CN107589253A - A kind of H9 subtype avian influenza virus fluorescent chromatographic test strips and its application - Google Patents

Abstract

A H9 subtype avian influenza virus fluorescent chromatography test strip, comprising a backing (1), an absorbent paper pad (7), a cellulose film (2), a sample pad (4), and a fluorescent microsphere mark H9 sprayed A fluorescent marker pad of subtype avian influenza virus monoclonal antibody (2G4); the upper surface of the nitrocellulose membrane (2) is provided with another H9 subtype avian influenza virus monoclonal antibody (1C3) layer and Goat anti-Balb/c mouse immunoglobulin layer, wherein a detection line (5) is set on the H9 subtype avian influenza virus monoclonal antibody (1C3) layer, and a control is set on the goat anti-Balb/c mouse immunoglobulin layer line (6). The H9 subtype avian influenza virus fluorescence chromatography test paper strip of the invention is simple to operate, extremely strong in practicability and high in detection efficiency.

Description

A kind of H9 subtype avian influenza virus fluorescent chromatography test strip and its application

technical field

The invention belongs to the field of virus detection, and in particular relates to a H9 subtype avian influenza virus fluorescence chromatography test strip and an application thereof.

Background technique

Avian influenza (Avian influenza, AI) is a highly contagious disease of poultry caused by influenza A virus of Orthomyxoviridae family. There are 16 HA subtypes and 10 NA subtypes. Avian influenza virus (AIV) It is one of the devastating diseases in the poultry industry. It belongs to the genus Influenza virus of the family Orthomyxoviridae. Sick avian influenza virus. Although the low-pathogenic H9 subtype avian influenza virus does not cause a large number of deaths of infected poultry, the virus has caused a large-scale epidemic in my country. Since the first report of H9N2 subtype AIV isolated from chicken flocks in my country in 1994, H9 subtype low pathogenicity AIV has widely existed in most parts of China, causing huge economic losses to my country's poultry industry.

At present, the detection of AIV is mainly through pathogen isolation and serological tests. These methods are time-consuming and laborious, and have certain limitations in the detection of mixed-infected AIV in practical applications. Real-time fluorescence quantitative PCR is a combination of ordinary PCR method and fluorescence detection method. By adding a probe that can specifically bind to the amplification template and labeled with a fluorescent group in the PCR reaction system, the PCR process can be monitored in real time through fluorescence accumulation. , the results can be directly observed by computer in real time without gel electrophoresis, and the corresponding virus content in the sample can be estimated according to the threshold value of the fluorescence curve, realizing the purpose of real-time and quantitative detection, but the required detection equipment is expensive and the laboratory conditions Strict, not easy to promote in grassroots veterinary departments. The detection sensitivity of colloidal gold detection method is too low, and it is easy to cause missed detection. Therefore, there is an urgent need for the detection of a H9 subtype avian influenza virus that can be fast and easy, low in detection cost, high in detection efficiency and high in sensitivity, and has become a major problem in the industry.

Contents of the invention

Compared with colloidal gold, the immunochromatographic detection technology based on fluorescent microspheres can increase the detection sensitivity by 10 times to 100 times. It can not only realize on-site detection, but also realize ultra-sensitive detection. It is an inevitable trend to replace the low-sensitivity detection of avian influenza colloidal gold. By formulating advanced and internationally leading standards for on-site fluorescence detection of avian influenza virus series, the ability to deal with avian influenza public emergencies can be effectively improved, and the impact of avian influenza public emergencies can be minimized.

That is, the object of the present invention is to provide a kind of H9 subtype avian influenza virus fluorescent chromatography test paper strip, comprise backing (1), absorbent paper pad (7), cellulose film (2), sample pad (4), And a fluorescent marker pad sprayed with fluorescent microspheres labeled H9 subtype avian influenza virus monoclonal antibody (2G4), the cellulose membrane (2) is located on the backing (1), and the right side of the fluorescent marker pad (3) is located on the cellulose On the left side of the upper surface of the membrane (2), the left side of the fluorescent marker pad (3) protrudes out of the cellulose membrane (2), the left side of the sample pad (4) is located on the fluorescent marker pad (3), and the sample pad ( The right side of 4) stretches out of the fluorescent marker pad (3), the left side of the absorbent paper pad (7) is located on the right side of the upper surface of the cellulose membrane (2), and the right side of the absorbent paper pad (7) stretches out to Outside the cellulose membrane (2); the upper surface of the cellulose membrane (2) is provided with another H9 subtype avian influenza virus monoclonal antibody (1C3) layer and goat anti-Balb/c mouse immune globules sequentially from left to right The protein layer, wherein the H9 subtype avian influenza virus monoclonal antibody (1C3) layer is provided with a detection line (5), and the goat anti-Balb/c mouse immunoglobulin layer is provided with a control line (6).

