Kit capable of simultaneously detecting mycoplasma pneumoniae / chlamydia pneumoniae / novel coronavirus / respiratory syncytial virus / influenza A virus / influenza B virus

Abstract

The invention relates to the technical field of immunodetection, in particular to a kit capable of simultaneously detecting mycoplasma pneumoniae / chlamydia pneumoniae / novel coronavirus / respiratory syncytial virus / influenza A virus / influenza B virus. The kit disclosed by the invention can be used for simultaneously detecting six pathogenic microorganisms in the respiratory tract, so that the accuracy of clinical respiratory disease examination is improved; the kit is simple in component and easy to produce and prepare; the kit has the advantages of convenience in detection operation, wide application scene, low cost and easiness in commercialization, and fills the technical blank that six pathogenic microorganisms in the respiratory tract cannot be simultaneously and quickly detected in the prior art.

Description

Technical Field

[0001] The present invention relates to the field of immunoassay technology, and in particular to a kit capable of simultaneously detecting Mycoplasma pneumoniae / Chlamydia pneumoniae / new coronavirus / respiratory syncytial virus / influenza A virus / influenza B virus. Background Art

[0002] The combined detection of novel coronavirus (SARS-CoV-2) antigen and other respiratory pathogens such as influenza A and B virus antigens, respiratory syncytial virus antigens, Mycoplasma pneumoniae and Chlamydia pneumoniae is of great significance. First, the combined detection can quickly distinguish different respiratory pathogens, which is of guiding significance for clinical diagnosis and treatment. It can effectively identify the types of pathogens, timely detect mixed infections, and select targeted drug treatments to make treatment more accurate and efficient. Secondly, the novel coronavirus antigen detection is fast and easy to operate. It can be performed without professional laboratories and equipment, and is suitable for large-scale screening in primary medical and health institutions.

[0003] Mycoplasma pneumoniae (MP) and Chlamydia pneumoniae (CP) are the main pathogens of community-acquired pneumonia (CAP). They can cause similar clinical symptoms, but require different detection methods and treatment plans. The significance of the examination lies in early diagnosis, guiding treatment and preventing transmission. The diagnosis of M. pneumoniae and C. pneumoniae infection mainly includes mycoplasma culture, serological methods, molecular diagnosis and rapid antigen detection methods. Among them, serological testing is the main diagnostic method, which assists diagnosis by detecting IgM and IgG antibodies. IgM can usually be detected one week after infection, while the positive rate of IgG antibodies is less than 50% in the acute phase and can reach 82% in the recovery phase. Molecular diagnostic methods such as PCR technology have high sensitivity and specificity, which can shorten the sample turnover time and facilitate clinical diagnosis and treatment. At present, the antigen detection reagents for M. pneumoniae are suitable for early screening because they are easy and fast to use, do not require the guidance of laboratory medical staff, and are more and more widely used. Therefore, the antigen detection of M. pneumoniae and C. pneumoniae also has important diagnostic reference value. Therefore, the examination of M. pneumoniae and C. pneumoniae has important clinical significance for the diagnosis, treatment and control of the spread of pneumonia. Through these tests, doctors can more accurately diagnose the condition, choose appropriate treatment options, and take preventive measures to reduce the spread of the disease.

[0004] Influenza virus antigen detection and novel coronavirus antigen detection have their own focuses and cannot replace each other. Influenza virus antigen detection mainly targets the nucleoprotein antigen of influenza virus, which mainly includes influenza A virus (Flu A) and influenza B virus (Flu B); while novel coronavirus antigen detection generally focuses on detecting N protein. The combined use of these detection methods can improve the detection rate of the disease, identify confirmed patients as much as possible, and is more conducive to the control of the epidemic.

[0005] Respiratory syncytial virus (RSV) belongs to the genus Pneumovirus of the family Paramyxoviridae. It has only one serotype and mainly causes lower respiratory tract infections such as bronchiolitis and pneumonia in infants under 6 months old, and upper respiratory tract infections such as rhinitis and colds in older children and adults. Respiratory syncytial virus (RSV) is a common respiratory pathogen, especially in infants and young children, and is one of the main viral pathogens causing acute lower respiratory tract infections (ALRTI). Clinically, RSV is the most important viral pathogen for acute lower respiratory tract infections in children under 5 years old, and almost every child has been infected with RSV before the age of 2. RSV infection can cause severe respiratory diseases such as bronchiolitis and pneumonia in infants and young children, especially in premature infants, infants with chronic lung disease, complex congenital heart disease or immunodeficiency. Severe RSV infection may cause dysfunction of multiple tissues and organs throughout the body and is closely related to repeated wheezing and asthma in infants and young children in the late stage of infection. In addition, the elderly are also a high-risk group for RSV infection. RSV infection in the elderly may lead to severe lower respiratory tract infections, increase hospitalization rates and mortality, especially in the elderly with chronic cardiopulmonary diseases.

