WO2023040026A1 - Immunochromatographic detection reagent strip and kit comprising same, and applications of both - Google Patents

Abstract

An immunochromatographic detection reagent strip and a kit comprising same, and applications of both, relating to the technical field of biological detection. According to the immunochromatographic detection reagent strip, fluorescent microspheres, latex, colloidal gold or colloidal carbon are coupled to a specific protein by using immunochromatography to obtain a specific protein complex. The specific protein comprises a novel coronavirus N protein first antibody, an influenza A virus NP protein first antibody and an influenza B virus NP protein first antibody, which are used to bind novel coronavirus N protein, influenza A virus NP protein and influenza B virus NP protein antigens. A nasopharyngeal swab, oropharyngeal swab, or saliva from a patient are then detected. The immunochromatographic detection reagent strip has the advantages of high sensitivity, high specificity and quantitative detection, and avoids weak positive sample missed detection and false positive sample false detection to the greatest extent.

Description

Immunochromatographic detection reagent strip, kit containing same and application of both

Cross References to Related Applications

This disclosure claims the priority of the Chinese patent application with the application number "202111084588.X" and the title "Immunochromatography Detection Reagent Strip Containing It and Its Application" submitted to the China Patent Office on September 15, 2021. The entire contents are incorporated by reference in this disclosure.

technical field

The disclosure relates to the technical field of biological detection, in particular to an immunochromatographic detection reagent strip, a kit containing the same and the application of the two.

Background technique

There is no clinically effective small-molecule chemical drug for the treatment of new coronavirus pneumonia. The accumulation of new coronavirus mutations has evolved into immune evasion, and therapeutic neutralizing antibodies are also ineffective. According to the existing technology, there are only about 10 glycosylation sites of influenza virus, and as long as there are 3-4 site mutations, the influenza virus will mutate, and our vaccination against influenza every year will be ineffective; the glycosylation site of the new coronavirus is 65- 70, which is 7 times that of influenza. If the glycosylation site of the new coronavirus mutates, new recombinant strains or hybrid recombinant strains will appear, and vaccines and antibodies will be completely ineffective. When the virus mutates to the point where protective antibodies produced by previous vaccinations or infections no longer work, this more lethal mutant strain "is "almost certainly going to happen" to some extent," causing fatalities. rate up to 35%.

Mutant novel coronaviruses continue to emerge, and their infectivity and pathogenicity continue to increase, paralyzing the world economy. The novel coronavirus Delta (Delta) mutant strain (B.1.617.2) has become the main pathogenic strain worldwide. The viral load of the delta mutant strain is 1260 times higher than that of the original strain, and it is highly contagious, fast in transmission and strong in vaccine resistance. People infected with the Delta mutant strain had a higher risk of hospitalization, severe pneumonia, and death than those infected with the non-mutated strain. The delta variant strain has caused a new wave of outbreaks around the world. Rapid screening and effective detection of the new coronavirus delta and lambda variant strains have become the key to the current epidemic prevention battle.

At the same time, influenza is another infectious disease that endangers human life and health. The mixed infection of new coronavirus/influenza virus has caused great troubles in clinical diagnosis and treatment. People with influenza infection have increased susceptibility to the new crown, and the aggravation of the mixed infection of new coronavirus influenza can easily develop into severe disease. pneumonia.

The joint detection of new coronavirus/influenza A virus/influenza B virus antigen is normalized, and its social effect and market scale are huge. Therefore, a new coronavirus/influenza A virus/influenza B virus antigen combined detection kit was developed to improve the sensitivity and early diagnosis rate, and one sample addition can realize the detection of new coronavirus/influenza A virus/influenza B virus The combined detection and differential diagnosis of influenza virus antigens has great clinical significance and social value.

Contents of the invention

The disclosure provides an immunochromatographic detection reagent strip, the reagent strip includes a bottom plate, and the bottom plate is provided with a sample pad, a marker binding pad, a chromatographic reaction membrane and absorbent paper;

Wherein, the marker binding pad is adsorbed with specific protein complexes, and the specific protein complexes are mainly obtained by coupling fluorescent microspheres, latex, colloidal gold or colloidal carbon with specific proteins; the specific proteins include Novel coronavirus N protein primary antibody, influenza A virus NP protein primary antibody and influenza B virus NP protein primary antibody, used to bind novel coronavirus N protein, influenza A virus NP protein and influenza B virus NP protein antigen;

The chromatographic reaction membrane is provided with a detection line, and the detection line contains the second antibody of the novel coronavirus N protein, the second antibody of the NP protein of the influenza A virus, and the second antibody of the NP protein of the influenza B virus, in order to bind the novel coronavirus. Antigens of coronavirus N protein, influenza A virus NP protein and influenza B virus NP protein.

In some embodiments, when the specific protein complex is mainly obtained by coupling latex with specific protein, the detection lines of influenza A and influenza B positive samples are blue, and the detection lines of novel coronavirus positive samples are red, which is convenient Visual recognition.

In some embodiments, the specific protein complex is obtained by coupling fluorescent microspheres, latex, colloidal gold or colloidal carbon to specific proteins.

In some embodiments, the amino acid sequence of the novel coronavirus N protein is shown in SEQ ID NO:01;

The amino acid sequence of the influenza A virus NP protein is shown in SEQ ID NO:02;

The amino acid sequence of the influenza B virus NP protein is shown in SEQ ID NO:03.

In some embodiments, the concentration of the second antibody to the novel coronavirus N protein in the detection line is 0.1-1 mg/mL;

The concentration of influenza A virus NP second protein antibody in the detection line is 0.1～1mg/mL;

The concentration of the second protein antibody of influenza B virus NP in the detection line is 0.1-1 mg/ml.

In some embodiments, the concentration of the specific protein complex in the marker-binding pad is 0.001-0.01 mg/mL.

In some embodiments, the chromatographic reaction membrane is arranged in the middle of the bottom plate, the marker binding pad and the sample pad are sequentially lapped on one end of the chromatographic reaction membrane, and the absorbent paper is lapped on the chromatographic reaction membrane on the other end.

In some embodiments, a quality control line is also provided on the chromatographic reaction membrane.

In some embodiments, when the specific protein complex is mainly obtained by coupling fluorescent microspheres with specific proteins, the LOD value for the new coronavirus delta strain is 20TCID ₅₀ /mL, corresponding to a nucleic acid Ct value of 34.97; The LOD value of the original strain of the novel coronavirus was 20TCID ₅₀ /mL, corresponding to a nucleic acid Ct value of 34.64.

In some embodiments, when the specific protein complex is mainly obtained by coupling latex with specific protein, the LOD value for the new coronavirus delta strain is 40TCID ₅₀ /mL, corresponding to the nucleic acid Ct value of 33.60; for the new coronavirus The LOD value of the original virus strain was 40TCID ₅₀ /mL, corresponding to the nucleic acid Ct value of 33.72.

In some embodiments, when the specific protein complex is mainly obtained by coupling colloidal gold and specific protein, the LOD value for the new coronavirus delta strain is 80 TCID ₅₀ /mL, corresponding to the nucleic acid Ct value of 32.35; The LOD value of the original coronavirus strain was 80TCID ₅₀ /mL, corresponding to a nucleic acid Ct value of 32.61.

