[0044] S4. Place the microfluidic chip into a nucleic acid amplification instrument for temperature-controlled amplification;

[0045] S5. Use a nucleic acid amplification instrument to perform optical detection on each chamber, and then process and analyze the data.

[0046] (III) Beneficial Effects

[0047] This invention designs a series of primers and probes for detecting nucleic acids of various common respiratory pathogens, and sets primer sets and probes for detecting extraction controls (GAPDH gene) and amplification controls (Bacillus atrophus DNA). These primers and probes are pre-coated in separate and closed chambers of a microfluidic chip. Only one sampling and pretreatment (preparation of liquid reagents for RT-PCR amplification system) is required to achieve simultaneous detection and analysis of multiple pathogens.

[0048] The microfluidic chip provided by this invention can detect a wide variety of pathogens in a single test, and has the advantages of high sensitivity and strong specificity. At the same time, the microfluidic chip provided by this invention can monitor the amplification status in real time through fluorescence amplification curves, avoiding the process of amplification and rehybridization in traditional gene chips. It is simple to operate, fast to detect, and the entire amplification and detection process is in a closed state, reducing the possibility of contamination. It is low in cost and fast. Attached Figure Description

[0049] Figure 1 This is a schematic diagram of the structure of a microfluidic chip according to the present invention.

[0050] Figure 2-20 After injecting a test sample containing 18 common respiratory pathogens into a microfluidic chip, the following amplification curves were detected using a nucleic acid amplification instrument: IFA (influenza A universal primer), IFB (influenza B universal primer), PIV1 (human parainfluenza), PIV2 (human parainfluenza), PIV3 (human parainfluenza), PIV4 (human parainfluenza), OC43 (coronavirus), HKU1 (coronavirus), 229E (coronavirus), NL63 (coronavirus), MP (Mycoplasma pneumoniae), RSV (respiratory syncytial virus), ADV (adenovirus), HMPV (human metapneumovirus), RV (human rhinovirus), 2019-nCOV-1ab (SARS-CoV-2 1ab gene), 2019-nCOV-1ab (SARS-CoV-2 N gene), GAPDH (EC) (internal standard), and Bacillus atrophicus DNA (amplification control).

[0051] Figure 21This is an exploded structural diagram of a microfluidic chip according to a preferred embodiment of the present invention.

[0052] Figure 22 This is a schematic diagram of the overall structure of the microfluidic chip according to a preferred embodiment of the present invention.

[0053] Figure 23 This is a schematic diagram of the chamber structure of a microfluidic chip according to a preferred embodiment of the present invention. Detailed Implementation

[0054] To better explain and facilitate understanding of the present invention, a detailed description of the invention is provided below with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the chemical reagents used in the following embodiments are all commercially available conventional reagents, and the experimental procedures are conventional methods well-known to those skilled in the art.

[0055] This invention mainly provides a microfluidic chip technology and detection method that can simultaneously detect multiple respiratory pathogens. It is not limited to application on a specific microfluidic chip with a certain structure. As long as the basic characteristic of a microfluidic chip that "integrates multiple independent chambers" is met, it meets the requirements of this invention: that is, a microfluidic chip that includes multiple independent chambers, each chamber being connected to a sample injection port and an exhaust port; the number of chambers is ≥3.

[0056] In these chambers, at least one chamber contains upstream and downstream primers and probes for detecting GAPDH, and at least one chamber contains upstream and downstream primers and probes for detecting Bacillus atrophus DNA; the remaining chambers are each independently coated with upstream and downstream primers and probes for detecting one or more of the following pathogen genes: novel coronavirus N gene, novel coronavirus 1ab gene, coronavirus 229E, coronavirus OC43, coronavirus NL63, coronavirus HKU1, adenovirus universal, respiratory syncytial virus A, respiratory syncytial virus B, human metapneumovirus, rhinovirus, influenza A virus, influenza B virus, human parainfluenza virus type 1, human parainfluenza virus type 2, human parainfluenza virus type 3, human parainfluenza virus type 4, and Mycoplasma pneumoniae.

[0057] The following are preferred embodiments of the present invention.

