CN117451992A - Fluorescent immunochromatography kit for detecting respiratory virus antigen and preparation method thereof - Google Patents

Abstract

The invention provides a fluorescence immunochromatography kit for detecting respiratory virus antigens and a preparation method thereof, belonging to the technical field of in-vitro detection. According to the invention, a double antibody sandwich method is utilized to carry out single detection or multiple detection of more than two types of novel coronaviruses, adenoviruses and influenza A viruses in respiratory viruses, compared with a conventional immunochromatography test strip, antibody probes on a binding region are separated from the test strip to prepare probe freeze-dried powder suitable for long-term normal-temperature storage, and the freeze-dried probes and samples are directly mixed and incubated when in use, so that the time for capturing a target object to be detected by the probes is increased, thereby improving the detection sensitivity and further improving the detection rate; meanwhile, the multiple detection test strip can realize identification detection and synchronous quantitative detection of various target detection objects, and effectively improves detection efficiency. The kit has simple and efficient detection operation flow, can report results within 15 minutes, and is suitable for on-site rapid detection and personal self-detection.

Description

Fluorescent immunochromatography kit for detecting respiratory virus antigen and preparation method thereof

The present application claims priority from the chinese patent office, application No. 202311460509.X, entitled "an ultrasensitive fluorescent immunochromatographic multiplex assay kit and method for its preparation", filed on day 11 and 06 of 2023, the entire contents of which are incorporated herein by reference.

Technical Field

The invention belongs to the technical field of in-vitro detection, and particularly relates to a fluorescence immunochromatography kit for detecting respiratory virus antigens and a preparation method thereof.

Background

Lateral Flow Immunochromatography (LFIA) is a novel immunoassay that emerged at the beginning of the 80 s of the 20 th century. Due to its simple, fast and low cost characteristics, it has become an increasingly important point-of-care testing (POCT) device in the medical testing and home testing fields. Lateral flow immunochromatography has been applied to various fields such as detection of progestins, drugs, stimulants, chronic diseases, infectious diseases, and the like. The application range of the method is continuously enlarged, and the method has important significance for rapid and convenient biomolecule detection.

The most widely used Point-of-care testing (POCT) technique is colloidal gold immunochromatography, which is regarded as the present technique with the advantages of simplicity, rapidness, semi-quantification and the likeThe most potential on-site rapid detection tool is simple and convenient to operate, can report results within about 15 minutes, and is very suitable for on-site rapid detection. However, the sensitivity of the colloidal gold immunochromatography is low, and in order to improve the detection sensitivity, in recent years, more and more novel nanomaterials are used as immune product markers, such as nano fluorescent dyes, up-conversion nanoparticles, quantum dot microspheres, time-resolved fluorescent microspheres, nano magnetic beads, and the like. Among the materials, the fluorescent quantum dot microsphere (Quantum Dot Microsphere Nanobeads, QBs) has excellent luminous performance such as good light stability, high fluorescence intensity, adjustable emission wavelength and the like, and is modified with-COOH, -NH on the surface of the quantum dot ₂ The coupling with biological macromolecules such as antibodies is easy after the different functional groups are the same. The time-resolved fluorescence microsphere combines the particle characteristics of microsphere technology with the high specificity and sensitivity of time-resolved fluorescence technology, and can be used for various biological and medical research applications, in particular for detecting low-concentration biomolecules and researching the dynamic behaviors of the biomolecules. Therefore, the sensitivity and stability of the immunochromatography product based on quantum dot/time-resolved fluorescence microsphere labeling are better than those of a colloidal gold immunochromatography product, and the quantitative detection can be realized by combining corresponding fluorescence detection equipment.

The traditional immunochromatography method is characterized in that a marked probe is fixed on a binding pad, and the to-be-detected object in a sample reacts with the probe along with the progress of chromatography after sample loading, but the reaction time is relatively short and the reaction cannot be completely performed, which is also an important reason for low sensitivity of the immunochromatography method. This makes increasing the sensitivity of LFIA a one of the problems to be solved in the field of real-time detection. At present, detection kits for external reaction exist in the market, but probes are in liquid form, so that products need to be stored at low temperature and have short effective period, and serious inconvenience is brought to users.

Disclosure of Invention

In view of the above, the present invention aims to provide a fluorescence immunochromatography kit for detecting respiratory virus antigens, which not only can realize the instant detection of various targets, but also can be preserved at a low temperature for a long period of time without affecting the detection effect.