In some embodiments, when the specific protein complex is mainly obtained by coupling latex with the specific protein, the detected LOD value for influenza A virus is 1.5×10 ³ TCID ₅₀ /mL, and the detected LOD value for influenza B virus It is 2×10 ⁴ TCID ₅₀ /mL.

The present disclosure provides an immunoassay kit, which includes the above-mentioned immunochromatography detection kit.

The application of the above-mentioned immunochromatography detection reagent strip or the above-mentioned immunochromatography detection kit provided in the present disclosure in the preparation of a joint detection product for novel coronavirus, influenza A virus and influenza B virus.

The present disclosure provides an immunochromatographic detection reagent strip according to any one of the above or the immunochromatographic detection kit described above in joint detection of novel coronavirus, influenza A virus and influenza B virus use.

The present disclosure provides an immunochromatographic detection reagent strip according to any one of the above or the immunochromatographic detection kit described above, which is used for joint detection of novel coronavirus, influenza A virus and influenza B virus use in .

The present disclosure provides a method for joint detection of novel coronavirus, influenza A virus and influenza B virus, including:

A) Use the immunochromatographic detection reagent strip or the immunochromatographic detection kit according to any of the above to detect novel coronavirus, influenza A virus and influenza B virus in the subject's sample ;and

B) Analyze the test results.

The present disclosure provides a method for diagnosing subjects infected with diseases related to novel coronavirus, influenza A virus and influenza B virus, comprising:

A) Use the immunochromatographic detection reagent strip described above or the immunochromatographic detection kit described above to detect the new coronavirus, influenza A virus and type B in the subject's sample influenza virus; and

B) Analyze the test results.

In some embodiments, the novel coronavirus-related diseases include: fever, dry cough, severe acute respiratory syndrome, shortness of breath, diarrhea, pneumonia;

The diseases related to influenza A virus include: influenza A H1N1 is an acute respiratory infectious disease, influenza A H2N2, and influenza A H3N2.

Description of drawings

In order to more clearly illustrate the specific embodiments of the present disclosure or the technical solutions in the prior art, the following will briefly introduce the drawings that need to be used in the description of the specific embodiments or the prior art. Obviously, the accompanying drawings in the following description The drawings are some implementations of the present disclosure, and those skilled in the art can also obtain other drawings according to these drawings without creative work.

Figure 1 is a schematic structural diagram of the immunochromatographic detection reagent strip provided in Example 1 of the present disclosure, wherein the arrow indicates the direction of chromatography;

Figure 2 is a schematic diagram of the structure of the immunoassay kit provided in Example 1 of the present disclosure, wherein, A represents: influenza A virus detection line, B represents: influenza B virus detection line, C represents: quality control line, SARS CoV-2 represents: COVID-19 testing line.

Reference signs: 100-reagent strip, 110-bottom plate, 120-sample pad, 140-marker binding pad, 160-chromatographic reaction membrane, 180-absorbent paper, 162-detection line, 164-quality control line.

Definition of Terms:

As used herein, the term "quality control line" refers to: the "C (Control) line" on the reagent strip, which is used to indicate whether the quality of the reagent strip is passed and whether the test result is valid. If there is no quality control line in the test result, it means that the test result is invalid.

As used herein, the term "subject" refers to a vertebrate, preferably a mammal, most preferably a human. Mammals include, but are not limited to, rodents, apes, humans, livestock, sport animals, and pets. Also included are tissues, cells and progeny of biological entities obtained in vivo or cultured in vitro.

As used herein, the term "antibody" refers to a protective protein produced by the body due to stimulation by an antigen. It is understood that, herein, the term "specific protein" may refer to an antibody and/or include a combination of two or more antibodies.

As used herein, the term "sensitivity" refers to the positive coincidence rate between the detection results of the detection method of the present disclosure and the detection results of the fluorescent PCR method in a statistical sense.

Detailed ways

The technical solution of the present disclosure will be clearly and completely described below in conjunction with the embodiments. Apparently, the described embodiments are part of the embodiments of the present disclosure, but not all of the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present disclosure.

The present disclosure provides an immunochromatographic detection reagent strip, the reagent strip includes a bottom plate 110, on which a sample pad 120, a marker binding pad 140, a chromatographic reaction membrane 160 and an absorbent paper 180 are arranged;

Wherein, the marker binding pad is adsorbed with specific protein complexes, and the specific protein complexes are mainly obtained by coupling fluorescent microspheres, latex, colloidal gold or colloidal carbon with specific proteins; the specific proteins include Novel coronavirus N protein, influenza A virus NP protein and influenza B virus NP protein;

A detection line is provided on the chromatographic reaction membrane, and the detection line contains antibodies to novel coronavirus N protein, influenza A virus NP protein, and influenza B virus NP protein.

The present disclosure provides an immunochromatographic detection reagent strip, the reagent strip includes a bottom plate 110, on which a sample pad 120, a marker binding pad 140, a chromatographic reaction membrane 160 and an absorbent paper 180 are arranged;

Wherein, the marker binding pad 140 is adsorbed with a specific protein complex, and the specific protein complex is mainly obtained by coupling fluorescent microspheres, latex, colloidal gold or colloidal carbon with specific proteins; the specific protein Including the first antibody of novel coronavirus N protein, the first antibody of influenza A virus NP protein and the first antibody of influenza B virus NP protein, which are used to bind the new coronavirus N protein, influenza A virus NP protein and influenza B virus NP protein antigen;

Chromatographic reaction membrane 160 is provided with detection line 162, and described detection line 162 contains novel coronavirus N protein second antibody, influenza A virus NP protein second antibody, influenza B virus NP protein second antibody, in order to combine Antigens of novel coronavirus N protein, influenza A virus NP protein and influenza B virus NP protein.

It is believed that, without being bound by theory, the sample to be tested is firstly contacted with the marker binding pad 140, and the viral antigen first interacts with the first antibody of the novel coronavirus N protein, the first antibody of the NP protein of the influenza A virus, and the first antibody of the influenza B virus. The first antibody of the virus NP protein specifically binds, and then the new coronavirus antigen, influenza A virus antigen, and influenza B virus antigen in the sample to be tested bind to the second antibody of the new coronavirus N protein of the detection line, and the Influenza virus NP protein secondary antibody, influenza B virus NP protein secondary antibody. Among them, the double-antibody sandwich method is adopted, the first antibody and the second antibody respectively act on different epitopes of the antigen, and the first antibody, the second antibody and the antigen form a sandwich structure.

