A dual-color coded microsphere, a multiplex respiratory pathogen detection system comprising the same and application

Abstract

This invention discloses a dual-color encoded microsphere, a multiplex respiratory pathogen detection system containing the microsphere, and its applications, belonging to the field of gene detection technology. This invention utilizes chemically modified groups on the surface of the microspheres to covalently couple with functional groups on mTagBFP and single-stranded DNA (ssDNA) strands, thereby achieving dual-color encoding of the microspheres. Furthermore, these dual-color encoded microspheres possess multi-channel discrimination characteristics; by encoding the microspheres with different fluorescence, multiple respiratory pathogen targets can be simultaneously detected in a single reaction system. This meets the high-throughput clinical demand for combined screening of multiple respiratory pathogens. Simultaneously, the simultaneous detection of multiple targets significantly reduces the amount of valuable clinical samples required, which is particularly important when sample volume is limited. This not only reduces the sampling burden on patients but also improves sample utilization efficiency.

Description

Technical Field

[0001] This invention belongs to the field of gene detection technology, specifically relating to a dual-color encoded microsphere, a multiplex respiratory pathogen detection system containing the microsphere, and its applications. Background Technology

[0002] Respiratory diseases pose a serious threat to human health globally, being a major contributor to high morbidity and mortality rates. The COVID-19 pandemic, caused by SARS-CoV-2, has further highlighted the urgent need for rapid and accurate detection of respiratory pathogens to enable timely clinical intervention and effective infection control. A core challenge in the diagnosis and management of respiratory infections lies in the complexity of their etiology. Pathogens causing respiratory infections (RTIs) include not only viruses (such as influenza A / B viruses, respiratory syncytial virus (RSV), and SARS-CoV-2) but also bacteria (such as Mycoplasma pneumoniae and Haemophilus influenzae type b). Furthermore, co-infections (involving two or more pathogens) occur in 19.7–34% of clinical cases. Delays or misdiagnosis of pathogens often lead to unnecessary antibiotic use. Approximately 37% of children with respiratory tract infections (RTIs) receive unnecessary antibiotic treatment, which impairs infection control and accelerates nosocomial transmission of respiratory pathogens and viral outbreaks.

[0003] Currently, the clinical diagnosis of respiratory diseases faces numerous serious challenges. First, the clinical symptoms caused by different pathogens are highly similar: common symptoms such as cough, fever, and dyspnea are prevalent, making it difficult to distinguish specific pathogens based solely on clinical manifestations. Second, co-infections are extremely common in respiratory diseases, with approximately 30-50% of hospitalized patients simultaneously infected with two or more pathogens. The heterogeneity of respiratory infections and frequent mixed infections have become a major clinical challenge. Traditional single-pathogen detection methods are prone to missing minor pathogens, thus affecting accurate diagnosis and effective treatment. Third, for highly infectious respiratory pathogens, clinical practice demands extremely high timeliness in detection. Traditional respiratory pathogen detection methods are insufficient in addressing these challenges. Furthermore, they are limited by factors such as sample quality and culture conditions, resulting in low sensitivity, which seriously delays clinical decision-making. While immunochromatography is rapid, it lacks sensitivity for low-titer infections and cannot distinguish closely related viruses, leading to a high false-negative rate. Single-particle polymerase chain reaction (PCR) improves sensitivity but cannot achieve multiplex detection. It requires setting up separate reaction systems for each target, making screening for 8-20 common pathogens associated with respiratory infections impractical. While antigen detection methods are rapid, their sensitivity is only 60-80% of PCR, making them prone to false negatives and missed diagnoses. Therefore, developing a multi-pathogen simultaneous detection technology that meets the requirements of high throughput, high sensitivity, and rapid detection has become a critical issue urgently needing to be addressed in clinical diagnosis and epidemiological surveillance.

[0004] Multiplex detection technologies for respiratory pathogens, which identify viral and bacterial pathogens through nucleic acid amplification or novel detection combinations, have demonstrated significant advantages in rapid and highly sensitive diagnosis. They have become promising solutions to address the dual challenges of rapid pathogen identification and comprehensive respiratory disease surveillance. Multiplex PCR-based pathogen detection offers significant advantages in rapid screening for respiratory infections, achieving high multiplex detection capabilities in a single reaction while exhibiting low reagent consumption, extremely low contamination risk, and low cost. Furthermore, it eliminates the need for fluorophores of different wavelengths or TaqMan probes (which may inhibit PCR amplification), thus avoiding problems such as optical congestion, signal suppression due to primer dimers, and preferential amplification of short amplicones. Given that multiple respiratory pathogen infections can easily lead to treatment failure and epidemic spread, there is an urgent need for a cost-effective and efficient multiplex PCR coupled suspension array technology capable of simultaneously identifying multiple respiratory pathogens, thereby enabling precision medicine and real-time infection control. Summary of the Invention

[0005] The purpose of this invention is to provide a dual-color coded microsphere, a multiplex respiratory pathogen detection system containing the microsphere, and its application. This addresses the problems of existing multiplex respiratory pathogen detection methods, such as high component density, low sensitivity, and small single-pass throughput.

[0006] In a first aspect, the present invention provides a dual-color encoded microsphere, comprising a microsphere and mTagBFP and fluorescently labeled anti-Tag ssDNA covalently coupled to the surface of the microsphere; wherein the fluorescently labeled anti-Tag ssDNA is used to capture a tag-labeled target to be detected.

[0007] In this invention, the inventors discovered that by covalently coupling chemical groups modified on the surface of microspheres with functional groups on mTagBFP and single-stranded DNA (ssDNA), dual-color encoding of the microspheres can be achieved. This design simplifies the overall microsphere encoding process, requiring only adjustment of the concentrations of mTagBFP and ssDNA, effectively avoiding the impact of complex operations on the microspheres and ensuring the uniformity of microsphere size. The encoding process is simple, reproducible, low-cost, has good detection compatibility, and fast signal acquisition, enabling mass production. Furthermore, these dual-color encoded microspheres possess multi-channel discrimination characteristics. By encoding the microspheres with different fluorescence, multiple respiratory pathogen targets can be simultaneously detected in a single reaction system. This meets the high-throughput clinical demand for combined screening of multiple respiratory pathogens. Simultaneously, the simultaneous detection of multiple targets significantly reduces the amount of valuable clinical samples used, which is particularly important when sample volume is limited. This not only reduces the sampling burden on patients but also improves sample utilization efficiency.