The invention provides a fluorescence immunochromatography kit for detecting respiratory virus antigens, which comprises a multiple detection test strip, wherein the multiple detection test strip comprises a back plate, and a sample adding area, a detection area and a water absorbing area which are sequentially arranged on one surface of the back plate from bottom to top; the detection area is provided with a plurality of detection lines according to the number of target viruses, and the respiratory tract infection viruses comprise at least one of the following viruses: novel coronaviruses (SARS-CoV-2), adenoviruses (ADV) and influenza A viruses (IVA); the detection lines are each coated with any one of the following different antibodies: anti-novel coronavirus antibody 1, anti-adenovirus antibody 1 and anti-influenza a virus antibody 1;

the kit also comprises fluorescent microsphere marked probe freeze-dried powder;

the fluorescent microsphere-labeled probe freeze-dried powder comprises an anti-novel coronavirus antibody 2, an anti-adenovirus antibody 2 and an anti-influenza A virus antibody 2 which are labeled by fluorescent microspheres.

Preferably, the fluorescent microsphere-labeled probe freeze-dried powder is prepared by mixing the fluorescent microsphere-labeled anti-novel coronavirus antibody 2, the fluorescent microsphere-labeled anti-adenovirus antibody 2, the fluorescent microsphere-labeled anti-influenza A virus antibody 2, a freeze-drying protective agent and a sample loading liquid, and sub-packaging the obtained mixed liquid and freeze-drying.

Preferably, the sample loading liquid is an aqueous solution comprising the following components in parts by weight:

0.05 to 0.15mol/L Tris-HCl, 0.5 to 2.5 percent of Tween-20 in volume concentration, 1.2 to 5.0 percent of sucrose in mass concentration, 1.0 to 4.6 percent of trehalose in mass concentration, 0.1 to 2.5 percent of bovine serum albumin in mass concentration and 0.1 to 0.9 percent of polyethylene glycol 20000 in mass concentration, and the pH value is 7.0 to 10.0.

Preferably, the volume of the mixed liquor is 30 mu l per tube;

the total mass concentration of the anti-novel coronavirus antibody 2, the anti-adenovirus antibody 2 and the anti-influenza A virus antibody 2 marked by the fluorescent microspheres in the mixed solution is 0.21-0.39%.

Preferably, the volume concentration of the freeze-drying protective agent in the mixed solution is 15-75%.

Preferably, the freeze-drying method is that the pre-freezing is carried out at the temperature of 4 ℃ for 0.5-2h; freezing at-50deg.C for 2-5h; sublimating for 5-12 h at the temperature of minus 30 ℃ once; and sublimating for 3-8 h at 25 ℃.

Preferably, the fluorescent microsphere is a quantum dot fluorescent microsphere and/or a time-resolved fluorescent microsphere.

Preferably, the coating concentration of the anti-novel coronavirus antibody 1 on the detection line is 0.2-1.0 mg/mL;

the coating concentration of the anti-adenovirus antibody 1 is 0.3 to 1.5mg/mL;

the coating concentration of the anti-influenza A virus antibody 1 is 0.3-1.2 mg/mL.

Preferably, the material of the sample area is treated with a treatment fluid before being attached to the back plate;

the treatment fluid is a Tris-HCl aqueous solution with the value of 7.4 and the concentration of 0.05 to 0.3mol/L, pH, wherein the treatment fluid comprises 0.5 to 3 percent of NaCl, 0.5 to 3 percent of BSA and 0.1 to 2 percent of PVP-10.

Compared with a conventional immunochromatography test strip, the invention separates the probe marker combined with the detection target on the binding area from the test strip independently, and prepares the probe freeze-dried powder suitable for long-term normal-temperature storage, so that the freeze-dried probe and a sample can be directly mixed and incubated when the kit is used, the time for capturing the target to be detected by the probe is increased, thereby improving the detection sensitivity and further improving the detection rate. Meanwhile, the multiple detection test strip can realize identification detection and synchronous quantitative detection of various target detection objects, and effectively improves detection efficiency. And the detection operation flow is simple and efficient, the result can be reported within 15 minutes, and the method is suitable for on-site rapid detection.