In an optional embodiment of the present disclosure, when the specific protein complex is mainly obtained by coupling latex with specific protein, the detection lines of influenza A and influenza B positive samples show blue, and the positive samples of novel coronavirus The detection line is displayed in red, which is easy to identify with the naked eye. The immunochromatographic detection reagent strip provided by the present disclosure, the reagent strip includes a bottom plate 110, on which a sample pad 120, a marker binding pad 140, a chromatographic reaction membrane 160 and an absorbent paper 180 are arranged; wherein the marker The binding pad 140 is adsorbed with specific protein complexes, which are mainly obtained by coupling fluorescent microspheres, latex, colloidal gold or colloidal carbon with specific proteins; the specific proteins include novel coronavirus N protein The first antibody, the first antibody of influenza A virus NP protein and the first antibody of influenza B virus NP protein; the chromatographic reaction membrane 160 is provided with a detection line 162, and the detection line 162 contains the first antibody of novel coronavirus N protein Secondary antibody, Influenza A virus NP protein secondary antibody, Influenza B virus NP protein secondary antibody. The above-mentioned detection reagent strip adopts immunochromatography technology, uses the new coronavirus N protein, influenza A virus NP protein and influenza B virus NP protein as detection antigens, and uses fluorescent microspheres, latex, colloidal gold or colloidal carbon and specific proteins Conjugation yields specific protein complexes, which are subsequently tested on patient nasopharyngeal swabs, oropharyngeal swabs, or saliva. The above-mentioned reagent strip has the advantages of high sensitivity, high specificity and quantitative detection, and avoids missed detection of weak positive samples and false positive samples to the greatest extent. At the same time, the immunochromatographic method used in the present disclosure is rapid in detection, and the result can be obtained within 15 minutes, and can be operated on site with simple training.

It should be noted that the above-mentioned reagent strips of the present disclosure adopt immunochromatography technology, and according to the actual application situation, latex method, fluorescence method, colloidal gold method or colloidal carbon method can be used to convert the N protein of the new coronavirus, type A and type B influenza virus As a detection antigen, the NP protein can be used to detect biological samples of suspected novel coronavirus or influenza A and B virus infections.

In an optional embodiment of the present disclosure, the amino acid sequence of the novel coronavirus N protein is shown in SEQ ID NO: 01, as follows:

The amino acid sequence of the influenza A virus NP protein is shown in SEQ ID NO:02, as follows:

The amino acid sequence of the influenza B virus NP protein is shown in SEQ ID NO:03, as follows:

It should be noted that the above amino acid sequences of SEQ ID NO:01, SEQ ID NO:02 and SEQ ID NO:03 in this disclosure may have mutations at individual points, and mutations at high-frequency mutation sites of amino acid sequences also occur in this disclosure. within the scope of the claims. Because there are various mutants in the amino acid sequence, but its conserved region is the main site for binding to our antibody. Amino acid sequences with mutations at the above-mentioned high-frequency mutation sites should also be within the protection scope of the present disclosure. For example, mutations in the N protein of the new coronavirus: R203K, G204R.

In an optional embodiment of the present disclosure, the concentration of the second antibody to the novel coronavirus N protein in the detection line 162 is 0.1-1 mg/mL;

The concentration of the second antibody to the influenza A virus NP protein in the detection line 162 is 0.1-1 mg/mL;

The concentration of the second antibody to the NP protein of influenza B virus in the detection line 162 is 0.1-1 mg/ml.

In an optional embodiment of the present disclosure, the concentration of the specific protein complex in the marker-binding pad 140 is 0.001-0.01 mg/mL.

In some embodiments, the novel coronavirus N protein first antibody and the second antibody are each independently selected from: novel coronavirus N protein polyclonal antibody (mouse source), novel coronavirus N protein monoclonal antibody (mouse source), Any one of the polyclonal antibody to the new coronavirus N protein (rabbit source) and the monoclonal antibody to the new coronavirus N protein (rabbit source).

In some embodiments, the first antibody to the NP protein of influenza A virus and the second antibody are each independently selected from: polyclonal antibody to NP protein of influenza A virus (mouse origin), monoclonal antibody to NP protein of influenza A virus (mouse) source), influenza A virus NP protein polyclonal antibody (rabbit source), any one of influenza A virus NP protein monoclonal antibody (rabbit source).

In some embodiments, the first antibody to the NP protein of influenza B virus and the second antibody are each independently selected from: polyclonal antibody to NP protein of influenza B virus (mouse origin), monoclonal antibody to NP protein of influenza B virus (mouse origin) source), influenza B virus NP protein polyclonal antibody (rabbit source), any one of influenza B virus NP protein monoclonal antibody (rabbit source).

In some embodiments, antibodies are obtained by:

(1) Using gene cloning technology, PCR amplifies the genes encoding novel coronavirus antigen N protein, influenza A virus NP protein, and influenza B virus NP protein; amino acid sequences such as SEQ ID NO:01, SEQ ID NO:02, Shown in SEQ ID NO:03.

(2) The above gene was transformed into Escherichia coli to express the novel coronavirus antigen N protein.

(3) Using the above-mentioned novel coronavirus antigen N protein, influenza A virus antigen NP protein and influenza B virus antigen NP protein to prepare novel coronavirus N protein antibody, influenza A virus NP protein antibody and B Antibody to influenza virus NP protein.

In an optional implementation manner of the present disclosure, the chromatographic reaction membrane 160 is disposed in the middle of the bottom plate 110 . The chromatographic reaction membrane 160 includes a proximal end (ie, the upper end in the chromatographic direction) and a distal end (ie, the lower end in the chromatographic direction). The marker binding pad 140 and the sample pad 120 are lapped on one end (the proximal end) of the chromatography reaction membrane 160 in turn, and the absorbent paper 180 is lapped on the other end (the far side end) of the chromatography reaction membrane 160 superior.

In an optional implementation manner of the present disclosure, a quality control line 164 is also provided on the chromatographic reaction membrane 160 .

In an optional embodiment of the present disclosure, when the specific protein complex is mainly obtained by coupling fluorescent microspheres with specific proteins, the LOD value for the new coronavirus delta strain is 20TCID ₅₀ /mL, corresponding to The nucleic acid Ct value is 34.97; the LOD value for the original strain of the new coronavirus is 20TCID ₅₀ /mL, corresponding to the nucleic acid Ct value of 34.64;

Alternatively, when the specific protein complex is mainly obtained by coupling latex with specific protein, the LOD value for the new coronavirus delta strain is 40TCID ₅₀ /mL, corresponding to the nucleic acid Ct value of 33.60; for the new coronavirus original The LOD value of the strain was 40TCID ₅₀ /mL, corresponding to the nucleic acid Ct value of 33.72;

Alternatively, when the specific protein complex is mainly obtained by coupling colloidal gold and specific protein, the LOD value for the new coronavirus delta strain is 80TCID ₅₀ /mL, corresponding to the nucleic acid Ct value of 32.35; for the new coronavirus The LOD value of the original strain was 80TCID ₅₀ /mL, corresponding to the nucleic acid Ct value of 32.61.

As an optional implementation mode, the new coronavirus clinical sample testing status of the new coronavirus/influenza A virus/influenza B virus one-card three-test antigen detection kit (fluorescence method, latex method) provided by the present disclosure is as follows: :

When Ct≤38, the positive coincidence rate of the new coronavirus/influenza A virus/influenza B virus one-card three-test antigen detection kit (fluorescence method) to the clinical positive samples of new coronavirus: 96.98%; negative Compliance rate: >99%; overall compliance rate: 98.21%;

When Ct≤35, the positive coincidence rate of the new coronavirus/influenza A virus/influenza B virus one-card three-test antigen detection kit (fluorescence method) to the clinical positive samples of new coronavirus: 97.96%; negative Compliance rate: >99%; Overall compliance rate: 98.85%.