[0008] In some implementations, the targets to be detected include respiratory pathogens, including respiratory pathogen Flu A, respiratory pathogen Flu B, respiratory pathogen RSV, respiratory pathogen SARS-CoV-2, and other respiratory pathogens. M. pneumoniae Used to capture tagged respiratory pathogens Flu A, Flu B, RSV, SARS-CoV-2, and others. M. pneumoniae The sequences of the anti-Tag ssDNA are shown in SEQ ID NO.16-20, respectively; the fluorescent label in the fluorescently labeled anti-Tag ssDNA includes Cy5.

[0009] In some embodiments, the microspheres include at least one of polystyrene microspheres, silica microspheres, agarose gel microspheres, dextran gel microspheres, magnetite magnetic microspheres, and ferric oxide microspheres, and the particle size of the microspheres is 1.0-30.0 μm.

[0010] In some embodiments, the chemical groups modified on the surface of the microspheres include at least one of carboxyl, amino, maleimide, haloacetamide, gold atom, alkynyl, and azido; the groups on mTagBFP include at least one of amino, carboxyl, mercapto, aldehyde, azido, and alkynyl; the chemical groups modified on the fluorescently labeled anti-Tag ssDNA molecule include at least one of amino, carboxyl, mercapto, thiol, aldehyde, epoxy, azido, alkynyl, hydroxyl, and cyano; the chemical groups modified on the surface of the microspheres are covalently coupled to the groups on mTagBFP and the chemical groups modified on the fluorescently labeled anti-Tag ssDNA molecule, respectively.

[0011] In a second aspect, the present invention provides a multiplex respiratory pathogen detection system, comprising the aforementioned dual-color coded microspheres.

[0012] In some implementations, the multiplex respiratory pathogen detection system further includes at least one of the following: A1) a primer set for amplifying a target to be detected that is doubly modified with a tag and biotin; A2) phycoerythrin, including streptavidin-modified phycoerythrin; and A3) a detection component, including at least one of a flow cytometer and a CCD imaging system.

[0013] In some implementations, A1), the upstream primers in the primer set all have the structure F1-F2-F3, and the downstream primers of the primer set include an R sequence complementary to the specific gene sequence of the target to be detected, and are labeled with biotin at the 5′ end; wherein, the nucleotide sequence of F1 is inversely complementary to the nucleotide sequence of the fluorescently labeled anti-Tag ssDNA; the nucleotide sequence of F3 is complementary to the specific gene sequence of the target to be detected; F2 includes a spacer arm, which includes at least one of iSpC3, iSpC6, iSpC12, poly-dT, poly-TTG, and oligotetraethylene glycol; in the primers for detecting the respiratory pathogen Flu A, the F1 sequence in the upstream primer is as shown in SEQ ID NO.11, the F3 sequence is as shown in SEQ ID NO.1, and the R sequence in the downstream primer is as shown in SEQ ID NO.2; in the primers for detecting the respiratory pathogen Flu B, the F1 sequence in the upstream primer is as shown in SEQ ID NO.12, the F3 sequence is as shown in SEQ ID NO.3, and the R sequence in the downstream primer is as shown in SEQ ID NO.12. As shown in NO.4; in primers for detecting the respiratory pathogen RSV, the F1 sequence of the upstream primer is shown in SEQ ID NO.13, and the F3 sequence is shown in SEQ ID NO.5; the R sequence of the downstream primer is shown in SEQ ID NO.6; in primers for detecting the respiratory pathogen SARS-CoV-2, the F1 sequence of the upstream primer is shown in SEQ ID NO.14, and the F3 sequence is shown in SEQ ID NO.7; the R sequence of the downstream primer is shown in SEQ ID NO.8; for detecting respiratory pathogens... M. pneumoniae In the primers, the F1 sequence of the upstream primer is shown in SEQ ID NO.15, and the F3 sequence is shown in SEQ ID NO.9; the R sequence of the downstream primer is shown in SEQ ID NO.10.

[0014] In a third aspect, the present invention provides the application of any of the above-described dual-color coded microspheres and any of the above-described multiplex respiratory pathogen detection systems in the detection of multiplex respiratory pathogens.

[0015] In a fourth aspect, the present invention provides a method for detecting multiple respiratory pathogens using any of the above-described multiplex respiratory pathogen detection systems, comprising the following steps: Chemical groups on the surface of microspheres were activated, and then mTagBFP solution and fluorescently labeled anti-Tag ssDNA molecular solution were added. After mixing and incubation, dual-color encoded microspheres were obtained. Multiplex PCR amplification of the target in the sample was performed using primer sets to obtain PCR amplification products with dual Tag and biotin modification. The PCR amplification products with dual Tag and biotin modification were mixed with dual-color encoded microspheres, and the amplification products were bound to the anti-Tag ssDNA on the surface of the dual-color encoded microspheres through complementary base pairing. Phycoerythrin modified with streptavidin was added, and after reaction, phycoerythrin bound to the surface of the dual-color encoded microspheres. After washing the dual-color encoded microspheres by applying an external magnetic field, the fluorescence signal on the surface of the dual-color encoded microspheres was detected by a detection component to achieve qualitative and quantitative analysis of the target.

[0016] In some embodiments, in the steps of activating the chemical groups on the surface of the microspheres and then adding the mTagBFP solution and the fluorescently labeled anti-Tag ssDNA molecule solution, the concentration of the mTagBFP solution is 0-1000 μg / mL and the concentration of the fluorescently labeled anti-Tag ssDNA molecule solution is 0-1000 nM.

