Test strip for combined immunochromatographic detection of multiple respiratory pathogenic microorganisms

Abstract

The present application belongs to the technical field of in-vitro diagnostic reagents. Provided is a test strip for the combined immunochromatographic detection of multiple respiratory pathogenic microorganisms. The detection test strip provided in the present application has an H-shaped structure, comprising two vertically arranged test strips, one on the left and one on the right, and one horizontally arranged sample-loading band; a sample-loading region is provided in the middle of test strip 1 and test strip 2, respectively; taking the sample-loading region as the center, conjugate regions, reaction regions and water absorption regions are sequentially overlapped in a symmetrical manner in both an upward direction and a downward direction; and one end of the sample-loading band is overlapped with the sample-loading region of test strip 1, and the other end is overlapped with the sample-loading region of test strip 2. The detection test strip has the characteristics of a high detection rate, preventing cross-reactions, and simple operation, demonstrating significant advantages compared with conventional detection test strips.

Description

A test strip for combined immunochromatographic detection of multiple respiratory pathogens

[0001] This application claims priority to Chinese Patent Application No. 202411737690.9, filed on November 29, 2024, entitled "A Combined Immunochromatographic Test Strip for Multiple Respiratory Pathogenic Microorganisms", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application belongs to the field of in vitro diagnostic reagent technology, specifically relating to a test strip for the combined immunochromatographic detection of multiple respiratory pathogenic microorganisms. Background Technology

[0003] Respiratory infections have a high incidence rate, with respiratory viruses such as influenza viruses (influenza A and B) and human respiratory syncytial virus (RSV) primarily affecting young children, pregnant women, the elderly, and patients with other complications. Influenza viruses and RSV are also major pathogens of acquired pneumonia. Besides influenza viruses and RSV, other common respiratory viruses include human metapneumovirus, rhinovirus, parainfluenza virus, coronavirus, adenovirus, and bocavirus. Other common respiratory pathogens include Mycoplasma pneumoniae and Streptococcus pneumoniae.

[0004] Currently, the commonly used methods for detecting respiratory viruses, Mycoplasma pneumoniae, and Streptococcus pneumoniae mainly involve separate detection to avoid cross-reactions during pathogen detection, which could affect the accuracy of the results. However, separate detection undoubtedly requires a large volume of sample, necessitating multiple sample additions. As the number of additions increases, insufficient sample processing solution becomes necessary, requiring additional volume of processing solution, which inevitably leads to a decrease in sample concentration and a reduction in detection rate. Summary of the Invention

[0005] In view of this, the purpose of this application is to provide a test strip for the combined immunochromatographic detection of multiple respiratory pathogens. By setting the test strip into an H-shaped structure, the sample application area is set at the symmetrical center of the H-shape. At the same time, the two test strips on the left and right sides of the H-shaped structure each have two binding areas, two reaction areas and two water absorption areas symmetrically distributed above and below the sample application area. This enables the combined detection of multiple respiratory pathogens, and ten pathogens can be detected simultaneously with only one sample application.

[0006] This application provides a test strip for the combined immunochromatographic detection of multiple respiratory pathogenic microorganisms, including test strip 1 and test strip 2, both placed vertically, and a sample application band placed horizontally.

[0007] Each of the test strips 1 and 2 has a sample application area in the middle, and a binding area, a reaction area, and a water absorption area are symmetrically connected vertically around each sample application area; one end of the sample application strip is attached to the sample application area of ​​the test strip 1, and the other end of the sample application strip is attached to the sample application area of ​​the test strip 2.

[0008] Each binding region is loaded with at least one latex microsphere-labeled first antibody; each reaction region is provided with at least one detection line and a control line; the detection line is coated with a second antibody; the first antibody and the second antibody can specifically bind to the same respiratory pathogenic microorganism.

[0009] Preferably, the test strip 1 has a first absorbent zone, a first reaction zone, a first binding zone, a first sample application zone, a second binding zone, a second reaction zone, and a second absorbent zone sequentially overlapped from top to bottom;

[0010] The test strip 2 has a third absorbent zone, a third reaction zone, a third binding zone, a second sample application zone, a fourth binding zone, a fourth reaction zone, and a fourth absorbent zone, which are sequentially overlapped from top to bottom;

[0011] The first binding region, the second binding region, the third binding region, and the fourth binding region are respectively loaded with different types of primary antibodies against respiratory pathogenic microorganisms labeled with latex microspheres;

[0012] Each of the first reaction zone, the second reaction zone, the third reaction zone, and the fourth reaction zone is provided with a control line and a detection line with the same number of first antibodies loaded on the binding region at the same end of the test strip; the detection line is coated with a second antibody against respiratory pathogenic microorganisms.

[0013] Preferably, the loading concentration of the primary antibody against respiratory pathogens labeled with latex microspheres on each binding region is independently 0.3% to 0.4%.

[0014] Preferably, the particle size of the latex microspheres includes 300-400 nm.

[0015] Preferably, on each detection line, the coating concentration of the secondary antibody against respiratory pathogens is independently 0.5–2.0 mg / mL.

[0016] Preferably, each quality control line is coated with goat anti-mouse IgG.

[0017] Preferably, the coating concentration of the goat anti-mouse IgG is 0.8–1.5 mg / mL.

[0018] Preferably, the sample pads of the sample application area and the sample application strip on the test strip 1 and test strip 2 have been pretreated with a treatment solution;

[0019] The treatment solution is an aqueous solution containing 0.5% to 1% polyvinylpyrrolidone, 0.5% to 1% casein, 0.5% to 0.8% Tween 20, and 0.8% to 1.2% Tris by weight.

[0020] Preferably, the first binding region is coated with a first antibody against influenza B virus labeled with blue latex microspheres, a first antibody against influenza A virus labeled with blue latex microspheres, and a first antibody against novel coronavirus labeled with red latex microspheres; the second binding region is coated with a first antibody against Mycoplasma pneumoniae labeled with red latex microspheres, a first antibody against respiratory adenovirus labeled with blue latex microspheres, and a first antibody against respiratory syncytial virus labeled with blue latex microspheres; the third binding region is coated with a first antibody against rhinovirus labeled with blue latex microspheres and a first antibody against parainfluenza virus labeled with red latex microspheres; and the fourth binding region is coated with a first antibody against group A streptococci labeled with red latex microspheres and a first antibody against Streptococcus pneumoniae labeled with blue latex microspheres.

