Combined immunochromatography detection test strip for multiple respiratory tract pathogenic microorganisms

Abstract

The invention provides a combined immunochromatography detection test strip for multiple respiratory tract pathogenic microorganisms, and belongs to the technical field of in-vitro diagnostic reagents. The detection test strip provided by the invention has an H-shaped structure and comprises a left test strip, a right test strip and a horizontal sample adding belt, a sample adding area is arranged in the middle of each of the test strip 1 and the test strip 2, and a binding area, a reaction area and a water absorption area are sequentially overlapped in an up-and-down symmetry manner by taking the sample adding areas as centers; one end of the sample adding belt is lapped on the sample adding area of the test strip 1, and the other end of the sample adding belt is lapped on the sample adding area of the test strip 2. The detection test strip has the characteristics of high detection rate, avoidance of cross reaction and simplicity and convenience in operation, and has obvious advantages compared with a traditional detection test strip.

Description

Technical Field

[0001] The invention belongs to the technical field of in vitro diagnostic reagents, and in particular relates to a test strip for combined immunochromatographic detection of multiple respiratory pathogenic microorganisms. Background Art

[0002] The incidence of respiratory tract infections is high, and infections with respiratory viruses such as influenza virus (influenza A virus and influenza B virus) and human respiratory syncytial virus are mainly concentrated in young children, pregnant women, the elderly and other patients with comorbidities. Influenza virus and human respiratory syncytial virus are also the main pathogens of acquired pneumonia. In addition to influenza virus and human respiratory syncytial virus, common viruses that can cause respiratory tract infections in clinical practice include human metapneumovirus, rhinovirus, parainfluenza virus, coronavirus, adenovirus and bocavirus. In addition, other common pathogenic microorganisms in the respiratory tract include Mycoplasma pneumoniae and Streptococcus pneumoniae.

[0003] At present, the commonly used detection methods for respiratory viruses, Mycoplasma pneumoniae and Streptococcus pneumoniae are mainly to detect them separately in order to avoid cross-reactions during pathogen detection, which affects the accuracy of the test results. However, separate detection undoubtedly requires a high volume of test samples and requires multiple sample additions. As the number of sample additions increases, the sample processing liquid is insufficient, and the volume of sample processing liquid needs to be increased, which will undoubtedly cause the sample concentration to decrease and reduce the detection rate. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a test strip for combined immunochromatographic detection of multiple respiratory pathogenic microorganisms. The test strip is arranged into an H-shaped structure, a sample addition area is arranged at the symmetrical center point of the H-shaped structure, and at the same time, the left and right test strips in the H-shaped structure are symmetrically distributed with a sample addition area, 2 binding areas, 2 reaction areas and 2 water absorption areas, respectively, to achieve combined detection of multiple respiratory pathogenic microorganisms, and simultaneous detection of ten pathogenic microorganisms can be achieved with only one sample addition.

[0005] The present invention provides a test strip for combined immunochromatographic detection of multiple respiratory pathogenic microorganisms, comprising a test strip 1 and a test strip 2 both placed in a vertical direction and a sample adding strip placed in a horizontal direction;

[0006] A sample adding area is respectively arranged in the middle of the test strip 1 and the test strip 2, and a binding area, a reaction area and a water absorption area are overlapped symmetrically in sequence with each sample adding area as the center; one end of the sample adding belt overlaps the sample adding area of ​​the test strip 1, and the other end of the sample adding belt overlaps the sample adding area on the test strip 2;

[0007] Each binding area is loaded with at least one first antibody labeled with latex microspheres; each reaction area is provided with at least one detection line and a quality 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.

[0008] Preferably, the test strip 1 is overlapped with a first water absorption area, a first reaction area, a first binding area, a first sample addition area, a second binding area, a second reaction area and a second water absorption area in sequence from top to bottom;

[0009] The test strip 2 is overlapped with a third water absorption area, a third reaction area, a third binding area, a second sample addition area, a fourth binding area, a fourth reaction area and a fourth water absorption area in sequence from top to bottom;

[0010] The first binding region, the second binding region, the third binding region and the fourth binding region are respectively loaded with different kinds of first antibodies against respiratory pathogenic microorganisms labeled with latex microspheres;

[0011] The first reaction zone, the second reaction zone, the third reaction zone and the fourth reaction zone are each provided with a quality control line and a detection line with the same number of first antibodies loaded as the binding zone at the same end of the test strip; the detection line is coated with a second antibody against respiratory pathogenic microorganisms.

[0012] Preferably, on each binding zone, the loading mass concentration of the first antibody against respiratory pathogenic microorganisms labeled with latex microspheres is independently 0.3% to 0.4%.

[0013] Preferably, the particle size of the latex microspheres is 300-400 nm.

[0014] Preferably, on each test line, the coating concentration of the second antibody against respiratory pathogens is independently 0.5-2.0 mg / mL.

[0015] Preferably, each quality control line is coated with goat anti-mouse IgG.

[0016] Preferably, the coating concentration of the goat anti-mouse IgG is 0.8-1.5 mg / mL.

[0017] Preferably, the sample adding pads in the sample adding area and the sample adding pads in the sample adding belt on the test strip 1 and the test strip 2 are treated with a treatment liquid in advance;

[0018] The treatment solution is a 0.8% to 1.2% Tris aqueous solution containing 0.5% to 1% polyvinyl pyrrolidone by mass, 0.5% to 1% casein by mass, 0.5% to 0.8% Tween20 by volume.

