CN116256510A - Pathogen detection system for viral pneumonia - Google Patents

Abstract

The invention discloses a pathogen detection system for viral pneumonia, which comprises a viral pneumonia antigen chip suitable for fluorescence detection, and detection reagents such as a sealing buffer solution, a reaction buffer solution and the like; the virus pneumonia antigen chip is provided with virus specific antigen probes derived from influenza A viruses H1N1, H3N2, H7N9, influenza B viruses, novel coronaviruses 2019-nCoV, SARS coronavirus, MERS coronavirus, parainfluenza virus, respiratory syncytial viruses A and B; can be used for analyzing the infection condition and typing of the virus pneumonia pathogen from serum sources.

Description

Pathogen detection system for viral pneumonia

Technical Field

The invention relates to a protein chip based on a specific antigen probe, in particular to a viral pneumonia pathogen detection chip and development of related detection reagents.

Background

Viral pneumonia is a major disorder with high morbidity and mortality worldwide. Viral pathogens such as influenza virus are an important cause of community-acquired pneumonia (CAP), and viruses are an important cause of hospital-acquired pneumonia (HAP).

Seasonal influenza is caused by influenza a/b viruses, mainly outbreaks in winter and pandemic worldwide, and causes influenza virus pneumonia. It is difficult to distinguish respiratory infections caused by influenza viruses from other pathogens by symptoms and signs alone. Influenza diagnostic tests can monitor influenza virus from respiratory tract specimens. These assays are highly specific and report results rapidly. But the sensitivity of the test is low, resulting in an increase in false negative rate.

Unlike human influenza viruses, animal-derived influenza viruses need to undergo many genetic changes to be transmitted to humans, generally without causing human-to-human transmission. Influenza viruses of animal origin, such as avian influenza virus, swine influenza virus, and influenza a/b virus are hardly distinguished, but the former often show more aggressive clinical processes and upper respiratory symptoms are often absent.

Respiratory Syncytial Virus (RSV) can cause acute respiratory illness in any age group. The virus is a common viral pathogen for children, but can also cause adult CAP, especially severe pneumonia caused by the elderly and immunocompromised patients, the death rate of the virus is equivalent to that of influenza pneumonia, clinical characteristics of the virus are not obviously different from those of pneumonia caused by other viruses, and patients often see the symptoms of upper respiratory tract infection, and the pneumonia is secondary.

Parainfluenza viruses (PIVs) have three serotypes: PIV-1, PIV-2, and PIV-3. PIV epidemic seasons in different regions are greatly different. Of these, PIV-3 is frequently epidemic in the outbreak of April to June and is most common in adult pneumonia hospitalized patients, while PIV-1 and PIV-2 are more common in autumn. PIV infection can lead to asthma attacks and acute exacerbations of chronic obstructive pulmonary disease. In healthy adult immunocompromised individuals, PIV infections may be manifested as asymptomatic or symptomatic causing mild upper respiratory tract infections. Comparison studies of clinical features of PIV pneumonia with non-PIV pneumonia show that wheezing is more common in the former.

In elderly and chronically cardiopulmonary disease and immunocompromised patients, the risk of pneumonia and severe symptoms caused by Human Metapneumovirus (HMPV) infection is significantly increased, and the rate of progression to pneumonia and death varies from study to study, but it can be determined that HMPV can cause pneumonia and has high disability rate and mortality rate. In lung transplant patients, some studies suggest that HMPV infection may lead to acute and chronic graft rejection.

Human adenoviruses (HAdVs) are a double stranded DNA virus. HAdVs can cause different clinical syndromes depending on the adenovirus serotype, viral tropism, invasion portal and host factors. Respiratory tract infections with HAdVs can be accompanied by gastrointestinal symptoms, and respiratory lesions are often mild and self-limiting in immunocompromised adult patients. Severe adenovirus infections can also be seen in HSCT and SOT patients, and can be asymptomatic or severe fatal pneumonia as part of systemic disease.

Human coronavirus (HCoV) strains ((HCoV-229E, HCoV-OC43, HCoV-NL63, coV-HKU 1) are fundamentally a respiratory virus that, by replication in nasopharyngeal epithelium, can cause common cold symptoms similar to rhinovirus infection, are one of the possible pathogens of CAP.