Preferably, in the H9 subtype avian influenza virus fluorescent chromatography test strip of the present invention, the cellulose membrane (2) is a nitrocellulose membrane.

Preferably, in the H9 subtype avian influenza virus fluorescent chromatography test strip of the present invention, the model of the nitrocellulose membrane is nitrocellulose membrane 140.

Preferably, in the H9 subtype avian influenza virus fluorescence chromatography test paper strip of the present invention, the thickness of the cellulose membrane (2) is 0.2mm and the width is 3.5mm.

Preferably, in the H9 subtype avian influenza virus fluorescent chromatography test strip of the present invention, the fluorescent microspheres in the fluorescent microspheres labeled H9 subtype avian influenza virus antibodies are carboxyl-modified fluorescent microspheres with an average diameter of 110 nm. ball.

Preferably, in the H9 subtype avian influenza virus fluorescent chromatography test strip of the present invention, the mass ratio of the fluorescent microspheres to the H9 subtype avian influenza virus hemagglutinin monoclonal antibody is 5:0.37.

Preferably, in the H9 subtype avian influenza virus fluorescence chromatography test paper strip of the present invention, the coating amount of the H9 subtype avian influenza virus antibody sprayed on the detection line is 0.5 μl/cm～1.5 μl/cm; The coating amount of the goat anti-mouse secondary antibody used for the control line is 0.5μl/cm-1.5μl/cm.

Preferably, in the H9 subtype avian influenza virus fluorescent chromatography test paper strip of the present invention, the coating concentration of the avian influenza virus monoclonal antibody (1C3) used in the detection line for delineation is 1.0mg/ml～ 2.5mg/ml; the spray volume of fluorescent microspheres and H9 subtype avian influenza virus hemagglutinin monoclonal antibody (2G4) conjugate is 2.0μl/cm-3.0μl/cm.

Preferably, in the H9 subtype avian influenza virus fluorescent chromatography test strip of the present invention, the concentration of the goat anti-mouse secondary antibody sprayed on the quality control line for filming is 0.5 mg/ml-2 mg/ml

Another object of the present invention is to provide the application of the above-mentioned H9 subtype avian influenza virus fluorescence chromatography test strip in the detection of H9 subtype avian influenza virus, that is, to use the above-mentioned H9 subtype avian influenza virus fluorescence chromatography test paper Strips are tested for samples:

If there is no band in the control line (6), the detection is invalid; if there is a band in the control line (6) and no band appears in the detection line (5), then it is negative; if a band appears in the control line (6) , a band appears on the detection line (5), then it is positive.

Compared with the prior art, the present invention has the following advantages:

When the fluorescent chromatography test strip of the present invention is in use, it is only necessary to drop the oral or cloacal mucus solution and tissue fluid of the animal to be detected on the sample pad. When the sample has H9 subtype avian influenza virus, the H9 subtype The antigen of avian influenza virus reacts with the fluorescently labeled H9 AIV monoclonal antibody (2G4) on the fluorescent labeling pad to generate a complex of H9 subtype avian influenza virus-fluorescently labeled H9 AIV monoclonal antibody, H9 subtype avian influenza virus-fluorescently labeled H9 AIV When the monoclonal antibody (2G4) complex moves to the right to the H9 AIV monoclonal antibody (1C3) layer, a complex of H9 AIV monoclonal antibody (1C3)-H9 subtype avian influenza virus-fluorescence-labeled H9 AIV monoclonal antibody (2G4) is formed and form an excited fluorescent band near the detection line, and at the same time, the unbound fluorescently labeled H9 AIV monoclonal antibody moves to the right and reacts with goat anti-Balb/c mouse immunoglobulin to generate goat anti-Balb/c mouse immunoglobulin Protein-fluorescence-labeled anti-H9 AIV monoclonal antibody (2G4) complex, thereby forming an excited fluorescent band near the control line, that is, when detected by a specific fluorescence chromatography analyzer, when the detection line and the control line appear fluorescent strips at the same time When it is detected, it means that the liquid of the animal to be tested contains H9 subtype avian influenza virus. The operation is simple, the practicability is strong, and the detection efficiency is high.