[0006] In summary, the combined detection of novel coronavirus antigens and other respiratory pathogens can guide doctors to carry out antiviral treatment as early as possible, especially within 48 hours after the onset of respiratory infection symptoms. Antiviral treatment can effectively reduce complications and reduce the risk of severe illness. At the same time, it can be used for epidemiological monitoring of influenza, helping to assess the epidemic trend and impact of influenza viruses, which plays an important role in improving detection efficiency, accuracy, and epidemic prevention and control.

[0007] The nucleic acid tests used in existing technologies cannot produce results in a timely manner, the operation requires nucleic acid extraction and nucleic acid detection instruments, the detection process is time-consuming and the fees are high. Some antigen detection reagents have a single detection item and cannot distinguish and identify which respiratory pathogen is infecting, thus failing to achieve the purpose of timely and accurate inspection. Summary of the invention

[0008] In order to solve the defects of the above-mentioned prior art, the present invention provides a kit that can simultaneously detect Mycoplasma pneumoniae / Chlamydia pneumoniae / new coronavirus / respiratory syncytial virus / influenza A virus / influenza B virus, including: a detection reagent strip and a sample diluent; The detection reagent strip includes two independent detection reagent strips, each detection reagent strip contains a reaction membrane and a binding pad, the reaction membrane contains an antigen detection line and a quality control line, and the antigen detection line is coated with a monoclonal antibody; wherein the Mycoplasma pneumoniae antigen detection line, the Chlamydia pneumoniae antigen detection line and the respiratory syncytial virus antigen detection line are located on the same detection reagent strip, and the new coronavirus antigen detection line, the influenza A virus antigen detection line and the influenza B virus antigen detection line are located on the same detection reagent strip; The sample diluent includes Tris solution, PC300, casein, heterophilic antibody blocking agent, S9 and Triton X-100.

[0009] The present invention optimizes the formula and finds that the use of the above sample diluent can improve the virus detection rate and effectively avoid the occurrence of false positives in the test samples, thereby ensuring the accuracy of clinical diagnosis results. At the same time, the present invention also finds that the combination of different types of antigen detection lines on two test strips will significantly affect the accuracy of the test results. Only when the above antigen detection lines are combined can the accuracy of the test results be guaranteed.

[0010] Preferably, the heterophilic antibody blocker is an active blocker.

[0011] In some embodiments, from the sample addition position to the quality control line, a Mycoplasma pneumoniae antigen detection line, a Chlamydia pneumoniae antigen detection line, a respiratory syncytial virus, and a quality control line are sequentially arranged on the reaction membrane of one of the detection reagent strips; a new coronavirus antigen detection line, an influenza A virus antigen detection line, an influenza B virus antigen detection line, and a quality control line are sequentially arranged on the reaction membrane of another detection reagent strip.

[0012] In some embodiments, the coating solution of the antigen detection line includes 0.01~0.1 mol / L Tris buffer, 0.5wt%~2wt% trehalose, 0.01%~0.05% PC300, 0.01wt%~0.1wt% BSA and 0.01~0.1wt% S9.

[0013] In some embodiments, the conjugate pad includes a monoclonal antibody that detects the target antigen and is labeled with a marker.

[0014] In some embodiments, the label is colloidal gold.

[0015] In some embodiments, the solution of the marker-labeled antibody includes: pH 7.5-8.5 (preferably 7.8-8.2), 0.01-0.1 mol / L Tris buffer, 0.05wt%-0.15wt% casein, 0.3-0.8wt% Triton X-100, 0.05-0.15 wt% sucrose, and 0.1-0.3 wt% polyvinylpyrrolidone (PVP-40).

[0016] The use of the above solution to prepare marker-labeled antibodies can further improve the efficiency of antigen-antibody reaction, shorten the detection time, reduce the detection limit, and shorten the detection window period.

[0017] In some embodiments, the quality control line is coated with IgG polyclonal antibodies.

[0018] Preferably, the quality control line is coated with goat anti-mouse IgG polyclonal antibody.