[0021] The first reaction zone is equipped with three detection lines and one first quality control line. The three detection lines are respectively coated with a second antibody against influenza B virus, a second antibody against influenza A virus, and a second antibody against novel coronavirus.

[0022] The second reaction zone is equipped with three detection lines and one second quality control line. The three detection lines are respectively coated with a second antibody against Mycoplasma pneumoniae, a second antibody against respiratory adenovirus, and a second antibody against respiratory syncytial virus.

[0023] The third reaction zone is equipped with two detection lines and one third quality control line. The two detection lines are respectively coated with anti-rhinovirus secondary antibody and anti-parainfluenza virus secondary antibody.

[0024] The fourth reaction zone is equipped with two detection lines and one fourth quality control line. The two detection lines are respectively coated with a second antibody against group A streptococcus and a second antibody against pneumococcus.

[0025] This application provides a test strip for the combined immunochromatographic detection of multiple respiratory pathogenic microorganisms, including the test strip, a card slot base plate, and a cover plate;

[0026] The shape of the slot on the slot base plate is adapted to the structure of the test strip and is used to fix the test strip.

[0027] The cover plate is provided with perforated sampling holes and several observation windows;

[0028] The sample spotting well is located above the sample application band; an observation window is provided above each reaction zone for observing the test results.

[0029] This application provides a combined immunochromatographic test strip for detecting multiple respiratory pathogens, comprising test strip 1 and test strip 2, both placed vertically, and a horizontally placed sample band. Each test strip 1 and test strip 2 has a sample application area in its center, with a binding area, a reaction area, and an absorbent area symmetrically overlapped vertically around each sample application area. One end of the sample band overlaps the sample application area of ​​test strip 1, and the other end overlaps the sample application area of ​​test strip 2. Each binding area is loaded with at least one latex microsphere-labeled first antibody. Each reaction area has at least one detection line and a control line. The detection line is coated with a second antibody. The first and second antibodies specifically bind to the same respiratory pathogen. The test strip of this application features a high detection rate. This application addresses the problem that traditional methods require multiple sample additions when the number of target analytes increases, leading to insufficient sample processing solution and a decrease in sample concentration. The H-type test strip provided in this application effectively avoids this issue. The test strip provided in this application can also avoid cross-reactions: some pathogens with high similarity may cause cross-reactions to a certain extent during detection, which previous two-dimensional chromatography methods could not completely avoid and could only be improved through process optimization. However, the test strip provided in this application has an H-shaped structure, which can avoid cross-reactions through physical barriers. In addition, the test strip provided in this application is easy to operate. Compared with traditional detection methods that require multiple sample additions as the target analyte increases, the H-shaped test strip only requires one sample addition, making the operation faster and simpler. Attached Figure Description

[0030] Figure 1 is a front view of the cover plate structure of the test card of this application;

[0031] Figure 2 is a structural diagram of the back of the cover plate of the test card of this application; where 1 is a boss and 2 is a protruding point;

[0032] Figure 3 is a structural diagram of the card slot base plate of the test card in this application;

[0033] Figure 4 is a schematic diagram of the H-type test strip provided in this application. Detailed Implementation

[0034] This application provides a combined immunochromatographic test strip for multiple respiratory pathogens, in an H-shape, comprising test strip 1 and test strip 2, both placed vertically, and a horizontally placed sample band; each of test strip 1 and test strip 2 has a sample application area in the middle, with a binding area, a reaction area, and an absorbent area symmetrically overlapped vertically around each sample application area; one end of the sample band overlaps the sample application area of ​​test strip 1, and the other end overlaps the sample application area of ​​test strip 2; each binding area is loaded with at least one latex microsphere-labeled first antibody; each reaction area has at least one detection line and a control line; the detection line is coated with a second antibody; the first antibody and the second antibody can specifically bind to the same respiratory pathogen.

[0035] In this application, the test strip includes a base plate and a sample application area, a binding area, a reaction area, and an absorbent area fixed on the base plate. The base plate preferably comprises a PVC board with adhesive backing on one side. The test strip 1 preferably has, from top to bottom, a first absorbent area, a first reaction area, a first binding area, a first sample application area, a second binding area, a second reaction area, and a second absorbent area. The test strip 2 preferably has, from top to bottom, a third absorbent area, a third reaction area, a third binding area, a second sample application area, a fourth binding area, a fourth reaction area, and a fourth absorbent area; the first, second, third, and fourth binding areas preferably each load at least one primary antibody labeled with latex microspheres for different types of respiratory pathogenic microorganisms. Each of the first, second, third, and fourth reaction areas is provided with a control line and a detection line having the same number of primary antibodies loaded in the binding area at the same end of the test strip. The number of test lines with the same number of primary antibodies loaded in the binding zone at the same end of the test strip refers to the number of test lines coated in the first reaction zone being the same as the number of primary antibodies against respiratory pathogens labeled with latex microspheres loaded in the first binding zone, the number of test lines coated in the second reaction zone being the same as the number of primary antibodies against respiratory pathogens labeled with latex microspheres loaded in the second binding zone, and so on. The test lines are coated with secondary antibodies against respiratory pathogens. The H-type test strip has a sample application area at a centrally symmetrical point. This structure facilitates simultaneous detection of multiple pathogens with a single application, greatly simplifying operation and reducing the required sample volume, meeting the need for low-sample-volume detection of multiple targets. The materials of each binding zone preferably include polyester membrane and / or glass fiber. The materials of each reaction zone preferably include nitrocellulose membrane. The materials of each absorbent zone preferably include filter paper. The materials of each sample application zone preferably include polyester membrane and / or glass fiber.

[0036] In this application, the loading concentration of the primary antibody against respiratory pathogens labeled with latex microspheres on each binding region is preferably 0.3% to 0.4%, more preferably 0.3%. The solvent for the primary antibody against respiratory pathogens labeled with latex microspheres is preferably Tris-Casein buffer. The Tris-Casein buffer is preferably an aqueous solution containing 1.5M Tris-HCl and 0.5% Casein, with a pH of 8.0 to 8.2. This application does not impose any particular limitation on the preparation method of the primary antibody against respiratory pathogens labeled with latex microspheres; any method well-known in the art for labeling antibodies with latex microspheres can be used. Primary antibodies against different types of respiratory pathogens can be labeled with latex microspheres of different colors. The colors of the latex microspheres preferably include red and blue. The particle size of the latex microspheres preferably includes 300 to 400 nm.