The types of pathogens causing viral pneumonia are various, the same pathogens are divided into different subtypes, and the viral pneumonia is mostly shown as slight respiratory tract infection symptoms in the early stage clinically, the respiratory tract symptoms caused by bacterial infection are difficult to distinguish from those caused by bacterial infection, and in epidemiological investigation, it is difficult for a virus infected person to determine the condition of infecting animal-derived viruses caused by contacting other animals, and the clinical diagnosis and disease control are greatly difficult.

After the pathogenic microorganism invades the human body, human humoral immunity can generate antibody molecules, namely immune globulin (Ig), wherein IgM is an antibody which is firstly generated by the human body when the human body receives antigen stimulation to perform humoral immunity, igG is an antibody with the largest content in the human body, different antigenic determinants on the surfaces of different pathogenic microorganisms can stimulate the human body to generate immune globulin IgG and IgM with antigen specificity in the immune reaction of the human body, and the immune globulin IgG and IgM can be used as indexes for detecting the infection of the pathogenic microorganism and can reflect the types and the types of the pathogenic microorganism specifically. At present, by taking virus antigens obtained by genetic engineering technology as probes, a kit capable of specifically detecting the level and the type of antibodies corresponding to the virus antigens in human serum and further judging the virus infection condition has been developed, such as Chinese patent CN108872608A. However, such a kit mainly adopts the conventional enzyme-linked immunosorbent assay principle, and in order to improve the detection efficiency, complex recombinant antigen design is often required.

Currently, the chip technology of capturing proteins by using probes has been applied to virus detection, such as chinese patent CN106645715a. However, these chips generally require the addition of a labeling reagent to treat the sample after incubation of the sample is completed, and only multiple sample detection of multiple classes of target molecules of the same virus is achieved. Besides protein chips, the sugar chips and the glycopeptide chips have simpler labeling (for generating fluorescence) modes, and are chip technologies with extremely high development and application values, such as establishment and application of a method for detecting glycoprotein by the glycopeptide chips. However, the structure of the antigen probe of the corresponding pathogen has obvious difference for different viral pneumonia, especially along with the recent large-scale popularity of some new viral pneumonia pathogens (such as novel HCoVs), the multiple point adsorption, fixation and reaction system construction of the new virus antigen probe and other original virus antigen probes by using the same substrate all need to be repeatedly searched through experiments, thus restricting the development and application efficiency of detection reagents, especially chip products.

Disclosure of Invention

The invention aims to provide a pathogen detection system for viral pneumonia.

In order to achieve the above purpose, the invention adopts the following technical scheme:

a viral pneumonia antigen chip suitable for fluorescent detection comprises a carrier, a plurality of virus specific antigen probes, negative quality control (BLANK) and positive quality control (Marker) which are arranged on the carrier, wherein the negative quality control is selected from Bovine Serum Albumin (BSA) or other protein molecules incapable of being combined with virus antibodies captured by the virus specific antigen probes, the positive quality control is a fluorescent Marker (namely fluorescent Marker protein molecule) of the protein molecules, and the virus specific antigen probes are derived from a plurality of viruses causing fever symptoms after respiratory tract infection.

Preferably, the virus-specific antigen probe is derived from a plurality of influenza a viruses (e.g., influenza a virus H1N1, influenza a virus H3N2, influenza a virus H7N 9), influenza B viruses (e.g., influenza B virus), beta coronaviruses (e.g., neocoronavirus 2019-nCoV, SARS coronavirus, MERS coronavirus), parainfluenza viruses, respiratory syncytial virus type a, respiratory syncytial virus type B, human Metapneumovirus (HMPV), human adenoviruses (HAdVs), and the like.

Preferably, the virus specific antigen probe is a specific virus antigen fragment obtained by expression of genetic engineering bacteria, such as nucleocapsid proteins of influenza A and B viruses and coronaviruses, hemagglutinin neuraminidase proteins of parainfluenza viruses and fusion glycoprotein of respiratory syncytial viruses.

Preferably, the virus-specific antigen probe is immobilized on a carrier via a spotting buffer, which is Phosphate Buffer (PBS) having a pH of 8-10, or Na having a pH of 8-10, to form an antigen chip for specifically detecting antibodies to the corresponding virus in serum ₂ CO ₃ Buffer, or NaAc buffer at pH8-10, wherein Phosphate Buffer (PBS) at pH8-10 contains 135-140mM NaCl and 2.5-3mM KCl (hereinafter).

Preferably, the carrier is selected from a glass slide or nitrocellulose membrane.

The preparation method of the viral pneumonia antigen chip suitable for fluorescence detection comprises the following steps:

carrying out surface epoxidation treatment on the carrier, and then fixing a virus specific antigen probe on the carrier; or directly immobilizing the virus-specific antigen probe on the carrier (the carrier is not subjected to surface epoxidation treatment).