[0019] Specifically, in the test strip containing the Mycoplasma pneumoniae antigen detection line, the Chlamydia pneumoniae antigen detection line and the respiratory syncytial virus antigen detection line, the conjugate pad includes Mycoplasma pneumoniae monoclonal antibody, Chlamydia pneumoniae monoclonal antibody and respiratory syncytial virus monoclonal antibody labeled with a marker (preferably colloidal gold). In the test strip containing the novel coronavirus antigen detection line, the influenza A virus antigen detection line and the influenza B virus antigen detection line, the conjugate pad includes the novel coronavirus monoclonal antibody, the influenza A virus monoclonal antibody and the influenza B virus monoclonal antibody labeled with a marker (preferably colloidal gold).

[0020] Preferably, the Mycoplasma pneumoniae antigen detection line is coated with Mycoplasma pneumoniae monoclonal antibodies at a concentration of 0.8-1.0 mg / mL, preferably 0.9 mg / mL.

[0021] Preferably, the Chlamydia pneumoniae antigen detection line is coated with Chlamydia pneumoniae monoclonal antibodies at a concentration of 0.9-1.1 mg / mL, preferably 1.0 mg / mL.

[0022] Preferably, the respiratory syncytial virus antigen detection line is coated with respiratory syncytial virus monoclonal antibody at a concentration of 0.9-1.2 mg / mL, preferably 1.1 mg / mL.

[0023] Preferably, the novel coronavirus antigen detection line is coated with novel coronavirus monoclonal antibodies at a concentration of 0.8-1.0 mg / mL, with a preferred concentration of 0.9 mg / mL.

[0024] Preferably, the influenza A virus antigen detection line is coated with influenza A virus monoclonal antibodies at a concentration of 1.0-1.3 mg / mL, preferably 1.2 mg / mL.

[0025] Preferably, the influenza B virus antigen detection line is coated with influenza B virus monoclonal antibodies at a concentration of 1.1-1.5 mg / mL, preferably 1.3 mg / mL.

[0026] Preferably, the concentration of the IgG polyclonal antibody is 0.8-1.2 mg / mL, and the preferred concentration is 1.0 mg / mL.

[0027] In some embodiments, the sample diluent includes a Tris solution with a pH of 7.5 to 8.5 and a concentration of 0.01 to 0.1 mol / L, PC300 with a concentration of 0.05% to 0.5%, casein with a concentration of 0.01 wt% to 0.3 wt%, a heterophilic antibody blocker with a concentration of 0.002 wt% to 0.05 wt%, S9 with a concentration of 0.01 wt% to 0.2 wt%, and Triton X-100 with a concentration of 0.1 wt% to 1wt%.

[0028] In some embodiments, the sample diluent includes a Tris solution with a pH of 7.5 to 8.5 and a concentration of 0.01 to 0.1 mol / L, PC300 with a concentration of 0.05% to 0.15%, casein with a concentration of 0.01 wt% to 0.3 wt%, a heterophilic antibody blocker with a concentration of 0.005 wt% to 0.02 wt%, S9 with a concentration of 0.01 wt% to 0.1 wt%, and Triton X-100 with a concentration of 0.1 wt% to 1wt%.

[0029] In some embodiments, the sample diluent includes a Tris solution with a pH of 7.8-8.2 and a concentration of 0.03-0.08 mol / L, PC300 with a concentration of 0.08%-0.12%, casein with a concentration of 0.15 wt%-0.3 wt%, a heterophilic antibody blocker with a concentration of 0.005 wt%-0.015 wt%, S9 with a concentration of 0.03 wt%-0.08 wt%, and Triton X-100 with a concentration of 0.3 wt%-0.8 wt%.

[0030] In some embodiments, the volume of the sample diluent is 500-1000 μL.

[0031] In some embodiments, the reaction membrane is a nitrocellulose membrane.

[0032] In some embodiments, the detection reagent strip comprises a sample pad, a conjugation pad, a reaction membrane, a water absorbent pad and a bottom plate; the sample pad, the conjugation pad, the reaction membrane and the water absorbent pad are sequentially adhered to the bottom plate.

[0033] Furthermore, the present invention provides the use of the kit in preparing a product; the product is used to detect at least one of Mycoplasma pneumoniae, Chlamydia pneumoniae, novel coronavirus, respiratory syncytial virus, influenza A virus, and influenza B virus.

[0034] Preferably, the product is used to simultaneously detect Mycoplasma pneumoniae, Chlamydia pneumoniae, novel coronavirus, respiratory syncytial virus, influenza A virus, and influenza B virus.