[0019] Preferably, the first binding region is coated with a first antibody to influenza B virus labeled with blue latex microspheres, a first antibody to influenza A virus labeled with blue latex microspheres, and a first antibody to the new coronavirus labeled with red latex microspheres; the second binding pad region is coated with a first antibody to Mycoplasma pneumoniae labeled with red latex microspheres, a first antibody to respiratory adenovirus labeled with blue latex microspheres, and a first antibody to respiratory syncytial virus labeled with blue latex microspheres; the third binding region is coated with a first antibody to rhinovirus labeled with blue latex microspheres, and a first antibody to parainfluenza virus labeled with red latex microspheres; the fourth binding region is coated with a first antibody to group A streptococcus labeled with red latex microspheres, and a first antibody to Streptococcus pneumoniae labeled with blue latex microspheres;

[0020] The first reaction area is provided with three detection lines and a first quality control line, and 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 the new coronavirus;

[0021] The second reaction area is provided with three detection lines and a second quality control line, and 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;

[0022] The third reaction zone is provided with two detection lines and a third quality control line, and the two detection lines are respectively coated with a second antibody against rhinovirus and a second antibody against parainfluenza virus;

[0023] The fourth reaction zone is provided with two detection lines and a fourth quality control line, and the two detection lines are respectively coated with a second antibody against group A streptococci and a second antibody against streptococci pneumoniae.

[0024] The present invention provides a test paper card for combined immunochromatographic detection of multiple respiratory pathogenic microorganisms, comprising the test paper strip, a card slot bottom plate and a cover plate;

[0025] The shape of the card slot on the card slot bottom plate is adapted to the structure of the test strip and is used to fix the test strip;

[0026] The cover plate is provided with hollow spotting holes and a plurality of observation windows;

[0027] The sample spotting hole is located above the sample adding belt; an observation window is arranged above each reaction area for observing the detection result.

[0028] The present invention provides a test strip for combined immunochromatographic detection of multiple respiratory pathogenic microorganisms, including a test strip 1 and a test strip 2 both placed in a vertical direction and a sample addition belt placed in a horizontal direction; a sample addition area is arranged in the middle of each test strip 1 and the test strip 2, and a binding area, a reaction area and a water absorption area are overlapped symmetrically in sequence with each sample addition area as the center; one end of the sample addition belt is overlapped on the sample addition area of ​​the test strip 1, and the other end of the sample addition belt is overlapped on the sample addition area on the test strip 2; each binding area is loaded with at least one first antibody labeled with latex microspheres; each reaction area is provided with at least one detection line and a quality 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. The test strip of the present invention has the characteristics of high detection rate. The present invention aims at the increase of traditional detection targets, and multiple sample additions are required, resulting in insufficient sample processing liquid, and the volume of the sample processing liquid needs to be increased, thereby causing the sample concentration to decrease. The H-type test strip provided by the present invention avoids this well. The test strip provided by the present invention can also avoid cross-reactions: some pathogens with high similarity will produce cross-reactions to a certain extent when they are detected. The previous two-way chromatography scheme cannot completely avoid this and can only be improved by optimizing the process. The test strip provided by the present invention has an H-shaped structure, which can avoid cross-reactions through physical barriers. In addition, the test strip provided by the present invention is easy to operate. Compared with the problem of multiple sample additions as the number of targets increases in traditional detection, the H-type test strip only needs one sample addition, which is faster and easier to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a front structural diagram of the cover plate of the detection card of the present invention;

[0030] Figure 2 The back side structure diagram of the cover plate of the detection card of the present invention; wherein 1 is a boss, and 2 is a convex point;

[0031] Figure 3 This is a structural diagram of the card slot bottom plate of the detection card of the present invention;

[0032] Figure 4 This is a schematic diagram of the structure of the H-type test strip provided by the present invention. DETAILED DESCRIPTION

[0033] The present invention provides a test strip for combined immunochromatographic detection of multiple respiratory pathogenic microorganisms, which is H-shaped and comprises a test strip 1 and a test strip 2 both placed in a vertical direction and a sample adding strip placed in a horizontal direction; a sample adding area is respectively arranged in the middle of the test strip 1 and the test strip 2, and a binding area, a reaction area and a water absorption area are overlapped in sequence symmetrically up and down with each sample adding area as the center; one end of the sample adding strip is overlapped on the sample adding area of ​​the test strip 1, and the other end of the sample adding strip is overlapped on the sample adding area on the test strip 2; each binding area is loaded with at least one first antibody labeled with latex microspheres; each reaction area is provided with at least one detection line and a quality control line; a second antibody is coated on the detection line; the first antibody and the second antibody can specifically bind to the same respiratory pathogenic microorganism.

[0034] In the present invention, the test strip comprises a bottom plate and a sample application area, a binding area, a reaction area and a water absorption area are fixed on the bottom plate. The bottom plate preferably comprises a PVC plate with adhesive on one side. The test strip 1 preferably overlaps the first water absorption area, the first reaction area, the first binding area, the first sample application area, the second binding area, the second reaction area and the second water absorption area in sequence from top to bottom. The test strip 2 preferably overlaps the third water absorption area, the third reaction area, the third binding area, the second sample application area, the fourth binding area, the fourth reaction area and the fourth water absorption area in sequence from top to bottom; the first binding area, the second binding area, the third binding area and the fourth binding area are preferably loaded with at least one first antibody of different types of respiratory pathogens labeled with latex microspheres. The first reaction area, the second reaction area, the third reaction area and the fourth reaction area are all provided with a quality control line and a detection line with the same number of first antibodies loaded as the binding area at the same end of the test strip. The detection line with the same number of first antibodies loaded in the binding area at the same end of the test strip means that the number of detection lines coated in the first reaction area is the same as the number of first antibodies of respiratory pathogens labeled by latex microspheres loaded in the first binding area, and the number of detection lines coated in the second reaction area is the same as the number of first antibodies of respiratory pathogens labeled by latex microspheres loaded in the second binding area, and so on. The detection line is coated with a second antibody against respiratory pathogens. The H-type test strip is provided with a spotting area at a central symmetrical point. This structure is conducive to spotting once, realizing the simultaneous detection of multiple pathogenic microorganisms, greatly simplifying the operation, and reducing the demand for sample volume, meeting the detection requirements of low sample volume and multiple targets at the same time. The material of each binding area preferably includes polyester film and / or glass fiber. The material of each reaction area preferably includes nitrocellulose membrane. The material of each water absorption area preferably includes filter paper. The material of each sample addition area preferably includes polyester film and / or glass fiber.