Description of drawings

Fig. 1 is the development and application technology roadmap of H9 subtype avian influenza virus fluorescence chromatography detection card of the present invention;

Fig. 2 is the longitudinal section and front schematic view of H9 subtype avian influenza virus fluorescence chromatography detection card of the present invention;

Fig. 3 is the schematic diagram of the detection reaction of the fluorescence chromatography detection card of the present invention;

Fig. 4 is the test result schematic diagram of the H9 subtype avian influenza virus fluorescent microsphere detection card in one embodiment of the present invention;

Fig. 5 is a graph showing test results of the H9 subtype avian influenza virus fluorescent detection card in an embodiment of the present invention.

detailed description

The present invention is described in further detail below in conjunction with accompanying drawing:

With reference to Fig. 2, fluorescent chromatography test strip structure of the present invention comprises backing 1, absorbent paper pad 7, cellulose membrane 2, sample pad 4 and the fluorescent label pad that contains fluorescent label H9 AIV monoclonal antibody (2G4) 3. The cellulose membrane 2 is located on the backing 1, the right side of the fluorescent marker pad 3 is located on the left side of the upper surface of the cellulose membrane 2, the left side of the fluorescent marker pad 3 protrudes out of the cellulose membrane 2, the sample pad 4 The left side is located on the fluorescent marker pad 3, the right side of the sample pad 4 protrudes out of the fluorescent marker pad 3, the left side of the absorbent paper pad 7 is located on the right side of the upper surface of the cellulose membrane 2, and the right side of the absorbent paper pad 7 extends out. Out of the cellulose membrane 2; the upper surface of the cellulose membrane 2 is sequentially provided with a H9 AIV monoclonal antibody (1C3) layer and a goat anti-Balb/c mouse immunoglobulin layer from left to right, wherein the H9 AIV monoclonal antibody (1C3 ) layer is provided with detection line 5, and the sheep anti-Balb/c mouse immunoglobulin layer is provided with control line 6.

It should be noted that the cellulose membrane 2 is a nitrocellulose membrane, the thickness of the cellulose membrane 2 is 0.2 cm, and the width of the cellulose membrane 2 is 3.5 mm.

The detection process of the present invention is:

It is only necessary to drop the mucus or tissue fluid of the animal to be detected on the sample pad 4. When the sample pad 4 has H9 subtype avian influenza virus, the antigen of the H9 subtype avian influenza virus and the fluorescent marker H9 on the fluorescent marker pad 3 The AIV monoclonal antibody (2G4) reacts to generate a complex of H9 subtype avian influenza virus-fluorescence-labeled H9 AIV monoclonal antibody (2G4), and at the same time, under the action of the absorbent paper pad 7, the H9 subtype avian influenza virus-fluorescence-labeled H9 AIV When the monoclonal antibody (2G4) complex moves to the right to the H9 AIV monoclonal antibody (1C3) layer, a complex of H9 AIV monoclonal antibody (1C3)-H9 subtype avian influenza virus-fluorescence-labeled H9 AIV monoclonal antibody (2G4) is formed and form a fluorescent band near the detection line 5, and at the same time, the unbound fluorescently labeled H9 AIV monoclonal antibody moves to the right and reacts with goat anti-Balb/c mouse immunoglobulin to generate goat anti-Balb/c mouse immunoglobulin Protein-fluorescence-labeled anti-H9 AIV monoclonal antibody (2G4) complex forms a fluorescent band near the control line 6. When detected by a fluorescence analyzer, when the fluorescent band appears on the detection line 5 and the control line 6 at the same time, Then it shows that the liquid of the animal to be tested contains H9 subtype avian influenza virus; when there is no fluorescent band within the control line 6, it means that the liquid of the animal to be tested does not contain H9 subtype avian influenza virus, and when the control line 6 does not contain When the fluorescent strips are displayed, it indicates that the fluorescent chromatography test strip of the present invention is invalid or incorrectly operated.