[0035] The working principle of the present invention is as follows: the present invention proposes a kit for in vitro qualitative detection of Mycoplasma pneumoniae, Chlamydia pneumoniae, novel coronavirus, respiratory syncytial virus, influenza A virus, and influenza B virus antigens in nasal swabs and nasopharyngeal swab samples, which adopts the process of double antibody sandwich method and can be used for medical institutions and family self-testing. The kit of the present invention comprises two reagent strips, namely Mycoplasma pneumoniae, Chlamydia pneumoniae and respiratory syncytial virus antigen detection reagent strips and novel coronavirus, influenza A virus and influenza B virus antigen detection reagent strips. One sample addition can simultaneously detect Mycoplasma pneumoniae, Chlamydia pneumoniae, novel coronavirus, respiratory syncytial virus, influenza A virus, and influenza B virus antigens present in human nasal swabs, nasopharyngeal swabs, and throat swab samples. The reagent can be stored at room temperature, and the test results can be judged by the naked eye, without the need for complex instrument operation, which greatly relieves the work pressure of medical staff, and sampling and detection also save time and reduce exposure risks. Moreover, the sample collection process of the present invention is simple and easy to operate. Six respiratory pathogens can be detected by sampling once, and the results can be read in 5 to 10 minutes. Compared with existing detection kits, the test results can be read in as fast as five minutes, shortening the detection time.

[0036] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a Mycoplasma pneumoniae / Chlamydia pneumoniae / new coronavirus / respiratory syncytial virus / influenza A virus / influenza B virus antigen combined detection kit, which can simultaneously detect six respiratory pathogenic microorganisms, thereby improving the accuracy of clinical respiratory disease examinations; the kit has simple components and is easy to produce and prepare; the detection operation is convenient, the applicable scenarios are wide, the cost is low, and it is easy to commercialize, filling the technical gap that the existing technology cannot simultaneously and quickly detect six respiratory pathogenic microorganisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of the detection reagent strip of Example 1 of the present invention.

[0038] Figure 2 It is a schematic diagram of the detection reagent strip of Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] If no specific techniques or conditions are specified in the examples, all are carried out according to conventional methods or the techniques or conditions described in the literature in the field, or according to the product instructions. If no manufacturer is specified for the reagents and instruments used, all are conventional products that can be purchased through regular channels.

[0041] Example 1

[0042] This embodiment provides a kit that can simultaneously detect Mycoplasma pneumoniae / Chlamydia pneumoniae / novel coronavirus / respiratory syncytial virus / influenza A virus / influenza B virus, including: a test strip and a sample diluent; a schematic diagram of the test strip is shown in Figure 1 As shown; The detection reagent strip includes two independent detection reagent strips, namely the first detection reagent strip and the second detection reagent strip; each detection reagent strip includes a sample pad, a conjugation pad, a reaction membrane, a water absorbent pad and a bottom plate; the sample pad, the conjugation pad, the reaction membrane and the water absorbent pad are sequentially pasted on the bottom plate; the reaction membrane is a nitrocellulose membrane; the first detection reagent strip includes a first reaction membrane and a first conjugation pad, the first reaction membrane includes Mycoplasma pneumoniae (marked as M line), Chlamydia pneumoniae (marked as C line), respiratory syncytial virus (marked as R line) and a first quality control line (marked as Z line), and the first conjugation pad includes colloidal gold-labeled Mycoplasma pneumoniae monoclonal antibody, Chlamydia pneumoniae monoclonal antibody and respiratory syncytial virus monoclonal antibody; the second detection reagent strip includes a second reaction membrane and a second conjugation pad, the second reaction membrane includes a new coronavirus (marked as S line), influenza A virus (marked as A line), influenza B virus (marked as B line) and a second quality control line (marked as Z line), and the second conjugation pad includes colloidal gold-labeled new coronavirus monoclonal antibody, influenza A virus monoclonal antibody and influenza B virus monoclonal antibody; The specification of the sample diluent is a single-person volume of 600 μL, and the sample diluent includes a Tris solution with a pH of 8.0 and a concentration of 0.05 mol / L, a PC300 with a concentration of 0.1%, a casein with a concentration of 0.2 wt%, a heterophilic antibody blocker with a concentration of 0.01 wt% (Wuhan Yingda Biological, Product No. CN302), a S9 with a concentration of 0.05 wt%, and a Triton X-100 with a concentration of 0.5 wt%.