[0058] Example 1

[0059] like Figure 1As shown, the microfluidic chip 1 includes a sample injection port 11 and multiple independent chambers 15. One end of each chamber 15 is connected to a branch sample inlet channel 132, and the other end of each chamber 15 is connected to an exhaust channel 121. Each branch sample inlet channel 132 is directly or indirectly connected to the sample injection port 11 (the branch sample inlet channel 132 is connected to the main channel 131, which in turn is connected to the sample injection port 11), and the exhaust channel 121 is connected to an exhaust port 12. Each chamber 15 is connected to at least one exhaust port 12. Each chamber 15 is coated with a mixture of probes containing primer sets of a pathogen, enabling simultaneous detection and analysis of nucleic acids from multiple pathogens. Of course, the structure of the microfluidic chip is not limited to the above. For example, multiple sample injection ports can be provided, with the number of ports corresponding to the number of chambers; that is, each chamber is independently connected to a sample injection port via an inlet channel and to an exhaust port via an exhaust channel. However, this structure is relatively complex and requires multiple sample injections during detection, making rapid detection inconvenient.

[0060] The microfluidic chip provided by this invention, capable of simultaneously detecting multiple respiratory pathogens, is prepared through the following steps:

[0061] (I) Selection of respiratory pathogens

[0062] The pathogen primer sets and probes coated in the microfluidic chip include at least the primer sets and probes for detecting the genes of the following pathogens: SARS-CoV-2-N, SARS-CoV-1ab, coronavirus 229E, coronavirus OC43, coronavirus NL63, coronavirus HKU1, adenovirus universal, respiratory syncytial virus A and B, human metapneumovirus, rhinovirus, influenza A virus, influenza B virus, human parainfluenza virus (HPIV1), human parainfluenza virus (HPIV2), human parainfluenza virus (HPIV3), human parainfluenza virus (HPIV4), and Mycoplasma pneumoniae.

[0063] It has been proven that most respiratory diseases are caused by pathogens other than bacteria, with respiratory viruses being the most common. The clinical symptoms and signs of respiratory infections are quite similar, mainly including rhinitis, pharyngitis, laryngitis, and tonsillitis. Severe cases can lead to tracheitis, bronchitis, and pneumonia. However, the treatment methods, efficacy, and course of the disease vary depending on the pathogen. To promptly identify the cause of the disease and treat the pathogen as early as possible, it is necessary to rapidly detect the type of pathogen causing the disease among multiple similar symptoms. When using primer sets and probes for known pathogens still yields no positive results, it indicates a potential new outbreak. Therefore, the microfluidic chip for nucleic acid detection of respiratory pathogens in this invention is of great significance for the simultaneous multi-pathogen detection of respiratory infections, enabling timely clinical diagnosis and outbreak reporting.

[0064] (II) Primer and probe design

[0065] By reviewing literature and analyzing pathogen genome sequences, specific target gene markers for each pathogen were identified. Then, using Primer Premier 5, Beacon Designer software, and Primer-BLAST (NCBI) analysis, primer and probe sequences were designed according to parameters such as an annealing temperature of 60℃, an amplification product length of 100-150 bp, and a probe Tm value at least 5 degrees higher than the primer. The corresponding primer and probe sequences for each pathogen are shown in Table 1. After design, the sequences were sent to a qualified synthetic company for synthesis. The probes were labeled with a FAM group at the 5' end and with BHQ1 or MGB at the 3' end.

[0066] In addition, in order to monitor the quality of samples and amplification reagents, extraction controls and amplification controls need to be set up. The extraction control is generally a housekeeping gene that is stably expressed in the human body. In this invention, the GAPDH gene is selected. The amplification control is a gene with low homology to the pathogen to be tested or no homology at all. In this invention, the Bacillus atrophus gene is selected. Primers and probes for these two genes are designed according to the same parameter requirements as the pathogen.

[0067] Finally, the primer sets and probe sequences pre-coated in different chambers of the microfluidic chip were determined as shown in Table 1.

[0068] Table 1: Pre-coated primer sets and probes in microfluidic chips

[0069]

[0070]

[0071]

[0072] Primer names containing F represent upstream primers, those containing R represent downstream primers, and those containing P represent probes; in the primer sequences of SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:39, Y represents C or T, K represents G or T, and R represents A or G.