Preferably, the fixing specifically includes the following steps: 1-2mol/L virus specific antigen probe solution is spotted on the surface of a carrier (which is subjected to epoxidation treatment or not) and the solvent adopted by the virus specific antigen probe solution is the spotting buffer solution, and the carrier is dried after spotting.

Preferably, the spotting conditions are: loading virus specific antigen probe solution by adopting a sample application instrument under the condition of 48% -52% of humidity, and incubating for 10-12h under the environment of 48% -52% of humidity after loading.

Preferably, the drying conditions are as follows: the spotted carrier was left to stand in a vacuum drier at 35-38℃for 2-3h.

A pathogen detection system for viral pneumonia comprises the viral pneumonia antigen chip suitable for fluorescence detection and a detection reagent.

Preferably, the detection reagent comprises an antigen-antibody reaction buffer solution, wherein the antigen-antibody reaction buffer solution is a solution containing 0.5-1% of NaCl, 1-2% of Bovine Serum Albumin (BSA) by mass and volume ratio and 0.01-0.1% of Tween-20 by volume ratio, and the solution adopts a Phosphate Buffer Solution (PBS) with pH of 8-10; the solution is used for preparing a sample (such as serum) solution containing fluorescent labeled antibodies as a reaction system of the chip, and the chip is cleaned after the reaction system is incubated for 3-3.5h at 35-38 ℃ and then can be used for scanning.

Preferably, the fluorescent-labeled antibody is obtained by labeling with a fluorescent dye Cy3 or Cy 5.

Preferably, the detection reagent further comprises a blocking buffer solution, wherein the blocking buffer solution is a solution containing Bovine Serum Albumin (BSA) with the mass volume ratio of 1-2%, 0.5-1mol/L glycine and Tween-20 with the volume fraction of 0.01-0.1%, and the solution adopts a Phosphate Buffer Solution (PBS) with the pH value of 8-10; the chip (which is cleaned in advance) is sealed by the solution, and the sealing conditions are as follows: incubating at 35-38deg.C for 0.75-1.5 hr.

The beneficial effects of the invention are as follows:

the antigen chip of the invention uses specific viral antigen fragments as probes, can detect different viral antibodies possibly existing in a sample at the same time, and provides a necessary material basis for high-throughput serological detection analysis of viral pneumonia pathogens. And the detection reagent is combined, so that the quantification and analysis of the obtained fluorescent signal value completely meet the actual detection requirement, and the method can be used for analyzing the infection condition of the viral pneumonia pathogen of serum sources (namely the virus type corresponding to the positive result) and the typing thereof.

Furthermore, through optimization experiments, the invention develops the sample application buffer solution which can not only stably fix the antigen probe in situ (ensure that the physical and chemical properties and the biological activity of the antigen probe exist stably and can be firmly attached to a carrier), but also effectively enhance the detection signal.

Furthermore, the invention develops a buffer system (namely antigen-antibody reaction buffer solution) which is used for stabilizing each reaction substance in the reaction process of the sample and the chip, promoting the reaction and not influencing the interpretation of the detection result, such as reducing the false positive rate of the detection result by using the reaction buffer solution without glycine.

Drawings

FIG. 1 is a schematic diagram of a sample array region of a chip product.

FIG. 2 is a graph of scan results annotation in chip product applications (negative validation: healthy human serum).

FIG. 3 is a graph of annotation of scan results in chip product applications (positive verification: healthy human serum+all detectable viral antibodies).

FIG. 4 is a diagram of annotation of scan results in chip product applications (specificity verification: healthy human serum +H2 virus antibodies).

FIG. 5 is a graph of annotation of scan results in chip product applications (specificity verification: healthy human serum +2019-nCoV virus antibodies).

Detailed Description

The invention is described in further detail below with reference to the drawings and examples. The examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention.

1. Reagent(s)

1.1 buffer solution

(1) PBS (ph=9.0) component and formulation: naCl (137 mM), KCl (2.7 mM), na ₂ HPO ₄ (4.3 mM) and KH ₂ PO ₄ (1.4 mM); and (5) using deionized water to fix the volume.

(2) 0.1M Tris-HCl (pH=9.0) formulation: 50mL of 0.1mol/L Tris solution and 5.7mL of 0.1mol/L hydrochloric acid were diluted to 100mL with water.