[0043] The preparation method of the above kit is as follows: 1. Reagent preparation

[0044] 1. Antibodies used for colloidal gold labeling: The brand number of the Mycoplasma pneumoniae monoclonal antibody is Henderson HDS-21214, the brand number of the Chlamydia pneumoniae monoclonal antibody is Maidian C01844M, the brand number of the respiratory syncytial virus monoclonal antibody is Henderson HDS-A5097, the brand number of the new coronavirus monoclonal antibody is Henderson HDS-A5091, the brand number of the influenza A virus monoclonal antibody is Feipeng Bio FLUA-REAB-G1-011, and the brand number of the influenza B virus monoclonal antibody is Feipeng Bio FLUB-REAB-G2-012.

[0045] 2. Gold label pad solution: 0.05 mol / L Tris solution adjusted to pH 8.0 with hydrochloric acid, 0.1 wt% casein, 0.5 wt% Triton X-100, 0.1 wt% sucrose, and 0.2 wt% polyvinylpyrrolidone (PVP-40).

[0046] 3. Coating solution of antigen detection line: 0.05 mol / L Tris buffer, 1 wt% trehalose, 0.03% PC300, 0.05wt% BSA, and 0.05wt% S9.

[0047] 4. Sample diluent: pH 8.0, 0.05 mol / L Tris solution, 0.1% PC300, 0.2 wt% casein, 0.01 wt% heterophilic antibody blocker (Wuhan Yingda Biological, Catalog No. CN302), 0.05 wt% S9 and 0.5 wt% Triton X-100.

[0048] 2. Preparation of test strips

[0049] (1) Add 20 μL / mL 0.1 mol / L potassium carbonate solution to colloidal gold and adjust the pH to 8.2, and mix for 5 min; (2) Add the antibody to the above solution at a ratio of 12 μg / mL colloidal gold, mix and react for 5 minutes; (3) Add 10 wt% BSA solution to the above solution at a ratio of 50 μL / mL colloidal gold, mix and react for 5 min; (4) The above solution was centrifuged at 9000 rpm, 10 min, and 6° C. using a high-speed refrigerated centrifuge, and the supernatant was discarded to obtain the precipitate, which was re-dissolved with a gold label pad solution to 20% of the original volume to obtain a labeled colloidal gold solution; (5) Setting the gold spraying instrument parameters to 5 μL / cm, spraying the labeled colloidal gold solution onto the conjugate pad, and placing it in a 37°C constant temperature drying oven to dry for 4 hours to obtain a gold-labeled conjugate pad; (6) Paste the nitrocellulose membrane (NC membrane model: Sartorius CN95) on the PVC plate; (7) The Mycoplasma pneumoniae monoclonal antibody Henderson HDS-21213, the Chlamydia pneumoniae monoclonal antibody Maidian C01843M, the respiratory syncytial virus monoclonal antibody Henderson HDS-A5098, the novel coronavirus monoclonal antibody HDS-A5092, the influenza A virus monoclonal antibody FLUA-REAB-G1-020, the influenza B virus monoclonal antibody FLUB-REAB-G2-011, and the sheep anti-mouse IgG polyclonal antibody Longi Bio PS00901 were prepared into antibody solutions of a certain concentration using the coating solution of the antigen detection line; among them, the concentration of the Mycoplasma pneumoniae monoclonal antibody (marked as the M line) was 0.9 mM. g / mL; the concentration of Chlamydia pneumoniae monoclonal antibody (marked as C line) is 1.0 mg / mL; the concentration of respiratory syncytial virus monoclonal antibody (marked as R line) is 1.1 mg / mL; the concentration of the first quality control line sheep anti-mouse IgG polyclonal antibody (marked as Z line) is 1.0 mg / mL; the concentration of the new coronavirus monoclonal antibody (marked as S line) is 0.9 mg / mL; the concentration of influenza A virus monoclonal antibody (marked as A line) is 1.2 mg / mL; the concentration of influenza B virus monoclonal antibody (marked as B line) is 1.3 mg / mL; the concentration of the second quality control line sheep anti-mouse IgG polyclonal antibody (marked as Z line) is 1.0 mg / mL; Then, the streaking parameter was set to 0.8 μL / cm, and the following lines were streaked on the first reaction membrane: Mycoplasma pneumoniae monoclonal antibody (marked as M line), Chlamydia pneumoniae monoclonal antibody (marked as C line), respiratory syncytial virus monoclonal antibody (marked as R line), and the first quality control line sheep anti-mouse IgG polyclonal antibody (marked as Z line); the streaking parameter was set to 0.8 μL / cm, and the following lines were streaked on the second reaction membrane: novel coronavirus monoclonal antibody (marked as S line), influenza A virus monoclonal antibody (marked as A line), influenza B virus monoclonal antibody (marked as B line), and the second quality control line sheep anti-mouse IgG polyclonal antibody (marked as Z line); after streaking, the membrane was placed in a 37°C constant temperature drying oven for 2 hours; (8) The sample pad, gold label binding pad, nitrocellulose membrane after streaking and drying, and absorbent pad were sequentially pasted on the bottom plate, cut into 3.0 mm wide test strips using a microcomputer chopper, and mounted on a plastic card to prepare a test reagent strip.