[0017] The beneficial effects of this invention are as follows: Unlike existing technologies, this invention utilizes chemically modified groups on the surface of microspheres to covalently couple with functional groups on mTagBFP and single-stranded DNA (ssDNA) strands, thereby achieving dual-color encoding of the microspheres. This design simplifies the overall microsphere encoding process, requiring only adjustment of the concentrations of mTagBFP and ssDNA, effectively avoiding the impact of complex operations on the microspheres, thus ensuring the uniformity of microsphere particle size. The encoding process is simple, reproducible, has low decoding costs, good detection compatibility, and fast signal acquisition speed, enabling batch preparation. Furthermore, this dual-color encoded microsphere has multi-channel discrimination characteristics; by encoding the microspheres with different fluorescence, multiple respiratory pathogen targets can be simultaneously detected in a single reaction system. This meets the high-throughput clinical demand for combined screening of multiple respiratory pathogens. Simultaneously, the simultaneous detection of multiple targets significantly reduces the amount of valuable clinical samples used, which is particularly important when sample volume is limited, reducing the sampling burden on patients and improving sample utilization efficiency. Attached Figure Description

[0018] Figure 1 This is a graph showing the forward scattering angle (FSC) and side scattering angle (SSC) signals of the substrate microspheres in Example 1 of the present invention after being processed by a flow cytometer. Figure 2Figure 1 shows a fluorescence microscope image of the dual-color encoded microspheres prepared in Example 1 of the present invention. Figure 1(a) is a confocal microscope image of mTagBFP on the dual-color encoded microspheres; Figure 2(b) is a confocal microscope image of fluorescently labeled anti-Tag ssDNA molecules on the dual-color encoded microspheres. Figure 3 This is a matrix diagram decoded by flow cytometry of the dual-color encoded microspheres prepared in Example 1 of the present invention; Figure 4 This is a gel electrophoresis image of the PCR products of five positive respiratory pathogens in Example 2 of the present invention; Figure 5 Figure 3 shows the detection results after amplification of the five targets in Example 3 of the present invention; Figure (a) shows the five suspension arrays encoded by mTagBFP and anti-Tag-Cy5 ssDNA; Figure (b) shows the detection results of the five targets. Detailed Implementation

[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] Experimental methods not specified in the examples are generally performed under conventional conditions and as described in the manual, or as recommended by the manufacturer. Unless otherwise specified, the general equipment, materials, reagents, etc. used are commercially available.

[0021] Currently, existing multiplex respiratory pathogen detection methods suffer from problems such as high component density, low sensitivity, and small single-sample processing capacity.

[0022] To address the problems of high component content, low sensitivity, and small single-processing capacity in existing multiplex respiratory pathogen detection methods, this invention provides a dual-color coded microsphere, a multiplex respiratory pathogen detection system containing the microsphere, and its application.

[0023] In a first aspect, the present invention provides a dual-color encoded microsphere, comprising a microsphere and mTagBFP and fluorescently labeled anti-Tag ssDNA covalently coupled to the surface of the microsphere; wherein the fluorescently labeled anti-Tag ssDNA is used to capture a tag-labeled target to be detected.

[0024] The dual-color encoded microspheres provided by this invention utilize chemically modified groups on the microsphere surface to covalently couple with functional groups on mTagBFP and single-stranded DNA (ssDNA) strands, thereby achieving dual-color encoding of the microspheres. This design simplifies the overall microsphere encoding process, requiring only adjustment of the concentrations of mTagBFP and ssDNA, effectively avoiding the impact of complex operations on the microspheres, thus ensuring the uniformity of microsphere particle size. The encoding process is simple, reproducible, has low decoding cost, good detection compatibility, and fast signal acquisition speed, enabling batch preparation. Furthermore, these dual-color encoded microspheres possess multi-channel discrimination characteristics; by encoding the microspheres with different fluorescence, multiple respiratory pathogen targets can be simultaneously detected in a single reaction system. This meets the high-throughput clinical demand for combined screening of multiple respiratory pathogens. Simultaneously, the simultaneous detection of multiple targets significantly reduces the amount of valuable clinical samples used, which is particularly important when sample volume is limited, reducing the sampling burden on patients and improving sample utilization efficiency.

[0025] In some implementations, the targets to be detected include respiratory pathogens, including respiratory pathogen Flu A, respiratory pathogen Flu B, respiratory pathogen RSV, respiratory pathogen SARS-CoV-2, and other respiratory pathogens. M. pneumoniae Used to capture tagged respiratory pathogens Flu A, Flu B, RSV, SARS-CoV-2, and others. M. pneumoniae The sequences of the anti-Tag ssDNA are shown in SEQ ID NO.16-20, respectively; the fluorescent label in the fluorescently labeled anti-Tag ssDNA includes Cy5.

[0026] It is understood that the types of targets to be detected can be conventionally selected according to actual needs, such as respiratory pathogens. Furthermore, the types of respiratory pathogens can be conventionally selected according to actual needs, as long as a corresponding fluorescently labeled anti-Tag ssDNA sequence is designed. For example, in this invention, respiratory pathogens preferably include respiratory pathogen Flu A, respiratory pathogen Flu B, respiratory pathogen RSV, respiratory pathogen SARS-CoV-2, and other respiratory pathogens. M. pneumoniae .

[0027] In some embodiments, the microspheres include at least one of polystyrene microspheres, silica microspheres, agarose gel microspheres, dextran gel microspheres, magnetite magnetic microspheres, and ferric oxide microspheres, preferably magnetite magnetic microspheres; and the particle size of the microspheres is 1.0-30.0 μm, preferably 10 μm.

[0028] It is understood that the type and particle size of the microspheres can be conventionally selected according to actual application needs, as long as they can be used for suspension array detection. For example, in this invention, the microspheres preferably include at least one of polystyrene microspheres, silica microspheres, agarose gel microspheres, dextran gel microspheres, magnetite magnetic microspheres, and ferric oxide microspheres, and the particle size of the microspheres is preferably 1.0-30.0 μm.