[0035] In the present invention, the loading mass concentration of the first antibody of respiratory pathogenic microorganisms labeled with latex microspheres loaded on each binding area is independently preferably 0.3% to 0.4%, and more preferably 0.3%. The solvent of the first antibody of respiratory pathogenic microorganisms labeled with latex microspheres is preferably Tris-Casein buffer. The Tris-Casein buffer is preferably an aqueous solution containing 1.5M Tris-HCl, 0.5% Casein, and the pH is 8.0 to 8.2. The present invention has no special restrictions on the preparation method of the first antibody of respiratory pathogenic microorganisms labeled with latex microspheres, and the method of labeling antibodies with latex microspheres well known in the art can be used. The first antibodies of different types of respiratory pathogenic microorganisms 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 400nm.

[0036] In the present invention, the material of the binding pad of each binding zone is preferably a polyester film. The binding pad is preferably treated with a treatment liquid in advance, which is beneficial to improve the solubility of the first antibody of each latex microsphere labeled respiratory pathogenic microorganism loaded in the binding zone and reduce its residue on the binding pad. The treatment liquid is preferably an aqueous solution containing the following components: 8% to 12% by mass percentage of 3-(N-morpholinyl)-2-hydroxypropanesulfonic acid, 4% to 6% by mass percentage of casein, 0.4% to 0.6% by volume percentage of Tween20 and 0.9% to 1.1% by volume percentage of Proclin300; it can be an aqueous solution containing the following components: 10% by mass percentage of 3-(N-morpholinyl)-2-hydroxypropanesulfonic acid, 5% by mass percentage of casein, 0.5% by volume percentage of Tween20 and 1% by volume percentage of Proclin300.

[0037] In the present invention, the coating concentration of the second 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 quality 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 of the second antibody against respiratory pathogens preferably includes 0.01M PBS solution.

[0038] In the present invention, the sample pads of the sample loading area and the sample loading band on the test strip 1 and the test strip 2 are preferably treated with a treatment solution in advance. The treatment solution is preferably an aqueous solution containing 0.5% to 1% polyvinyl pyrrolidone by weight, 0.5% to 1% casein by weight, 0.5% to 0.8% Tween20 by volume, and 0.8% to 1.2% Tris by weight, and can be an aqueous solution containing 0.5% polyvinyl pyrrolidone by weight, 0.5% casein by weight, 0.5% Tween20 by volume, and 1.0% Tris by weight. Using the treatment solution to treat the sample pad is beneficial to eliminating the difference in different pH values ​​of the sample, while changing the flowability of the sample and improving the reaction system, improving the detection sensitivity and eliminating non-specific binding.