[0050] 3. Kit Assembly

[0051] The test reagent strip and desiccant are placed in an aluminum foil bag and sealed, and a 600 μL / tube single-person sample diluent is placed, and the finished product is packaged to obtain a test kit.

[0052] IV. How to use the kit

[0053] Place the swab after collecting the sample immediately into the sampling tube containing the sample diluent, break the swab rod, immerse the swab head in the sample diluent, squeeze the sampling tube to allow the sample to dissolve in the sample diluent as much as possible, and then soak it for 30 seconds; gently squeeze the sampling tube to add 3 drops of sample to the sample holes of each of the two test reagent strips; start the timer after adding the sample, and judge the result in 5 to 10 minutes.

[0054] Result judgment: (1) On the reaction membrane of the first test reagent strip: a red strip appears at the position of the M line / C line / R line and the Z line is also red, indicating that the test result of Mycoplasma pneumoniae / Chlamydia pneumoniae / Respiratory syncytial virus is positive; On the reaction membrane of the second test strip: a red strip appears at the position of the S line / A line / B line and the Z line is also red, indicating that the test result of the new coronavirus / influenza A virus / influenza B virus is positive; (2) Negative: Only a red stripe appears on the Z line, indicating a negative result; (3) Invalid: If there is no red stripe at the Z line position after the test, the result is considered invalid.

[0055] Comparative Example 1

[0056] This comparative example provides a kit for simultaneously detecting Mycoplasma pneumoniae / Chlamydia pneumoniae / new coronavirus / respiratory syncytial virus / influenza A virus / influenza B virus, which is different from Example 1 only in that: the first reaction membrane includes Mycoplasma pneumoniae (marked as M line), Chlamydia pneumoniae (marked as C line), influenza B virus (marked as B line) and a first quality control line (marked as Z line), and the first conjugate pad includes colloidal gold-labeled Mycoplasma pneumoniae monoclonal antibody, Chlamydia pneumoniae monoclonal antibody and influenza B virus monoclonal antibody; the second reaction membrane includes new coronavirus (marked as S line), influenza A virus (marked as A line) and respiratory syncytial virus (marked as R line) and a second quality control line (marked as Z line), and the second conjugate pad includes colloidal gold-labeled new coronavirus monoclonal antibody, influenza A virus monoclonal antibody and respiratory syncytial virus monoclonal antibody; the schematic diagram of the detection reagent strip is shown in FIG. Figure 2 shown.

[0057] Test Example 1

[0058] The kits of Example 1 and Comparative Example 1 were used to detect five weak positive samples of dilutions of Mycoplasma pneumoniae, Chlamydia pneumoniae, respiratory syncytial virus, novel coronavirus, influenza A virus and influenza B virus, and five negative samples, respectively. The test results are shown in Table 1.

[0059] Table 1

[0060] From the test results, it can be seen that in Comparative Example 1, a false positive result was detected in the negative sample, and a false negative result was detected in the influenza B virus; while in Example 1, no false positive or false negative results occurred, so adjusting the combination of test items will cause inaccurate test results.

[0061] Comparative Example 2

[0062] This comparative example provides a kit that can simultaneously detect Mycoplasma pneumoniae / Chlamydia pneumoniae / new coronavirus / respiratory syncytial virus / influenza A virus / influenza B virus, the only difference from Example 1 is that PC300 in the sample diluent is replaced with NaN3 of the same concentration.

[0063] Comparative Example 3

[0064] This comparative example provides a kit that can simultaneously detect Mycoplasma pneumoniae / Chlamydia pneumoniae / new coronavirus / respiratory syncytial virus / influenza A virus / influenza B virus, the only difference from Example 1 is that the casein in the sample diluent is replaced with BSA of the same concentration.

