Kit for joint detection of four respiratory virus antigens and application thereof

Abstract

The invention provides a kit for joint detection of four respiratory virus antigens and application of the kit. The respiratory viruses include a new crown virus, an influenza A virus, an influenza B virus and a respiratory syncytial virus. The kit comprises a test strip, wherein the test strip comprises a first marker pad, a second marker pad and a nitrocellulose membrane. The nitrocellulose membrane comprises five lines, four lines are different virus antibody lines, and one line is a quality control line. The method has the advantages that cross influence and false positive are avoided, and the detection accuracy, sensitivity and specificity are improved; 2, the loading workload is reduced, and the detection efficiency of four items is improved; 3, the sampling frequency is reduced, the repeated contact between medical personnel and patients in multiple sampling processes is reduced, and the epidemic situation spreading risk is reduced; and fourthly, when a nose swab or a throat swab and other tools are used for detection, the injury to a detected person is obviously reduced, and the safety is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a kit for jointly detecting four respiratory virus antigens and an application thereof. Background Art

[0002] The new coronavirus (2019-nCOV) belongs to the β-genus coronavirus, has an envelope, and the particles are round or oval with a diameter of 60 to 140nm. After infection with the 2019 new coronavirus, some patients have decreased or lost sense of smell and taste as the first symptoms, and a few patients have symptoms such as nasal congestion, runny nose, sore throat, conjunctivitis, myalgia and diarrhea. Severe patients often experience dyspnea and (or) hypoxemia one week after onset. Severe cases can quickly progress to acute respiratory distress syndrome, septic shock, difficult to correct metabolic acidosis and coagulation dysfunction and multiple organ failure. A very small number of patients may also have manifestations such as involvement of the central nervous system and ischemic necrosis of the extremities.

[0003] Influenza A (Flu A) and influenza B (Flu B) are acute respiratory infectious diseases caused by influenza A and B viruses infecting the human body respectively. Influenza viruses are divided into three types: A, B and C. Among them, influenza caused by influenza A and B viruses is the most common. Influenza is a Class C infectious disease in my country. The disease has the characteristics of rapid transmission, severe condition and high mortality rate. Influenza virus is mainly transmitted through respiratory droplets, and can also be transmitted through direct contact with the patient's oral and nasal secretions, or indirect contact with daily utensils such as towels and clothes contaminated by the virus. Typical influenza has an acute onset, usually with systemic poisoning symptoms such as high fever, chills, headache, fatigue, loss of appetite and muscle aches. Upper respiratory tract symptoms are relatively mild or not obvious. Most patients have a good prognosis. A few patients may suffer from bacterial upper respiratory tract infections, tracheitis, bronchitis, bacterial pneumonia and other diseases, which may lead to death in severe cases.

[0004] Respiratory syncytial virus (RSV) is a common cause of respiratory infections worldwide. It is the most common pathogen for respiratory infections in infants and young children, and the most common cause of lung infections in infants under 1 year old. It can cause respiratory diseases such as infections of the trachea, lungs and middle ear. After infection, there will be fever and other symptoms such as chills, headache, loss of appetite, body aches or weakness.

[0005] Most of the existing testing products (test kits) on the market can only perform single tests for COVID-19, influenza or respiratory syncytial virus. When both need to be tested simultaneously clinically, repeated sampling is usually required. The operation is cumbersome and there is an exposure risk, which increases the burden on patients and the risk of infection for medical staff.

[0006] The Chinese utility model patent with the authorization announcement number CN 214895338U discloses a triple test card for respiratory syncytial virus, influenza A and influenza B, which can simultaneously detect respiratory syncytial virus, influenza A and influenza B. However, on the one hand, the test card can only detect three respiratory viruses at the same time; on the other hand, the test card is provided with two nitrocellulose membranes, and the three virus antibodies are respectively arranged on three gold label pads, which will absorb part of the sample. The increase in the number of gold label pads increases the demand for samples; on the other hand, the test card does not reflect the sensitivity of the test.