[0029] In some embodiments, the chemical groups modified on the surface of the microspheres include at least one of carboxyl, amino, maleimide, haloacetamide, gold atom, alkynyl, and azido, preferably carboxyl; the groups on mTagBFP include at least one of amino, carboxyl, mercapto, aldehyde, azido, and alkynyl, preferably amino; the chemical groups modified on the fluorescently labeled anti-Tag ssDNA molecule include at least one of amino, carboxyl, mercapto, thiol, aldehyde, epoxy, azido, alkynyl, hydroxyl, and cyano, preferably amino; the chemical groups modified on the surface of the microspheres are covalently coupled to the groups on mTagBFP and the chemical groups modified on the fluorescently labeled anti-Tag ssDNA molecule, respectively.

[0030] In this invention, chemical groups modified on the surface of microspheres are covalently coupled with groups on mTagBFP and chemical groups modified on fluorescently labeled anti-Tag ssDNA molecules, respectively, to achieve dual-color encoding of microspheres, which facilitates efficient subsequent detection.

[0031] In a second aspect, the present invention provides a multiplex respiratory pathogen detection system, comprising the aforementioned dual-color coded microspheres.

[0032] In some implementations, the multiplex respiratory pathogen detection system further includes at least one of the following: A1) a primer set for amplifying a target to be detected that is doubly modified with a tag and biotin; A2) phycoerythrin, including streptavidin-modified phycoerythrin; and A3) a detection component, including at least one of a flow cytometer and a CCD imaging system.

[0033] In this invention, the above-mentioned multiplex respiratory pathogen detection system can perform efficient and large-scale detection of the target and has the advantage of high sensitivity.

[0034] In some implementations, A1), the upstream primers in the primer set all have the structure F1-F2-F3, and the downstream primers of the primer set include an R sequence complementary to the specific gene sequence of the target to be detected, and are labeled with biotin at the 5′ end; wherein, the nucleotide sequence of F1 is inversely complementary to the nucleotide sequence of the fluorescently labeled anti-Tag ssDNA; the nucleotide sequence of F3 is complementary to the specific gene sequence of the target to be detected; F2 includes a spacer arm, which includes at least one of iSpC3, iSpC6, iSpC12, poly-dT, poly-TTG, and oligotetraethylene glycol; in the primers for detecting the respiratory pathogen Flu A, the F1 sequence in the upstream primer is as shown in SEQ ID NO.11, the F3 sequence is as shown in SEQ ID NO.1, and the R sequence in the downstream primer is as shown in SEQ ID NO.2; in the primers for detecting the respiratory pathogen Flu B, the F1 sequence in the upstream primer is as shown in SEQ ID NO.12, the F3 sequence is as shown in SEQ ID NO.3, and the R sequence in the downstream primer is as shown in SEQ ID NO.12. As shown in NO.4; in primers for detecting the respiratory pathogen RSV, the F1 sequence of the upstream primer is shown in SEQ ID NO.13, and the F3 sequence is shown in SEQ ID NO.5; the R sequence of the downstream primer is shown in SEQ ID NO.6; in primers for detecting the respiratory pathogen SARS-CoV-2, the F1 sequence of the upstream primer is shown in SEQ ID NO.14, and the F3 sequence is shown in SEQ ID NO.7; the R sequence of the downstream primer is shown in SEQ ID NO.8; for detecting respiratory pathogens... M. pneumoniae In the primers, the F1 sequence of the upstream primer is shown in SEQ ID NO.15, and the F3 sequence is shown in SEQ ID NO.9; the R sequence of the downstream primer is shown in SEQ ID NO.10.

[0035] In this invention, when using primer sets for multiplex PCR to simultaneously detect five respiratory pathogens, there is no cross-reactivity between the five primer pairs, resulting in high specificity and sensitivity. It has the advantages of high throughput, high speed, low cost, high sensitivity, good specificity, good repeatability, and a wide linear range, and has great potential for application in the detection of respiratory pathogens. Furthermore, after PCR amplification, there is no need to process the PCR products, which can be directly hybridized with microspheres, saving time and simplifying the process.

[0036] In a third aspect, the present invention provides the application of any of the above-described dual-color coded microspheres and any of the above-described multiplex respiratory pathogen detection systems in the detection of multiplex respiratory pathogens.

[0037] In a fourth aspect, the present invention provides a method for detecting multiple respiratory pathogens using any of the above-mentioned multiplex respiratory pathogen detection systems, comprising the following steps: activating chemical groups on the surface of microspheres, then adding mTagBFP solution and fluorescently labeled anti-Tag ssDNA molecular solution, mixing and incubating to obtain dual-color encoded microspheres; performing multiplex PCR amplification of the target in the sample to be detected using primer sets to obtain PCR amplification products doubly modified with Tag and biotin; mixing the PCR amplification products doubly modified with Tag and biotin with dual-color encoded microspheres, and binding the amplification products to the anti-Tag ssDNA on the surface of the dual-color encoded microspheres through complementary hybridization of base pairing; adding streptavidin-modified phycoerythrin, and after reaction, binding phycoerythrin to the surface of the dual-color encoded microspheres; then separating and washing the dual-color encoded microspheres by an external magnetic field, and detecting the fluorescence signal on the surface of the dual-color encoded microspheres using a detection component to achieve qualitative and quantitative analysis of the target to be detected.

[0038] In this invention, multiple pairs of specific primers for respiratory pathogens with tags are designed. Anti-tags immobilized on a suspension array capture the tag-labeled products after multiplex PCR amplification, achieving accurate detection. This allows for the simultaneous detection of five respiratory pathogens (Flu A, Flu B, RSV, SARS-CoV-2, ...) in the same reaction system. M. pneumoniae It has good specificity and high sensitivity, and has the advantages of high throughput, high speed, good repeatability and wide linear range. After PCR amplification, no special treatment is required for the PCR product, which can be directly hybridized with microspheres, reducing reagent consumption, lowering costs and shortening detection time.