[0065] Comparative Example 4

[0066] This comparative example provides a kit that can simultaneously detect Mycoplasma pneumoniae / Chlamydia pneumoniae / new coronavirus / respiratory syncytial virus / influenza A virus / influenza B virus, the only difference from Example 1 is that S9 in the sample diluent is replaced by S7 of the same concentration.

[0067] Comparative Example 5

[0068] This comparative example provides a kit that can simultaneously detect Mycoplasma pneumoniae / Chlamydia pneumoniae / new coronavirus / respiratory syncytial virus / influenza A virus / influenza B virus, the only difference from Example 1 is that Triton X-100 in the sample diluent is replaced with Tween-20 of the same concentration.

[0069] Test Example 2

[0070] Prepare medium positive and weak positive samples and negative samples of Mycoplasma pneumoniae, Chlamydia pneumoniae, respiratory syncytial virus, novel coronavirus, influenza A virus and influenza B virus respectively, and use the sample diluents in Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4 and Comparative Example 5 to treat the swab samples after collection, and test according to the method of Example 1, and repeat each sample 3 times. The test results are shown in Table 2.

[0071] Table 2

[0072] It can be seen that the sample diluent of Example 1 has the best effect.

[0073] Comparative Example 6

[0074] This comparative example provides a kit that can simultaneously detect Mycoplasma pneumoniae / Chlamydia pneumoniae / new coronavirus / respiratory syncytial virus / influenza A virus / influenza B virus, the only difference from Example 1 is that no heterophilic antibody blocker is added to the sample diluent.

[0075] Test Example 3

[0076] The same 40 normal human throat swab samples were tested using the kits of Example 1 and Comparative Example 6, and the test results are shown in Table 3.

[0077] Table 3

[0078] It can be seen that adding heterophilic antibody blockers to the sample diluent can eliminate the false positive reaction of the kit to non-specific samples and improve the detection accuracy of the kit.

[0079] Test Example 4

[0080] The minimum detection limit of the kit of Example 1 was tested.

[0081] (1) Verification of the minimum detection limit of Mycoplasma pneumoniae

[0082] Select 1 inactivated Mycoplasma pneumoniae virus strain: Use a negative nasal swab sample for serial dilution, and the diluted concentration is 56 TCID 50 / mL, each sample was tested 20 times using the kit, and the minimum detection limit of Mycoplasma pneumoniae could reach 95% detection rate.

[0083] (2) Verification of the minimum detection limit of Chlamydia pneumoniae

[0084] Select 1 inactivated Chlamydia pneumoniae virus strain: Use a negative nasal swab sample for serial dilution, and the diluted concentration is 112 TCID 50 / mL, each sample was tested 20 times using the kit, and the minimum detection limit of Chlamydia pneumoniae could reach 95% detection rate.

[0085] (3) Verification of the minimum detection limit of respiratory syncytial virus

[0086] Select two RSV inactivated virus strains: respiratory syncytial virus type A and respiratory syncytial virus type B, and use nasal swab negative samples for gradient dilution. The diluted concentration is 4.2×10 2 TCID 50 / mL, 2.3×10 2 TCID 50 / mL, each sample was tested 20 times using the kit, and the minimum detection limit of respiratory syncytial virus could reach 95% detection rate.

[0087] (4) Verification of the minimum detection limit of the new coronavirus

[0088] Five inactivated novel coronavirus strains were selected: Wuhan wild strain 7559, Indian Delta strain B.1.617.2, South African Beta strain B.1.351, Omicron B.1.1.529 strain 7610, and Omicron BA.5.2. Negative nasal swab samples were used for gradient dilution, and the concentration after dilution was 1.8×10 2 TCID 50 / mL, 2.0×10 2 TCID 50 / mL, 2.0×10 2 TCID 50 / mL, 1.8×10 2 TCID 50 / mL, 2.0×10 2 TCID 50 / mL, 1.7×10 2 TCID 50 / mL, and each sample was tested 20 times using the test kit. The minimum detection limit of the new coronavirus could reach 95%.

[0089] (5) Verification of the minimum detection limit of influenza A virus

[0090] Five inactivated Flu A virus strains were selected: the new influenza A (H1N1) virus (2009), seasonal influenza A (H1N1) virus, H3N2, H5N1, and H7N9. The negative nasal swab samples were used for gradient dilution, and the concentration after dilution was 2.8 × 10 2 TCID 50 / mL, 3.0×10 2 TCID 50 / mL, 2.5×10 2 TCID 50 / mL, 1.5×10 2 TCID 50 / mL, 4.0×10 2 TCID 50 / mL, each sample was tested 20 times using the kit, and the minimum detection limit of influenza A virus could reach 95%.