[0039] In an embodiment of the present invention, when respiratory pathogenic microorganisms include influenza B virus, influenza A virus, new coronavirus, Mycoplasma pneumoniae, respiratory adenovirus, respiratory syncytial virus, rhinovirus, parainfluenza virus, Streptococcus pneumoniae and group A streptococcus, the first binding area is coated with a first antibody for influenza B virus labeled with blue latex microspheres, a first antibody for influenza A virus labeled with blue latex microspheres, and a first antibody for the new coronavirus labeled with red latex microspheres; the second binding area is coated with a first antibody for Mycoplasma pneumoniae labeled with red latex microspheres, a first antibody for respiratory adenovirus labeled with blue latex microspheres, and a first antibody for respiratory syncytial virus labeled with blue latex microspheres; the third binding area is coated with a first antibody for rhinovirus labeled with blue latex microspheres, and a first antibody for parainfluenza virus labeled with red latex microspheres; the fourth binding area is coated with a first antibody for group A streptococcus labeled with red latex microspheres and a first antibody for Streptococcus pneumoniae labeled with blue latex microspheres. The first reaction zone is provided with three detection lines and a first quality control line, and the three detection lines are coated with the second antibody against influenza B virus, the second antibody against influenza A virus, and the second antibody against the new coronavirus; the second reaction zone is provided with three detection lines and a second quality control line, and the three detection lines are coated with the second antibody against Mycoplasma pneumoniae, the second antibody against respiratory adenovirus, and the second antibody against respiratory syncytial virus; the third reaction zone is provided with two detection lines and a third quality control line, and the two detection lines are coated with the second antibody against rhinovirus and the second antibody against parainfluenza virus; the fourth reaction zone is provided with two detection lines and a fourth quality control line, and the two detection lines are coated with the second antibody against group A streptococcus and the second antibody against streptococcus pneumoniae. The first antibody against the new coronavirus was purchased from Beijing Baixinyi Biotechnology Co., Ltd., item number 2020TN15. The first antibody against influenza A virus was purchased from Shenzhen Aiweidi Biotechnology Co., Ltd., item number ABFLUA05. The first antibody against influenza B virus was purchased from Nanjing Songtian Shengke Biotechnology Co., Ltd., item number S-1TFB2. The first antibody against respiratory syncytial virus was purchased from Guangzhou Feipeng Biotechnology Co., Ltd., with the catalog number RSV-REAB-G1-012. The first antibody against adenovirus was purchased from Beijing Yinghe Lingyuan Biotechnology Co., Ltd., with the catalog number Baa001. The first antibody against Mycoplasma pneumoniae was purchased from Xiamen Huasheng Times Biotechnology Co., Ltd., with the catalog number HSA02. The first antibody against parainfluenza virus was purchased from Sino-US Xinxin Biotechnology Co., Ltd., with the catalog number ACT-mAb-PIVS1-002. The first antibody against group A streptococcus was purchased from Shandong Zhuokang Biotechnology Co., Ltd., with the catalog number STA-Ab1. The first antibody against rhinovirus was purchased from Sino-US Xinxin Biotechnology Co., Ltd., with the catalog number ACT-mAb-RhV-002. The first antibody against Streptococcus pneumoniae was purchased from Sino-US Xinxin Biotechnology Co., Ltd., with the catalog number ACT-mAb-SP-003.The second antibody against the novel coronavirus was purchased from Beijing Baixinyi Biotechnology Co., Ltd., with the catalog number 2020TN8. The second antibody against influenza A virus was purchased from Shenzhen Aiweidi Biotechnology Co., Ltd., with the catalog number ABFLUA06. The second antibody against influenza B virus was purchased from Nanjing Songtian Shengke Biotechnology Co., Ltd., with the catalog number S-1TFB1. The second antibody against respiratory syncytial virus was purchased from Guangzhou Feipeng Biological Co., Ltd., with the catalog number RSV-REAB-G1-011. The second antibody against adenovirus was purchased from Beijing Yinghe Lingyuan Biotechnology Co., Ltd., with the catalog number Baa001-2. The second antibody against Mycoplasma pneumoniae was purchased from Xiamen Huasheng Times Biotechnology Co., Ltd., with the catalog number HSA01. The second antibody against parainfluenza virus was purchased from Sino-US Xinxin Biotechnology Co., Ltd., with the catalog number ACT-mAb-PIVS1-001. The second antibody against group A streptococci was purchased from Shandong Zhuokang Biotechnology Co., Ltd., with the catalog number STA-Ab1. The second antibody against rhinovirus was purchased from Sino-US Xinxin Biotechnology Co., Ltd., with the catalog number ACT-mAb-RhV-001. The secondary antibody against Streptococcus pneumoniae was purchased from Sino-US Xinxin Biotechnology Co., Ltd., catalog number ACT-mAb-SP-001.

[0040] The present invention provides a multiple respiratory pathogenic microorganisms combined immunochromatographic detection test paper card, comprising the test paper strip, a card slot bottom plate and a cover plate; the shape of the card slot on the card slot bottom plate is adapted to the structure of the test paper strip and is used to fix the test paper strip; the cover plate is provided with a hollow spot hole and four observation windows; the spot hole is located above the sample addition strip in the vertical direction; 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.

[0041] In the present invention, the length and width of the card slot bottom plate and the cover plate are consistent. There are several protruding points distributed around the back of the cover plate, and several concave points are distributed at the corresponding positions on the front of the card slot bottom plate. The card slot bottom plate and the cover plate are sealed and buckled by inserting the protruding points into the concave points; there are also several one-shaped bosses distributed on the back of the cover plate, which are used to fix the test strip in the groove of the card slot bottom plate. The distribution position of the boss is between two vertically distributed observation windows. The number of the bosses is preferably 2 to 4, and can be 3. The protruding points, bosses and cover plate are preferably formed in one piece. In one embodiment of the present invention, the overall length of the card shell formed by the cover plate and the card slot bottom plate after buckling is preferably 118mm and the width is 35.7mm. The width of the card slot in the card slot bottom plate is 4.6mm. The height of the cover plate is 2.1mm, the height of the boss in the cover plate is 1.85mm, and the length of the boss close to the reaction area from the nearest edge of the observation window is 1.8mm.

[0042] In the present invention, the method for preparing the test paper card preferably comprises the following steps:

[0043] pretreating the sample loading pad with a treatment liquid, and drying to obtain a pretreated sample loading pad;

[0044] Directly or mixedly spraying different kinds of first antibodies labeled with latex microspheres of different colors on the conjugate pad, and drying, to obtain a conjugate pad loaded with the first antibodies labeled with latex microspheres;

[0045] The corresponding second antibodies are sprayed on the nitrocellulose membrane to form a detection line, and the mouse anti-IgG is sprayed on the nitrocellulose membrane to form a quality control line, thereby obtaining nitrocellulose membranes coated with different second antibodies;

[0046] The sample loading pad, the binding pad loaded with the first antibody labeled with latex microspheres, the nitrocellulose membrane coated with different secondary antibodies and the water absorption pad are overlapped and assembled in sequence to obtain the detection plate strips and the detection test paper strips; the detection test paper strips are placed on the bottom plate of the card slot, and the cover plate is buckled to obtain the detection test paper card.

[0047] In the present invention, the detection method of the test paper card is preferably based on the double antibody sandwich principle, and specifically comprises the following steps:

[0048] Treat the sample with a sample extraction solution to obtain a treated sample;

[0049] Add the treated sample to the spot hole of the test paper card, let it stand and observe, and judge whether the sample is infected with the target pathogenic microorganism according to the color development at the observation window:

[0050] When the quality control line in the observation window turns color and the test line turns color at the same time, it means the sample is positive;

[0051] When the quality control line in the observation window shows color, but the test line does not show color, it means the sample is negative;

[0052] When the quality control line in the observation window does not show color, retest regardless of whether the test line shows color or not.