When Ct≤38, the positive coincidence rate of the new coronavirus/influenza A virus/influenza B virus one-card three-test antigen detection kit (latex method) provided by the disclosure to the clinical positive samples of new coronavirus: 95.47%; negative Compliance rate: >99%; overall compliance rate: 97.42%;

When Ct≤35, the positive coincidence rate of the new coronavirus/influenza A virus/influenza B virus one-card three-test antigen detection kit (latex method) provided by the disclosure to the clinical positive samples of new coronavirus: 96.43%; negative Compliance rate: >99%; Overall compliance rate: 98.16%.

In an optional embodiment of the present disclosure, when the specific protein complex is mainly obtained by coupling latex with specific protein, the detected LOD value for influenza A virus is 1.5×10 ³ TCID ₅₀ /mL, and for influenza B The detected LOD value of influenza virus was 2×10 ⁴ TCID ₅₀ /mL.

As an optional embodiment, the LOD value of the above-mentioned latex method reagent strip for detecting influenza A virus is 1.5×10 ³ TCID ₅₀ /mL, the detection sensitivity of clinical samples is 90.00%, the specificity is >99%, and the accuracy is 98.57%; The LOD value of the latex method reagent strip for detecting influenza B virus was 2×10 ⁴ TCID ₅₀ /mL, the sensitivity was 92.86%, the specificity was >99%, and the accuracy was 98.44%.

According to one aspect of the present disclosure, an immunoassay kit includes the above-mentioned immunochromatographic detection reagent strip.

The immunoassay kit provided by the present disclosure, the kit includes the above-mentioned immunochromatography detection reagent strip. Determined by the characteristics of the above-mentioned reagent strips, the disclosed kit has the advantages of high sensitivity, high specificity, and quantitative detection, and can quickly detect novel coronavirus, influenza A virus, and influenza B virus antigens by adding a sample. At the same time, the missed detection of weak positive samples and the false detection of false positive samples can be avoided to the greatest extent.

Optionally, the latex method novel coronavirus/influenza A virus/influenza B virus antigen one-card-three test reagent Influenza A and influenza B positive sample T line shows blue, and the new coronavirus positive sample T line shows Red, easy to identify with the naked eye.

According to one aspect of the present disclosure, an application of the above-mentioned immunochromatographic detection reagent strip or the above-mentioned immunochromatographic detection kit in the preparation of a joint detection product for novel coronavirus, influenza A virus and influenza B virus.

The above-mentioned immunochromatographic detection reagent strips or the above-mentioned immunochromatographic detection kits provided in the present disclosure can be widely used in the preparation process of joint detection products for novel coronavirus, influenza A virus and influenza B virus.

In one or more embodiments, the sample is dropped onto an immunochromatographic detection reagent strip to detect viruses. After the sample is dropped on the sample pad 120 , the antigen in the sample runs toward the absorbent paper 180 through the suction of the absorbent paper 180 , and binds to the primary antibody on the marker-binding pad 140 . Then, the combination of the antigen and the first antibody continues to run on the plate, moves to the detection line 162, combines with the second antibody coated with anti-"antigen" on the detection line 162 and develops color, wherein the first antibody and the second antibody Acting on different epitopes of the antigen respectively, the first antibody, the second antibody and the antigen form a sandwich structure.

One embodiment of the present disclosure discloses the use of an immunochromatographic detection reagent strip or an immunochromatographic detection kit in joint detection of novel coronavirus, influenza A virus and influenza B virus.

One embodiment of the present disclosure discloses a method for joint detection of novel coronavirus, influenza A virus and influenza B virus, including:

A) Use immunochromatographic detection reagent strips or immunochromatographic detection kits to detect novel coronavirus, influenza A virus and influenza B virus in the subject's samples; and

B) Analyze the test results.

One embodiment of the present disclosure discloses a method for diagnosing subjects infected with diseases related to novel coronavirus, influenza A virus and influenza B virus, including:

A) using an immunochromatographic detection reagent strip or an immunochromatographic detection kit to detect novel coronavirus, influenza A virus and influenza B virus in the subject's sample; and

B) Analyze the test results.

In an optional embodiment, diseases related to the novel coronavirus include: fever, dry cough, severe acute respiratory syndrome, shortness of breath, diarrhea, pneumonia;

The diseases related to influenza A virus include: influenza A H1N1 is an acute respiratory infectious disease, influenza A H2N2, and influenza A H3N2.

The reagent strip provided by the present disclosure has the advantages of high sensitivity, high specificity and quantitative detection, and avoids missed detection of weak positive samples and false positive samples to the greatest extent. At the same time, the immunochromatographic method used in the present disclosure is rapid in detection, and the result can be obtained within 15 minutes, and can be operated on site with simple training.

In the immunochromatographic detection reagent strip provided by the present disclosure, the reagent strip includes a bottom plate on which a sample pad, a marker binding pad, a chromatographic reaction membrane, and an absorbent paper are arranged; wherein, the marker binding pad is adsorbed with a specific Protein complexes, the specific protein complexes are mainly obtained by coupling fluorescent microspheres, latex, colloidal gold or colloidal carbon with specific proteins; the specific proteins include the first antibody of the new coronavirus N protein, influenza A Viral NP protein first antibody and influenza B virus NP protein first antibody; a detection line is provided on the chromatographic reaction membrane, and the detection line contains the new coronavirus N protein second antibody, influenza A virus NP protein first antibody Secondary antibody, influenza B virus NP protein secondary antibody. The above-mentioned detection reagent strip adopts immunochromatography technology, uses the new coronavirus N protein, influenza A virus NP protein and influenza B virus NP protein as detection antigens, and uses fluorescent microspheres, latex, colloidal gold or colloidal carbon and specific proteins Conjugation yields specific protein complexes, which are then tested on patient nasopharyngeal swabs, oropharyngeal swabs, or saliva. The above-mentioned reagent strip has the advantages of high sensitivity, high specificity and quantitative detection, and avoids missed detection of weak positive samples and false positive samples to the greatest extent. At the same time, the immunochromatographic method used in the present disclosure is rapid in detection, and the result can be obtained within 15 minutes, and can be operated on site with simple training.

The immunoassay kit provided by the present disclosure, the kit includes the above-mentioned immunochromatography detection reagent strip. Determined by the characteristics of the above-mentioned reagent strips, the disclosed kit has the advantages of high sensitivity, high specificity, and quantitative detection, and can quickly detect novel coronavirus, influenza A virus, and influenza B virus antigens by adding a sample. At the same time, the missed detection of weak positive samples and the false detection of false positive samples can be avoided to the greatest extent.

The above-mentioned immunochromatographic detection reagent strips or the above-mentioned immunochromatographic detection kits provided in the present disclosure can be widely used in the preparation process of joint detection products for novel coronavirus, influenza A virus and influenza B virus.

The technical solution of the present disclosure will be further described below in combination with embodiments.

Acquisition of sensitivity, specificity, and precision:

The data relating to sensitivity (i.e. positive coincidence rate), specificity (i.e. negative coincidence rate) and precision (i.e. overall coincidence rate) in the context of the present disclosure are obtained from the following table and the following formula:

Among them, A, B, C, and D represent the test results.