[0091] (6) Verification of the minimum detection limit of influenza B virus

[0092] Two inactivated Flu B virus strains were selected: influenza B Yamagata and influenza B Victoria. Negative nasal swab samples were used for serial dilution, and the concentration after dilution was 1.8 × 10 2 TCID 50 / mL, 3.4×10 2 TCID 50 / mL, each sample was tested 20 times using the kit, and the minimum detection limit of influenza B virus could reach 95% detection rate.

[0093] Test Example 5

[0094] The high-dose hook effect (HOOK) of the kit of Example 1 was tested.

[0095] Inactivated cultures of Mycoplasma pneumoniae, Chlamydia pneumoniae, respiratory syncytial virus, novel coronavirus, influenza A virus, and influenza B virus were diluted using negative nasal swab sample matrix to a concentration above the detection limit of 1000 times (1×10 7 TCID 50 / mL, and then 10-fold serial dilution (1×10 6 TCID 50 / mL, 1×10 5 TCID 50 / mL, 1×10 4 TCID 50 / mL), using 3 batches of kits for testing, and testing each concentration 3 times. The results of the HOOK test are shown in Table 4 below. The test results are all strongly positive, indicating that the virus concentration is not higher than 1×10 7 TCID 50 / mL, the kit of Example 1 can detect it and no HOOK reaction occurs.

[0096] Table 4

[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A kit capable of simultaneously detecting Mycoplasma pneumoniae / Chlamydia pneumoniae / new coronavirus / respiratory syncytial virus / influenza A virus / influenza B virus, characterized in that: include: Test strips and sample diluents; The detection reagent strip includes two independent detection reagent strips, each detection reagent strip contains a reaction membrane and a binding pad, the reaction membrane contains an antigen detection line and a quality control line, and the antigen detection line is coated with a monoclonal antibody; wherein the Mycoplasma pneumoniae antigen detection line, the Chlamydia pneumoniae antigen detection line and the respiratory syncytial virus antigen detection line are located on the same detection reagent strip, and the new coronavirus antigen detection line, the influenza A virus antigen detection line and the influenza B virus antigen detection line are located on the same detection reagent strip; The sample diluent includes Tris solution, PC300, casein, heterophilic antibody blocking agent, S9 and Triton X-100.

2. The kit according to claim 1, characterized in that From the sample addition position to the quality control line, the Mycoplasma pneumoniae antigen detection line, the Chlamydia pneumoniae antigen detection line, the respiratory syncytial virus and the quality control line are arranged in sequence on the reaction membrane of one of the test reagent strips; the new coronavirus antigen detection line, the influenza A virus antigen detection line, the influenza B virus antigen detection line and the quality control line are arranged in sequence on the reaction membrane of the other test reagent strip.

3. The kit according to claim 1, characterized in that The conjugate pad includes a monoclonal antibody for detecting a target antigen that is labeled with a marker.

4. The kit according to claim 3, characterized in that The label is colloidal gold.

5. The kit according to claim 1, characterized in that The quality control line is coated with IgG polyclonal antibody.

6. The kit according to claim 1, characterized in that The sample diluent includes a Tris solution with a pH of 7.5 to 8.5 and a concentration of 0.01 to 0.1 mol / L, a PC300 with a concentration of 0.05% to 0.5%, a casein with a concentration of 0.01 wt% to 0.3 wt%, a heterophilic antibody blocker with a concentration of 0.002 wt% to 0.05 wt%, S9 with a concentration of 0.01 wt% to 0.2 wt%, and Triton X-100 with a concentration of 0.1 wt% to 1wt%.

7. The kit according to claim 1, characterized in that The volume of the sample diluent is 500-1000 μL.

8. The kit according to claim 1, characterized in that The reaction membrane is a nitrocellulose membrane.

9. The kit according to claim 1, characterized in that The detection reagent strip comprises a sample pad, a conjugation pad, a reaction membrane, a water absorbent pad and a bottom plate; the sample pad, the conjugation pad, the reaction membrane and the water absorbent pad are sequentially pasted on the bottom plate.

10. Use of the kit according to any one of claims 1 to 9 in the preparation of a product; the product is used to detect at least one of Mycoplasma pneumoniae, Chlamydia pneumoniae, novel coronavirus, respiratory syncytial virus, influenza A virus, and influenza B virus.

Images