Drawings

FIG. 1 is a schematic diagram of a multiple test strip in a kit of the present invention;

FIG. 2 is a diagram of a fluorescent microsphere-labeled probe lyophilized powder;

FIG. 3 is a graph of other optimization factor results; a is the optimization of NC film; b is the optimization of the concentration of the detection antibody on the T line; c is the optimization of the incubation time of the sample; d is the optimization of the chromatographic time of the test strip;

FIG. 4 is a sensitivity test result; (a) Detecting fluorescence images of test strips of SARS-CoV-2, ADV and IAV and (b) corresponding signal intensities on T-line; (c-e) a calibration curve corresponding to detection of SARS-CoV-2, ADV and IAV;

FIG. 5 is a graph showing the results of repeatability tests; (a) a reproducible assay result for a multiplex assay kit; (b) a kit-specific assay; (c) detection of a clinical sample of the new coronavirus by the kit; (d) stability assay of multiplex assay kit.

Detailed Description

The invention provides a fluorescence immunochromatography kit for detecting respiratory virus antigens, which comprises a multiple detection test strip, wherein the multiple detection test strip comprises a back plate, and a sample adding area, a detection area and a water absorbing area which are sequentially arranged on one surface of the back plate from bottom to top; the detection area is provided with a plurality of detection lines according to the number of target viruses, and the respiratory tract infection viruses comprise at least one of the following viruses: novel coronaviruses, adenoviruses and influenza a viruses; the detection lines are each coated with any one of the following different antibodies: anti-novel coronavirus antibody 1, anti-adenovirus antibody 1 and anti-influenza a virus antibody 1;

the kit also comprises fluorescent microsphere marked probe freeze-dried powder;

the fluorescent microsphere marked probe freeze-dried powder comprises a fluorescent microsphere marked anti-novel coronavirus antibody 2, an anti-adenovirus antibody 2 and an anti-influenza A virus antibody 2.

In the invention, the fluorescent microsphere-marked probe freeze-dried powder is preferably prepared by mixing fluorescent microsphere-marked anti-novel coronavirus antibody 2, fluorescent microsphere-marked anti-adenovirus antibody 2, fluorescent microsphere-marked anti-influenza A virus antibody 2, freeze-drying protective agent and sample loading liquid, and the volume of each tube of the obtained mixed liquid is 30 mu l after sub-packaging. The total mass concentration of the fluorescent microsphere marked anti-novel coronavirus antibody 2, anti-adenovirus antibody 2 and anti-influenza A virus antibody 2 in the mixed solution is preferably 0.21% -0.39%, more preferably 0.3%. The mass ratio of the fluorescent microsphere marked anti-novel coronavirus antibody 2, the anti-adenovirus antibody 2 and the anti-influenza A virus antibody 2 is preferably 1:1:1. The preparation method of the anti-novel coronavirus antibody 2, the anti-adenovirus antibody 2 and the anti-influenza A virus antibody 2 marked by the fluorescent microspheres preferably comprises the following steps: coupling reaction is carried out on fluorescent microspheres, anti-novel coronavirus antibody 2, anti-adenovirus antibody 2 or anti-influenza A virus antibody 2 under the action of EDC and NHS. The fluorescent microspheres are preferably quantum dot fluorescent microspheres and/or time-resolved fluorescent microspheres, more preferably quantum dot fluorescent microspheres. The sample loading liquid is preferably an aqueous solution comprising the following components in percentage by weight: the sample loading liquid is an aqueous solution comprising the following components in parts by weight: 0.05 to 0.15mol/L Tris-HCl, 0.5 to 2.5 percent of Tween-20 in volume concentration, 1.2 to 5.0 percent of sucrose in mass concentration, 1.0 to 4.6 percent of trehalose in mass concentration, 0.1 to 2.5 percent of bovine serum albumin in mass concentration and 0.1 to 0.9 percent of polyethylene glycol 20000 in mass concentration, wherein the pH value is 7.0 to 10.0, more preferably 0.07 to 0.08mol/L pH 8.3 to 8.7Tris-HCl, 1.8 to 2.2 percent of Tween-20 in volume concentration, 2.5 to 3.5 percent of sucrose in mass concentration, 0.8 to 1.2 percent of trehalose in mass concentration, 0.4 to 0.6 percent of bovine serum albumin in mass concentration and 0.2 to 0.3 percent of trehalose in mass concentration, more preferably 0.075mol/LTris-HCl, 2 percent of Tween-20, 3 percent of sucrose, 1 percent of sucrose in mass concentration, 0.5wt% of bovine serum albumin and 0.25 percent of polyethylene glycol in mass concentration are 20008.5. The final volume concentration of the lyoprotectant in the loading solution is preferably 15% -75%, more preferably 25% -50%. The freeze-drying protective agent is an aqueous solution comprising the following components in mass concentration: 3 to 6 percent of sucrose, 4 to 6 percent of mannitol and 3 to 8 percent of glycine, and most preferably 4 percent of sucrose, 5 percent of mannitol and 5 percent of glycine. The lyophilization procedure is preferably pre-frozen at 4 ℃ for 0.5-2 hours; freezing at-50deg.C for 2-5h; sublimating for 5-12 h at the temperature of minus 30 ℃ once; secondary sublimation is carried out for 3-8 hours at 25 ℃, and prefreezing is carried out for 1 hour at 4 ℃ more preferably; freezing at-50deg.C for 3 hr; primary sublimation is carried out for 8 hours at the temperature of minus 30 ℃; and sublimating for 5 hours at 25 ℃. The three antibody probes are subjected to freeze-drying treatment in the presence of the loading liquid and the freeze-drying protection liquid, so that the long-term preservation of the antibodies can be ensured, the binding activity is not reduced, the reaction time with a sample is convenient to control during detection, the full combination of the target antigen and the antibody probes is facilitated, and the final detection sensitivity is improved. When the probe freeze-dried powder is used, the sample solution is adopted for dissolution. The working concentration of the novel fluorescent microsphere-labeled coronavirus antibody 2, the fluorescent microsphere-labeled adenovirus antibody 2 or the fluorescent microsphere-labeled influenza A virus antibody 2 is preferably 0.035-0.065%, and most preferably 0.05%.