[0053] In the present invention, the sample extract is preferably a physiological saline solution containing 0.5% Tween-20 and 0.5% TritonX-100 in a volume concentration. The sample collection method is preferably nasal fluid collection.

[0054] In the embodiment of the present invention, the test paper card is used for detection. The results show that compared with conventional test cards, the detection efficiency is high, the number of sample additions is reduced, and the color development effect is more obvious. The color development for low-concentration samples is stronger, indicating that the test paper card provided by the present invention has higher detection sensitivity.

[0055] The following is a detailed description of a multiple respiratory pathogenic microorganisms combined immunochromatographic detection test strip provided by the present invention in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0056] Example 1

[0057] Structure and preparation method of a test strip for combined immunochromatographic detection of multiple respiratory pathogenic microorganisms

[0058] 1. The test strip is an H-shaped structure (see Figure 3 ), wherein the test strip 1 is overlapped with a first water absorption area, a first reaction area, a first binding area, a first sample adding area, a second binding area, a second reaction area and a second water absorption area in sequence from top to bottom; the test strip 2 is overlapped with a third water absorption area, a third reaction area, a third binding area, a second sample adding area, a fourth binding area, a fourth reaction area and a fourth water absorption area in sequence from top to bottom, and the two ends of the sample adding belt are overlapped with the sample adding area of ​​the test strip 1 and the sample adding area on the test strip 2 respectively. When the target pathogenic microorganisms include influenza B virus, influenza A virus, new coronavirus, Mycoplasma pneumoniae, respiratory adenovirus, respiratory syncytial virus, rhinovirus, parainfluenza virus, Streptococcus pneumoniae and group A streptococcus, the first binding area is coated with a first antibody for influenza B virus labeled with blue latex microspheres, a first antibody for influenza A virus labeled with blue latex microspheres, and a first antibody for new coronavirus labeled with red latex microspheres; the second binding pad is coated with a first antibody for Mycoplasma pneumoniae labeled with red latex microspheres, a first antibody for respiratory adenovirus labeled with blue latex microspheres, and a first antibody for respiratory syncytial virus labeled with blue latex microspheres; the third binding pad is coated with a first antibody for rhinovirus labeled with blue latex microspheres, and a first antibody for parainfluenza virus labeled with red latex microspheres; the fourth binding pad is coated with a first antibody for group A streptococcus labeled with red latex microspheres and a first antibody for Streptococcus pneumoniae labeled with blue latex microspheres. The first reaction zone is provided with three detection lines and a first quality control line, and the first detection line, which is sequentially distributed from bottom to top among the three detection lines, 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 the new coronavirus; the second reaction zone is provided with three detection lines and a second quality control line, and the first detection line, which is sequentially distributed from bottom to top among the three detection lines, 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 is provided with two detection lines and a third quality control line, and the first detection line, which is sequentially distributed from bottom to top among the two detection lines, 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 is provided with two detection lines and a fourth quality control line, and the first detection line, which is sequentially distributed from bottom to top among the two detection lines, is coated with a second antibody against Group A Streptococcus, and the second detection line is coated with a second antibody against Streptococcus pneumoniae.

[0059] 2. Preparation method

[0060] 2.1 Taking the preparation method of the first antibody of influenza B virus labeled with blue latex microspheres as an example, the preparation method of the first antibody against pathogenic microorganisms labeled with latex microspheres is as follows:

[0061] 2.1.1. Cleaning of latex microspheres

[0062] (1) Calculate the required volume of blue latex microspheres according to formula I.

[0063] Blue latex microsphere volume = final volume × 1% ÷ 4% Formula I.

[0064] In actual preparation, final volume: 0.6 mL.

[0065] (2) Calculate the volume of diluted blue latex microspheres added to 1% borax buffer (pH 8.0 ± 0.1) according to Formula II:

[0066] The volume of borax buffer = total volume - the volume of blue latex microspheres Formula II.

[0067] In actual preparation, final volume: 1.5 mL.

[0068] (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.

[0069] (4) Wash the blue latex microsphere particles again. Add borax buffer to the blue latex microspheres and sonicate for a total of 20 seconds, with ultrasound on for 0.3 seconds and off for 0.2 seconds.

[0070] 2.1.2. Activate latex microspheres and prepare EDC solution (the prepared EDC solution must be used up within 10 minutes).

[0071] (1) Calculate the volume of EDC according to formula III. The minimum volume is 0.5 mL.

[0072] The volume of EDC = final volume of solution × 0.07 mL / ml Formula III.

[0073] (2) Calculate the mass of EDC according to Formula IV and weigh EDC;

[0074] The mass of EDC = the volume of EDC × 10 mg / mL Formula IV.

[0075] (3) Calculate the required volume of water according to formula V;

[0076] The volume of water = the volume of EDC ÷ 10 mg / mL formula V.

[0077] 2.1.3. Preparation of primary antibody for influenza B virus labeled with blue latex microspheres

[0078] (1) Calculating the required first antibody against influenza B virus according to formula VI;

[0079] The mass of the first antibody to be labeled = final volume × labeling ratio 1.0 mg / mL ÷ raw material concentration formula VI.

[0080] In actual preparation, final volume: 0.6 mL.