Sensitivity=A/(A+C)*100% (Formula 1)

Specificity = B/(B+D)*100% (Formula 2)

Accuracy = (A+D)/(A+B+C+D)*100% (Formula 3)

Antibody preparation:

The novel coronavirus N protein monoclonal antibody can be prepared by the following:

Reagents required:

The mouse myeloma cell line SP2/0 is preserved by our company;

BALB/c mice, provided by Shanghai Slack Animal Center;

PEG1500, hypoxanthine, thymine, aminopterin, and DMSO were purchased from Sigma;

RPMI1640 basal medium was purchased from Gibco;

Fetal bovine serum was purchased from LONSASCIENCESRL;

Monoclonal antibody preparation:

According to the conventional hybridoma preparation method, 6-week-old female Balb/C mice (Shanghai Slack Animal Center) were selected, and the target protein was subcutaneously injected with an immunization dose of 100 μg/mouse, and an equal amount of Freund's complete adjuvant was used for initial immunization. , Immunization was boosted with the same amount of incomplete Freund's adjuvant, boosted immunization 4 times, immunization interval was 2 weeks, and 100 μg protein was injected intravenously 3 days before fusion. Collect splenic B cells from immunized mice and mouse myeloma cell lines (Sp2/0-Ag14, Sp2/0) for cell fusion with PEG1500, and the resulting hybridoma cells are obtained through ELISA detection, cloning operation, ascites induction and purification monoclonal antibody.

The above methods are only exemplary, and the antibodies herein can be obtained by methods known in the art, or obtained commercially.

Embodiment 1 prepares immunochromatography detection reagent strip:

As shown in Figure 1, a kind of immunochromatographic detection reagent strip, the preparation method of described reagent strip comprises the following steps:

Coupling fluorescent microspheres, latex, colloidal gold or colloidal carbon with specific proteins to obtain specific protein complexes, and adsorbing the specific protein complexes on the marker binding pad 140, wherein the specific protein complexes contain Novel coronavirus N protein primary antibody (material code: N26), influenza A virus NP protein primary antibody (material code: FA6), influenza B virus NP protein primary antibody (material code: FB14).

The detection line 162 will contain the second antibody of novel coronavirus N protein (material code: N34), the second antibody of influenza A virus NP protein (material code: FA12), the second antibody of type B influenza virus NP protein (material code: The chromatographic reaction membrane 160 of FB11); The marker binding pad 140 prepared above, and the sample pad 120 and the absorbent paper 180 are lapped and adhered to the PVC backboard (ie the bottom plate 110);

Wherein, the chromatographic reaction membrane 160 includes a proximal end (ie, the upper end in the chromatographic direction) and a distal end (ie, the lower end in the chromatographic direction). The sample pad 120 and the quantum dot marker binding pad 140 are lapped successively on one end (the proximal end) of the chromatography reaction membrane 160, and the absorbent paper 180 is lapped on the other end (the far side end) of the chromatography reaction membrane 160, and then adjusted The parameters of the strip cutting machine are to cut into strips and cut into test strips with a width of 3-4mm.

As shown in FIG. 2 , an immunoassay kit, the immunoassay kit includes the above-mentioned immunochromatographic detection reagent strip.

Example 2 Sensitivity test of novel coronavirus/influenza A virus/influenza B virus antigen one card three test kit to novel coronavirus antigen:

Take 3×10 ⁵ TCID ₅₀ /ml samples of the original strain of novel coronavirus and inactivated virus samples from Delta (source of original strain and virus samples: P3 Laboratory of State Key Laboratory of Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital of Zhejiang University School of Medicine ), diluted 10-fold with PBS buffer, and then serially diluted 2-fold, for a total of 11 dilutions. All dilution gradient samples were checked with the fluorescent PCR detection kit of Daan Gene Company, and the operation was carried out according to the instructions to determine the corresponding Ct value of each sample.

Take 300 μL samples of each dilution gradient and 300 μL sample detection kit buffer and mix 5 times. After standing for 2 minutes, take 90 μL of the above buffer-treated samples and add them to the reagent injection wells. Each diluted sample and each reagent are repeated for 1 person. Take the reading after 15 minutes of dropping the sample, use the instrument to read the fluorescence method, and use the color card to read the latex method and colloidal gold method. In this example, the kits prepared in Example 1 were used to test the novel coronavirus/influenza A virus/influenza B virus antigen in one card and three tests by fluorescence method, latex method and colloidal gold method respectively, and the reagents were selected from fluorescence method, latex method and The colloidal gold method 2019-nCoV antigen one card single test reagent was used as the control reagent.

For the original strain of the novel coronavirus, the latex method, the fluorescence method and the colloidal gold method have one card with three tests (that is, one reagent strip detects three viruses at the same time) and one card with a single test for the new crown antigen reagent. The sensitivity is similar, and the corresponding nucleic acid detection Ct value is 33.72. , 34.64, and 32.61; for the new coronavirus delta strain, the latex method, fluorescence method, and colloidal gold method have similar sensitivities to one-card three-test and one-card single-test new crown antigen reagents, corresponding to nucleic acid detection Ct values of 33.60, 34.97 and 32.35, respectively. The test results are shown in Table 1 and Table 2:

Table 1: Sensitivity test of the original strain of new coronavirus/influenza A virus/influenza B virus antigen one card three test reagents:

Wherein, "+" means: positive, "-" means negative, and the symbols in Table 2 below have the same meaning as Table 1.

Table 2: Sensitivity test of new coronavirus/influenza A virus/influenza B virus antigen one card three test reagents for new coronavirus delta strain:

From the above, it can be seen that the fluorescence method, latex method and colloidal gold method of the present disclosure have the same sensitivity for one card three tests and one card single test for the new crown antigen reagent. For the Wuhan original strain and the Indian mutant strain, the sensitivity of the new crown antigen latex method reagent is higher than that of the colloidal gold method , the sensitivity of the fluorescence method is higher than that of the latex method.

Example 3 Novel Coronavirus/Influenza A/Influenza B Antigen One Card Three Test Reagent Influenza A Virus and Influenza B Virus Antigen Sensitivity Test:

Experimental steps:

Take two inactivated virus samples of influenza A virus and influenza B virus respectively (Flu A/GUANGDONG/2019, Flu A/NYMC X-223A, Flu B/Washington/02/2019, Flu B/phuket/3073/ 2013), the initial concentration of Flu A was 7.5×10 ⁵ TCID ₅₀ /ml, and the initial concentration of Flu B was 1.0×10 ⁶ TCID ₅₀ /ml.

Dilute each 10 times with normal saline to make 500ul, then dilute 8 concentrations with 5 times ratio of test buffer, that is, dilute the original solution by 10, 50, 2.5×10 ² , 1.25×10 ³ , 6.25×10 ³ , 3.125 ×10 ⁴ , 1.5625×10 ⁵ and 7.8125×10 ⁵ times, a total of 4×8=32 different concentrations of inactivated virus sample solutions to be tested; take out the new coronavirus/influenza A virus/influenza B virus antigen joint detection reagent 100ul of each of the 8 concentrations of the above-mentioned 4 strains of inactivated influenza virus were loaded, and the interpretation was performed after 15 minutes.