[0007] Therefore, there is an urgent need to find a test kit that can simultaneously detect four respiratory viruses with high accuracy, sensitivity, and specificity and requires a small amount of sample to meet people's needs. Summary of the invention

[0008] In order to solve the problems existing in the prior art, the present invention provides a kit for joint detection of four respiratory virus antigens and its application. On the one hand, it significantly reduces the possibility of cross-influence and false positives, and improves the accuracy, sensitivity and specificity of detection; on the other hand, it reduces the workload of loading samples and improves the detection efficiency of the four items; on the other hand, it reduces the number of sampling times, reduces the repeated contact between medical staff and patients during multiple sampling processes, and reduces the risk of epidemic transmission; at the same time, when using tools such as nasal swabs or throat swabs for detection, it will significantly reduce the harm to the test subjects and improve safety.

[0009] In order to achieve the above object, the present invention adopts the following scheme:

[0010] On the one hand, the present invention provides a kit for jointly testing four respiratory virus antigens, wherein the respiratory viruses include the new coronavirus, influenza A, influenza B and respiratory syncytial virus; the kit includes a test strip, and the test strip includes two layers of marking pads and a nitrocellulose membrane, from left to right, respectively, a first marking pad, a second marking pad and a nitrocellulose membrane; the first marking pad contains a labeled mouse anti-Flu A antibody and a mouse anti-Flu B antibody, and the second marking pad contains a labeled mouse anti-COV antibody and a mouse anti-RSV antibody.

[0011] In some embodiments, when the novel coronavirus antibody and the respiratory syncytial virus antibody are on the first marking pad, COV has a risk of false positive and its sensitivity is reduced. The reason may be that the properties of COV itself have changed in some way, thereby increasing the risk of false positive, and the COV reaction time is prolonged, thereby reducing sensitivity. Therefore, COV antibodies and RSV antibodies are set on the second marking pad, and Flu A and Flu B antibodies are set on the first marking pad.

[0012] Furthermore, the nitrocellulose membrane includes 5 lines, which are RSV detection line, COV detection line, Flu A detection line, Flu B detection line and C line (quality control line) in order from the direction close to the second marking pad.

[0013] In some embodiments, when the Flu A test line and the Flu B test line are in front and the COV test line is in the back, FluA produces a false positive phenomenon, and Flu B has a risk of false positive, and COV sensitivity decreases at the same time. The reason may be that the properties of Flu A and Flu B themselves have changed in a certain way, so a false positive phenomenon occurs or a false positive risk is generated, and the COV reaction time is extended, so the sensitivity is reduced. And when the COV test line is in the first place, COV has a false positive phenomenon, and the possible reason is that the COV itself has changed in a certain way, so a false positive phenomenon occurs. Therefore, the test line sequence is set to be RSV test line, COV test line, Flu A test line, Flu B test line and C line (quality control line) in turn from the direction close to the second marking pad.

[0014] Furthermore, the nitrocellulose membrane (NC membrane) includes one or more of PALL 120, CN 140, and MDI 150.

[0015] Preferably, the nitrocellulose membrane is an MDI 150 membrane.

[0016] Under the same conditions, PALL 120 and CN 140 membranes will both have a certain probability of false positives when used, while when MDI 150 membrane is used, false positives basically do not occur. Therefore, NC membrane is preferably MDI 150 membrane, which greatly reduces the possibility of cross-influence and false positives and improves the accuracy and specificity of detection.

[0017] Furthermore, the antibody on the labeling pad is labeled with latex microspheres.

[0018] Experiments have shown that antibodies can be labeled with colloidal gold or latex microspheres. Under the same conditions, antibodies labeled with latex microspheres ultimately display a darker color on the NC membrane, and the numbers are larger when compared with the standard colorimetric card. Therefore, latex microsphere labeling is preferred to further improve the sensitivity of detection.

[0019] Furthermore, the concentrations of the antibodies are: FluA: 0.15-0.3 ml / ml; FluB: 0.15-0.3 ml / ml; COV: 0.3 ml / ml; RSV: 0.3 ml / ml.

[0020] Preferably, the concentrations of the antibodies are: FluA: 0.3 ml / ml; FluB: 0.3 ml / ml; COV: 0.3 ml / ml; RSV: 0.3 ml / ml.

[0021] According to the experimental results, when the antibody concentration on the marking pad is within a certain range, false positive phenomenon will not occur, especially when the preferred antibody concentration is FluA: 0.3ml / ml; FluB: 0.3ml / ml; COV: 0.3ml / ml; RSV: 0.3ml / ml, the color development result on the NC membrane is compared with the standard colorimetric card, and the number is larger, so the concentration is preferred to improve the sensitivity and accuracy of the detection.