In the present invention, the material of the detection region is nitrocellulose membrane (NC). The nitrocellulose membrane of the detection zone is preferably CN95, CN140 and Milipeire 135, more preferably CN95. The nitrocellulose membrane optimization experiment shows that CN95 has obvious advantages compared with CN140 and Miltieire 135 in detecting three respiratory tract infection viruses, and can be used for multiple detection of respiratory tract infection viruses. The CN95 was purchased from certolis (germany).

In the present invention, the coating concentration of the anti-novel coronavirus antibody 1 on the detection line is preferably 0.2 to 1.0mg/mL, more preferably 0.45 to 0.55mg/mL, and most preferably 0.5mg/mL. The coating concentration of the anti-adenovirus antibody 1 is preferably 0.3 to 1.5mg/mL, more preferably 0.75 to 0.85mg/mL, and most preferably 0.8mg/mL. The coating concentration of the anti-influenza A virus antibody 1 is preferably 0.3 to 1.2mg/mL, more preferably 0.7 to 0.9mg/mL, and most preferably 0.8mg/mL. The order of the three detection lines is not particularly limited. The quality control line of the detection area is one, and the goat anti-mouse IgG polyclonal antibody is coated, and the coating concentration is preferably 0.3mg/mL. According to the invention, a detection line antibody concentration optimization experiment is carried out, coating concentrations of 0.3mg/mL, 0.5mg/mL, 0.8mg/mL and 1.0mg/mL are respectively adopted for three antibodies to be sprayed on a nitrocellulose membrane to prepare a test strip, and the result shows that the coating concentrations of antibodies of different viruses coated on the nitrocellulose membrane are inconsistent, and the highest signal-to-noise ratio (SNR) for detecting the corresponding target viruses can be obtained by respectively spraying 0.5mg/mL of anti-novel coronavirus antibody, 0.8mg/mL of anti-adenovirus antibody and 0.8mg/mL of anti-influenza A virus antibody on an NC membrane.

In the invention, the multiple detection test strip is compared with the conventional multiple detection test strip, and the setting of a binding area is omitted. The material of the sample area is preferably treated with a treatment fluid before being applied to the back plate. The treatment solution preferably comprises 0.5 to 3% of NaCl, 0.5 to 3% of BSA and 0.05 to 0.3mol/L, pH value 7.4Tris-HCl aqueous solution of PVP-10 with a mass concentration of 0.1 to 2%, more preferably 0.05mol/L, pH value 7.4Tris-HCl solution containing 1.5wt% of NaCl,1wt% of BSA and 1wt% of PVP-10. The treatment solution is preferably applied by spraying or immersing the material in the sample area with the treatment solution and then drying at 37 ℃. The material of the sample area is preferably a glass fiber or a polyester film. Treatment with the treatment fluid is advantageous in that the material in the sample area reduces the residual of the target analyte and intercepts impurities in the sample.