③Na ₂ CO ₃ Buffer (ph=9.0) components and formulation: naHCO (NaHCO) ₃ (13.65 mg/mL) and Na ₂ CO ₃ (1 mg/mL); and (5) using deionized water to fix the volume.

(4) NaAc buffer (ph=9.0) components and formulation: naAc (8.2 mg/mL); and (5) using deionized water to fix the volume.

(5) PBS (ph=7.0) formulation: 6mL 11.876g/L Na ₂ HPO ₄ And 4mL of 9.078g/L KH ₂ PO ₄ 。

(6) 0.1M Tris-HCl (pH=7.0) formulation: 50mL of 0.1mol/L Tris solution and 46.7mL of 0.1mol/L hydrochloric acid were diluted to 100mL with water.

⑦Na ₂ CO ₃ Buffer (ph=7.0) formulation: 0.1mol/L NaHCO ₃ Solution with 0.1mol/L H ₂ CO ₃ The solution was titrated to ph=7.0.

(8) NaAc buffer (ph=7.0) components and formulation: naAc (3 mol/L); and (5) using deionized water to fix the volume.

1.2PBS-T

A0.1% (v/v) Tween-20 solution was prepared using PBS (pH=9.0) as a solvent.

1.3GPTS working fluid

240mL of absolute ethanol, 450. Mu.L of glacial acetic acid and 10mL of GPTS ((3-glycidoxy) trimethoxysilane) are taken and mixed.

1.4 1mol/L BSA solution

Purchased BSA (sigma) was dissolved in deionized water and the volume was set to the target concentration.

1.5 antigen Probe

For the viral pneumonia antigen chip to be developed, a relatively common virus specific antigen fragment is selected as a probe, the sequence of which is shown in table 1, and the antigen probes are all purchased from manufacturers (such as sinobiolologic).

TABLE 1 antigen probe design

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2. Epoxidation modification of slides

(1) Selecting a clean glass slide with no scratch on the surface; placing the slide in 10% (w/w) HCl solution, and performing water bath heat preservation at 60 ℃ for 1h; taking out the glass slide, and shaking and washing with ultrapure water for 20min, wherein the shaking speed is 70-80r/min, and water is changed every 5min; and then the absolute ethyl alcohol is shaken and washed for 15min, wherein the shaking speed is 70-80r/min, and the absolute ethyl alcohol is replaced every 5 min. Spin-drying at room temperature at 500rpm for 10 min.

(2) After the step 1, placing the slide glass in 15% (w/w) NaOH solution, and shaking and washing overnight, wherein the shaking speed is 70-80r/min; the slide was sonicated in the 15% (w/w) NaOH solution for a further 20min; taking out the glass slide, and shaking and washing with ultrapure water for 20min, wherein the shaking speed is 70-80r/min, and water is changed every 5min; and then the absolute ethyl alcohol is shaken and washed for 15min, wherein the shaking speed is 70-80r/min, and the absolute ethyl alcohol is replaced every 5 min. Spin-drying at room temperature at 500rpm for 10 min.

(3) After step 2, 250mL of GPTS working solution is taken, the slide is placed in the GPTS working solution and is protected from light, and the slide reacts for 3 hours (60 r/min) on a shaking table at room temperature.

(4) Continuing to carry out ultrasonic treatment in the GPTS working solution for 15min; taking out the slide, and shaking and washing with absolute ethyl alcohol for 15min, wherein the shaking speed is 70-80r/min, and the absolute ethyl alcohol is replaced every 5 min. Spin-drying at room temperature at 500rpm for 10 min.

(5) Vacuum drying in a 37 ℃ oven for 3 hours to obtain the epoxidized chip base, and sealing and preserving at 4 ℃.

3. Spot-making of antigen chip

3.1 flow

Each antigen probe in Table 1 was prepared as a 1mol/L spotting solution (the solvent was a spotting buffer), i.e., the spotting solution contained as an active ingredient a recombinant antigen having the same structure as the viral surface antigenic determinant.

Negative quality control (BLANK) was spotted with 1mol/L BSA solution and Marker was spotted with formulated Cy 3-labeled BSA (1 mg/mL). The negative quality control solution, the sample application solution and the Marker solution are added into a 384-well plate for standby. The array of zones 4 was loaded onto the epoxidised substrate with a spotter as shown in figure 1. The loaded epoxidised chip base (i.e. the sample application liquid is added to the epoxidised chip base according to the sample application points shown in the array after the sample application needle dips the sample application liquid, etc.), and incubated overnight in an environment with a certain humidity (at room temperature). And then drying in a vacuum dryer to fix the sample points (related to antigen probes, negative quality control and markers) on the epoxidation substrate, and hermetically storing the prepared antigen chip at 4 ℃ in a dark place.