[0022] In another aspect, the present invention provides a use of latex microspheres for preparing a highly sensitive kit for joint detection of four respiratory virus antigens, wherein the latex microspheres are respectively labeled on antibodies of two layers of labeling pads and are 400 nm red carboxyl latex microspheres.

[0023] In another aspect, the present invention provides a use of a nitrocellulose membrane for preparing a highly specific kit for joint detection of four respiratory virus antigens, wherein the nitrocellulose membrane comprises one or more of PALL 120, CN 140, and MDI 150.

[0024] Preferably, the nitrocellulose membrane is an MDI 150 membrane.

[0025] In another aspect, the present invention provides a method for jointly detecting four respiratory virus antigens, using the kit as described in any one of claims 1 to 7 to detect four respiratory viruses.

[0026] In another aspect, the present invention provides a method for preparing a kit for jointly testing four respiratory virus antigens, wherein the kit comprises a test strip, and the test strip comprises a first marking pad and a second marking pad.

[0027] The beneficial effects of the present invention are:

[0028] 1. Two layers of marking pads and NC membranes are arranged from left to right, the first marking pad contains labeled mouse anti-Flu A antibody and mouse anti-Flu B antibody, the second marking pad contains labeled mouse anti-COV antibody and mouse anti-RSV antibody, RSV detection line, COV detection line, Flu A detection line, Flu B detection line and C line (quality control line) are arranged on the NC membrane in sequence from the direction close to the second marking pad to prevent false positive or reduced sensitivity, significantly reduce the possibility of cross-influence and false positive, and improve the accuracy, sensitivity and specificity of detection.

[0029] 2. Setting up two layers of marking pads allows the use of higher antibody concentrations to improve detection sensitivity; on the other hand, the thickness of the two layers of marking pads makes the card shell extrusion tightness appropriate, which promotes the flow of samples; on the other hand, the two layers of marking pads require an appropriate amount of sample, which reduces the sampling workload, reduces the repeated contact between medical staff and patients during multiple sampling processes, and reduces the risk of epidemic transmission; the reduction in the required sample volume also reduces the sampling workload and improves the detection efficiency of the four items; at the same time, when using tools such as nasal swabs or throat swabs for testing, it will significantly reduce the harm to the subjects and improve safety.

[0030] 3. The use of MDI 150 membrane greatly reduces the possibility of false positives and further improves the specificity and accuracy of detection.

[0031] 4. The appropriate antibody concentration range on the marking pad reduces the possibility of false positives and improves the specificity and accuracy of the test; at the same time, the optimal antibody concentration increases in numbers on the standard colorimetric card, further improving the sensitivity of the test.

[0032] 5. Use latex microspheres to label the antibodies on the labeling pad to further improve the sensitivity of the detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a schematic diagram of the structure of a quadruple test strip.

[0034] Figure 2 This is a schematic diagram of the standard colorimetric card. DETAILED DESCRIPTION

[0035] The present invention will be further described in detail below in conjunction with the examples. It should be pointed out that the examples described below are intended to facilitate the understanding of the present invention and do not have any limiting effect on the present invention.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0037] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0038] In the following experiments, "numbers indicate the depth of color. The larger the number, the darker the color. The numbers from left to right indicate the results of Flu B, Flu A, COV, and RSV, respectively. Colors above 3 are considered positive, and negative results are preferably 1, and 2 indicates a certain risk."

[0039] Example 1 Preparation of quadruple test strips

[0040] The four-test strip provided in this embodiment is the optimal combination and is prepared as follows:

[0041] Refer to the attached Figure 1A four-in-one test strip comprises a base plate, a sample pad, a first marking pad (Flu AFlu B marking pad), a second marking pad (COV RSV marking pad), a nitrocellulose membrane (NC membrane) and a water-absorbing filter paper; the upper end surface of the base plate is provided with the first marking pad, the second marking pad, the nitrocellulose membrane and the water-absorbing filter paper, one side of the nitrocellulose membrane is provided with the water-absorbing filter paper, the other side is provided with the second marking pad, and the other side of the second marking pad is provided with the first marking pad, one end of the lower end surface of the second marking pad is connected to one end of the upper end surface of the nitrocellulose membrane, one end of the lower end surface of the water-absorbing filter paper is connected to the other end of the upper end surface of the nitrocellulose membrane, and one end of the lower end surface of the first marking pad is connected to the upper end surface of the corresponding end of the second marking pad; one side of the lower end surface of the sample pad is bonded to the upper end surface of the base plate, and the lower end surface of the sample pad is connected to the upper end surface of the corresponding side of the first marking pad.