[0039] In some embodiments, in the steps of activating the chemical groups on the surface of the microspheres and then adding the mTagBFP solution and the fluorescently labeled anti-Tag ssDNA molecule solution, the concentration of the mTagBFP solution is 0-1000 μg / mL and the concentration of the fluorescently labeled anti-Tag ssDNA molecule solution is 0-1000 nM.

[0040] In this invention, by controlling the concentrations of mTagBFP solution and fluorescently labeled anti-Tag ssDNA molecule solution within a specific range, high-performance dual-color encoded microspheres can be obtained.

[0041] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0042] In this invention, mTagBFP can be purchased directly from commercial manufacturers, such as Evrogen (Russia, website: https: / / evrogen.com / products / TagBFP / TagBFP_Detailed_description.shtml). Alternatively, it can be expressed and purified in our laboratory, as follows: The bacterial culture of mTagBFP positive clones was inoculated into LB medium containing 50 μg / mL kanamycin at 37°C for 12 h, and then inoculated into fresh LB medium (50 μg / mL kanamycin) at a 1% inoculation rate and cultured until OD600 = 0.5-0.6. 0.25 mmol / L IPTG was added, and the culture was incubated at 16°C for 22 h. Bacteria were collected (10000 rpm, 10 min). Cell disruption was performed using a high-pressure cell homogenizer, and crude protein was extracted by centrifugation (11000 rpm, 30 min). mTagBFP protein was further purified by nickel affinity chromatography. All proteins were frozen in PBS buffer (10 mmol / L, pH 7.4) at -80°C for subsequent experiments. The target protein containing the His tag in the supernatant was further purified by Ni affinity chromatography. The entire experiment was performed at low temperature on ice. The specific steps of the gravity-based protein purification method are as follows: 1) Take 2-4 mL of the mixed Ni-NTA gel purification resin and pack it into a nickel column.

[0043] 2) Use 5-10 column volumes of Bingding Buffer to thoroughly mix the buffer and resin to equilibrate the column.

[0044] 3) Mix the protein sample with Binding Buffer at a 1:1 ratio to prepare the sample solution, so that the total volume of the sample solution is twice the column volume.

[0045] 4) Add the sample solution to the column, maintain a flow rate of approximately 1 mL / min, and collect the effluent into a centrifuge tube. The effluent sample can be loaded again and allowed to pass through once more to improve the binding force between the protein sample and the packing material.

[0046] 5) Wash the column with twice the column volume of Binding / Wash Buffer and collect the flowthrough. Repeat this step using a new collection tube until the absorbance of the flowthrough at 280 nm is close to the baseline.

[0047] 6) Elute the impurities with Native Washing Buffer containing imidazole at concentrations of 25, 50 and 75 mmol / L, from low to high concentration, and collect the eluent.

[0048] 7) Elute the target protein with Native Washing Buffer containing 150 and 250 mmol / L imidazole and collect the eluent.

[0049] 8) Elute all proteins in the column with high-concentration imidazole buffer, then add 20% ethanol protection solution to preserve the column after balancing the column with native binding buffer and water.

[0050] 9) Using an ultrafiltration tube with a molecular weight cutoff of 10 kDa, the purified mTagBFP was ultrafiltered with PBS buffer to remove imidazole and salt ions. After ultrafiltration, the mTagBFP solution was concentrated to a certain volume. After adding sterile glycerol, it was aliquoted and stored at -20°C for later use.

[0051] 10) The obtained mTagBFP was analyzed by SDS-PAGE and fluorescence spectroscopy.

[0052] Example 1: Preparation of mTagBFP and fluorescently labeled anti-Tag ssDNA molecularly encoded microspheres In this embodiment, the aim is to prepare mTagBFP and fluorescently labeled anti-Tag ssDNA molecularly encoded microspheres (two-color encoded microspheres). Specifically, the process includes the following steps: 1. Activate the functional groups on the surface of magnetic beads Commercially available carboxylated magnetic beads (Fe3O4-polymer microspheres, 10.0 μm in diameter) were used as substrate microspheres for encoding microspheres. Figure 1 The image shows the forward scattering angle (FSC) and side scattering angle (SSC) signals of a 10.0 μm diameter magnetic bead analyzed by flow cytometry. The surface carboxyl groups of the bead were activated through the following steps: 1) First, mix the carboxyl magnetic beads thoroughly, then take 1×10 10 Add microspheres to an EP tube, add 200 μL of 100 mM sodium 2-(N-morpholine) ethanesulfonate (MEST, pH 5.0) solution to resuspend the magnetic beads, place the EP tube on a magnetic separator and let it stand for 2 min. After the magnetic beads are completely adsorbed, remove the supernatant and wash 3 times to obtain the treated magnetic beads.

[0053] 2) Quickly add 200 μL of freshly prepared 10 mg / mL 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide (EDC / NHS, MEST as solvent) to the treated magnetic beads, and vortex mix to fully suspend and disperse the magnetic beads to obtain activated magnetic beads for later use.