3.2 Condition optimization

(1) Determination of optimal spotting pH

The pH of the spotting solution and the type of salt ions in the spotting solution will have a certain effect on the binding of the antigen probe to the epoxidised platelet. Therefore, firstly, the sample application pH is determined by selecting the homologous antigen probe sample application liquid with pH7.0 and pH9.0 (the sample application liquid is respectively composed of 0.1M Tris-HCl, PBS, na with corresponding pH) ₂ CO ₃ Buffer solution and NaAc buffer solution. As a result, it was found that the antigen chip prepared at pH7.0 was washed off the antigen probe spots after the first PBS-T washing, indicating that the antigen probe spots did not bind well to the epoxidized chip base. And at pH9.0, the antigen probe sample spots are completely reserved after two times of PBS-T cleaning, which is more beneficial at pH9.0Binding of antigen probes to the epoxidised substrate.

(2) Determination of optimal sample application humidity

The humidity of 60%, 50% and 40% are selected as experimental conditions, and observation is carried out during and after the spot-making process. As a result, it was found that when the humidity was 60%, the spreading phenomenon occurred after loading the spotting solution (e.g., spotting solution); at 40% humidity, the loaded solution (e.g., spotting solution) was allowed to stand overnight and shrink and dry. The humidity of 50% can well maintain the form of the sample after loading the solution (such as sample application liquid). The above results indicate that the spotting humidity of 50% is more suitable for loading the spotting liquid on the epoxidized substrate.

(3) Determination of optimal drying time

The loaded epoxidation substrate is incubated overnight in an environment with 50% humidity, and after incubation, the antigen probe is immobilized on the epoxidation substrate to form a corresponding sample point by vacuumizing in a vacuum drier at 60 ℃ for 1 hour (marked as a drying condition I) or in a vacuum drier at 37 ℃ for 3 hours (marked as a drying condition II). The results show that drying condition one resulted in a reduced antigen probe spot compared to drying condition two. The above results indicate that the second drying condition is more suitable for drying the spotting solution loaded on the spotting solution after overnight incubation.

4. Closure and use of chips

4.1 procedure

(1) Cleaning: washing antigen chip with PBS-T for 2 times on shaking table, wherein shaking speed is 80-85r/min for 5min each time; shake-wash 2 times with PBS (ph=9.0), wherein shake speed is 80-85r/min for 5min each. Spin-dry at 500rpm at room temperature.

(2) Closing: 1mL of sealing buffer solution is added in an array area of the antigen chip after cleaning (in actual use, the sealing buffer solution is firstly added on a special slide, a liquid adding area which is formed by a large rubber ring and is integrally matched with the chip array area is arranged on the slide, the antigen chip is bonded with the special slide after the sealing buffer solution is added in the area and is fixed in a metal reaction box), and the antigen chip is incubated for 1h in a 37 ℃ environment (the metal reaction box slowly rotates in an incubator, so that the sealing buffer solution fully contacts and reacts with the part of the chip array area where antigen probes are not fixed); the well incubated antigen chip is shaken and washed for 1-2 times by PBS-T, wherein the shaking speed is 80-85r/min for 5min each time; then shake-washed 1-2 times with PBS (ph=9.0), wherein the shaking speed is 80-85r/min for 5min each time. Spin-drying at 500rpm at room temperature, and sealing and preserving at 4deg.C in dark.

(3) Detection reaction: the sample solution containing Cy 3-labeled antibody was added to the antigen-antibody reaction buffer to prepare 180. Mu.L of a reaction system. Different prepared reaction systems (such as 4 reaction systems) are added in an array area of a sealed antigen chip (in actual use, the reaction systems are firstly added on a special glass slide, the glass slide is provided with four liquid adding areas which are formed by four small rubber rings and are respectively matched with the array area of the chip, the antigen chip is bonded with the special glass slide and is fixed in a metal reaction box after the reaction systems are added in the areas), and the mixture is incubated for 3 hours in a 37 ℃ environment (the metal reaction box slowly rotates in an incubator, so that the reaction systems fully contact and react with antigen probes in the array area of the chip); the well incubated antigen chip is shaken and washed for 2 times by PBS-T, wherein the shaking speed is 60-70r/min for 5min each time; then shake-washed 2 times with PBS (ph=9.0), wherein the shaking speed is 60-70r/min for 5min each time. Spin-dry at 400rpm at room temperature.