[0037] In this application, the binding pad material for each binding region is preferably a polyester film. The binding pad is preferably pre-treated with a second treatment solution, which helps improve the solubility of the primary antibodies against respiratory pathogens labeled with each latex microsphere loaded in the binding region and reduces their residue on the binding pad. The second treatment solution is preferably an aqueous solution containing the following components: 8%–12% by mass of 3-(N-morpholino)-2-hydroxypropanesulfonic acid, 4%–6% by mass of casein, 0.4%–0.6% by volume of Tween 20, and 0.9%–1.1% by volume of Proclin 300; or it can be an aqueous solution containing the following components: 10% by mass of 3-(N-morpholino)-2-hydroxypropanesulfonic acid, 5% by mass of casein, 0.5% by volume of Tween 20, and 1% by volume of Proclin 300.

[0038] In this application, the coating concentration of the secondary antibody against respiratory pathogens is preferably 0.5–2.0 mg / mL, more preferably 0.8–1.5 mg / mL, and most preferably 1.0 mg / mL. The control line is preferably coated with goat anti-mouse IgG. The coating concentration of the goat anti-mouse IgG is preferably 0.8–1.5 mg / mL, and can be 1.0 mg / mL. The solvent for the secondary antibody against respiratory pathogens preferably includes 0.01 M PBS solution.

[0039] In this application, the sample pads of the sample application areas and the sample application strips on test strips 1 and 2 are preferably pre-treated with a first treatment solution. The first treatment solution is preferably an aqueous solution containing 0.5%–1% polyvinylpyrrolidone (PVP), 0.5%–1% casein (CUP), 0.5%–0.8% Tween 20 (TW2), and 0.8%–1.2% Tris (TW2), or an aqueous solution containing 0.5% PVP, 0.5% casein, 0.5% TW20 (TW2), and 1.0% Tris (TW2). Treating the sample pads with this solution helps eliminate differences in pH values ​​between samples, while also altering the sample flowability and improving the reaction system, thereby enhancing detection sensitivity and eliminating non-specific binding.

[0040] In this embodiment, when the respiratory pathogenic microorganisms include influenza B virus, influenza A virus, novel coronavirus, mycoplasma pneumoniae, respiratory adenovirus, respiratory syncytial virus, rhinovirus, parainfluenza virus, Streptococcus pneumoniae, and group A streptococcus, the first binding region is coated with a first antibody against influenza B virus labeled with blue latex microspheres, a first antibody against influenza A virus labeled with blue latex microspheres, and a first antibody against novel coronavirus labeled with red latex microspheres; the second binding region is coated with a first antibody against mycoplasma pneumoniae labeled with red latex microspheres, a first antibody against respiratory adenovirus labeled with blue latex microspheres, and a first antibody against respiratory syncytial virus labeled with blue latex microspheres; the third binding region is coated with a first antibody against rhinovirus labeled with blue latex microspheres and a first antibody against parainfluenza virus labeled with red latex microspheres; and the fourth binding region is coated with a first antibody against group A streptococcus labeled with red latex microspheres and a first antibody against Streptococcus pneumoniae labeled with blue latex microspheres. The first reaction zone has three detection lines and one first control line. The three detection lines are coated with a second antibody against influenza B virus, an second antibody against influenza A virus, and a second antibody against the novel coronavirus. The second reaction zone has three detection lines and one second control line. The three detection lines are coated with a second antibody against Mycoplasma pneumoniae, a second antibody against respiratory adenovirus, and a second antibody against respiratory syncytial virus. The third reaction zone has two detection lines and one third control line. The two detection lines are coated with a second antibody against rhinovirus and a second antibody against parainfluenza virus. The fourth reaction zone has two detection lines and one fourth control line. The two detection lines are coated with a second antibody against group A streptococcus and a second antibody against Streptococcus pneumoniae. The first antibody against the novel coronavirus was purchased from Beijing Baixinyi Biotechnology Co., Ltd., catalog number 2020TN15. The first antibody against influenza A virus was purchased from Shenzhen Aiweidi Biotechnology Co., Ltd., catalog number ABFLUA05. The first antibody against influenza B virus was purchased from Nanjing Songtianshengke Biotechnology Co., Ltd., catalog number S-1TFB2. The primary antibody against respiratory syncytial virus (RSV) was purchased from Guangzhou Feipeng Biotechnology Co., Ltd., catalog number RSV-REAB-G1-012. The primary antibody against adenovirus was purchased from Beijing Yinghe Lingyuan Biotechnology Co., Ltd., catalog number Baa001. The primary antibody against Mycoplasma pneumoniae was purchased from Xiamen Huasheng Times Biotechnology Co., Ltd., catalog number HSA02. The primary antibody against parainfluenza virus was purchased from Sino-American Xinxin Biotechnology Co., Ltd., catalog number ACT-mAb-PIVS1-002. The primary antibody against Group A Streptococcus was purchased from Shandong Zhuokang Biotechnology Co., Ltd., catalog number STA-Ab1. The primary antibody against rhinovirus was purchased from Sino-American Xinxin Biotechnology Co., Ltd., catalog number ACT-mAb-RhV-002. The primary antibody against Streptococcus pneumoniae was purchased from Sino-American Xinxin Biotechnology Co., Ltd., catalog number ACT-mAb-SP-003.The following secondary antibodies were purchased: Anti-novel coronavirus (NCoV) from Beijing Baixinyi Biotechnology Co., Ltd. (Catalog No. 2020TN8); Anti-influenza A virus (IIVV) from Shenzhen Aiweidi Biotechnology Co., Ltd. (Catalog No. ABFLUA06); Anti-influenza B virus (IIVV) from Nanjing Songtianshengke Biotechnology Co., Ltd. (Catalog No. S-1TFB1); Anti-respiratory syncytial virus (RSV) from Guangzhou Feipeng Biotechnology Co., Ltd. (Catalog No. RSV-REAB-G1-011); Anti-adenovirus (ADNA) from Beijing Yinghe Lingyuan Biotechnology Co., Ltd. (Catalog No. Baa001-2); Anti-Mycoplasma pneumoniae (MOP) from Xiamen Huasheng Times Biotechnology Co., Ltd. (Catalog No. HSA01); Anti-parainfluenza virus (PIV) from Sino-American Xinxin Biotechnology Co., Ltd. (Catalog No. ACT-mAb-PIVS1-001); Anti-Group A Streptococcus (GTA) from Shandong Zhuokang Biotechnology Co., Ltd. (Catalog No. STA-Ab1); and Anti-rhvirus (ARV) from Sino-American Xinxin Biotechnology Co., Ltd. (Catalog No. ACT-mAb-RhV-001). The secondary antibody against Streptococcus pneumoniae was purchased from Sino-American Xinxin Biotechnology Co., Ltd., catalog number ACT-mAb-SP-001.