[0081] (2) Weigh the required anti-influenza B virus primary antibody and add it to the washed latex microspheres. Immediately add the required volume of borax buffer and then add the required volume of EDC. Mix by shaking for 1 minute.

[0082] (3) Stir overnight at room temperature to obtain a latex antibody solution.

[0083] 2.1.4. Preparation of ethanolamine solution

[0084] (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.

[0085] The volume of ethanolamine = the final volume of latex antibody solution × 0.14 mL / ml Formula VII.

[0086] (2) Centrifuge at 14,000 rpm for 15 minutes and remove the supernatant.

[0087] 2.1.5. Preparation of Tris-Casein Buffer

[0088] (1) Add Tris-Casein buffer to the primary antibody labeled with latex microspheres, sonicate for a total of 20 seconds, turn on for 0.3 seconds, turn off for 0.2 seconds, and mix for 4 hours.

[0089] (2) Centrifuge at 14,000 rpm for 15 minutes and remove the supernatant.

[0090] Tris-Casein buffer (1.5M Tris-HCl, 0.5% Casein, pH 8.0-8.2) was added to the washed latex microspheres labeled primary antibody, and water bath sonication was performed for 15 minutes to obtain a latex microspheres labeled influenza B virus primary antibody solution.

[0091] 2.2 Preparation of conjugate pad

[0092] 2.2.1 The pretreatment steps of polyester film are as follows:

[0093] ① Take the untreated polyester pad and soak it in the preparation box filled with treatment liquid. The polyester should be completely immersed in the treatment liquid and the liquid level should be higher than the polyester pad.

[0094] ② Place the preparation box in a shaker and shake slowly for 5 hours.

[0095] ③ Pick up the polyester piece by piece and drain off the excess treatment liquid until the treatment liquid is in the form of drops.

[0096] ④Put the drained polyester mat in a drying room or drying box for drying at a temperature of 40°C and a relative humidity of ≤20% for 12 hours.

[0097] The formula of the treatment solution is as follows: 10% by weight of 3-(N-morpholinyl)-2-hydroxypropanesulfonic acid, 5% by weight of casein, 0.5% by volume of Tween 20 and 1% by volume of Proclin 300.

[0098] The concentration of the first antibody solution of each pathogenic microorganism labeled with latex microspheres prepared above is adjusted to 0.3% with a solvent to obtain a first antibody working solution labeled with latex microspheres; the first antibody working solution labeled with a specific type of latex microspheres is mixed and sprayed on a pretreated polyester film, and dried to obtain a polyester film loaded with the first antibody labeled with latex microspheres.

[0099] 2.3. Preparation of nitrocellulose membrane coated with secondary antibody

[0100] A 1 mg / mL specific anti-pathogenic microorganism second antibody and a 1.0 mg / mL anti-mouse IgG antibody working solution were streaked onto a nitrocellulose membrane using a streak instrument, with a coating amount of 30 μL / 30 cm, and dried to obtain a nitrocellulose membrane coated with the second antibody.

[0101] 2.4. Sample pad processing

[0102] The polyester film was immersed in the sample treatment solution for 1 hour, taken out and dried to obtain a pretreated sample pad, wherein the sample treatment solution is an aqueous solution containing 0.5% polyvinyl pyrrolidone by weight, 0.5% casein by weight, 0.5% Tween 20 by volume and 1.0% Tris by weight.

[0103] 2.5. Assembly and cutting of test strips

[0104] The sample pad is attached to the middle of the PVC base plate. In a vertically symmetrical manner, the binding pad loaded with 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 board is cut into strips with a width of 4 mm each. The cut test strips are spliced ​​with the sample pad according to the type of the test object to obtain H-shaped test strips.

[0105] 2.6 Preparation of test paper cards

[0106] Prepare the cover plate for the jam ( Figure 1 and Figure 2 ) and card slot bottom plate ( Figure 3 ), wherein the overall length of the card shell formed by the cover plate and the card slot bottom plate being buckled together is 118mm, the width is 35.7mm, and the width of the card slot in the card slot bottom plate is 4.6mm. The height of the cover plate is 2.1mm, the height of the boss in the cover plate is 1.85mm, and the length of the boss close to the reaction zone from the nearest edge of the observation window is 1.8mm. First, place the two test strips vertically on the card slot bottom plate, and then place the sample pad with a small PVC bottom plate attached to the bottom horizontally (i.e., the sample adding tape connecting the test strip 1 and the test strip 2), and the sample adding tape is built to connect the test strip 1 and the test strip 2 and is placed horizontally in the groove in the middle of the card slot bottom plate to obtain a test paper card.

[0107] 3. Detection method of test paper card

[0108] Immediately place the swab after collecting the sample into 0.5 ml of sample extract in the sample extraction tube. The swab head should be rotated and mixed in the sample extract for at least 30 seconds. At the same time, squeeze the swab head at least 5 times through the outer wall of the sample extraction tube to ensure that the sample is fully eluted in the sampling tube. After squeezing the swab head liquid dry through the outer wall of the sample extraction tube, discard the swab and cover the extraction tube cap.

[0109] Add the treated sample liquid droplets to the spot hole of the test paper card, let it stand for 10 minutes, and observe the test results:

[0110] When both the test line and the quality control line are colored, the sample is positive, indicating that the sample is infected with the pathogenic microorganism corresponding to the second antibody on the test line;

[0111] When the quality control line shows color but the test line does not, the sample is negative, indicating that the sample does not contain the pathogenic microorganism corresponding to the second antibody on the test line;

[0112] If the quality control line does not show color, retest.