[0042] The first marking pad includes mouse anti-Flu A antibody (the antigen is the N protein in Flu A, purchased from Hangzhou Boyin Biotechnology Co., Ltd.) and mouse anti-Flu B antibody (the antigen is the N protein in Flu B, purchased from Hangzhou Boyin Biotechnology Co., Ltd.) labeled with latex microspheres, and the second marking pad includes mouse anti-COV antibody (the antigen is the N protein in COV, purchased from Hangzhou Boyin Biotechnology Co., Ltd.) and mouse anti-RSV antibody (the antigen is the N protein in RSV, purchased from Hangzhou Boyin Biotechnology Co., Ltd.) labeled with latex microspheres. The antibody concentrations are Flu A: 0.3ml / ml; Flu B: 0.3ml / ml on the first marking pad; COV: 0.3ml / ml; RSV: 0.3ml / ml on the second marking pad; the latex microspheres are 400nm red carboxyl latex microspheres purchased from MAGSPHERE INC, and the antibodies are labeled antibodies.

[0043] At the same time, the NC membrane is provided with RSV detection line, COV detection line, Flu A detection line, FluB detection line and quality control line C line from the direction close to the marking pad; the RSV detection line is a detection line containing mouse anti-RSV antibody II, and the antibody concentration is 2 mg / mL, 0.7 ul / cm; the COV detection line is a detection line containing mouse anti-COV antibody II, and the antibody concentration is 2 mg / mL, 0.7 ul / cm; the Flu A detection line is a detection line containing mouse anti-Flu A antibody II, and the antibody concentration is 2 mg / mL, 0.7 ul / cm; the Flu B detection line is a detection line containing mouse anti-Flu B antibody II, and the antibody concentration is 2 mg / mL, 0.7 ul / cm; the quality control line C line is a detection line containing goat anti-mouse IgG, and the antibody concentration is 2 mg / mL, 0.7 ul / cm; the antibody is a coated antibody; the NC membrane is selected as MDI 150 membrane, which is purchased from the company ADVANCED MICRODEVICES PVT, membrane model is 150CNPH-N-SS40.

[0044] When in use, add the sample from the front of the test card at one time, and the same sample flows to the respiratory syncytial virus, new coronavirus, influenza A, and influenza B combined test strips through the bridged sample pad. After the reaction time is over, the test results of four different test items can be read from it; that is, one sample is loaded and four items of influenza A, influenza B, respiratory syncytial virus, and new coronavirus are tested at the same time. This not only greatly reduces the workload of loading, but also can improve the detection efficiency of the four items. On the other hand, the reduction in the number of sample loadings will also reduce the number of sampling times. In particular, when using tools such as nasal swabs or throat swabs for testing, the harm to the person being tested will be significantly reduced.

[0045] This embodiment provides the optimal composition and preparation of a quadruple test strip. The use of the quadruple test strip significantly reduces the risk of false positives and cross-influence, greatly improves the specificity, accuracy and sensitivity of the test, and at the same time improves the detection efficiency of the four items, reduces the sample demand, reduces the workload of sampling and loading, and increases the safety of use.

[0046] Example 2 Sequential arrangement of marking pads and sequential arrangement of detection lines on NC membrane

[0047] In order to obtain the best effect in Example 1, this example combined and screened the sequence of the label pad and the detection line on the NC membrane in the following ways (the other unmentioned operation processes and conditions are the optimal conditions in Example 1, which are the same; at the same time, the antibody is stated in different ways, which are all abbreviations of the complete statement in Example 1, such as: mouse anti-Flu A antibody is abbreviated as Flu A antibody):

[0048] 1. The first marking pad includes Flu A and Flu B antibodies, the second marking pad includes COV and RSV antibodies, and the order of the detection lines from left to right is RSV detection line, COV detection line, Flu A detection line, Flu B detection line and C line;

[0049] 2. The first marking pad includes Flu A and Flu B antibodies, the second marking pad includes COV and RSV antibodies, and the order of the detection lines from left to right is Flu A detection line, Flu B detection line, RSV detection line, COV detection line and C line;

[0050] 3. The first marking pad includes Flu A and Flu B antibodies, the second marking pad includes COV and RSV antibodies, and the order of the detection lines from left to right is COV detection line, RSV detection line, Flu A detection line, Flu B detection line, and C line;

[0051] 4. The first marking pad includes COV and RSV antibodies, the second marking pad includes Flu A and Flu B antibodies, and the order of the detection lines from left to right is RSV detection line, COV detection line, Flu A detection line, Flu B detection line and C line;

[0052] 5. The first marking pad includes COV and RSV antibodies, the second marking pad includes Flu A and Flu B antibodies, and the order of the detection lines from left to right is Flu A detection line, Flu B detection line, RSV detection line, COV detection line and C line.