[0041] This application provides a multi-respiratory pathogenic microorganism combined immunochromatographic test strip, including the test strip, a slot base plate, and a cover plate; the slot shape on the slot base plate is adapted to the structure of the test strip for fixing the test strip; the cover plate is provided with a perforated sample dispensing hole and four observation windows; the sample dispensing hole is located above the vertical direction of the sample application strip; the four observation windows are located above the first reaction zone, the second reaction zone, the third reaction zone, and the fourth reaction zone in the vertical direction.

[0042] In this application, the card slot base plate and the cover plate have the same length and width. Several raised points are distributed around the back of the cover plate, and several recessed points are distributed at corresponding positions on the front of the card slot base plate. The card slot base plate and the cover plate are sealed and fastened by inserting the raised points into the recessed points. Several straight protrusions are also distributed on the back of the cover plate for fixing the test strip in the grooves of the card slot base plate. The protrusions are positioned between two vertically distributed observation windows. The number of protrusions is preferably 2 to 4, and can be 3. The raised points, protrusions, and cover plate are preferably integrally formed. In one embodiment of this application, the overall length of the card case formed after the cover plate and card slot base plate are fastened is preferably 118 mm, and the width is 35.7 mm. The width of the card slot in the card slot base plate is 4.6 mm. The height of the cover plate is 2.1 mm, the height of the protrusions in the cover plate is 1.85 mm, and the distance from the protrusion near the reaction area to the nearest edge of the observation window is 1.8 mm.

[0043] In this application, the method for preparing the test strip preferably includes the following steps:

[0044] The sample pads of the sample application area and the sample application strip on test strip 1 and test strip 2 are pretreated with the treatment solution and dried to obtain pretreated sample pads;

[0045] Different types of primary antibodies labeled with different colored latex microspheres were directly or mixed and sprayed onto the conjugate pad, and dried to obtain a conjugate pad loaded with primary antibodies labeled with latex microspheres.

[0046] The corresponding second antibodies were sprayed onto the nitrocellulose membrane to form detection lines, and mouse anti-IgG was sprayed onto the nitrocellulose membrane to form control lines, thus obtaining nitrocellulose membranes coated with different second antibodies.

[0047] The sample application pad, the binding pad loaded with latex microsphere-labeled first antibody, and the nitrocellulose membrane and absorbent pad coated with different second antibodies are overlapped and assembled to obtain the detection plate strips, which are then cut into test strips.

[0048] Place the test strip on the card slot base plate, with one end of the sample application strip overlapping the sample application area of ​​the test strip 1 and the other end of the sample application strip overlapping the sample application area of ​​the test strip 2. Close the cover plate to obtain the test strip card.

[0049] Alternatively, the sample pad and sample strip can be molded as a single unit. During assembly, the nitrocellulose membrane is first symmetrically pasted onto the base plate, followed by the binding pad and absorbent pad. The strips are then cut, and the integrally molded sample pad is pasted onto each strip. The resulting test strip is placed on the card slot base plate and the cover plate is closed to obtain the test strip card.

[0050] In this invention, the overlap distance between the conjugate pad and the nitrocellulose membrane can be 1.3–1.7 mm or 1.5 mm. The overlap distance between the sample pad and the conjugate pad can be 2–2.7 mm or 2.5 mm. The overlap distance between the absorbent paper and the nitrocellulose membrane can be 2.8–3.2 mm or 3 mm.

[0051] In this application, the detection method of the test strip is preferably based on the double antibody sandwich principle, and specifically includes the following steps:

[0052] The sample is treated with a sample extraction solution to obtain a treated sample;

[0053] Add the processed sample to the well of the test strip, let it stand and observe. Determine whether the sample is infected with the target pathogenic microorganism based on the color development at the observation window:

[0054] When both the control line and the test line show color in the observation window, the sample is positive.

[0055] When the control line in the observation window shows color but the test line does not, it indicates that the sample is negative.

[0056] If the control line in the observation window does not show color, retest regardless of whether the test line shows color.

[0057] In this application, the sample extraction solution is preferably physiological saline containing 0.5% Tween-20 and 0.5% Triton X-100 by volume. The preferred method for collecting the processed sample is nasal fluid collection.

[0058] In the embodiments of this application, the test strip was used for detection. The results showed that compared with conventional test strips, the detection efficiency was higher, the number of sample additions was less, and the color development effect was more obvious. The color development was stronger for low concentration samples, indicating that the test strip provided in this application has higher detection sensitivity.

[0059] The following detailed description, in conjunction with embodiments, of a multi-respiratory pathogenic microorganism combined immunochromatographic test strip provided in this application, should not be construed as limiting the scope of protection of this application.

[0060] Example 1

[0061] Structure and preparation method of a multi-respiratory pathogenic microorganism combined immunochromatographic test strip