[0073] (III) Preparation of primer-probe mixture

[0074] After synthesizing the upstream and downstream primer sets and probes designed for each pathogen as listed in Table 1, they were dissolved in TE solution to prepare 10 μmol / L stock solutions. Then, they were prepared according to the proportions shown in Table 2 to make a primer-probe mixture with a total concentration of 1 μmol / L, and stored at -20℃ for later use.

[0075] When preparing the primer-probe mixture for the amplification control (Bacillus atrophicus), replace the water in Table 2 with 1*10^5 copies / μL of Bacillus atrophicus genomic DNA.

[0076] Table 2 Preparation of primer-probe mixture

[0077] Components Volume (μL) Upstream primer (10 μmol / L) 0.1 Downstream primer (10 μmol / L) 0.1 Probe (10 μmol / L) 0.1 TE 0.7 total 1

[0078] (iv) Coating primer-probe mixtures in different chambers of the microfluidic chip

[0079] (1) Take the microfluidic chip (the second side of the detection chip body is sealed with a membrane, and the first side is open), first ultrasonically clean it in acetone for 10 minutes, then ultrasonically clean it in anhydrous ethanol for 10 minutes, then ultrasonically clean it in deionized water for 10 minutes, and finally take it out and dry it in an oven for later use.

[0080] (2) Place the microfluidic chip on a clean workbench, use a pipette to apply 1 μL of primer-probe mixture to different chambers, and allow it to air dry naturally. The pathogens and controls are arranged in the chip as follows: Figure 1 As shown in Table 3.

[0081] (3) The hot press is used to coat the chip, forming a closed microfluidic chip;

[0082] (4) After vacuum packaging the microfluidic chip using a vacuum device, store it away from light for later use.

[0083] Table 3 Pathogens Detected by Chip Reactor

[0084]

[0085]

[0086] Note: Figure 1 The lower row of chambers in the chip shown corresponds to chambers 1-10 from left to right, and the upper row corresponds to chambers 11-20 from right to left. Chamber #3 is coated with a mixture of primers and probes for detecting syncytial virus types A and B to ensure detection rate.

[0087] The method for pathogen detection using a microfluidic chip with the aforementioned structure for respiratory pathogen nucleic acid detection comprises the following steps:

[0088] Step 1: Sample processing and template extraction

[0089] Taking a throat swab as an example, take 200uL of freshly collected throat swabs, use the viral RNA extraction kit (CW3127-S) produced by Jiangsu Kangwei Century Co., Ltd. or the QIAamp Viral RNA Mini Kit (52904) from QIAGEN, and strictly follow the instructions for extraction. Finally, elute with 50uL of nuclease-free water.

[0090] Step 2: Preparation of the amplification system

[0091] The RT-PCR amplification system was prepared using the Novizan One Step RT-PCR Kit (Q223-1). The reaction mixture was prepared (on ice), vortexed, and briefly centrifuged.

[0092] Step 3: Chip Sample Loading

[0093] Use a 200μL pipette to draw up the amplification system and inject the liquid into the microfluidic chip. After the microfluidic chip is filled with liquid, remove the pipette tip and seal the sample well with sealant.

[0094] Step 4: PCR amplification

[0095] Place the microfluidic chip into the nucleic acid amplification analyzer (device model or brand), and set the cycling parameters as shown in Table 4 for amplification.

[0096] Table 4 PCR amplification cycle parameters

[0097]

[0098] Step 5: Results Analysis

[0099] After amplification, the data is processed by the software built into the nucleic acid amplification analyzer. Compared with the blank control well, the detection well is considered positive if an S-shaped curve is observed on the nucleic acid amplification analyzer, and negative if no S-shaped curve is observed.

[0100] To verify the detection effectiveness of the microfluidic chip, clinical positive samples were collected. For pathogens for which clinical positive samples could not be collected, plasmid DNA or pseudoviruses were used to prepare simulated pharyngeal swab positive samples. After all samples were mixed, the above method was used for detection. The detection results are as follows: Figure 2-20 As shown: Figure 2 The result showed a positive result for FluA. Figure 3 The result showed a positive result for FluB. Figure 4 The result showed a positive PIV1 test. Figure 5 The result showed a positive PIV2 test. Figure 6 The result showed a positive result for PIV3. Figure 7 The result showed a positive result for PIV4. Figure 8 The result showed a positive result for HcoV OC43. Figure 9 The result showed a positive HcoV HKU1 test. Figure 10 The result showed a positive result for HCOV 229E. Figure 11 The result showed a positive result for HcoVNL63. Figure 12 The result showed that MP was positive. Figure 13 The result showed a positive RSV test. Figure 14 The result showed a positive result for AdvV. Figure 15 The result showed a positive HMPV test. Figure 16 The result showed that RV was positive. Figure 17 The test result for 2019-nCoV-ORF 1ab was positive. Figure 18 The result showed a positive result for 2019-nCoV-N.