In the invention, the fluorescence immunochromatography kit for detecting respiratory virus antigens is based on a double-antibody sandwich method. The multiplex detection kit is not particularly limited, and a preparation method of the multiplex detection kit known in the art is adopted, wherein the difference is that the setting of a binding region is omitted and the fluorescent probe is prepared into freeze-dried powder which is easy to store at normal temperature.

In the present invention, the method of using the kit preferably comprises the steps of: and (3) dissolving and incubating the fluorescent microsphere marked probe freeze-dried powder by using a sample to be tested, dripping the incubated mixture into a sample adding area of a multiple immunity detection test strip, and performing chromatography for 5-10 min, directly performing qualitative observation under ultraviolet irradiation or performing chromatography for 15min, and then performing concentration measurement result judgment of viruses after measuring a fluorescent value by using an instrument.

In the embodiment of the invention, the detection principle and the result of the sandwich method are judged as follows: when the sample contains the target virus antigen, the antigen is combined with the antibody of the freeze-dried powder probe to form an antibody 2-antigen complex, the antibody is captured by the target antigen specific antibody on a corresponding detection line after sample adding chromatography to form an antibody 2-antigen-antibody 1 complex for color development, and the redundant probe is continuously chromatographed to a quality control line and is captured by the secondary antibody for color development. If the sample does not contain the target antigen, the antigen cannot be captured by the antibody on the detection line, so that the color is not developed, and the quality control line captures the antibody of the probe and then develops the color. If the quality control line does not develop color, the test strip is invalid and needs to be detected again. Experimental results show that the kit prepared by the method is used for detecting novel coronaviruses, adenoviruses and influenza A viruses, and the detection limits are 56, 120 and 41copies/mL in sequence. The kit is stable for more than 8 months under the normal temperature condition, and the performance is not changed.

The following describes in detail a fluorescent immunochromatographic kit for detecting respiratory viral antigen and a method for preparing the same according to the present invention with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

A preparation method of an ultrasensitive immunochromatography multiplex detection kit comprises the following steps:

1. method of

(1) And preparing quantum dot microsphere fluorescent probes of different antibodies by adopting an activated ester method.

25 μLMES (20 mmol/L, pH 6.0) was mixed with 25 μL quantum dot microspheres (QBs, 1 μmol/L), and 20mg/mL of each of EDC and NHS in DMSO was added and activated at 37deg.C in the absence of light for 15min;10000g of centrifugal supernatant is removed after 20min, 25 mu L of MES (10 mmol/L, pH 6.0) solution is added for re-suspending and precipitating, and vortex mixing is carried out; adding 10 μg of novel coronavirus (SARS-CoV-2) antibody Ab604 (purchased from Ningbo-leap biotechnology Co., ltd., preparation of SARS-CoV-2 fluorescent probe QBs-Ab 604), 10 μg of Influenza A Virus (IAV) antibody Ab07 (purchased from Phpeng biological Co., ltd., preparation of IAV fluorescent probe QBs-Ab 07) and 10 μg of Adenovirus (ADV) antibody Ab02 (purchased from Zhongmei novel biotechnology Co., ltd., preparation of ADV fluorescent probe QBs-Ab 02) respectively, mixing by vortex, and incubating at 37deg.C under shaking at 800rpm in the dark for 1 hr; then 25 mu L of 10% BSA solution is added, the mixture is stirred and uniformly mixed, the mixture is sealed for 30min at 37 ℃ in a dark place, 10000g of the mixture is centrifuged for 15min, and the supernatant is discarded; then 50. Mu.L of boric acid buffer (5 mmol/L, pH 8.0) and 1% BSA solution were added to the mixture and the mixture was subjected to resuspension washing 1 time, 10000g of the mixture was centrifuged for 15min, and the supernatant was discarded; finally, 25. Mu.L of boric acid buffer (5 mmol/L, pH 8.0) and 1% BSA solution were added to resuspend, and three types of fluorescent probes were obtained in a volume ratio of 1:1:1, mixing and preserving at 4 ℃ for standby.

(2) And preparing fluorescent probe freeze-dried powder.

Preparing a sample liquid and a freeze-drying protective agent, wherein the sample liquid comprises the following components in percentage by weight: 0.075mol/L Tris-HCl, 1vt%Tween-20, 3 wt%sucrose, 1% trehalose, 1 wt%bovine serum albumin and 0.25 wt%polyethylene glycol 20000, and the pH value is 8.5. The lyoprotectant formulation was 5wt% sucrose, 4wt% mannitol, 8wt% glycine.