[0062] 1. The test strip has an H-shaped structure (see Figure 3). Test strip 1 has the following layers from top to bottom: first absorbent zone, first reaction zone, first binding zone, first sample application zone, second binding zone, second reaction zone, and second absorbent zone. Test strip 2 has the following layers from top to bottom: third absorbent zone, third reaction zone, third binding zone, second sample application zone, fourth binding zone, fourth reaction zone, and fourth absorbent zone. The two ends of the sample application strip overlap the sample application zones of test strip 1 and test strip 2, respectively. When the target pathogenic microorganisms include influenza B virus, influenza A virus, novel coronavirus, mycoplasma pneumoniae, respiratory adenovirus, respiratory syncytial virus, rhinovirus, parainfluenza virus, Streptococcus pneumoniae, and group A streptococcus, the first binding region is coated with a first antibody against influenza B virus labeled with blue latex microspheres, a first antibody against influenza A virus labeled with blue latex microspheres, and a first antibody against novel coronavirus labeled with red latex microspheres; the second binding pad is coated with a first antibody against mycoplasma pneumoniae labeled with red latex microspheres, a first antibody against respiratory adenovirus labeled with blue latex microspheres, and a first antibody against respiratory syncytial virus labeled with blue latex microspheres; the third binding pad is coated with a first antibody against rhinovirus labeled with blue latex microspheres and a first antibody against parainfluenza virus labeled with red latex microspheres; and the fourth binding pad is coated with a first antibody against group A streptococcus labeled with red latex microspheres and a first antibody against Streptococcus pneumoniae labeled with blue latex microspheres. The first reaction zone has three detection lines and one first control line. The first detection line, arranged sequentially from bottom to top, is coated with a second antibody against influenza B virus; the second detection line is coated with a second antibody against influenza A virus; and the third detection line is coated with a second antibody against novel coronavirus. The second reaction zone has three detection lines and one second control line. The first detection line, arranged sequentially from bottom to top, is coated with a second antibody against Mycoplasma pneumoniae; the second detection line is coated with a second antibody against respiratory adenovirus; and the third detection line is coated with a second antibody against respiratory syncytial virus. The third reaction zone has two detection lines and one third control line. The first detection line, arranged sequentially from bottom to top, is coated with a second antibody against rhinovirus; and the second detection line is coated with a second antibody against parainfluenza virus. The fourth reaction zone has two detection lines and one fourth control line. The first detection line, arranged sequentially from bottom to top, is coated with a second antibody against group A streptococci; and the second detection line is coated with a second antibody against Streptococcus pneumoniae.

[0063] II. Preparation Method

[0064] 2.1 Taking the preparation method of primary antibody against influenza B virus labeled with blue latex microspheres as an example, the preparation method of primary antibody against pathogenic microorganisms labeled with latex microspheres is illustrated as follows:

[0065] 2.1.1. Cleaning the latex microspheres

[0066] (1) Calculate the required volume of blue latex microspheres according to Formula I.

[0067] Blue latex microsphere volume = final volume × 1% ÷ 4% Formula I.

[0068] In actual preparation, the final volume is 0.6 mL.

[0069] (2) Calculate the required volume of diluted blue latex microspheres to be added to 1% borax buffer (pH 8.0 ± 0.1) according to Formula II:

[0070] The volume of borax buffer solution = total volume - volume of blue latex microspheres (Formula II).

[0071] In actual preparation, the final volume is 1.5 mL.

[0072] (3) Add blue latex microspheres to the borax buffer and mix thoroughly. Centrifuge at 14,000 rpm for 15 minutes. Remove the supernatant using a pipette.

[0073] (4) Clean the blue latex microspheres again. Add borax buffer to the blue latex microspheres, and sonicate for a total of 20 seconds, with sonication on for 0.3 seconds and off for 0.2 seconds.

[0074] 2.1.2. Activate the latex microspheres and prepare the EDC solution (the prepared EDC solution must be used within 10 minutes).

[0075] (1) Calculate the volume of EDC according to Formula III. The minimum volume is 0.5 mL.

[0076] EDC volume = final solution volume × 0.07 mL / ml (Formula III).

[0077] (2) Calculate the mass of EDC according to formula IV, and weigh EDC;

[0078] Mass of EDC = Volume of EDC × 10 mg / mL (Formula IV)

[0079] (3) Calculate the required volume of water according to formula V;

[0080] The volume of water = the volume of EDC ÷ 10 mg / mL. Formula V.

[0081] 2.1.3. Preparation of primary antibody against influenza B virus labeled with blue latex microspheres

[0082] (1) Calculate the required primary antibody against influenza B virus according to formula VI;

[0083] Mass of the primary antibody to be labeled = final volume × labeling ratio 1.0 mg / mL ÷ raw material concentration (Formula VI)

[0084] In actual preparation, the final volume is 0.6 mL.

[0085] (2) Weigh the required primary antibody against influenza B virus, add it to the cleaned latex microspheres, immediately add the required volume of borax buffer, then add the required volume of EDC, and shake to mix for 1 minute.

[0086] (3) Stir overnight at room temperature to obtain latex antibody solution.

[0087] 2.1.4. Preparation of ethanolamine solution

[0088] (1) Calculate the required amount of ethanolamine according to formula VII, add the required ethanolamine solution to the latex antibody solution, and mix for 30 minutes.

[0089] Volume of ethanolamine = final volume of latex antibody solution × 0.14 mL / ml (Formula VII).

[0090] (2) Centrifuge at 14000 rpm for 15 minutes and discard the supernatant.

[0091] 2.1.5. Prepare Tris-Casein buffer.

[0092] (1) Add Tris-Casein buffer to the first antibody labeled with latex microspheres, sonicate for a total time of 20s, sonicate on for 0.3s and off for 0.2s, and mix for 4h.

[0093] (2) Centrifuge at 14000 rpm for 15 minutes and discard the supernatant.

[0094] Tris-Casein buffer (1.5M Tris-HCl, 0.5% Casein, pH 8.0-8.2) was added to the washed latex microsphere-labeled primary antibody and sonicated in a water bath for 15 min to obtain the latex microsphere-labeled influenza B virus primary antibody solution.

[0095] 2.2 Preparation of the binding pad

[0096] 2.2.1 The pretreatment steps for polyester film are as follows:

[0097] ① Take an untreated polyester pad and immerse it in a preparation tank containing the treatment solution. The polyester pad should be completely submerged in the treatment solution, and the liquid level should be higher than the polyester pad.

[0098] ② Place the preparation box in a shaker and shake it slowly for 5 hours.

[0099] ③ Lift up a single sheet of polyester and drain off the excess treatment liquid until it is in droplets.

[0100] ④ Place the asphalt-coated polyester pads in a drying room or drying oven to dry. The drying temperature is 40℃, the relative humidity is ≤20%, and the drying time is 12 hours.

[0101] The treatment solution is formulated as follows: 10% by mass of 3-(N-morpholino)-2-hydroxypropanesulfonic acid, 5% by mass of casein, 0.5% by volume of Tween20, and 1% by volume of Proclin300.

[0102] The concentration of the first antibody solutions of pathogenic microorganisms labeled with latex microspheres prepared above was adjusted to 0.3% with solvent to obtain the working solution of the first antibody labeled with latex microspheres; the working solutions of the first antibody labeled with specific types of latex microspheres were mixed and sprayed onto the pretreated polyester membrane and dried to obtain the polyester membrane loaded with the first antibody labeled with latex microspheres.