[0054] 2. mTagBFP and fluorescently labeled anti-Tag ssDNA molecules are loaded onto the surface of magnetic beads. 1) Concentration gradient configuration of mTagBFP: The mTagBFP used (whose ε-amino group on the lysine residue side chain can directly participate in the EDC / NHS coupling reaction) was expressed and purified in our laboratory; Concentration gradient configuration of anti-Tag ssDNA: Five anti-Tag ssDNAs (synthesized by Shanghai Sangon Biotech) were used, which were respectively used to amplify five respiratory pathogens (Flu A, Flu B, RSV, SARS-CoV-2, and...) M. pneumoniae The F1 sequences of the upstream primers are complementary, corresponding to SEQ ID NO. 16-20 respectively, and are all labeled with the fluorescent dye Cy5 (i.e., anti-Tag-Cy5 ssDNA) and modified with amino groups. The specific sequences are as follows: The anti-Tag ssDNA sequence complementary to the F1 sequence in the upstream primer of the respiratory pathogen Flu A is shown in SEQ ID NO.16: 5′-ATTGATATTTGAATGTTTGTTTG-3′; The anti-Tag ssDNA sequence complementary to the F1 sequence in the upstream primer of the respiratory pathogen Flu B is shown in SEQ ID NO.17: 5′-AAATTAGTTGAAAGTATGAGAAAG-3′; The anti-Tag ssDNA sequence complementary to the F1 sequence in the upstream primer of the respiratory pathogen Flu RSV is shown in SEQ ID NO.18: 5′-GTTGTAAATTGTAGTAAAGAAGTA-3′; The anti-Tag ssDNA sequence complementary to the F1 sequence in the upstream primer of the respiratory pathogen Flu SARS-CoV-2 is shown in SEQ ID NO.19: 5′-ATTAAGTAAGAATTGAGAGTTTGA-3′; The anti-Tag ssDNA sequence complementary to the F1 sequence in the upstream primer of the respiratory pathogen M. pneumoniae is shown in SEQ ID NO.20: 5′-GTATGTTGTAATGTATTAAGAAAG-3′.

[0055] The mTagBFP was diluted with PBS buffer to four concentrations: 0, 1.5, 4.5, and 25 μg / mL (denoted as L1-L4); the fluorescently labeled anti-Tag ssDNA was diluted to seven concentrations: 0, 1.5, 5, 12, 25, 100, and 250 nM (denoted as R1-R7). Then, the mTagBFP and fluorescently labeled anti-Tag ssDNA were paired according to the concentration gradient (LxRy) to construct a two-color encoded microsphere matrix containing 28 coding units.

[0056] 2) Coupling reaction of mTagBFP and fluorescently labeled ssDNA with activated magnetic beads: Take the activated magnetic beads, aliquot them, and add 200 μL of mTagBFP and fluorescently labeled anti-Tag ssDNA solutions of corresponding concentration gradients, and mix gently; place the mixture in a constant temperature shaker at 25℃ and 140 rpm for 5 h. The amino groups labeled by the mTagBFP and anti-Tag ssDNA molecular chains will undergo a coupling reaction with the carboxyl groups modified on the surface of the magnetic beads, thereby assembling mTagBFP and fluorescently labeled anti-Tag ssDNA molecules onto the surface of the magnetic beads.

[0057] 3) Washing and purification of the coupling products: After the reaction, the EP tube was placed on a magnetic separator and allowed to stand for 2 min. The supernatant was aspirated, and 500 μL of phosphate buffer (0.01 M PBS, pH 7.4) was added to fully suspend the magnetic beads. The reaction tube was then placed on a magnetic separator and allowed to stand for 2 min. The supernatant was aspirated, and the tube was washed three times with PBS to obtain mTagBFP and fluorescently labeled anti-Tag ssDNA molecularly encoded microspheres. See [link to relevant documentation]. Figure 2 The amount of magnetic beads used for each coding group (concentration group) is approximately 3.5 × 10⁻⁶. 4 indivual.

[0058] 3. Flow cytometry analysis and decoding verification of coded microspheres Flow cytometry was used to analyze and decode the mTagBFP and fluorescently labeled anti-Tag ssDNA molecularly encoded microspheres. In the mTagBFP and fluorescently labeled anti-Tag ssDNA molecularly encoded microspheres, the two-color microspheres encoded by mTagBFP and anti-Tag-Cy5ssDNA were detected in the FL8-PB and FL5-APC channels of the flow cytometer and could be divided into 28 suspension arrays (see...). Figure 3 This enables the coding differentiation of different target detections.

[0059] Example 2 Preparation of PCR amplification products dually modified with Tag and biotin In this embodiment, the aim is to prepare a PCR amplification product doubly modified with both Tag and biotin, captured by a fluorescently labeled anti-Tag ssDNA molecule on the dual-color encoded microspheres of Example 1. Specifically, the steps include: 1. Primer screening and design for detecting multiple respiratory pathogens Primer screening for the detection of multiple respiratory pathogens was performed, selecting RSV (Gene Bank number: MK733768.1) from GenBank. M. pneumoniaeUsing the following reference sequences (Gene Bank number: PV037333.1), Flu A (Gene Bank number: CY085004.1), SARS-CoV-2 (Gene Bank number: ON594991.1), and Flu B (Gene Bank number: MT056628.1), sequence homology comparisons were performed to select conserved regions. Candidate primers were designed using PrimerPlex2 multiplex primer design software, and after extensive optimization of reaction conditions, comparative experiments, and validation experiments, a liquid chip primer set for multiple respiratory pathogen detection was obtained. The primer set was synthesized by Sangon Biotech (Shanghai) Co., Ltd. The upstream primers of the primer set all have the structure F1-F2-F3, where the F1 sequence is identical to the anti-Tag sequence on the dual-color encoded microspheres prepared in Example 1. The ssDNA sequence is reverse complementary, the F3 sequence is complementary to the specific gene sequence of the respiratory pathogen to be tested, and F2 is the iSpC12 spacer arm; the downstream primers of the primer set include an R sequence complementary to the specific gene sequence of the respiratory pathogen to be tested, and biotin is labeled at the 5′ end.

[0060] Among them, the primers for detecting the respiratory pathogen Flu A have the following sequences: F1 sequence in the upstream primer as shown in SEQ ID NO.11: 5′-CAAACAAACATTCAAATATCAAT-3′, F3 sequence as shown in SEQ ID NO.1: 5′-GGAATGGTGACCCGAACAACA-3′; and R sequence in the downstream primer as shown in SEQ ID NO.2: 5′-CACCTCAGTGGTGACAGTCC-3′.

[0061] In the primers for detecting the respiratory pathogen Flu B, the F1 sequence of the upstream primer is shown in SEQ ID NO.12: 5′-CTTTCTCATACTTTCAACTAATTT-3′, and the F3 sequence is shown in SEQ ID NO.3: 5′-CCGGTCAAGACCGCCTAAACA-3′; the R sequence of the downstream primer is shown in SEQ ID NO.4: 5′-TGAGGAACTGTTCATACAGT-3′.