[0101] Example 2

[0102] To improve detection accuracy, the present invention can further design the microfluidic chip structure as follows: Figure 21-22 As shown, the primer set and probe mixture are both described in Example 1.

[0103] In this embodiment, the microfluidic chip includes a detection chip body 1, on which a plurality of chambers 11 and a plurality of vent holes 12 are formed, each corresponding to and connected to the other. The detection chip body 1 also has a sample injection port 13, which communicates with the chambers 11. A first sealing membrane 2 is provided on the first side of the detection chip body 1 to seal the first side of the chambers 11, the first side of the vent holes 12, and the first side of the sample injection port 13. A second sealing membrane 3 is provided on the second side of the detection chip body 1 to seal the second side of the chambers 11. A hydrophobic and breathable membrane 4 is also provided on the second side of the detection chip body 1 to seal the second side of the vent holes 12. The horizontal cross-section of the chambers 11 is crescent-shaped. The hydrophobic and breathable membrane 4 is a multilayer composite material, preferably a non-absorbent material, to prevent the hydrophobic and breathable membrane 4 from absorbing liquid from the vent holes 12 during the amplification reaction, thus preventing the formation of bubbles in the chambers 11 and affecting the detection accuracy.

[0104] Preferably, such as Figure 21 and Figure 23 As shown, chamber 11 is not a regular crescent shape; the length-to-width ratio of a single chamber 11 is between 3:1.4 and 2, and the length-to-thickness ratio is between 3:1 and 1.5. (As shown...) Figure 23In the illustrated embodiment, chamber 11 has a length of 3 mm, a width of 1.7 mm, and a thickness of 1.2 mm. The chamber 11 is designed with a relatively long length to provide sufficient optical detection surface for each chamber, while the thickness needs to be as thin as possible while still meeting the requirements of chip injection molding, thus adapting to instruments with double-sided heating and improving heating efficiency. The width of the chamber is adjusted appropriately based on the designed volume of chamber 11, but preferably has an aspect ratio between 3:1.4 and 2 to ensure sufficient curvature of the arc-shaped sidewalls, which helps reduce residual air bubbles in the liquid reagent, maximizes the focusing effect of the convex lens, and enhances the optical detection signal. The volume of chamber 11 is 5-10 μL (e.g., 5 μL, 6 μL, 8 μL, or 10 μL), but this invention does not limit the specific volume size and can be designed according to requirements.

[0105] The injection port 13, chamber 11, and vent 12 are all through holes, facilitating manufacturing. The through hole in chamber 11, combined with the first sealing membrane 2 and the second sealing membrane 3, facilitates rapid heating and cooling of the sample on both sides within chamber 11. The microfluidic chip also includes a cover plate 6. A slot 17 protrudes from the second side of the detection strip body 1, located in the center of the second side. The cover plate 6 is fastened and sealed to the slot 17 of the detection strip body 1 by a sealing film 7 to close the injection port 13. A protruding locking structure is provided on the edge of the slot 17 to facilitate the positioning and installation of the cover plate 6. The double-fixed assembly, achieved by fastening the cover plate to the slot 17 of the detection strip body 1 and sealing it with the sealing film 7, prevents sample overflow and leakage during the heating and cooling reaction process.