[0103] 2.3 Preparation method of nitrocellulose membrane coated with secondary antibody

[0104] A specific anti-pathogenic microorganism secondary antibody with a concentration of 1 mg / mL and an anti-mouse IgG antibody working solution with a concentration of 1.0 mg / mL were streaked onto a nitrocellulose membrane using a streak scribing apparatus. The coating volume was 30 μL / 30 cm. After drying, a nitrocellulose membrane coated with the secondary antibody was obtained.

[0105] 2.4 Sample pad treatment

[0106] The polyester film was immersed in the sample processing solution for 1 hour, then removed and dried to obtain a pretreated sample pad. The sample processing solution was an aqueous solution containing 0.5% polyvinylpyrrolidone, 0.5% casein, 0.5% Tween 20, and 1.0% Tris by mass.

[0107] 2.5 Assembly and cutting of test strips

[0108] The sample pad is attached to the center of the PVC base plate. Following a symmetrical arrangement, the binding pad of the first antibody labeled with latex microspheres, the nitrocellulose membrane coated with the second antibody, and the absorbent paper are overlapped and attached to both sides of the sample pad. The assembly plate is then cut into strips, each 4 mm wide. Depending on the type of test, the cut test strips are assembled using the sample pad to obtain an H-shaped test strip.

[0109] 2.6 Preparation of Test Strips

[0110] Prepare the cover plate (Figures 1 and 2) and the card slot base plate (Figure 3) of the card set. The overall length of the card set formed by the cover plate and the card slot base plate after fastening is 118 mm and the width is 35.7 mm. The width of the card slot in the card slot base plate is 4.6 mm. The height of the cover plate is 2.1 mm, the height of the boss in the cover plate is 1.85 mm, and the distance from the boss near the reaction area to the nearest edge of the observation window is 1.8 mm. First, place the two test strips vertically on the card slot base plate. Then, place the sample pad (i.e., the sample application strip connecting test strip 1 and test strip 2) with a small PVC base plate attached to the bottom horizontally. The sample application strip connects test strip 1 and test strip 2 and is placed horizontally in the groove in the middle of the card slot base plate to obtain the test strip card.

[0111] 3. Testing methods for test strips

[0112] Immediately after collecting the sample, place the swab into 0.5 ml of sample extraction solution in the sample extraction tube. Swirl the swab head in the sample extraction solution to mix thoroughly for at least 30 seconds. Simultaneously, squeeze the swab head at least 5 times through the outer wall of the sample extraction tube to ensure the sample is fully eluted into the tube. After squeezing the liquid from the swab head through the outer wall of the sample extraction tube, discard the swab and cap the extraction tube.

[0113] Add the processed sample solution to the sampling well of the test strip, let it stand for 10 minutes, and observe the test results:

[0114] When both the test line and the control line show color, the sample is positive, indicating that the sample contains an infection of the pathogenic microorganism corresponding to the second antibody on the test line.

[0115] When the control line shows color but the test line does not, the sample is negative, indicating that there is no infection of the pathogenic microorganism corresponding to the second antibody on the test line in the sample;

[0116] If the control line does not show color, retest.

[0117] Example 2

[0118] Example 1: Clinical Sample Detection Using Test Strips

[0119] Using recombinant protein control samples of the target pathogenic microorganism as samples, the samples were processed according to the method in Example 1, and diluted 2-fold to obtain sample solutions with concentrations of 10 pg / ml, 20 pg / ml, 40 pg / ml, 80 pg / ml, and 160 pg / ml. The sample solutions of different concentrations were then added to the test strips of Example 1, and the color development was observed. Simultaneously, nasal swab sample buffer from healthy individuals with negative PCR results was used as a negative matrix for detection.

[0120] The source information of the recombinant protein control products for the target pathogenic microorganisms is shown in Table 1.

[0121] Table 1. List of sources of recombinant protein controls for target pathogenic microorganisms

[0122] Using the same sample solution as the test subjects, conventional test cards were used to detect influenza B virus (Jiangsu ShuoShi Biotechnology Co., Ltd., SC10101), influenza A virus (Jiangsu ShuoShi Biotechnology Co., Ltd., SC10101), novel coronavirus (Wuhan Mingde Biotechnology Co., Ltd., AgH-20A), mycoplasma pneumoniae (Hangzhou Innovation Biotechnology Co., Ltd., P111005), respiratory adenovirus (Hangzhou Innovation Biotechnology Co., Ltd., P111004), respiratory syncytial virus (Hangzhou Innovation Biotechnology Co., Ltd., P111003), rhinovirus (Guangzhou DaAn Gene Co., Ltd., RP2020-1), parainfluenza virus (Shandong Kanghua Biomedical Technology Co., Ltd., k48595), Streptococcus pneumoniae (Beijing Bell Biotechnology Co., Ltd., 303633), and group A streptococcus (Beijing Jinwofu Biotechnology Co., Ltd., 55296).

[0123] Table 2 Detection Results Note: "-" indicates no color development; "±" indicates weak positive, with a color development intensity weaker than "+"; "+" indicates different color development intensities of the test lines, and the more "+" signs there are, the darker the test line color.

[0124] As shown in Table 2, compared to conventional test strips, Example 1 has higher production efficiency, fewer sample additions, and more obvious and clear color development, making it more user-friendly. Testing with recombinant protein revealed that Example 1, with the binding pad removed, has higher sensitivity and stronger color development at low concentrations compared to conventional strips.

[0125] Comparative Example 1

[0126] The test card was prepared according to the method of Example 1, except that the sample pad was treated with sample pad treatment solution 2. Sample pad treatment solution 2 was a 0.8% Tris aqueous solution containing 0.5% polyvinylpyrrolidone and 1% bovine serum albumin by mass.

[0127] Table 3 Detection Results Note: "-" indicates no color development; "+" indicates different color development intensities of the test lines. The more "+" signs, the darker the color of the test line.

[0128] As shown in Table 3 above, the poor hydrophilicity of the sample pad resulted in slow liquid flow. After the sample was dropped into the sample application hole of the test strip, the sample pad was located directly below the hole. The insufficient hydrophilicity of the sample pad obstructed liquid flow, causing the liquid to accumulate on the pad and diffuse slowly, leading to uneven sample flow and resulting in unilateral chromatography and slow chromatography. Furthermore, the unoptimized sample pad treatment solution contained an excessively high concentration of the blocking agent bovine serum albumin, resulting in poor sensitivity.