[0053] 6. The first marking pad includes COV and RSV antibodies, the second marking pad includes Flu A and Flu B antibodies, and the order of the detection lines from left to right is COV detection line, RSV detection line, Flu A detection line, Flu B detection line, and C line;

[0054] The test samples are blank buffer and Flu A, Flu B, COV, RSV antigens. Figure 2 The standard colorimetric card is used, and the test results are shown in Table 1. The blank buffer solution includes 12g / L Tris (trishydroxymethylaminomethane), 1g / LCasin-Na (sodium casein), 7ml / L Triton-X100 (Triton X-100), 9g / L NaCl (sodium chloride), 20g / LKCl (potassium chloride), 10g / L NaCO3 (sodium carbonate), 5ml / L Proclin300 (a preservative), 2ml / L Niusha BND-10 (an antibacterial agent, the main component of which is 10% 5-bromo-5-nitro-1,3-dioxane propanediol solution), and the sample solution is the four antigens added to the buffer. (The preparation of the buffer and sample solution described below is the same as this)

[0055] Table 1 The influence of different arrangement orders on the results

[0056]

[0057]

[0058] Note: "The numbers indicate the depth of the color. The larger the number, the darker the color. The numbers from left to right indicate the results of FluB, Flu A, COV, and RSV. Colors above 3 are considered positive, and negative results are best 1, and 2 indicates a certain risk."

[0059] According to the results in Table 1, it can be seen that only the first arrangement has no effect on the Buffer, and Flu A, Flu B, COV, and RSV do not show false positives and there is no risk, while the sensitivity is high;

[0060] In the second arrangement, Flu A in the buffer shows a false positive phenomenon, Flu B has a false positive risk, and the sensitivity of COV decreases. The reason may be that when the Flu A and Flu B detection lines are in front, their own properties have changed, thus producing a false positive phenomenon or a false positive risk; at the same time, when the Flu A and Flu B detection lines are in front and COV is in the back, the sensitivity of COV decreases, which may be due to the order of the membrane detection lines, which prolongs the reaction time of COV, so the sensitivity of COV decreases. Combined with the first result, it can be seen that the Flu A and Flu B detection lines should be ranked in the last order, and COV should be ranked in the first or second order.

[0061] In the third arrangement, COV in Buffer shows a false positive phenomenon. The reason may be that when the COV test line is in the first order, its own properties have changed in some way, thus producing a false positive phenomenon. Combining the results of the first and second methods, it can be seen that the COV test line should be in the second order.

[0062] In the fourth arrangement, there is a risk of false positives for COV in the buffer, and the sensitivity of COV decreases. The reason may be that when COV and RSV antibodies are on the front marker pad (the first marker pad) and Flu A and Flu B antibodies are on the back marker pad (the second marker pad), the nature of COV itself changes, increasing the risk of false positives; at the same time, the COV reaction time is prolonged, so the sensitivity is reduced. Combined with the first result, it can be seen that Flu A and Flu B antibodies should be on the first marker pad, and COV and RSV antibodies should be on the second marker pad.

[0063] In the fifth arrangement, there is a risk of false positives for Flu B and COV in the buffer, and Flu A shows a false positive phenomenon, while the sensitivity of COV is reduced. Combined with the second and fourth results, the reason may be that when COV and RSV antibodies are on the front marking pad (first marking pad) and Flu A and Flu B antibodies are on the back marking pad (second marking pad), the nature of COV itself has changed, increasing the risk of false positives; and when the Flu A and Flu B detection lines are in front, their own properties have changed, so false positives or the risk of false positives are generated; at the same time, when the COV detection line is in the back, the sensitivity of COV decreases, which may be due to the order of the membrane detection line, which prolongs the reaction time of COV, so the sensitivity of COV decreases. Combined with the above results, it further verifies that Flu A and Flu B antibodies should be on the first marking pad, COV and RSV antibodies should be on the second marking pad, Flu A and Flu B detection lines should be in the back sequence, and COV should be in the first and second sequence positions.