[0062] In the primers for detecting the respiratory pathogen RSV, the F1 sequence of the upstream primer is shown in SEQ ID NO.13: 5′-TACTTCTTTACTACAATTTACAAC-3′, and the F3 sequence is shown in SEQ ID NO.5: 5′-GACAGAAGTTGATCTTTGTT-3′; the R sequence of the downstream primer is shown in SEQ ID NO.6: 5′-AATACAACCATGGCTCTTAGC-3′.

[0063] The primers for detecting the respiratory pathogen SARS-CoV-2 have the following sequences: F1 in the upstream primer (SEQ ID NO.14): 5′-TCAAACTCTCAATTCTTACTTAAT-3′, F3 in the upstream primer (SEQ ID NO.7): 5′-CAATCCCTTTGAGTGCGTGAC-3′; and R in the downstream primer (SEQ ID NO.8): 5′-GGCTTTGTGTGCTGACTCTATC-3′.

[0064] Detection of respiratory pathogens M. pneumoniae In the primers, the F1 sequence of the upstream primer is shown in SEQ ID NO.15: 5′-CTTTCTTAATACATTACAACATAC-3′, and the F3 sequence is shown in SEQ ID NO.9: 5′-ACACCTCCTCCACCAACAACCT-3′; the R sequence of the downstream primer is shown in SEQ ID NO.10: 5′-AGATCGGTGACTGGGTGGGT-3′.

[0065] The specific primer sequences and amplified fragment lengths are shown in Table 1 below.

[0066] Table 1 Primer sequences and amplified fragment lengths

[0067] Note: In the table, lowercase letters represent F1 sequences, iSpC12 represents spacer arms, uppercase letters connected to iSpC12 represent F3 sequences, Biotin represents biotin, and uppercase letters connected to Biotin represent R sequences.

[0068] 2. Primer pretreatment and PCR amplification Based on their respective molar amounts, the five primer pairs were dissolved in double-distilled water to prepare 100 μM stock solutions for later use. Using these five primer pairs, five target genes (Flu A, Flu B, RSV, SARS-CoV-2, and...) were synthesized. M. pneumoniae Using DNA as a template, PCR amplification was performed separately, followed by 2% agarose gel electrophoresis. The results are shown in the figure. Figure 4Lane M contains the DL2000 DNA marker produced by Wuhan Qingke Biotechnology Co., Ltd.; lanes 1-5 correspond to RSV, Flu A, Flu B, SARS-CoV-2, and... M. pneumoniae Nucleic acid samples.

[0069] As can be seen from Figure 4, each primer pair can amplify a clear target band, and the size of the target band is in the corresponding position.

[0070] Example 3: Application of mTagBFP and fluorescently labeled anti-Tag ssDNA molecularly encoded microspheres as carriers in the detection of multiple respiratory pathogens. This embodiment aims to validate the ability of the mTagBFP and fluorescently labeled anti-Tag ssDNA molecularly encoded microspheres prepared in Example 1 to detect multiple respiratory pathogens. Specifically, it includes the following steps: 1) Preprocessing of encoded microspheres Take 3.5 × 10⁻⁶ of each. 4 10.0 μm (L2R2-L2R6) mTagBFP and fluorescently labeled anti-Tag ssDNA molecules were mixed in the same EP tube. Magnetic beads were adsorbed by an external magnetic field, the supernatant was removed, and unbound impurities were removed to obtain pretreated encoded microspheres for later use.

[0071] 2) Multiplex PCR amplification of respiratory pathogens Select Flu A, Flu B, RSV, SARS-CoV-2 and M. pneumoniae Positive plasmids were mixed and then subjected to a uniform concentration gradient dilution (10⁻⁶) in the same tube. 8 -10 1 The following reagents (copies / μL) and their corresponding primers were used to prepare a multiplex PCR reaction system: including Flu AF 0.25μL, Flu AR 0.25μL, Flu BF 0.25μL, Flu BR 0.25μL, RSV-F 0.25μL, RSV-R 0.25μL, SARS-CoV-2-F 0.25μL, SARS-CoV-2-R 0.25μL. M. pneumoniae -F 0.25μL and M. pneumoniae -R 0.25μL, 2×PCR Mix buffer 12.5μL, template 1μL, add H2O to make up to 50μL. PCR reaction conditions: 94℃ pre-denaturation for 1 min; 94℃ denaturation for 30 s, 56℃ annealing for 1 min, 30 cycles; 72℃ extension for 10 s.

[0072] 3) Hybridization reaction Take 3 μL of amplification product and pretreated encoded microspheres (3.5 × 10⁻⁶). 4 Mix mTagBFP and fluorescently labeled anti-TagssDNA molecules encoding microspheres L2R2-L2R6 with 1 μg / mL SAPE, add hybridization buffer (0.01M PBS buffer, pH 7.4 and 0.05% Tween-20), and incubate for hybridization in a 25°C metal bath for 60 min.

[0073] 4) Flow cytometry detection and result interpretation After the reaction was completed, the fluorescence signal of SAPE (streptavidin-modified phycoerythrin) on the microspheres was detected by flow cytometry. The detection channel for SAPE fluorescence signal in the flow cytometer was FL2-PE. The results are as follows: Figure 5 As shown, where, Figure 5 a represents five suspension arrays encoded by mTagBFP and anti-Tag-Cy5 ssDNA. Figure 5 b represents the detection results of five targets. The results show that the microspheres of this invention can achieve simultaneous detection and accurate identification of five targets.

[0074] In summary, the dual-color encoded microspheres provided by this invention have multi-channel differentiation characteristics. By encoding the microspheres with different fluorescence, multiple respiratory pathogen targets can be detected simultaneously in a single reaction system, which can meet the high-throughput requirements of clinical joint screening for multiple respiratory pathogens.

[0075] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.