[0129] Comparative Example 2

[0130] The test card was prepared according to the method of Example 1, the only difference being that the H-type test strip lacked the support of the base plate under the sample pad in the middle. The test card prepared above was tested according to the method of Example 2, and the results are shown in Table 4.

[0131] Table 4 Test Results Note: "-" indicates no color development; "+" indicates different color development intensities of the test lines. The more "+" signs, the darker the color of the test line.

[0132] The results above show that the H-type test strip has only a thin sample pad in the middle, which cannot guarantee a sufficient amount of liquid to flow to the test strip from side to side. The glass fiber / polyester membrane is also thin; after the sample is added, liquid remains at the bottom of the glass fiber / polyester membrane, reducing the amount of sample that flows to the test strip for chromatography. This results in decreased test sensitivity and affects the accuracy and reliability of the detection results.

[0133] To address this issue, this application adds a PVC base plate adapted to the size of the sample pad at the bottom of the central sample pad of the H-type test strip. The small PVC base plate measures 7×4mm, while the H-type central sample pad measures 18×4mm. The small base plate is attached directly to the center of the bottom of the sample pad, with the excess portion at both ends resting on the test strips at the left and right ends. Adding the small PVC base plate to the bottom of the sample pad not only effectively prevents excessive liquid from accumulating at the bottom of the sample pad, thus reducing the liquid volume, but also makes the overall structure of the test strip card more stable. The small PVC base plate provides a stable support platform for the H-type central sample pad, allowing the sample to flow more smoothly to the test strips on both sides, effectively improving liquid flow.

[0134] Comparative Example 3

[0135] The test card was prepared according to the method of Example 1, with the only difference being that the height of the protrusion in the card cover was 2.2 mm, and the distance from the protrusion near the reaction zone to the nearest edge of the observation window was 1.3 mm. The test card prepared above was tested according to the method of Example 2, and the results are shown in Table 5.

[0136] Table 5 Test Results Note: "-" indicates no color development; "+" indicates different color development intensities of the detection lines, the more "+" signs there are, the darker the color of the detection lines; " / " indicates no detection.

[0137] The results above show that an excessively high protrusion will severely compress the test strip, causing deformation of the flow channel and even damage to the test strip. The protrusion height was 2.2 mm, and the adjusted height was 1.85 mm. In Example 1, moving the protrusion closest to the observation window 0.5 mm away from the observation window helps reduce the local pressure of the protrusion on the nitrocellulose membrane, thus preventing inaccurate test results. This indicates that the design of the test card housing structure is not reasonable enough, resulting in uneven pressure distribution. The test strip is compressed during fixation, causing deformation of the flow channel and obstructed liquid flow, leading to poor repeatability of the test results.

[0138] Comparative Example 4

[0139] The test strips were prepared according to the method in Example 1, and the tests were performed according to the method in Example 2, the only difference being that the sample volume added to each test strip was 3 drops (approximately 80 μL). The results showed that 3 drops of sample were far from sufficient; after adding the sample, the liquid could not move, resulting in the inability to perform chromatography and thus preventing the test strips from achieving good chromatographic results.

[0140] The test card described in this application, when the sample volume is tested, adds 6 drops (approximately 160 μL). The results show that the sample moves at a uniform speed on the test card. While increasing the sample migration speed, the reaction area of ​​the test strip is sufficiently wetted, allowing the antigen-antibody complex to react fully. This avoids signal weakening and false negatives caused by insufficient sample volume.

[0141] The above description is only a preferred embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

Claims

1. A test strip for the combined immunochromatographic detection of multiple respiratory pathogenic microorganisms, characterized in that, It includes test strip 1 and test strip 2, both placed vertically, and a sample application strip placed horizontally; Each of the test strips 1 and 2 has a sample application area in the middle, and a binding area, a reaction area, and a water absorption area are symmetrically connected vertically around each sample application area; one end of the sample application strip is attached to the sample application area of ​​the test strip 1, and the other end of the sample application strip is attached to the sample application area of ​​the test strip 2. Each binding region is loaded with at least one latex microsphere-labeled first antibody; each reaction region is provided with at least one detection line and a control line; the detection line is coated with a second antibody; the first antibody and the second antibody can specifically bind to the same respiratory pathogenic microorganism.

2. The combined immunochromatographic test strip for detecting multiple respiratory pathogenic microorganisms according to claim 1, characterized in that, The test strip 1 has, from top to bottom, a first absorbent zone, a first reaction zone, a first binding zone, a first sample application zone, a second binding zone, a second reaction zone, and a second absorbent zone. The test strip 2 has a third absorbent zone, a third reaction zone, a third binding zone, a second sample application zone, a fourth binding zone, a fourth reaction zone, and a fourth absorbent zone, which are sequentially overlapped from top to bottom; The first binding region, the second binding region, the third binding region, and the fourth binding region are respectively loaded with different types of primary antibodies against respiratory pathogenic microorganisms labeled with latex microspheres; Each of the first reaction zone, the second reaction zone, the third reaction zone, and the fourth reaction zone is provided with a control line and a detection line with the same number of first antibodies loaded on the binding region at the same end of the test strip; the detection line is coated with a second antibody against respiratory pathogenic microorganisms.

3. The combined immunochromatographic test strip for detecting multiple respiratory pathogenic microorganisms according to claim 2, characterized in that, On each binding region, the loading concentration of the primary antibody against respiratory pathogens labeled with latex microspheres was independently 0.3%–0.4%.

4. The combined immunochromatographic test strip for detecting multiple respiratory pathogenic microorganisms according to claim 2, characterized in that, The particle size of the latex microspheres includes 300–400 nm.

5. The combined immunochromatographic test strip for detecting multiple respiratory pathogenic microorganisms according to claim 2, characterized in that, On each testing line, the coating concentration of the secondary antibody against respiratory pathogens is independently 0.5–2.0 mg / mL.

6. The combined immunochromatographic test strip for detecting multiple respiratory pathogenic microorganisms according to claim 2, characterized in that, Each quality control line is coated with goat anti-mouse IgG.

7. The combined immunochromatographic test strip for detecting multiple respiratory pathogenic microorganisms according to claim 6, characterized in that, The coating concentration of the goat anti-mouse IgG was 0.8–1.5 mg / mL.