Record the lowest detection concentration that can be interpreted as positive at the end, and according to the detection results, set 3 concentrations of 2 times the ratio near this concentration, and repeat each concentration 3 times to ensure that at least one concentration is positive for three repetitions, and at least one concentration The three test repetitions were all negative, and the concentration that could be detected positive in all three repetitions was set as the LOD. According to the LOD obtained in the above steps, use the detection buffer (buffer) (composed of PBS and TWEEN20 volume ratio: 200:1) to dilute the 4 strains of inactivated influenza virus to their respective LOD, each diluted 3ml; take out the example 1 Novel coronavirus/influenza A virus/influenza B virus antigen combined detection kit, respectively detect 4 strains of inactivated influenza virus solutions diluted to their respective LODs, each solution is repeated 20 times, and interpreted separately. The test results are shown in Table 3.

Table 3: Fluorescence method and latex method Novel coronavirus/influenza A virus/influenza B virus antigen one card three test reagents Influenza A virus and influenza B virus LOD test:

Further, the LOD concentration of inactivated virus samples of influenza A virus strain Flu A/GUANGDONG/2019 and influenza B virus strain Flu B/Washington/02/2019 was taken to make mixed standard 1, and influenza A virus strain Flu A/ The LOD concentration of NYMC X-223A and influenza B virus strain Flu B/phuket/3073/2013 inactivated virus samples was prepared as a mixed standard 2, and this product was compared with Japan's competitor products (QuickNavi-Flu A&B, latex method) experiment. According to the sample volume and running time of the respective product manuals, test and interpret respectively, and judge the brightness of the ribbon according to the color chart, and the results are shown in Table 4. Experimental conclusion: The experimental data of this example proves that the latex method new coronavirus/influenza A virus/influenza B virus antigen combined detection reagent product provided by the present disclosure has both the detection sensitivity of influenza A virus and the detection sensitivity of influenza B virus. It is about 1 times higher than that of Shengyan's competing products (QuickNavi-Flu A&B, latex method). The test results are shown in Table 4.

Table 4: New coronavirus/influenza A virus/influenza B virus antigen one card three test reagent (latex method) and Sanken influenza A virus/influenza B virus antigen one card dual test reagent (QuickNavi-Flu A&B, latex method) method) sensitivity comparison:

Among them, "+" means: weakly positive, "++" means: positive, and "-" means negative.

Example 4 Multiple Influenza A/Influenza B Virus Subtype Antigen Detection Limit Test:

Fluorescence method, latex method and colloidal gold method The limit of detection (LOD) of influenza A virus and influenza B virus in the 2019-nCoV/Influenza A virus/Influenza B virus antigen one-card three test reagent is a gradient using virus samples Measured in diluent.

In this embodiment, inactivated sample stock solutions of 10 types of influenza A virus and 7 types of influenza B virus were selected for this test. The initial material concentration was 3.0×10 ⁵ TCID ₅₀ /mL. The initial detection range of the samples was determined using three PBS buffer gradient dilution series, and then the LOD was obtained by further refining the test through a 2-fold dilution series.

Mix 3000 μL samples of each dilution gradient with 3000 μL sample detection kit buffer, let stand for 2 minutes, then take 90 μL of the above-mentioned buffer-treated samples and add them to the reagent injection wells, and repeat 1 person for each diluted sample and each reagent. After dropping the sample for 15 minutes, take a reading, use an instrument for the fluorescence method, and use a color card for the latex method and colloidal gold method. The results are shown in Table 5. The detection limit of the detection reagent provided by the disclosure is about 1.5×10 ³ for influenza A virus TCID ₅₀ /mL, the detection limit of influenza B virus is about 2.0×10 ⁴ TCID ₅₀ /mL.

Table 5: Antigen detection limit test results of various influenza A/influenza B virus subtypes:

Embodiment 5 multiple novel coronavirus/influenza A virus/influenza B virus antigen cross-reaction test:

In order to verify the cross-reactivity of the influenza A virus/influenza B virus part in the new coronavirus/influenza A virus/influenza B virus antigen one-card three test reagent, this embodiment designs experiments for multiple subtypes of A Cross-reactivity was assessed between Type 2 and Type B influenza virus strains.

Take 500 μL of the sample solution of each sample and 500 μL of the buffer solution of the sample detection kit and mix it. After standing for 2 minutes, take 90 μL of the above-mentioned buffer-treated sample and add it to the reagent injection hole. Repeat for 3 people for each diluted sample and each reagent. Take the reading after 15 minutes of dropping the sample, use the instrument to read the fluorescence method, and use the color card to read the latex method and colloidal gold method. The reading results are shown in Table 6, and the test results showed no cross-reaction.

Table 6: Antigen cross-reactivity test results of various influenza A/Influenza B virus subtypes:

It should be noted that, in the test results, the reagent strips used for coupling specific protein complexes with latex (latex method), fluorescent microspheres (fluorescence method), colloidal gold (colloidal gold method) and specific proteins were obtained through The results of the reagent strips prepared by the three methods are consistent and there is no difference at all. The same is true in Tables 7 and 8 below.

Example 6 Crossover and Interference Test of Common Respiratory Tract Pathogens of Novel Coronavirus/Influenza A/Influenza B Virus Antigen Detection Kit:

This example designs an experiment to study the cross-interference and non-specific interference of the latex method, fluorescence method, and colloidal gold method for novel coronavirus/influenza A/influenza B virus antigen detection reagents provided by this disclosure.

Cross-interference experiment design for common respiratory pathogens: use inactivated positive samples of novel coronavirus, influenza A virus and influenza B virus for cross-interference test, and confirm the following 12 virus samples (1.0×10 ⁵ Pfu/mL), 8 There is no cross-interference between bacteria (1.0×10 ⁶ Pfu/mL), Chlamydia pneumoniae (1.0×10 ⁶ Pfu/mL) and Mycoplasma pneumoniae (1.0×10 ⁶ Pfu/mL) and this reagent.

Operation steps: Take 500 μL of the sample solution of each sample and 500 μL of the buffer solution of the sample detection kit and mix it. After standing for 2 minutes, take 90 μL of the above buffer solution-treated sample and add it to the reagent injection hole. Repeat for 1 person for each diluted sample and each reagent. Take the reading after 15 minutes of dropping the sample, use the instrument to read the fluorescence method, and use the color card to read the latex method and colloidal gold method. Table 7 shows the cross-interference and interference test results of common respiratory pathogens in the novel coronavirus/influenza A/influenza B virus antigen detection kit.

Table 7: Influenza A/Influenza B virus multi-subtype antigen cross-interference test results (latex method, fluorescence method, colloidal gold method)

Example 7 Crossover and interference test of common non-specific substances in the respiratory tract of the new coronavirus/influenza A/influenza B virus antigen detection kit:

Cross-interference experimental design of common non-specific substances in the respiratory tract: use inactivated positive samples of novel coronavirus, influenza A virus, and influenza B virus for cross-interference test, and confirm that the following 21 sample substances have no cross-interference with this reagent. Take 500 μL of the sample solution of each sample and 500 μL of the buffer solution of the sample detection kit and mix it. After standing for 2 minutes, take 90 μL of the above-mentioned buffer-treated sample and add it to the reagent injection hole. Repeat for 1 person for each diluted sample and each reagent. Take the reading after 15 minutes of dropping the sample, use the instrument to read the fluorescence method, and use the color card to read the latex method and colloidal gold method. According to the test results, at the indicated concentrations, no interference from non-specific substances as shown in Table 8 was observed.