[0076] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A dual-color coded microsphere, characterized in that, Includes microspheres and mTagBFP and fluorescently labeled anti-Tag ssDNA covalently coupled to the surface of the microspheres; The fluorescently labeled anti-Tag ssDNA is used to capture the target to be detected, which is labeled with a tag.

2. The dual-color coded microsphere according to claim 1, characterized in that, The targets to be detected include respiratory pathogens, including respiratory pathogen Flu A, respiratory pathogen Flu B, respiratory pathogen RSV, respiratory pathogen SARS-CoV-2, and other respiratory pathogens. M. pneumoniae ; Used to capture tagged respiratory pathogens Flu A, Flu B, RSV, SARS-CoV-2, and other respiratory pathogens. M. pneumoniae The anti-Tag ssDNA sequences are shown in SEQ ID NO.16-20, respectively; The fluorescent marker in the anti-Tag ssDNA includes Cy5.

3. The dual-color coded microsphere according to claim 1, characterized in that, The microspheres include at least one of polystyrene microspheres, silica microspheres, agarose gel microspheres, dextran gel microspheres, magnetite magnetic microspheres, and ferric oxide microspheres, and the particle size of the microspheres is 1.0-30.0 μm.

4. The dual-color coded microsphere according to claim 1, characterized in that, The chemical groups modified on the surface of the microspheres include at least one of carboxyl, amino, maleimide, haloacetamide, gold atom, alkynyl, and azido; the groups on the mTagBFP include at least one of amino, carboxyl, mercapto, aldehyde, azido, and alkynyl; the chemical groups modified on the fluorescently labeled anti-Tag ssDNA molecule include at least one of amino, carboxyl, mercapto, thiol, aldehyde, epoxy, azido, alkynyl, hydroxyl, and cyano. The chemical groups modified on the surface of the microspheres are covalently coupled to the groups on the mTagBFP and the chemical groups modified on the fluorescently labeled anti-Tag ssDNA molecule.

5. A multiplex respiratory pathogen detection system, characterized in that, Includes the dual-color coded microspheres according to any one of claims 1-4.

6. The multiplex respiratory pathogen detection system according to claim 5, characterized in that, It also includes at least one of the following: A1) Primer set, which is used to amplify the target to be detected with Tag label and biotin dual modification; A2) Phycoerythrin, wherein the phycoerythrin includes streptavidin-modified phycoerythrin; A3) Detection components, the detection components including at least one of flow cytometer and CCD imaging system.

7. The multiplex respiratory pathogen detection system according to claim 6, characterized in that, In A1), the upstream primers in the primer set all have the structure F1-F2-F3, and the downstream primers of the primer set include an R sequence complementary to the specific gene sequence of the target to be detected, and are labeled with biotin at the 5′ end; Wherein, the nucleotide sequence of F1 is reverse complementary to the nucleotide sequence of the fluorescently labeled anti-Tag ssDNA; the nucleotide sequence of F3 is complementary to the specific gene sequence of the target to be detected; F2 includes a spacer arm, wherein the spacer arm includes at least one of iSpC3, iSpC6, iSpC12, poly-dT, poly-TTG, and oligotetraethylene glycol. In the primers for detecting the respiratory pathogen Flu A, the F1 sequence of the upstream primer is shown in SEQ ID NO.11, the F3 sequence is shown in SEQ ID NO.1, and the R sequence of the downstream primer is shown in SEQ ID NO.

2. In the primers for detecting the respiratory pathogen Flu B, the F1 sequence of the upstream primer is shown in SEQ ID NO.12, the F3 sequence is shown in SEQ ID NO.3, and the R sequence of the downstream primer is shown in SEQ ID NO.

4. In the primers for detecting the respiratory pathogen RSV, the F1 sequence of the upstream primer is shown in SEQ ID NO.13, the F3 sequence is shown in SEQ ID NO.5, and the R sequence of the downstream primer is shown in SEQ ID NO.

6. In the primers for detecting the respiratory pathogen SARS-CoV-2, the F1 sequence of the upstream primer is shown in SEQ ID NO.14, the F3 sequence is shown in SEQ ID NO.7, and the R sequence of the downstream primer is shown in SEQ ID NO.

8. Detection of respiratory pathogens M. pneumoniae In the primers, the F1 sequence of the upstream primer is shown in SEQ ID NO.15, the F3 sequence is shown in SEQ ID NO.9, and the R sequence of the downstream primer is shown in SEQ ID NO.

10.

8. The application of the dual-color coded microspheres as described in any one of claims 1-4 and the multiplex respiratory pathogen detection system as described in any one of claims 5-7 in the detection of multiple respiratory pathogens.

9. A method for detecting multiple respiratory pathogens using the multiplex respiratory pathogen detection system according to any one of claims 5-7, characterized in that, Includes the following steps: The chemical groups on the surface of the microspheres were activated, and then mTagBFP solution and fluorescently labeled anti-Tag ssDNA molecule solution were added. After mixing and incubation, dual-color encoded microspheres were obtained. Multiplex PCR amplification of the target in the sample was performed using a primer set to obtain PCR amplification products with dual modification of Tag and biotin. The PCR amplification product, which is dual-modified with Tag and biotin, is mixed with the dual-color encoded microspheres. Through complementary hybridization of base pairing, the amplification product is bound to the anti-Tag ssDNA on the surface of the dual-color encoded microspheres. Phycoerythrin modified with streptavidin was added, and after reaction, the phycoerythrin bound to the surface of the two-color coded microspheres. Then, the two-color coded microspheres were separated and washed by an external magnetic field, and the fluorescence signal on the surface of the two-color coded microspheres was detected by a detection component to achieve qualitative and quantitative analysis of the target to be detected.

10. The method according to claim 9, characterized in that, In the step of activating the chemical groups on the surface of the microspheres and then adding the mTagBFP solution and the fluorescently labeled anti-Tag ssDNA molecule solution, the concentration of the mTagBFP solution is 0-1000 μg / mL and the concentration of the fluorescently labeled anti-Tag ssDNA molecule solution is 0-1000 nM.

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