8. The combined immunochromatographic test strip for detecting multiple respiratory pathogenic microorganisms according to claim 2, characterized in that, The sample pads in the sample application areas and the sample application strips on test strip 1 and test strip 2 have been pretreated with the first treatment solution; The first treatment solution is an aqueous solution containing 0.5% to 1% polyvinylpyrrolidone, 0.5% to 1% casein, 0.5% to 0.8% Tween 20, and 0.8% to 1.2% Tris by mass.

9. The combined immunochromatographic test strip for detecting multiple respiratory pathogenic microorganisms according to at least one of claims 2 to 8, characterized in that, The first binding region is coated with a first antibody against influenza B virus labeled with blue latex microspheres, a first antibody against influenza A virus labeled with blue latex microspheres, and a first antibody against novel coronavirus labeled with red latex microspheres; the second binding region is coated with a first antibody against Mycoplasma pneumoniae labeled with red latex microspheres, a first antibody against respiratory adenovirus labeled with blue latex microspheres, and a first antibody against respiratory syncytial virus labeled with blue latex microspheres; the third binding region is coated with a first antibody against rhinovirus labeled with blue latex microspheres and a first antibody against parainfluenza virus labeled with red latex microspheres; the fourth binding region is coated with a first antibody against group A streptococci labeled with red latex microspheres and a first antibody against Streptococcus pneumoniae labeled with blue latex microspheres. The first reaction zone is equipped with three detection lines and one first quality control line. The three detection lines are respectively coated with a second antibody against influenza B virus, a second antibody against influenza A virus, and a second antibody against novel coronavirus. The second reaction zone is equipped with three detection lines and one second quality control line. The three detection lines are respectively coated with a second antibody against Mycoplasma pneumoniae, a second antibody against respiratory adenovirus, and a second antibody against respiratory syncytial virus. The third reaction zone is equipped with two detection lines and one third quality control line. The two detection lines are respectively coated with anti-rhinovirus secondary antibody and anti-parainfluenza virus secondary antibody. The fourth reaction zone is equipped with two detection lines and one fourth quality control line. The two detection lines are respectively coated with a second antibody against group A streptococcus and a second antibody against pneumococcus.

10. A test strip for combined immunochromatographic detection of multiple respiratory pathogens, characterized in that, Includes the multi-respiratory pathogenic microorganism combined immunochromatographic test strip as described in any one of claims 1 to 9, a card slot base plate, and a cover plate that can be fastened to the card slot base plate; The shape of the slot on the slot base plate is adapted to the structure of the test strip and is used to fix the test strip. The cover plate is provided with perforated sampling holes and several observation windows; The sample spotting hole is located above the sample application strip; an observation window is provided above each reaction zone of the test strip for observing the test results.

11. The combined immunochromatographic test strip for detecting multiple respiratory pathogenic microorganisms according to claim 10, characterized in that, The back of the cover plate is also provided with several straight protrusions; the straight protrusions are arranged vertically between the two observation windows.

12. The combined immunochromatographic test strip for detecting multiple respiratory pathogenic microorganisms according to claim 11, characterized in that, The number of the straight bosses is 2 to 4.

13. The combined immunochromatographic test strip for detecting multiple respiratory pathogenic microorganisms according to claim 11, characterized in that, The overall length of the retaining shell formed by the fastening of the cover plate and the bottom plate of the retaining slot is 118mm and the width is 35.7mm.

14. The combined immunochromatographic test strip for detecting multiple respiratory pathogenic microorganisms according to claim 13, characterized in that, The height of the cover plate is 2.1 mm; The height of the straight protrusion on the back of the cover plate is 1.85mm, and the length of the straight protrusion near the reaction zone from the nearest edge of the observation window is 1.8mm.

15. A method for preparing the combined immunochromatographic test strip for detecting multiple respiratory pathogenic microorganisms according to any one of claims 10 to 14, characterized in that, Includes the following steps: The sample pads in the sample application areas and the sample application strips in test strips 1 and 2 are pretreated with a first treatment solution and dried to obtain pretreated sample pads; the first treatment solution is an aqueous solution containing 0.5% to 1% polyvinylpyrrolidone, 0.5% to 1% casein, 0.5% to 0.8% Tween 20, and 0.8% to 1.2% Tris by mass. First antibodies against different types of respiratory pathogenic microorganisms labeled with different colored latex microspheres were directly or mixed and sprayed onto the conjugate pad, and dried to obtain a conjugate pad loaded with the first antibody labeled with latex microspheres. Secondary antibodies against different types of respiratory pathogens were sprayed onto nitrocellulose membranes to form detection lines, and mouse anti-IgG was sprayed onto nitrocellulose membranes to form control lines, thus obtaining nitrocellulose membranes coated with different secondary antibodies. The sample application pad, the binding pad loaded with latex microsphere-labeled first antibody, and the nitrocellulose membrane and absorbent pad coated with different second antibodies are overlapped and assembled to obtain the detection plate strips, which are then cut into test strips. Place the test strip on the card slot base plate, with one end of the sample application strip overlapping the sample application area of ​​the test strip 1 and the other end of the sample application strip overlapping the sample application area of ​​the test strip 2. Close the cover plate to obtain the test strip card.

16. The preparation method according to claim 15, characterized in that, The conjugate pad is pretreated with a second treatment solution; The second treatment solution is an aqueous solution containing the following components: 8%–12% by mass of 3-(N-morpholino)-2-hydroxypropanesulfonic acid, 4%–6% by mass of casein, 0.4%–0.6% by volume of Tween20, and 0.9%–1.1% by volume of Proclin300.

17. The use of the test strip according to any one of claims 1 to 9, the multi-respiratory pathogenic microorganism combined immunochromatographic test strip according to claim 10, or the multi-respiratory pathogenic microorganism combined immunochromatographic test strip prepared by the preparation method according to any one of claims 11 to 16 in detecting multiple respiratory pathogenic microorganisms or diagnosing diseases caused by multiple respiratory pathogenic microorganisms.

18. The application according to claim 17, characterized in that, The multiple respiratory pathogenic microorganisms include at least four of the following: influenza B virus, influenza A virus, novel coronavirus, mycoplasma pneumoniae, respiratory adenovirus, respiratory syncytial virus, rhinovirus, parainfluenza virus, Streptococcus pneumoniae, and group A streptococcus.

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