Diluting the mixed probe with the loading solution, wherein the total concentration of the mixed probe is 0.15%, adding 25vt percent of freeze-drying protective agent, subpackaging into centrifuge tubes (30 μl/tube) for freeze-drying, and the cold drying procedure is as follows: pre-freezing at 4 ℃ for 0.5h, freezing at-50 ℃ for 3h, primary sublimating at-30 ℃ for 8h, and secondary sublimating at 25 ℃ for 6h to prepare the probe freeze-dried powder (see figure 2).

(3) Sample pad treatment.

The sample pad treatment solution was formulated as a 0.05mol/LpH value 7.4Tris-HCl solution containing 1.5wt% NaCl,1wt% BSA,1wt% PVP-10. And uniformly spraying the sample pad treatment liquid onto the sample pad, putting the sample pad treatment liquid into an oven at 37 ℃ for 3 hours, taking out, drying and preserving for standby.

(4) Assembly of test paper

A nitrocellulose membrane (NC membrane) was stuck on a base plate, and SARS-CoV-2 capture antibody Ab602 (0.5 mg/mL, purchased from Ningbo mei biotechnology limited), ADV capture antibody Ab01 (0.8 mg/mL, purchased from new technology limited in Zhongmei) and IAV capture antibody Ab08 (0.8 mg/mL, purchased from fei peng biosystems limited) were coated on the NC membrane as detection lines (T1, T2, T3 lines) and goat anti-mouse IgG polyclonal antibody (0.3 mg/mL) was coated on the NC membrane as a quality control line (C line) using an automatic metal-spraying and film-drawing instrument. And (3) after the film is drawn, placing the plate in an oven for drying at 37 ℃ for 3 hours, sequentially assembling the sample pad and the absorbent paper on a bottom plate after the drying is finished, compacting, cutting into strips with the width of 3mm by using a numerical control strip cutting machine, and drying and keeping the strips away from light for later use.

(5) Detection method

A tube of probe lyophilized powder was dissolved using 60. Mu.L of the labeled sample and incubated at 37℃for 5min. And (3) dripping the incubated sample onto a sample pad, performing chromatography for 5-10 min, observing the color development condition of the strip under an ultraviolet light source, performing chromatography for 15min, and reading and recording the fluorescence signal value of the strip by using a fluorescence signal reader.

2. System optimization

(1) Sample loading liquid optimization

Optimization of the loading solution fractions using orthogonal assays, wherein the concentration of SARS-CoV-2, ADV and IAV detected by each of the three viruses was 2.0X10 ⁴ copies/mL，5.0×10 ⁵ Copies/mL and 2.5X10 ⁴ copies/mL). Factors were set to pH, ion concentration, sucrose content, trehalose content, tween-20 content, PEG-20000 content and BSA content. The experimental observation index is the absolute signal intensity (the difference between the total signal intensity of the three T-lines and the total negative control signal intensity). Table 1 lists the factors and levels of the orthogonal tests, and the 18 sets of orthogonal tests generated using SPSS software and their test results are shown in table 2. The final selected component of the loading solution was a 0.075M Tris-HCl system containing 3wt% sucrose, 1wt% trehalose, 2vt% Tween-20, 0.25wt% PEG-20000 and 0.5wt% BSA, pH 8.5.

TABLE 1 orthogonal test factors and levels

Table 2 orthogonal test (L18 (3) ⁷ ) Design and test results

(2) Other parameter optimization

NC membrane optimization experiments, preparing a sample liquid according to the optimized sample liquid formula, and simultaneously preparing test strips by respectively taking CN140, CN95 and Miltipeire 135 as NC membranes, wherein other parameters are recorded as above. The results are shown in fig. 3 a, and compared with other NC membranes in the immunochromatographic test strip based on QBs, the CN95 membrane has excellent detection performance, especially in the aspect of multiple detection of respiratory viruses.