[0064] In the sixth arrangement, COV in Buffer shows a false positive phenomenon. The reason may be that when the COV test line is in the first position, its own properties have changed, thus producing a false positive phenomenon. Combined with the above results, it further verifies that FluA and Flu B antibodies should be on the first marking pad, COV and RSV antibodies should be on the second marking pad, Flu A and Flu B test lines should be in the last order, and COV should be in the second order.

[0065] Combined with the above results, we determined the optimal order of antibodies on the marker pad and detection lines on the NC membrane. When the first marker pad includes Flu A and Flu B antibodies, the second marker pad includes COV and RSV antibodies, and the NC membrane is in the order of RSV detection line, COV detection line, Flu A detection line, Flu B detection line and C line from the direction close to the second marker pad, the false positive phenomenon and risk are eliminated, and the sensitivity of the test is high, which improves the specificity, accuracy and sensitivity of the test.

[0066] Example 3 Selection of the number of marking pads on the quadruple test strip

[0067] In order to obtain the best effect in Example 1, the number of labeling pads was screened in the present example in the following ways (the rest of the operation processes and conditions not mentioned are the optimal conditions in Example 1 and are the same; at the same time, the antibody is described in different ways and is an abbreviation of the complete description in Example 1, such as: mouse anti-Flu A antibody is abbreviated as Flu A antibody):

[0068] 1. Two-layer labeling pad, the first labeling pad includes Flu A and Flu B antibodies, and the second labeling pad includes COV and RSV antibodies;

[0069] 2. A layer of labeling pad, which includes four antibodies: Flu A, Flu B, COV and RSV;

[0070] 3. Three-layer marking pad, the first marking pad includes Flu A and Flu B antibodies, the second marking pad includes COV antibodies, and the third marking pad includes RSV antibodies.

[0071] The test samples are blank buffer and Flu A, Flu B, COV, RSV antigens. Figure 2 The test results are shown in Table 2. (The preparation of the buffer and the sample solution is the same as in Example 2)

[0072] Table 2 Effect of different number of marking pads on the results

[0073]

[0074] Note: "The numbers indicate the depth of the color. The larger the number, the darker the color. The numbers from left to right indicate the results of FluB, Flu A, COV, and RSV. Colors above 3 are considered positive, and negative results are best 1, and 2 indicates a certain risk."

[0075] According to the results in Table 2, it can be seen that as shown in ② and ③, when one layer of marking pad is used and the antibody concentration on the marking pad is 0.2ml / ml, no false positive occurs; when the antibody concentration on the marking pad is 0.25ml / ml, Flu A has a false positive phenomenon, and both Flu B and COV have a false positive risk, indicating that when one layer of marking pad is used, the antibody concentration increases and the possibility of false positive increases; at the same time, as shown in ①, when two layers of marking pads are used and the antibody concentrations on the marking pads are 0.3ml / ml, no false positive occurs, and compared with ②, the corresponding numbers of Flu A, Flu B, and COV on the standard colorimetric card are larger, indicating that the detection sensitivity of FluA, Flu B, and COV is higher.

[0076] It can be seen that when using two layers of labeling pads, higher antibody concentrations can be used to improve the detection sensitivity and accuracy of Flu A, Flu B, and COV.

[0077] As shown in ① and ④, when two or three layers of marking pads are used, no false positive phenomenon occurs, and the detection sensitivity is the same, indicating that there is no difference in detection when using two or three layers of marking pads. However, the thickness of the two-layer marking pad makes the card shell squeeze tightness appropriate. If three layers of marking pads are used, the card shell will be pressed too tightly, affecting the flow of the sample; at the same time, the marking pad will absorb part of the sample. The more marking pads there are, the larger the sample volume required, which increases the sampling workload.

[0078] It can be seen from this that the use of two layers of marking pads is the best choice, which further reduces the possibility of false positives and improves the sensitivity, accuracy and specificity of detection; at the same time, it reduces the workload of sampling and loading, reduces the risk of epidemic transmission, improves detection efficiency, and improves the safety of detection.