[0113] Example 2

[0114] Example 1 Clinical sample detection of test paper card

[0115] The recombinant protein control of the target pathogenic microorganism was used as a sample, and the sample was treated according to the method of Example 1, and was diluted 2 times to obtain sample solutions with concentrations of 10pg / ml, 20pg / ml, 40pg / ml, 80pg / ml, and 160pg / ml, respectively; the sample solutions of different concentrations were dripped onto the test paper card of Example 1, and the color development was observed. At the same time, the front nasal swab sample buffer of healthy people with negative PCR results was used as a negative matrix for detection.

[0116] The source information of the recombinant protein control products of the target pathogenic microorganisms is shown in Table 1.

[0117] Table 1 List of sources of recombinant protein controls of target pathogenic microorganisms

[0118]

[0119] The same sample liquid was used as the test object, and 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), new coronavirus (Wuhan Mingde Biotechnology Co., Ltd., AgH-20A), Mycoplasma pneumoniae (Hangzhou Innovation Biological Inspection Technology Co., Ltd., P111005), respiratory adenovirus (Hangzhou Innovation Biological Inspection Technology Co., Ltd., P111004), respiratory syncytial virus (Hangzhou Innovation Biological Inspection Technology Co., Ltd., P111003), rhinovirus (Guangzhou Daan Gene Co., Ltd., RP2020-1), parainfluenza virus (Shandong Kanghua Biomedical Technology Co., Ltd., k48595), Streptococcus pneumoniae (Beijing Bell Bioengineering Co., Ltd., 303633) and group A streptococcus (Beijing Jinwolf Bioengineering Technology Co., Ltd., 55296).

[0120] Table 2 Test results

[0121]

[0122] Note: “-” means no color development; “±” means weak positive, and the color intensity is weaker than “+”; “+” means different color intensity of the test line. The more “+” there are, the darker the color of the test line is.

[0123] As can be seen from Table 2, compared with conventional test cards, Example 1 has higher production efficiency, fewer sample additions, more obvious and clear color development, and is more user-friendly. By using recombinant proteins to test the two, it was found that Example 1 without the conjugate pad has higher sensitivity than conventional card strips, and stronger color development at low concentrations.

[0124] Comparative Example 1

[0125] The test card was prepared according to the method of Example 1, except that the sample pad was treated with sample pad treating solution 2. The sample pad treating solution 2 was a 0.8% Tris aqueous solution containing 0.5% by weight polyvinyl pyrrolidone and 1% by weight bovine serum albumin.

[0126] Table 3 Test results

[0127]

[0128] Note: “-” means no color; “+” means different color intensity of the test line. The more “+”, the darker the color of the test line.

[0129] From the results in Table 3 above, it can be seen that the sample pad has poor hydrophilicity, which leads to slow liquid flow. After the sample is dripped into the sample well of the test paper card, the sample pad is located below the sample well. The sample pad is not hydrophilic enough, which leads to obstruction of liquid flow. The liquid gathers on the sample pad and diffuses slowly, resulting in uneven sample flow, unilateral chromatography and slow chromatography. In addition, the sample pad treatment solution before optimization contains too high a concentration of blocking agent bovine serum albumin, resulting in poor sensitivity.

[0130] Comparative Example 2

[0131] The test card was prepared according to the method of Example 1, except that the support of the bottom plate was missing under the sample pad in the middle of the H-type test paper strip. The test card prepared above was tested according to the method of Example 2, and the results are shown in Table 4.

[0132] Table 4 Test results

[0133]

[0134]

[0135] Note: “-” means no color; “+” means different color intensity of the test line. The more “+”, the darker the color of the test line.

[0136] From the above results, it can be seen that only a thin layer of sample pad is placed in the middle of the H-type test strip, which cannot ensure that a sufficient amount of liquid flows to the test strip. The glass fiber membrane / polyester membrane is thin, and after the sample is dripped into it, the liquid remains at the bottom of the glass fiber membrane / polyester membrane, resulting in a reduction in the amount of sample flowing to the test strip chromatography, resulting in a decrease in the sensitivity of the test results, affecting the accuracy and reliability of the test results.

[0137] In response to this problem, the present invention adds a PVC base plate that matches the size of the sample pad at the bottom of the middle sample pad of the H-type test paper strip. The size of the small PVC base plate is 7×4mm, and the size of the H-type middle sample pad is 18×4mm. The small base plate is attached to the middle directly below the sample pad, and the extra parts on the left and right ends of the sample pad just rest on the test strips on the left and right ends. Adding a small PVC base plate at the bottom of the sample pad can not only effectively prevent excessive liquid from gathering at the bottom of the sample pad and reducing the amount of liquid, but also make the overall structure of the test paper card more stable. The small PVC base plate provides a stable support platform for the H-type middle sample pad, allowing the sample to flow more smoothly to the test strips on the left and right sides, effectively improving the liquid flow.

[0138] Comparative Example 3

[0139] The test card was prepared according to the method of Example 1, except that the height of the boss in the card cover was 2.2 mm, and the length of the boss near the reaction area from 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.

[0140] Table 5 Test results

[0141]

[0142]

[0143] Note: “-” indicates no color development; “+” indicates different color development intensities of the test line. The more “+” there are, the darker the color of the test line will be. “ / ” indicates no detection.

[0144] It can be seen from the above results that if the boss is too high, it will severely squeeze the test strip, causing the flow channel to deform or even damage the test strip. The boss height is 2.2mm, and the height after adjustment is 1.85mm. In Example 1, the position of the boss closest to the observation window is moved 0.5mm away from the observation window, which is beneficial to reduce the local pressure of the boss on the nitrocellulose membrane, thereby causing inaccurate test results. This shows that the design of the test card shell structure is not reasonable enough, the pressure distribution is uneven, the test strip is squeezed during the fixation process, the flow channel is deformed, and the liquid flows unsmoothly, resulting in poor repeatability of the test results.