And (3) performing T-line detection antibody coating concentration optimization experiments, preparing a sample solution according to the optimized sample solution formula, preparing a test strip by taking CN95 as an NC film, wherein the working concentrations of SARS-CoV-2 antibody, ADV antibody and IAV antibody sprayed on the NC film are respectively 0.3, 0.5, 0.8 and 1.0mg/mL, and other parameters are the same as those described above. As can be seen in FIG. 3 b, the highest signal-to-noise ratio (SNR) for detecting their corresponding target viruses was obtained by spraying the NC membrane with 0.5mg/mLSARS-CoV-2 antibody, 0.8mg/mLADV antibody and 0.8mg/mL IAV antibody, respectively.

Sample incubation time optimization experiment, preparing test strips according to the optimization parameters, wherein the sample incubation time is respectively 0, 2, 5, 10, 15 and 20min during detection. By analyzing the time required to reach saturation signal intensity, the optimal incubation time for the sample can be determined to be 5min, as shown in fig. 3 c.

And (3) optimizing test of chromatographic time of the test strip, preparing the test strip according to the optimized parameters, wherein the chromatographic time is respectively 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 30min during detection. The optimal chromatographic time of the strip was evaluated as indicated by d in 3, indicating that a chromatographic time of 15 minutes was optimal.

3. Performance investigation

3.1 sensitivity

To evaluate the performance of the kit for detection of various respiratory viruses, mixed virus samples containing SARS-CoV-2, ADV and IAV were tested. Figure 4 shows a bar graph and corresponding fluorescent intensity statistics for detection of these three target respiratory viruses. With the decrease of the mixed virus concentration, the fluorescence intensity of the three test lines gradually decreases, and finally cannot be detected. The lowest detectable virus concentrations visible to the naked eye for the three viruses were 160copies/mL, 1000copies/mL and 200copies/mL. The signal intensity of all bands was recorded using a fluorescence signal reader (b in fig. 4). A standard curve was constructed based on the correspondence between virus concentration and signal intensity (c-e in FIG. 4). The S-shaped calibration curves for SARS-CoV-2, ADV and IAV show a broad detection range spanning four orders of magnitude. The LOD of the kit for three virus assays was calculated to be approximately 56copies/mL,120copies/mL and 41copies/mL, respectively.

(2) Repeatability of

Evaluation by six independent testsThe reproducibility of the kit for the detection of three respiratory viruses was estimated. FIG. 5 a shows the test strip pair high concentration (SRS-CoV-2 is 8.0X10) ⁴ copies/mL ADV 5.0X10 ⁶ copies/mL, IAV of 1.0X10 ⁵ cobies/mL), medium concentration (8.0X10 virus concentration respectively) ³ copies/mL、4.0×10 ⁵ Copies/mL and IAV 4.0X10 ³ copies/mL) and low concentrations (8.0X10 of each of the three viruses ² copies/mL, 2.0X10 for ADV ⁴ copies/mL and 1.0X10 for IAV ³ copies/mL) of the virus sample. The fluorescence intensities of the 6 independent detection bands at the same concentration can be found to be basically consistent, and the Relative Standard Deviation (RSD) is lower than 8%, which indicates that the prepared kit has good repeatability.

(3) Specificity (specificity)

Other important respiratory viruses including Influenza B Virus (IBV), parainfluenza virus (PIV), respiratory Syncytial Virus (RSV), streptococcus Pneumoniae (SP), staphylococcus Aureus (SA) and Neisseria Meningitidis (MC) were tested using the prepared kit, and only three corresponding viruses (SARS-CoV-2 concentration 2.0X10 ⁴ copies/mL ADV concentration is 5.0X10 ⁵ copies/mL, IAV concentration 2.5X10 ⁴ The detection result of the copies/mL was positive, and other viruses (concentration 5.0X10) ⁵ The detection results of the copies/mL) are negative. Therefore, the prepared kit has good specificity.

(4) New crown clinical sample detection

32 clinical nasal swab samples of 2019 coronavirus (new coronavirus) cases were tested using the prepared kit. As shown in fig. 5 c, all samples tested positive, consistent with the clinical test. Therefore, the kit has enough sensitivity for detecting the coronavirus type 2 antigen of the clinical severe acute respiratory syndrome.