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

Example 1 Preparation of fluorescent microsphere-labeled avian influenza virus antibody

Use carboxyl-modified fluorescent microspheres with an average diameter of 110 nm (Bangs Laboratories, Inc., catalog number FC02F/10930), and two monoclonal antibodies against H9 subtype avian influenza virus hemagglutinin (2G4 and 1C3 ) (purchased from Yangzhou University School of Veterinary Medicine, Key Laboratory of Infectious Diseases and Preventive Veterinary Medicine), prepared fluorescent microsphere-labeled avian influenza virus-labeled antibody according to the following method:

Take 5 mg of the above-mentioned carboxy-modified fluorescent microspheres and wash them with MES (2-(N-morpholine) ethanesulfonic acid) buffer (0.1M, pH4.7) .7) Resuspend, add 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) to a final concentration of 5mM, add NHS (N-hydroxysuccinimide) to a final concentration 10mM at room temperature, protected from light, reacted for half an hour to obtain activated carboxyl-modified fluorescent microspheres.

Wash the activated carboxy-modified fluorescent microspheres with 50 mM pH8.5 borax buffer, take 0.37 mg of the above-mentioned H9 subtype avian influenza virus antibody (2G4) to be labeled and 5 mg of the above-mentioned activated carboxy-modified fluorescent microspheres and mix to 50 mM pH8 .5 in borax buffer and mix thoroughly. React for 2 hours at room temperature in the dark, let the antibody and fluorescent microspheres form a stable peptide bond, and covalently bond to obtain a conjugate of fluorescent microspheres and H9 subtype avian influenza virus antibody. After the reaction is over, add a BSA (bovine serum albumin) solution with a final concentration of 1% (mass percentage content) to block the remaining active carboxyl sites on the conjugate of the fluorescent microspheres and the avian influenza virus antibody, and keep it away from room temperature. Light reaction for 0.5 hours. After completion, wash and resuspend with 0.02M PBS buffer solution with pH 7.4 to obtain 5mg/ml fluorescent microsphere-labeled avian influenza virus antibody liquid, which is stored at 4°C until use.

Example 2 Preparation of Fluorescence Detection Card for H9 Subtype Avian Influenza Virus

The H9 subtype avian influenza virus antibody solution labeled with fluorescent microspheres obtained in Example 1 was sprayed onto the treated glass cellulose membrane (purchased from waterman and Schleicher & Schuell companies) according to a certain amount by using the XYZ3050 spray film system of BioDot to prepare Form a fluorescent antibody binding pad, then move it into a vacuum drying oven with a grid tray, dry it in vacuum for 2 hours, add a desiccant or color-changing silica gel, and store it sealed in a refrigerator at 4°C until use.

Select the coating volume of the test line and the quality control line, that is, the spray film amount ranges from 0.5 μl/cm to 1.5 μl/cm, select the appropriate protein coating concentration according to experience, and spray the test line with an automatic film spraying machine. The spray film volume is respectively It is 0.5μl/cm, 0.75μl/cm, 1.0μl/cm, 1.5μl/cm, and compares the marking effect obtained by different spray film amounts.

Antigen and secondary antibody diluent were used to dilute the H9 subtype avian influenza virus antibody (1C3) to different concentrations: 1.0mg/ml, 1.5mg/ml, 2.0mg/ml, 2.5mg/ml for detection line coating. Use an automatic film spraying machine to combine the nitrocellulose membranes that have been sprayed with the above four concentration detection lines with different spray volumes of bonding pads to prepare test strip samples and paste them in turn with other auxiliary materials to make quick test cards for detection tests.

Using 0.02M PBS buffer solution with a pH of 7.4, after diluting the AI test antigen of the H9N2 subtype of avian influenza virus, the detection experiment was performed on the quick test card.

On the basis of determining the coating concentration of the H9 subtype avian influenza virus antibody (1C3) and the spray volume of the fluorescently labeled conjugate, the goat anti-mouse secondary antibody was diluted into different concentrations with antigen and secondary antibody diluent: 2mg/ml, 1.5 mg/ml, 1mg/ml, 0.5mg/ml, the quality control line coating is carried out, and the quality control line coating concentration is determined by detecting the H9 subtype avian influenza cell culture virus.

Antigen and secondary antibody diluent were used to prepare goat anti-mouse secondary antibody and H9 subtype avian influenza virus monoclonal antibody (1C3) to a certain concentration solution, and BioDot’s XYZ3050 spraying system was used to apply the goat anti-mouse secondary antibody to the film. At the position of the quality control line (C line) of the coated membrane (nitrocellulose membrane), draw the H9 subtype avian influenza virus monoclonal antibody (1C3) to the position of the detection line (T line), and label the fluorescent microspheres with the H9 subtype The antibody solution of avian influenza virus is sprayed onto the treated glass cellulose membrane according to a certain spray amount, and is dehumidified in a drying workshop with a relative humidity below 30% for 24 hours and then dried for use.