[0079] Example 4 Screening of nitrocellulose membrane

[0080] In order to obtain the best effect in Example 1, the NC membranes were screened in this example, and different NC membranes were used, among which PALL 120 was purchased from Pall Corporation, and the membrane model was Vivid 120; CN 140 was purchased from Sartorius, and the membrane model was CN 140; MDI 150 was purchased from ADVANCED MICRODEVICES PVT, and the membrane model was 150CNPH-N-SS40; the sample was Buffer, and the results of 0 days, 3 days and accelerated 15 days were observed respectively. Figure 2 The experimental results are shown in Table 3 (the other operating processes and conditions not mentioned are the optimal conditions in Example 1, which are the same; the Buffer is the same as in Example 2):

[0081] Table 3 Results of using different NC membranes

[0082] NC membrane Day 0 3 days Accelerate 15 days PALL 120 2 / 3.5 / 3.5 / 1 2 / 4 / 4 / 1 3 / 6 / 6 / 1 CN 140 1 / 2 / 2 / 1 1 / 3 / 3 / 1 1 / 5 / 5 / 1 MDI 150 1 / 1 / 1 / 1 1 / 1 / 1 / 1 1 / 2 / 2 / 1

[0083] Note: "The numbers indicate the depth of the color. The larger the number, the darker the color. The numbers from left to right indicate the results of FluB, Flu A, COV, and RSV. Colors above 3 are considered positive, and negative results are best 1, and 2 indicates a certain risk."

[0084] According to the results in Table 3, when using PALL 120 membrane and CN 140 membrane, Flu A, Flu B and COV are prone to false positive phenomenon or risks, while when using MDI 150 membrane, Flu A and COV will have the risk of false positive only when accelerated for 15 days.

[0085] It can be seen that the MDI 150 membrane is the optimal NC membrane choice, which significantly reduces the risk of crossover and false positives, further improves the specificity and accuracy of the test, and the test results are highly stable.

[0086] Example 5 Screening of antibody concentration on the marker pad

[0087] In order to obtain the best effect in Example 1, the antibody concentration on the labeling pad was screened in the following ways:

[0088] 1. The concentration of the first labeling pad antibody: Flu A: 0.15ml / ml; Flu B: 0.15ml / ml; The concentration of the second labeling pad antibody: COV: 0.3ml / ml; RSV: 0.3ml / ml;

[0089] 2. The first marker pad antibody concentration: Flu A: 0.2ml / ml; Flu B: 0.2ml / ml; The second marker pad antibody concentration: COV: 0.3ml / ml; RSV: 0.3ml / ml;

[0090] 3. The concentration of the first labeling pad antibody: Flu A: 0.3ml / ml; Flu B: 0.3ml / ml; The concentration of the second labeling pad antibody: COV: 0.3ml / ml; RSV: 0.3ml / ml;

[0091] 4. The concentration of the first labeling pad antibody: Flu A: 0.3ml / ml; Flu B: 0.3ml / ml; The concentration of the second labeling pad antibody: COV: 0.6ml / ml; RSV: 0.3ml / ml;

[0092] Add sample solution and observe the results displayed on the NC membrane. Figure 2 The experimental results are shown in Table 4 (the other operating procedures and conditions not mentioned are the optimal conditions in Example 1, which are the same; the preparation of the buffer and sample solution is the same as in Example 2; at the same time, the antibody is stated in a different way, which is an abbreviation of the complete statement in Example 1, such as: mouse anti-Flu A antibody is abbreviated as Flu A antibody):

[0093] Table 4 Effect of antibody concentration on marker pad on false positive phenomenon

[0094]

[0095] Note: "The numbers indicate the depth of the color. The larger the number, the darker the color. The numbers from left to right indicate the results of FluB, Flu A, COV, and RSV. Colors above 3 are considered positive, and negative results are best 1, and 2 indicates a certain risk."

[0096] According to the results in Table 4, when the concentration of Flu A and Flu B antibodies on the first marking pad was 0.15-0.3 ml / ml, and the concentration of COV and RSV antibodies on the second marking pad was 0.3 ml / ml, there was no false positive in the accelerated test for 28 days.

[0097] It can be seen that the concentration of Flu A and Flu B antibodies on the first marking pad is within the range of 0.15-0.3 ml / ml, and the concentration of COV and RSV antibodies on the second marking pad is within the range of 0.3 ml / ml, which can further reduce the possibility of crossover and false positives, and further improve the specificity and accuracy of the detection. At the same time, when the antibody concentrations are Flu A on the first marking pad: 0.3 ml / ml; Flu B: 0.3 ml / ml; COV on the second marking pad: 0.3 ml / ml; RSV: 0.3 ml / ml, the detection sensitivity is optimal, which further improves the sensitivity of the detection.