(4) Scanning chip and analysis: photomultiplier tube (PMT) was set to 70%; the antigen chip reacted with the reaction system is pre-scanned, and then the reaction area is selected for fine scanning. The scan results were plotted using GenePix3.0 software.

4.2 Condition optimization

(1) Determination of optimal spotting buffer class

In order to optimize the detection signal, experiments were performed on the optimal salt ion species of the spotted solution based on the above determination of the optimal spotting pH, i.e., 0.1M Tris-HCl, PBS, na at pH9.0 was selected, respectively ₂ CO ₃ And (3) after spotting four solutions of the buffer solution and the NaAc buffer solution, sealing the antigen chip, and adding a reaction system for reaction. In the scan results, tris-HCl spotting solution showed the weakest detection signal (probably due to the fact that the amino group on Tris-HCl consumes part of epoxy groups and competitive binding with antigen molecules), while PBS, na ₂ CO ₃ Buffer solutionNaAc buffer showed a stronger detection signal. The results showed that PBS, na, pH9.0 ₂ CO ₃ Either buffer or NaAc buffer can be used as spotting buffer for preparing antigen chips, where Na ₂ CO ₃ The buffer works best.

In addition, by comparing the detection signals of different experiments, it is also found that in the case of using the above first drying condition in preparing the antigen chip, the corresponding detection signal is weaker, and using the second drying condition, a stronger detection signal can be obtained.

(2) Determination of optimal closure conditions

In the preparation process of the chip, epoxidation modification is needed on a glass slide, after antigen probes are fixed (namely after antigen chips are prepared), the part of the chip array area which does not contain the antigen probes is blocked, so that the influence of excessive background signals caused by nonspecific adsorption of antibodies and epoxidation chip bases in a sample on detection results is prevented (a large number of exposed epoxy groups are also arranged on blank parts of the epoxidation chip bases after sample application, and when the antigen probes react with the antibodies, the epoxy groups can be combined with a marker, namely Cy3 labeled antibodies or free fluorescence, namely a small amount of unremoved fluorescent dye Cy3, so that the detection results cannot be accurately observed). Although BSA can be generally used to bind to epoxy groups on non-spotted portions of the substrate so that the excess epoxy groups do not bind to labels or free fluorescence during sample reactions, BSA alone has poor blocking effects for antigen chips. Four different solutions (PBS with the solvent of pH 9.0) containing 1% (g/100 mL) BSA or 2% BSA, 0.05% (v/v) Tween-20, 0.5mol/L or 1mol/L glycine were used as blocking buffers for blocking the antigen chip, respectively, and then detection reaction was performed. As a result, it was found that the corresponding chips after blocking with four blocking buffers showed lower background levels in the scan results, wherein the background values of the chips blocked with the solutions containing 1% BSA, 0.05% Tween-20 and 1mol/L glycine were smaller than the background values of the chips blocked with the solutions containing 1% BSA, 0.05% Tween-20 and 0.5mol/L glycine, the solutions containing 2% BSA, 0.05% Tween-20 and 0.5mol/L glycine, or the solutions containing 2% BSA, 0.05% Tween-20 and 1mol/L glycine. The results show that the blocking effect of the solution containing 1% BSA, 0.05% Tween-20 and 1mol/L glycine is superior to the blocking effect of the other three blocking buffers.

(3) Determination of optimal antigen-antibody reaction buffer

Four different solutions (PBS with the solvent of pH 9.0) containing 0.6% (w/w) NaCl, 1% (g/100 mL) BSA or 2% BSA, 0.05% (v/v) Tween-20, 0mol/L or 1mol/L glycine were used as antigen-antibody reaction buffers, and the reaction system was prepared to incubate the antigen chip, and the effect of the four reaction buffers on the final detection signal was compared. As a result, it was found that a solution containing 0.6% NaCl, 0.05% Tween-20 and 1% BSA (i.e., containing 0mol/L glycine) or a solution containing 0.6% NaCl, 0.05% Tween-20 and 2% BSA (i.e., containing 0mol/L glycine) was used as an antigen-antibody reaction buffer, and that a desired detection signal could be obtained from the reacted chip, and that the detection signal could not be obtained from the reacted chip once 1mol/L glycine (or 0.5mol/L glycine) was introduced into the two solutions, indicating that glycine could affect the specific binding of antigen probes to antibodies. Meanwhile, the comparison shows that the solution containing 0.6% NaCl, 0.05% Tween-20 and 1% BSA is used as an antigen-antibody reaction buffer solution, and the formed detection signal is higher than that of the solution containing 0.6% NaCl, 0.05% Tween-20 and 2% BSA. The above results indicate that, among the four antigen-antibody reaction buffers, a solution containing 0.6% NaCl, 0.05% Tween-20 and 1% BSA was the optimal reaction buffer.