[0106] In practical applications, before sample injection, the detection reagents (primer-probe mixture) are pre-embedded and dried in each chamber 11. After the sample to be tested is injected into the injection hole 13 of the detection strip body 1, the sample flows into each chamber 11. The air in the chamber 11 is discharged through the vent hole 12 to eliminate air bubbles and improve PCR detection results. The first sealing film 2 and the second sealing film 3 are heat-sealed together on the first and second sides of the detection strip body 1 to prevent the reagents or samples from overflowing during thermal reactions. Because the first sealing film 2 is provided on the first side of the vent hole 12 and the hydrophobic and breathable film 4 is provided on the second side of the vent hole 12, the vent hole 12 is breathable but waterproof. After the pathogen sample to be tested is injected into the injection hole 13 of the detection strip body 1, the cover 6 is fastened and sealed on the slot 17 of the detection strip body 1 to close the injection hole 13 and the vent hole 12. Since the cover plate 6 and the test strip body 1 cooperate to seal the injection hole 13, the interior of the chamber 11 of the microfluidic chip is sealed after the sample is injected. During the temperature rise and fall process of the PCR reaction, the sample and reagent will not leak, causing environmental pollution. This ensures that there are no air bubbles in the chamber 11 during injection, and that the sample can fill the entire chamber 11 without leakage.

[0107] Based on multiple experiments, when equal amounts of samples were introduced into circular, elliptical, rectangular, and crescent-shaped chambers of the same capacity under the same experimental environment, the average number of bubbles in the circular chamber was greater than 2, the average number of bubbles in the elliptical chamber was 1.4, the average number of bubbles in the rectangular chamber was 8, and the average number of bubbles in the crescent-shaped chamber was 0.

[0108] When the horizontal cross-section of chamber 11 is crescent-shaped, as liquid flows into chamber 11, the liquid inlet side of chamber 11 gradually expands while the gas outlet side gradually contracts. This facilitates the complete filling of liquid while the gas is completely discharged, preventing residual air from forming bubbles and thus improving the accuracy of subsequent optical detection results. Simultaneously, in conventional optical detection equipment, the width of each chamber 11, perpendicular to the arrangement direction of multiple chambers 11, cannot exceed a specified size. Since the crescent-shaped structure of chamber 11 in this invention maximizes the number of chambers 11 on a detection strip body 1 of the same size, it enables PCR detection of a wider variety of respiratory pathogens. The crescent-shaped structure of chamber 11 includes a concave arc surface and a convex arc surface, with its convex arc surface facing the outer peripheral edge of the detection body 1. This structure promotes fluorescence refraction and aggregation, thereby improving the accuracy of subsequent optical detection results. Furthermore, in this embodiment, since both the upper and lower sides of chamber 11 serve as heating surfaces simultaneously, the heat transfer area is large, resulting in faster and more uniform heat transfer. All chambers 11 are located at the edge of the microfluidic chip, which facilitates the optical detection module located on the outer periphery of the microfluidic chip to utilize the light-gathering effect of the arc-shaped surface of the chamber 11 to enhance the optical signal, thereby facilitating the subsequent acquisition of fluorescence signals by the optical detection module. It should be noted that the structure of the chamber 11 can also be replaced by elliptical, spindle-shaped, or other structures, but the convex arc surface should preferably face the outer peripheral side of the detection pad body 1.

[0109] The shape of the test strip body 1 is designed according to actual needs. The test strip body 1 can be made of transparent material, preferably polypropylene, polycarbonate, polystyrene, or polymethyl methacrylate. Furthermore, because the microfluidic chip in this application pre-encapsulates the test reagents within the chamber 11, a pipette can be used to inject the sample to be tested into the microfluidic chip. The sample enters the chamber 11 through various channels and mixes with the pre-sealed reagents. Only one sample extraction is required, greatly reducing the number of pipetting operations required by the operator.

[0110] Chamber 11 has a crescent-shaped structure, wider in the middle and gradually narrowing at both ends, making the liquid reagent inlet gradually expanding and the gas outlet gradually narrowing. When the liquid enters the chamber, it will expel the gas inside the chamber. If the air is not expelled in time, it can easily form residual bubbles. The gradually expanding and narrowing structure can avoid the formation of right angles, acute angles or other parts that obstruct fluid flow in the chamber, facilitating the entry of liquid into the chamber and the expulsion of air from the chamber.