[0098] Example 6 Screening of Antibody Labeling Materials on Labeling Pads

[0099] In order to obtain the best effect in Example 1, the materials for antibody labeling on the labeling pad were screened in this example, and latex microspheres and colloidal gold were selected to label the antibodies, respectively. The latex microspheres were 400nm red carboxyl latex microspheres purchased from MAGSPHERE INC; the colloidal gold was homemade and had a particle size of 40nm.

[0100] according to Figure 2 The experimental results are shown in Table 5 (the other operating processes and conditions not mentioned are the optimal conditions in Example 1, which are the same; the preparation of the buffer and the sample solution is the same as in Example 2):

[0101] Table 5 Effect of using different material markings

[0102] Marking material Buffer Flu A, Flu B, COV, RSV antigen Latex Microspheres 1 / 1 / 1 / 1 6+ / 7 / 8 / 5 Colloidal Gold 1 / 1 / 1 / 1 4 / 4 / 5 / 3

[0103] Note: "The numbers indicate the depth of the color. The larger the number, the darker the color. The numbers from left to right indicate the results of FluB, Flu A, COV, and RSV. Colors above 3 are considered positive, and negative results are best 1, and 2 indicates a certain risk."

[0104] According to Table 5, compared with colloidal gold, the number displayed by using latex microspheres to label antibodies on the label pad is larger than that of the standard colorimetric card. Therefore, it can be seen that using latex microspheres to label antibodies can further improve the sensitivity of detection.

[0105] In summary, the kit for joint detection of four respiratory virus antigens and its application provided by the present invention are the optimal combination in the above embodiments. The use of the optimal combination can significantly reduce the possibility of cross-influence and false positives, and improve the accuracy, sensitivity and specificity of detection; on the other hand, reduce the workload of sampling and improve the detection efficiency of the four items; on the other hand, reduce the number of sampling times, reduce the repeated contact between medical staff and patients during multiple sampling processes, and reduce the risk of epidemic transmission; at the same time, when using tools such as nasal swabs or throat swabs for detection, it will significantly reduce the harm to the subject and improve safety.

[0106] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A kit for joint detection of four respiratory virus antigens, characterized in that: The respiratory viruses include COVID-19, influenza A, influenza B and respiratory syncytial virus; the test kit includes a test strip, which includes two layers of marking pads and a nitrocellulose membrane, from left to right, a first marking pad, a second marking pad and a nitrocellulose membrane; the first marking pad contains labeled mouse anti-Flu A antibody and mouse anti-Flu B antibody, and the second marking pad contains labeled mouse anti-COV antibody and mouse anti-RSV antibody.

2. The kit according to claim 1, characterized in that The nitrocellulose membrane includes 5 lines, which are RSV detection line, COV detection line, Flu A detection line, Flu B detection line and C line in order from the direction close to the second marking pad.

3. The kit according to claim 2, characterized in that The nitrocellulose membrane includes one or more of PALL 120, CN 140, and MDI 150.

4. The kit according to claim 3, characterized in that The nitrocellulose membrane is MDI 150.

5. The kit according to claim 4, characterized in that The antibody on the labeling pad is labeled with latex microspheres.

6. The kit according to claim 5, characterized in that The concentrations of the antibodies are: Flu A: 0.15-0.3 ml / ml; Flu B: 0.15~0.3ml / ml; COV: 0.3ml / ml; RSV: 0.3ml / ml.

7. The kit according to claim 6, characterized in that The concentrations of the antibodies are: Flu A: 0.3 ml / ml; FluB: 0.3 ml / ml; COV: 0.3 ml / ml; RSV: 0.3 ml / ml.

8. Use of latex microspheres for preparing a highly sensitive kit for joint detection of four respiratory virus antigens, characterized in that: The latex microspheres are respectively labeled on the antibodies of the two layers of labeling pads and are 400nm red carboxyl latex microspheres.

9. A use of a nitrocellulose membrane for preparing a highly specific kit for joint detection of four respiratory virus antigens, characterized in that: The nitrocellulose membrane includes one or more of PALL 120, CN 140, and MDI 150.

10. A method for detecting four respiratory virus antigens, characterized in that: The kit according to any one of claims 1 to 7 is used to detect four respiratory viruses.

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