(5) Stability of

And (3) filling the probe freeze-dried powder and the detection test paper filled into the clamping shell into an aluminum foil bag, and carrying out vacuum plastic packaging. Then placing the plastic package bag into a baking oven at 37 ℃ for accelerating, taking out the plastic package bag at the same time every week, and mixingVirus (concentration of SARS-CoV-2, ADV and IAV was 2.0X10, respectively) ⁴ copies/mL，5.0×10 ⁵ Copies/mL and 2.5X10 ⁴ cobies/mL). Stability experiments combined with the arrhenius acceleration model showed that the kit could be stored at room temperature for up to 8 months with little degradation in performance (as shown in fig. 5 d). The freeze-dried powder probe is convenient to store and transport due to the volatilization of the moisture, so that the shelf life of the product is prolonged. Therefore, the multiple respiratory tract virus detection kit has great potential in clinical application.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (9)

1. The fluorescence immunochromatography kit for detecting respiratory tract virus antigens comprises a multiple detection test strip, wherein the multiple detection test strip comprises a back plate, and a sample adding area, a detection area and a water absorbing area which are sequentially arranged on one surface of the back plate from bottom to top; the detection area is provided with a plurality of detection lines according to the number of target viruses, and is characterized in that the respiratory tract infection viruses comprise at least one of the following viruses: novel coronaviruses, adenoviruses and influenza a viruses; the detection lines are each coated with any one of the following different antibodies: anti-novel coronavirus antibody 1, anti-adenovirus antibody 1 and anti-influenza a virus antibody 1;

the kit also comprises fluorescent microsphere marked probe freeze-dried powder;

the fluorescent microsphere marked probe freeze-dried powder comprises a fluorescent microsphere marked anti-novel coronavirus antibody 2, an anti-adenovirus antibody 2 and an anti-influenza A virus antibody 2.

2. The fluorescence immunochromatography kit for detecting respiratory virus antigens according to claim 1, wherein the fluorescence microsphere-labeled probe freeze-dried powder is prepared by mixing fluorescence microsphere-labeled anti-novel coronavirus antibody 2, fluorescence microsphere-labeled anti-adenovirus antibody 2, fluorescence microsphere-labeled anti-influenza A virus antibody 2, freeze-drying protective agent and loading liquid, and sub-packaging the obtained mixed liquid and freeze-drying.

3. The fluorescence immunochromatographic kit for detecting respiratory viral antigens according to claim 2, wherein the loading solution is an aqueous solution comprising the following components in content:

0.05 to 0.15mol/L Tris-HCl, 0.5 to 2.5 percent of Tween-20 in volume concentration, 1.2 to 5.0 percent of sucrose in mass concentration, 1.0 to 4.6 percent of trehalose in mass concentration, 0.1 to 2.5 percent of bovine serum albumin in mass concentration and 0.1 to 0.9 percent of polyethylene glycol 20000 in mass concentration, and the pH value is 7.0 to 10.0.

4. A kit for detecting an antigen of a respiratory virus according to claim 2, wherein the volume of the mixture per tube is 30 μl;

the total mass concentration of the anti-novel coronavirus antibody 2, the anti-adenovirus antibody 2 and the anti-influenza A virus antibody 2 marked by the fluorescent microspheres in the mixed solution is 0.21-0.39%.

5. The kit for detecting respiratory virus antigens according to claim 3, wherein the volume concentration of said lyoprotectant in said mixed solution is 15% -75%.

6. The fluorescent immunochromatographic kit for detecting respiratory viral antigens according to claim 2, wherein the lyophilization method is prefreezing at 4 ℃ for 0.5-2h; freezing at-50deg.C for 2-5h; sublimating for 5-12 h at the temperature of minus 30 ℃ once; and sublimating for 3-8 h at 25 ℃.

7. The fluorescent immunochromatographic kit for detecting respiratory viral antigens according to claim 1, wherein the fluorescent microsphere is a quantum dot fluorescent microsphere and/or a time-resolved fluorescent microsphere.

8. The fluorescence immunochromatographic kit for detecting respiratory virus antigens according to claim 1, wherein the coating concentration of the anti-novel coronavirus antibody 1 on the detection line is 0.2-1.0 mg/mL;

the coating concentration of the anti-adenovirus antibody 1 is 0.3 to 1.5mg/mL;

the coating concentration of the anti-influenza A virus antibody 1 is 0.3-1.2 mg/mL.

9. The fluorescence immunochromatographic kit for detecting a respiratory viral antigen according to any one of claims 1 to 8, in which a material of the sample zone is treated with a treatment liquid before being attached to the back plate;

the treatment fluid is a Tris-HCl aqueous solution with the value of 7.4 and the concentration of 0.05 to 0.3mol/L, pH, wherein the treatment fluid comprises 0.5 to 3 percent of NaCl, 0.5 to 3 percent of BSA and 0.1 to 2 percent of PVP-10.