[0111] like Figure 21-22As shown, a main flow channel 14 and a branch sample inlet flow channel 15 are recessed on the first side of the test strip body 1 extending to the second side of the test strip body 1. That is, the main flow channel 14, the branch sample inlet flow channel 15, and the exhaust flow channel 16 are only perforated on one side of the test strip body 1, facilitating the installation of the hydrophobic and breathable membrane 4 on the subsequent exhaust port 12 on the test strip body 1. The injection port 13 is connected to the main flow channel 14, each chamber 11 is connected to the main flow channel 14 via the branch sample inlet flow channel 15, and the chamber 11 is connected to the exhaust port 12 via the exhaust flow channel 16. In this application, the end of the exhaust flow channel 16 near the exhaust port 12 has a sloping structure, so that the liquid, guided by a certain slope, flows into the exhaust port 12, forming a vortex that discharges the liquid bubbles. It should be noted that the inner walls of the main flow channel 14, the branch sample inlet flow channel 15, and the exhaust flow channel 16 are all smooth transition channels to facilitate sample flow during injection. For example... Figure 23 As shown, chamber 11 has through holes on both sides to facilitate the holding of a large amount of reagents in a small volume and rapid heating from both sides. However, the branch sample inlet channel 15 and exhaust channel 16, which communicate with chamber 11, are not penetrating the detection strip body 1, and a gentle slope transition 110 is provided at the connection with chamber 11. This structure not only facilitates the rapid entry of reagents into chamber 11, reducing turbulence and residual air bubbles in chamber 11, but also saves reagent consumption. The through holes in chamber 11, in conjunction with the first sealing membrane 2 and the second sealing membrane 3, enable dual-sided heating, facilitating rapid heating and cooling of the sample within chamber 11, and avoiding the formation of a temperature gradient inside the liquid in chamber 11 caused by unilateral heating, thereby improving the reaction rate and reaction efficiency.

[0112] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A microfluidic chip for nucleic acid detection of respiratory pathogens, characterized in that, It includes multiple independent chambers, each connected to the injection port and the vent port; the number of chambers is greater than 3; The microfluidic chip includes a detection plate body, which is a sheet-like plate with a first side and a second side. The first side has recessed and formed multiple chamber holes, branch sample inlet channels communicating with a first end of each chamber hole, an exhaust channel communicating with the second end, exhaust holes communicating with each exhaust channel, a main channel communicating with each branch sample inlet channel, and a sample injection port located at the beginning of the main channel. The chamber holes are through-hole structures; the branch sample inlet channels, exhaust channels, and exhaust holes are non-through-hole structures; the opening side of the exhaust hole is covered with a hydrophobic and breathable membrane. The first side of the test piece body is sealed with a first sealing sheet to block the branch sample inlet channel, the exhaust channel, and the first side of the chamber orifice; the second side of the test piece body is sealed with a second sealing sheet to block the second side of the chamber orifice, making the chamber orifice a sealed chamber; the horizontal cross-section of the chamber is crescent-shaped, and the width of each chamber gradually narrows from the middle to both ends; the chamber has a concave arc surface and a convex arc surface; the convex arc surface faces the outer peripheral edge of the test piece body; the length-to-width ratio of the chamber is between 3:1.4 and 2, and the length-to-thickness ratio is between 3:1 and 1.5; At least one chamber in the multiple chambers contains primers and probes for detecting GAPDH upstream and downstream, and at least one chamber contains primers and probes for detecting Bacillus atrophus DNA. The remaining chambers are each independently coated with upstream and downstream primers and probes for detecting one or more of the following pathogen genes: novel coronavirus N gene, novel coronavirus 1ab gene, coronavirus 229E, coronavirus OC43, coronavirus NL63, coronavirus HKU1, adenovirus universal, respiratory syncytial virus A, respiratory syncytial virus B, human metapneumovirus, rhinovirus, influenza A virus, influenza B virus, human parainfluenza virus 1, human parainfluenza virus 2, human parainfluenza virus 3, human parainfluenza virus 4, and mycoplasma pneumoniae.

2. The microfluidic chip for nucleic acid detection of respiratory pathogens according to claim 1, characterized in that, The upstream and downstream primers for detecting GAPDH are shown in SEQ ID NO: 55 and SEQ ID NO: 56, and the probe is shown in SEQ ID NO: 57; the upstream and downstream primers for detecting Bacillus atrophus DNA are shown in SEQ ID NO: 58 and SEQ ID NO: 59, and the probe is shown in SEQ ID NO:

60.