Table 8: Non-specific interference test results of novel coronavirus/influenza A/influenza B virus antigen detection reagents:

Example 8

Novel coronavirus/influenza A/influenza B virus antigen detection kit (fluorescence method, latex method) new coronavirus clinical positive sample test:

In this embodiment, a clinical sample test experiment is designed to verify the performance of the detection reagent provided by the present disclosure.

Novel coronavirus clinical sample testing: a total of 579 samples. 358 clinical samples of patients with new crown virus, including 23 nasopharyngeal swab samples, 93 sputum samples, 236 saliva samples and 6 alveolar lavage fluid samples; 96 new coronavirus negative volunteer saliva samples, negative volunteer throat swab The sub-sample is 125 cases.

Sputum or alveolar lavage fluid sample processing method: After the sputum or alveolar lavage fluid sample is centrifuged at high speed to remove coarse particles, the centrifuged supernatant is mixed with buffer solution 1:1 (v/v); the nasopharyngeal swab is processed Method: take the virus preservation solution containing nasopharyngeal swab, and mix it with the detection buffer 1:1 (v/v); saliva sample processing method: take 100 μL of the saliva sample, and mix it with the detection buffer 1:3 (v/v) Mix well. All samples were checked with the fluorescent PCR detection kit of Daan Gene Company, and the operation was carried out according to the instructions to determine the corresponding Ct value of each sample.

For clinical samples, refer to hospital clinical laboratory diagnostic standards and reagent sensitivity, and determine negative or positive according to the Ct value of fluorescent PCR and the non-amplification curve. With reference to hospital clinical laboratory diagnostic standards and reagent sensitivity, the Ct value of fluorescent PCR > 40 and no amplification curve were judged as negative. The following will be graded and statistically evaluated according to the Ct value results of PCR.

Take 100 μL of the buffer-treated sample prepared in the above steps, and add it dropwise to the sample well of the new coronavirus/influenza A virus/influenza B virus one-card three-test antigen detection reagent (fluorescence method), read after 15 minutes, and the fluorescence Instrument readings.

The test results are shown in Table 9, Table 10 and Table 11.

When Ct≤38, the positive coincidence rate of the new coronavirus/influenza A virus/influenza B virus one-card three-test antigen detection kit (fluorescence method) to the clinical positive samples of new coronavirus: 96.98%; negative Compliance rate: >99%; overall compliance rate: 98.21%;

When Ct≤35, the positive coincidence rate of the new coronavirus/influenza A virus/influenza B virus one-card three-test antigen detection kit (fluorescence method) to the clinical positive samples of new coronavirus: 97.96%; negative Compliance rate: >99%; Overall compliance rate: 98.85%.

When Ct≤38, the positive coincidence rate of the new coronavirus/influenza A virus/influenza B virus one-card three-test antigen detection kit (latex method) provided by the disclosure to the clinical positive samples of new coronavirus: 95.47%; negative Compliance rate: >99%; overall compliance rate: 97.42%;

When Ct≤35, the positive coincidence rate of the new coronavirus/influenza A virus/influenza B virus one-card three-test antigen detection kit (latex method) provided by the disclosure to the clinical positive samples of new coronavirus: 96.43%; negative Compliance rate: >99%; Overall compliance rate: 98.16%.

Table 9-1 (fluorescence method, Ct≤38) novel coronavirus/influenza A virus/influenza B virus one-card three-test antigen detection reagent clinical detection results of new coronavirus:

Positive coincidence rate: 96.98%; Negative coincidence rate: >99%; Overall coincidence rate: 98.21%; 95% confidence interval.

Table 9-2 (latex method, Ct≤38) novel coronavirus/influenza A virus/influenza B virus one-card three-test antigen detection reagent clinical detection results of new coronavirus:

Positive coincidence rate: 95.47%; Negative coincidence rate: >99%; Overall coincidence rate: 97.42%; 95% confidence interval.

Table 10-1, (fluorescence method, Ct≤35) new coronavirus/influenza A virus/influenza B virus one-card three-test antigen detection reagent clinical detection results of new coronavirus:

Positive coincidence rate: 97.96%; Negative coincidence rate: >99%; Overall coincidence rate: 98.85%; 95% confidence interval.

Table 10-2, (latex method, Ct ≤ 35) new coronavirus/influenza A virus/influenza B virus one-card three-test antigen detection reagent new coronavirus clinical test results:

Positive coincidence rate: 96.43%; Negative coincidence rate: >99%; Overall coincidence rate: 98.16%; 95% confidence interval.

Table 11. (Fluorescence method, latex method) novel coronavirus/influenza A virus/influenza B virus one-card three-test antigen detection kit novel coronavirus clinical new crown patient positive sample detection results (Ct≤38) raw data (fluorescence T/C greater than 0.01 is judged as positive, and latex method color card greater than 3 is judged as positive).

Example 9 Novel coronavirus/influenza A virus/influenza B virus antigen detection kit (latex method) influenza A virus/influenza B clinical positive sample test:

In this embodiment, a clinical sample test experiment is designed to verify the performance of the detection reagent provided by the present disclosure.

Influenza A virus/influenza B virus clinical sample test: a total of 20 cases of influenza A virus positive clinical samples (18 cases of nasopharyngeal swabs, 2 cases of saliva), 28 cases of influenza B virus positive clinical samples (nasopharyngeal swabs 19 cases, 11 cases of saliva). Nasopharyngeal swab sample processing method: take the virus preservation solution containing the nasopharyngeal swab sample, and mix it with the detection buffer 1:1; saliva sample processing method: after the saliva sample is centrifuged at high speed to remove coarse particles, take the centrifuged supernatant and Buffer 1:3 was mixed well. For clinical samples, refer to hospital clinical laboratory diagnostic standards and reagent sensitivity, and determine negative or positive according to the Ct value of fluorescent PCR and the non-amplification curve. Take 100 μL of the buffer-treated sample prepared in the above step, drop it into the sample hole of the new coronavirus/influenza A virus/influenza B virus one-card three-test antigen detection reagent (latex method), read after 15 minutes, and detect The results are shown in Tables 12 and 13.

Table 12. Clinical detection results of influenza A virus with one card and three antigen detection reagents for novel coronavirus/influenza A virus/influenza B virus:

Sensitivity: 90.00% (93.37-96.63%)*; specificity: >99% (98.37-101.63%)*; precision: 98.57% (97.54-100.79%)*; *95% confidence interval.

Table 13. Clinical detection results of influenza B virus with one card and three antigen detection reagents for novel coronavirus/influenza A virus/influenza B virus:

Sensitivity: 92.86% (90.71-95.01%)*; specificity: >99% (97.85-102.15%)*; precision: 98.44% (96.29-100.59%)*; *95% confidence interval.

It can be seen from the above experiments that the sensitivity: 90.00%, specificity: 100.00%, and accuracy: 98.57% for 20 clinical samples of influenza A patients. 28 clinical samples of influenza B patients, sensitivity: 92.86%, specificity: 100.00%, precision: 98.44%.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present disclosure, not to limit them; although the present disclosure has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: It is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the various embodiments of the present disclosure. scope.