[0145] Comparative Example 4

[0146] The test card was prepared according to the method of Example 1, and the test was performed according to the method of Example 2, except that the amount of sample added to each test card was 3 drops (about 80 μL). The results showed that 3 drops of sample was far from enough, and the liquid could not flow after adding the sample, resulting in the phenomenon of inability to chromatographically separate, which could not allow the test paper card to have a good chromatographic result.

[0147] When testing the sample volume, the test card of the present invention added 6 drops (about 160 μL). The results showed that the sample moved at a uniform speed on the test card. While increasing the sample migration speed, the reaction area of ​​the test strip was sufficiently infiltrated, so that the antigen-antibody complex could react fully, avoiding signal attenuation and false negative problems caused by insufficient sample volume.

[0148] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A test strip for multiple respiratory pathogenic microorganisms combined with immunochromatographic detection, characterized in that: It includes a test strip 1 and a test strip 2 both placed in a vertical direction and a sample adding strip placed in a horizontal direction; A sample adding area is respectively arranged in the middle of the test strip 1 and the test strip 2, and a binding area, a reaction area and a water absorption area are overlapped symmetrically in sequence with each sample adding area as the center; one end of the sample adding belt overlaps the sample adding area of ​​the test strip 1, and the other end of the sample adding belt overlaps the sample adding area on the test strip 2; Each binding area is loaded with at least one first antibody labeled with latex microspheres; each reaction area is provided with at least one detection line and a quality 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 multiple respiratory pathogenic microorganisms combined immunochromatographic test strip according to claim 1, characterized in that: The test strip 1 is overlapped with a first water absorption area, a first reaction area, a first binding area, a first sample addition area, a second binding area, a second reaction area and a second water absorption area in sequence from top to bottom; The test strip 2 is overlapped with a third water absorption area, a third reaction area, a third binding area, a second sample addition area, a fourth binding area, a fourth reaction area and a fourth water absorption area in sequence 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 kinds of first antibodies against respiratory pathogenic microorganisms labeled with latex microspheres; The first reaction zone, the second reaction zone, the third reaction zone and the fourth reaction zone are each provided with a quality control line and a detection line with the same number of first antibodies loaded as the binding zone at the same end of the test strip; the detection line is coated with a second antibody against respiratory pathogenic microorganisms.

3. The multiple respiratory pathogenic microorganisms combined immunochromatographic detection test strip according to claim 2, characterized in that: On each binding area, the loading mass concentration of the first antibody of respiratory pathogenic microorganisms labeled with latex microspheres is independently 0.3% to 0.4%.

4. The multiple respiratory pathogenic microorganisms combined immunochromatographic detection test strip according to claim 2, characterized in that: The particle size of the latex microspheres ranges from 300 to 400 nm.

5. The multiple respiratory pathogenic microorganisms combined immunochromatographic test strip according to claim 2, characterized in that: On each test line, the coating concentration of the second antibody against respiratory pathogenic microorganisms is independently 0.5-2.0 mg / mL.

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

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

8. The multiple respiratory pathogenic microorganisms combined immunochromatographic test strip according to claim 2, characterized in that: The sample adding pads in the sample adding area and the sample adding pads in the sample adding belt on the test strip 1 and the test strip 2 are treated with a treatment liquid in advance; The treatment solution is a 0.8% to 1.2% Tris aqueous solution containing 0.5% to 1% polyvinyl pyrrolidone by mass, 0.5% to 1% casein by mass, 0.5% to 0.8% Tween20 by volume.

9. The test strip for combined immunochromatographic detection of 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 of influenza B virus labeled with blue latex microspheres, a first antibody of influenza A virus labeled with blue latex microspheres, and a first antibody of the new coronavirus labeled with red latex microspheres; the second binding pad region is coated with a first antibody of Mycoplasma pneumoniae labeled with red latex microspheres, a first antibody of respiratory adenovirus labeled with blue latex microspheres, and a first antibody of respiratory syncytial virus labeled with blue latex microspheres; the third binding region is coated with a first antibody of rhinovirus labeled with blue latex microspheres, and a first antibody of parainfluenza virus labeled with red latex microspheres; the fourth binding region is coated with a first antibody of group A streptococcus labeled with red latex microspheres, and a first antibody of Streptococcus pneumoniae labeled with blue latex microspheres; The first reaction area is provided with three detection lines and a first quality control line, and 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 the new coronavirus; The second reaction area is provided with three detection lines and a second quality control line, and 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 provided with two detection lines and a third quality control line, and the two detection lines are respectively coated with a second antibody against rhinovirus and a second antibody against parainfluenza virus; The fourth reaction zone is provided with two detection lines and a fourth quality control line, and the two detection lines are respectively coated with a second antibody against group A streptococci and a second antibody against streptococci pneumoniae.

10. A test paper card for multiple respiratory pathogenic microorganisms combined with immunochromatographic detection, characterized in that: It comprises the test strip, the card slot bottom plate and the cover plate as described in any one of claims 1 to 9; The shape of the card slot on the card slot bottom plate is adapted to the structure of the test paper strip and is used to fix the test paper strip; The cover plate is provided with hollow spotting holes and a plurality of observation windows; The sample spot hole is located above the sample adding belt; an observation window is arranged above each reaction area in the test strip for observing the test result.

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