3. A microfluidic chip for nucleic acid detection of respiratory pathogens according to claim 1, characterized in that, The upstream and downstream primers used for detecting influenza A virus are shown in SEQ ID NO:1 and SEQ ID NO:2, and the probe is shown in SEQ ID NO:3; The upstream and downstream primers used for detecting influenza B virus are shown in SEQ ID NO:4 and SEQ ID NO:5, and the probe is shown in SEQ ID NO:6; The upstream and downstream primers for detecting human parainfluenza virus type 1 are shown in SEQ ID NO:7 and SEQ ID NO:8, and the probe is shown in SEQ ID NO:9; The upstream and downstream primers for detecting human parainfluenza virus type 2 are shown in SEQ ID NO:10 and SEQ ID NO:11, and the probe is shown in SEQ ID NO:12; The upstream and downstream primers for detecting human parainfluenza virus type 3 are shown in SEQ ID NO:13 and SEQ ID NO:14, and the probe is shown in SEQ ID NO:15; The upstream and downstream primers for detecting human parainfluenza virus type 4 are shown in SEQ ID NO:16 and SEQ ID NO:17, and the probe is shown in SEQ ID NO:18; The upstream and downstream primers for detecting adenovirus universally are shown in SEQ ID NO:19 and SEQ ID NO:20, and the probe is shown in SEQ ID NO:21; The upstream and downstream primers used to detect coronavirus 229E are shown in SEQ ID NO:22 and SEQ ID NO:23, and the probe is shown in SEQ ID NO:24; The upstream and downstream primers for detecting coronavirus NL63 are shown in SEQ ID NO:25 and SEQ ID NO:26, and the probe is shown in SEQ ID NO:27; The upstream and downstream primers used to detect coronavirus HKU1 are shown in SEQ ID NO:28 and SEQ ID NO:29, and the probe is shown in SEQ ID NO:30; The upstream and downstream primers for coronavirus OC43 are shown in SEQ ID NO:31 and SEQ ID NO:32, and the probe is shown in SEQ ID NO:33; The upstream and downstream primers for detecting respiratory syncytial virus type A are shown in SEQ ID NO:34 and SEQ ID NO:35, and the probe is shown in SEQ ID NO:

36. The upstream and downstream primers for detecting respiratory syncytial virus type B are shown in SEQ ID NO:37 and SEQ ID NO:38, and the probe is shown in SEQ ID NO:

39. The upstream and downstream primers used for detecting Mycoplasma pneumoniae are shown in SEQ ID NO:40 and SEQ ID NO:41, and the probe is shown in SEQ ID NO:42; The upstream and downstream primers used to detect human metapneumovirus are shown in SEQ ID NO:43 and SEQ ID NO:44, and the probe is shown in SEQ ID NO:

45. The upstream and downstream primers used for detecting rhinovirus are shown in SEQ ID NO:46 and SEQ ID NO:47, and the probe is shown in SEQ ID NO:48; The upstream and downstream primers used to detect the N gene of the novel coronavirus are shown in SEQ ID NO:49 and SEQ ID NO:50, and the probe is shown in SEQ ID NO:

51. The upstream and downstream primers used to detect the novel coronavirus 1ab gene are shown in SEQ ID NO:52 and SEQ ID NO:53, and the probe is shown in SEQ ID NO:

54.

4. A microfluidic chip for nucleic acid detection of respiratory pathogens according to claim 1, characterized in that, At least one of the multiple chambers is empty and not coated with any test reagents.

5. A microfluidic chip for nucleic acid detection of respiratory pathogens according to claim 1, characterized in that, During the process of coating primers and probes into each chamber, 1 μL of the primer set and probe mixture is applied to the corresponding chamber using a pipette, dried, and then the chamber is sealed with a membrane; wherein, the total concentration of primer set and probe in the mixture is 0.4-1 μmol / L.

6. A microfluidic chip for nucleic acid detection of respiratory pathogens according to claim 1, characterized in that, The number of chambers is ≥20.

7. A microfluidic chip for nucleic acid detection of respiratory pathogens according to claim 1, characterized in that, The microfluidic chip also includes a third sealing sheet and a cover plate for sealing the vent and injection holes after injection is completed.

8. A microfluidic chip for nucleic acid detection of respiratory pathogens according to claim 1, characterized in that, The volume of the chamber is 5-10 μL.

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