Industrial Applicability

The present disclosure provides an immunochromatographic detection reagent strip and a kit containing the same and applications thereof. The reagent strip provided by the present disclosure has the advantages of high sensitivity, high specificity and quantitative detection, and avoids missed detection of weak positive samples and false positive samples to the greatest extent. At the same time, the immunochromatographic method used in the present disclosure is rapid in detection, and the result can be obtained within 15 minutes, and can be operated on site with simple training. The immunoassay kit provided by the present disclosure has the advantages of high sensitivity, high specificity, and quantitative detection. One-time sample addition can quickly detect novel coronavirus, influenza A virus, and influenza B virus antigens, and at the same time avoid The missed detection of weak positive samples and the false detection of false positive samples were eliminated.

The above-mentioned immunochromatographic detection reagent strip or the above-mentioned immunochromatographic detection kit provided in the present disclosure can be widely used in the preparation process of joint detection products for novel coronavirus, influenza A virus and influenza B virus, and has wide application value.

Claims (16)

1. An immunochromatographic detection reagent strip, characterized in that the reagent strip comprises a bottom plate, on which a sample pad, a marker binding pad, a chromatographic reaction membrane and absorbent paper are arranged;

   Wherein, the marker binding pad is adsorbed with specific protein complexes, and the specific protein complexes are mainly obtained by coupling fluorescent microspheres, latex, colloidal gold or colloidal carbon with specific proteins; the specific proteins include Novel coronavirus N protein primary antibody, influenza A virus NP protein primary antibody and influenza B virus NP protein primary antibody, used to bind novel coronavirus N protein, influenza A virus NP protein and influenza B virus NP protein antigen;

   The chromatographic reaction membrane is provided with a detection line, and the detection line contains the second antibody of the novel coronavirus N protein, the second antibody of the NP protein of the influenza A virus, and the second antibody of the NP protein of the influenza B virus, in order to bind the novel coronavirus. Coronavirus N protein, influenza A virus NP protein and influenza B virus NP protein antigen;

   Preferably, when the specific protein complex is mainly obtained by coupling latex with a specific protein, the detection lines of influenza A and influenza B positive samples are blue, and the detection lines of novel coronavirus positive samples are red, which is convenient for visual identification.
2. The immunochromatographic detection reagent strip according to claim 1, wherein the specific protein complex is obtained by coupling a specific protein with fluorescent microspheres, latex, colloidal gold or colloidal carbon.
3. The immunochromatographic detection reagent strip according to claim 1 or 2, wherein the amino acid sequence of the novel coronavirus N protein is as shown in SEQ ID NO:01;

   The amino acid sequence of the influenza A virus NP protein is shown in SEQ ID NO:02;

   The amino acid sequence of the influenza B virus NP protein is shown in SEQ ID NO:03.
4. The immunochromatographic detection reagent strip according to any one of claims 1 to 3, wherein the concentration of the second antibody to the novel coronavirus N protein in the detection line is 0.1 to 1 mg/mL;

   The concentration of the second antibody of influenza A virus NP protein in the detection line is 0.1～1mg/mL;

   The concentration of the second antibody to the influenza B virus NP protein in the detection line is 0.1-1 mg/ml.
5. The immunochromatographic detection reagent strip according to any one of claims 1-4, characterized in that the concentration of the specific protein complex in the marker-binding pad is 0.001-0.01 mg/mL.
6. The immunochromatographic detection reagent strip according to claim 1, wherein the chromatographic reaction membrane is arranged in the middle of the bottom plate, and the marker binding pad and the sample pad are successively overlapped on one end of the chromatographic reaction membrane , the absorbent paper is lapped on the other end of the chromatographic reaction membrane.
7. The immunochromatographic detection reagent strip according to claim 1 or 6, wherein a quality control line is also arranged on the chromatographic reaction membrane.
8. The immunochromatographic detection reagent strip according to claim 1 or 2, characterized in that, when the specific protein complex is mainly obtained by coupling fluorescent microspheres and specific proteins, the LOD for the new coronavirus delta strain The value is 20TCID ₅₀ /mL, corresponding to the nucleic acid Ct value of 34.97; the LOD value for the original strain of the novel coronavirus is 20TCID ₅₀ /mL, corresponding to the nucleic acid Ct value of 34.64;

   Preferably, when the specific protein complex is mainly obtained by coupling latex with specific protein, the LOD value for the new coronavirus delta strain is 40TCID ₅₀ /mL, corresponding to the nucleic acid Ct value of 33.60; for the new coronavirus original strain The LOD value is 40TCID ₅₀ /mL, and the corresponding nucleic acid Ct value is 33.72;

   Preferably, when the specific protein complex is mainly obtained by coupling colloidal gold and specific protein, the LOD value for the new coronavirus delta strain is 80TCID ₅₀ /mL, corresponding to the nucleic acid Ct value of 32.35; for the new coronavirus original The LOD value of the strain was 80TCID ₅₀ /mL, corresponding to the nucleic acid Ct value of 32.61.
9. The immunochromatographic detection reagent strip according to claim 1 or 2, wherein when the specific protein complex is mainly obtained by coupling latex with specific protein, the LOD value for detection of influenza A virus is 1.5×10 ³ TCID ₅₀ /mL, the detection LOD value of influenza B virus is 2×10 ⁴ TCID ₅₀ /mL.
10. An immunoassay kit, characterized in that the kit includes the immunochromatographic detection reagent strip according to any one of claims 1-9.
11. An immunochromatographic detection reagent strip according to any one of claims 1 to 9 or the immunochromatographic detection kit according to claim 10 in the preparation of joint detection of novel coronavirus, influenza A virus and influenza B virus application in the product.
12. An immunochromatographic detection reagent strip according to any one of claims 1 to 9 or an immunochromatographic detection kit according to claim 10 or 11 in joint detection of novel coronavirus, influenza A virus and influenza B use in viruses.
13. An immunochromatographic detection reagent strip according to any one of claims 1 to 9 or an immunochromatographic detection kit according to claim 10 or 11, for joint detection of novel coronavirus, influenza A virus and B virus use in influenza viruses.
14. A method for joint detection of novel coronavirus, influenza A virus and influenza B virus, comprising:

    A) Use the immunochromatographic detection reagent strip according to any one of claims 1 to 9 or the immunochromatographic detection kit according to claim 10 or 11 to detect novel coronavirus, A Influenza Type B and Influenza B viruses; and

    B) Analyze the test results.
15. A method of diagnosing a subject's infection with a disease associated with novel coronavirus, influenza A virus, and influenza B virus, comprising:

    A) Use the immunochromatographic detection reagent strip according to any one of claims 1 to 9 or the immunochromatographic detection kit according to claim 10 or 11 to detect novel coronavirus, A Influenza Type B and Influenza B viruses; and

    B) Analyze the test results.
16. The method according to claim 15, characterized in that,

    The diseases related to the novel coronavirus include: fever, dry cough, severe acute respiratory syndrome, shortness of breath, diarrhea, pneumonia;

    The diseases related to influenza A virus include: influenza A H1N1 is an acute respiratory infectious disease, influenza A H2N2, and influenza A H3N2.

Images