5. Chip application instance

5.1 collection, handling and labelling of serum samples

Standing the collected whole blood at room temperature for 0.5h, collecting supernatant, centrifuging at 12000rpm at 4deg.C in a refrigerated centrifuge for 10min, and collecting supernatant to obtain serum. Serum sample protein concentrations were determined with a micro nucleic acid protein meter. 100 μg serum samples were taken and an equal volume of 0.1M Na ₂ CO ₃ The solutions were mixed (pH 0.9), and 5. Mu.L of the fluorescent dye Cy3 solution was added thereto, followed by mixing, and a slight shaking reaction was performed at room temperature for 3 hours to complete the labeling of fluorescent dye Cy3, wherein the fluorescent dye Cy3 solution was prepared: 10mg of the fluorescent dye Cy3 (Siamitraz) was dissolved in 1.2mL of DMSO (sigma). Fluorescence by Cy3After the optical dye is marked, an ultrafiltration centrifuge tube is used for removing free fluorescent dye, and the marked protein solution in the ultrafiltration tube is collected. The concentration of the collected labeled protein solution was roughly measured with a trace nucleic acid protein meter using PBS with ph=7.0 as a blank for detecting the protein concentration, for later use.

5.2 antigen chip for detecting virus antibody in serum sample

(1) Shaking the blocked antigen chip on a shaking table by PBS-T for 5min (shaking speed is 80-85 r/min); then, the mixture was shaken with PBS (pH=9.0) for 5min (shaking speed: 80-85 r/min), and dried.

(2) Preparing 180 mu L of reaction system (containing 10-20 mu g of labeled protein) by using the labeled protein solution (containing Cy3 fluorescent labeled antibody) and the optimal reaction buffer; the reaction system was added and incubated at 37℃for 3h. Shaking and washing the incubated antigen chip for 2 times by using PBS-T; then, the mixture was shaken with PBS (pH=9.0) for 2 times and dried.

(3) Setting a photomultiplier tube (PMT) to 70% prior to scanning the antigen chip; the antigen chip after the pre-scanning reaction is firstly scanned (namely, all array areas and blank areas on the chip are scanned at one time), then the selected reaction area is finely scanned (namely, the selected single array area is scanned, each array area on the chip is required to be scanned one by one in actual application, and the aim of fine scanning is to reduce the influence of the blank area on signal value calculation). The scan results were plotted using GenePix3.0 software (FIGS. 2, 3, 4, 5).

5.3 judgment of results

The information such as the detection signal (fluorescence signal intensity value of the antigen probe spot position) and the background value is obtained from the scan result diagram by using GenePix3.0 software and then analyzed. 3 samples are repeated in one experiment for each antigen probe, the signal value of each sample is subtracted from the background value and then averaged to obtain the actual fluorescence signal value of the corresponding probe, the actual fluorescence signal value is compared with the background value, the samples with the values smaller than the background value are negative (antibodies of the corresponding viruses are not contained in serum), and the samples with the values higher than the background value are positive (antibodies of the corresponding viruses are contained in serum).

Criterion analysis principle: when the antigen chip detection signal is read, in order to reduce the influence of background and sample original signals (detection signals obtained by directly scanning without adding a reaction system, and the detection signals can be slightly higher than signals of blank background parts in the scanning result), the reliability of the signal values is improved, and the signal values with the background values being larger than 1.5 times or 2 times are generally adopted as effective signals (positive), so that the above steps adopt the method of subtracting the background values, averaging and comparing with the background values, namely, the method is equivalent to taking the background values being larger than 2 times as the effective signals.

6. Features of the invention

(1) Compared with the traditional serological detection, the method has the advantages that the demand for detection samples is small, the detection speed is higher, and the information obtained by single detection is more abundant;

(2) Compared with an immunochromatography method, the sensitivity and the accuracy are higher, and the quantitative requirement can be met more easily;

(3) Compared with the method using an antibody as a probe, the method using a recombinant antigen (i.e. without using a virus complete protein shell) does not need to obtain a specific antibody through an antigen immune model animal in the preparation process of the product, saves the development cost of the product, and can finish the development of the product more quickly when a detection product is developed aiming at a sudden novel virus pathogen.

(4) The sample application pH and sample application buffer class, sample application humidity and drying condition of the antigen chip, the sealing buffer, reaction buffer and the like of the antigen chip are optimized, and the same substrate can be utilized to carry out the multi-point adsorption, fixation and reaction system construction of a new virus (such as new coronavirus 2019-nCoV) antigen probe and other original virus antigen probes, so that the method provides a guarantee for rapidly expanding or adjusting the chip virus antigen detection class and practical application.

(5) Methods and techniques have been developed that can stably and accurately detect and interpret fluorescent signals, thereby effectively reading the detected signals and excluding unwanted signals from the results of the detection of the antigen.

(6) Luminescent signal substances (e.g., cy 3) capable of binding to the antibody to be detected by physical adsorption or non-specific binding are selected. For the analysis and detection of antibodies in serum, the luminous signal substances do not change the structure of the antibodies, do not influence the combination of the antibodies and specific antigen probes, and have wide application, namely, the luminous signal substances can be used for marking a plurality of detected antibodies.

Claims (10)

1. A viral pneumonia antigen chip, characterized in that: the chip comprises a carrier, a plurality of virus specific antigen probes arranged on the carrier, negative quality control and positive quality control, wherein the negative quality control is selected from bovine serum albumin or other protein molecules which are not combined with virus antibodies captured by the virus specific antigen probes, the positive quality control is a fluorescent marker of the protein molecules, and the virus specific antigen probes are derived from viruses causing fever symptoms after respiratory tract infection.

2. The viral pneumonia antigen chip according to claim 1, wherein: the virus-specific antigen probe is derived from a plurality of viruses in influenza A virus, influenza B virus, beta coronavirus, parainfluenza virus, respiratory syncytial virus, human metapneumovirus and human adenovirus.

3. The viral pneumonia antigen chip according to claim 1, wherein: the virus specific antigen probe is immobilized on a carrier through a sample application buffer solution, so as to form an antigen chip for specifically detecting antibodies of the corresponding viruses in serum, wherein the sample application buffer solution is phosphate buffer solution with pH of 8-10 and Na ₂ CO ₃ Buffer or NaAc buffer, wherein the phosphate buffer contains 135-140mM NaCl and 2.5-3mM KCl.

4. A viral pneumonia antigen chip according to claim 3, wherein: the carrier is selected from a glass slide or a nitrocellulose membrane.

5. The method for preparing the viral pneumonia antigen chip according to any one of claims 1-4, wherein: the method comprises the following steps:

carrying out surface epoxidation treatment on the carrier, and then fixing a virus specific antigen probe on the carrier; or directly immobilizing virus-specific antigen probes on a carrier.

6. The method of manufacturing according to claim 5, wherein: the fixing specifically comprises the following steps: spotting 1-2mol/L virus specific antigen probe solution on the surface of a carrier, wherein the virus specific antigen probe solution adopts a solvent of phosphate buffer solution with pH of 8-10 and Na ₂ CO ₃ Buffer or NaAc buffer, wherein the phosphate buffer contains 135-140mM NaCl and 2.5-3mM KCl, and drying after spotting.

7. The method of manufacturing according to claim 6, wherein: the spotting conditions are as follows: loading virus specific antigen probe solution by adopting a sample application instrument under the condition of 48% -52% of humidity, and incubating for 10-12h in the environment of 48% -52% of humidity after loading; the drying conditions are as follows: the spotted carrier was left to stand in a vacuum drier at 35-38℃for 2-3h.

8. A pathogen detection system for viral pneumonia, characterized by: the system comprising the viral pneumonia antigen chip as claimed in any one of claims 1-4 and a detection reagent.

9. The pathogen detection system of claim 8, wherein: the detection reagent comprises an antigen-antibody reaction buffer solution, wherein the antigen-antibody reaction buffer solution is a solution containing 0.5% -1% of NaCl, 1% -2% of bovine serum albumin and 0.01% -0.1% of Tween-20, and a solvent adopted by the solution is phosphate buffer solution with the pH value of 8-10.

10. The pathogen detection system of claim 8, wherein: the detection reagent also comprises a blocking buffer solution, wherein the blocking buffer solution is a solution containing 1% -2% of bovine serum albumin, 0.5-1mol/L glycine and 0.01% -0.1% of Tween-20, and the solution adopts a phosphate buffer solution with pH of 8-10.

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