In a 100,000-level clean and dry workshop, the dried coating film with test line and quality control line, the above-mentioned sample pad, water-absorbing pad, and back plate are assembled and assembled, and the pasted film is cut by BioDot's CM4000 cutting system. Cut the cardboard and put it into the test clip for use.

Experimental results:

1 Determination results of coating volume of detection line and quality control line of H9 subtype avian influenza virus fluorescence detection card

According to experience, the H9 subtype avian influenza virus monoclonal antibody (1C3) with a concentration of 3mg/ml and the goat anti-mouse secondary antibody with a concentration of 1.5mg/ml were respectively selected according to the coating volume of 0.5μl/cm, 0.75μl/cm, 1.0μl/cm and 1.5μl/cm scribing film, the scribing effect is shown in Table 1:

Table 1 Determination of coating volume of test line and quality control line

Remarks: + indicates that the bands are clear, concentrated, and non-diffused; - indicates that the color of the bands is not concentrated, unclear, and diffuse; ± indicates that it is between + and -.

The results show that when the coating volume of the test line and the quality control line is 1.0 μl/cm, the edge of the drawn line is neat, the line is fine, and the raw material is saved the most.

2 Determination results of H9 subtype avian influenza virus fluorescent detection card detection line coating concentration and binding pad injection volume

The quick test card was tested by the prepared sample, and the test results are shown in Table 2.

Table 2 Determination of antibody coating concentration and injection volume

Remarks: + indicates that the bands are clear, concentrated, and non-diffused; - indicates that the color of the bands is not concentrated, unclear, and diffuse; ± indicates that it is between + and -.

The results showed that when the coating concentration of the test line was 2.0 mg/ml and the injection volume of the binding pad was 2.5 μl/cm, the color bands were clear and concentrated.

3 Determination results of the coating concentration of the quality control line of the H9 subtype avian influenza virus fluorescent detection card

Use the H9 subtype avian influenza cell culture virus sample to test the quick test card, and the test results are shown in Table 3.

Table 3 Determination of coating concentration of quality control line

Remarks: + indicates that the bands are clear, concentrated, and non-diffused; - indicates that the color of the bands is not concentrated, unclear, and diffuse; ± indicates that it is between + and -.

The experimental results show that when the coating concentration is 1 mg/ml, the color bands in the H9 subtype avian influenza cell culture virus samples are clear and concentrated.

Therefore, the detection line of the detection card has been determined, the coating concentration of the quality control line and the spray volume of the binding pad, the detection line (T line) H9 subtype avian influenza virus monoclonal antibody concentration 2mg/ml, the quality control line (C line) sheep The concentration of anti-mouse secondary antibody is 1mg/ml, and the volume of spraying volume of the avian influenza virus antibody solution on the binding pad is 2.5μl/cm. In the following examples 3-4, the application experiments were carried out with the fluorescent detection card of the determined H9 subtype avian influenza virus.

Example 3 Application Test of H9 Subtype Avian Influenza Virus Fluorescent Detection Card

Positive serum and negative serum of H9 subtype AIV, avian influenza virus H7N9 antigen, H7 subtype antigen, H5N1 (Re-6) antigen, H5N2 antigen, H9N2 antigen, H1N1 antigen, H3N2 antigen, NDV antigen, IBDV antigen, IBV antigen , ILTV antigens were purchased from the National Animal Engineering Center of Harbin Veterinary Research Institute.

With the virus antigen or positive sample of cell culture, the H9 subtype avian influenza virus fluorescent detection card prepared in embodiment 2 is carried out the result test, and its result judgment is as shown in Figure 4, wherein, there is no band in the quality control line, then detects Invalid; if there is a band on the quality control line and no band on the test line, it is negative; if there is a band on the quality control line and there is a band on the test line, it is positive.

The test results are shown in Figure 5: (p7 and p8 are H9 subtype avian influenza virus strains).

Example 4 Performance Test of Fluorescence Detection Card for H9 Subtype Avian Influenza Virus

Positive serum and negative serum of H9 subtype AIV, avian influenza virus H7N9 antigen, H7 subtype antigen, H5N1 (Re-6) antigen, H5N2 antigen, H9N2 antigen, H1N1 antigen, H3N2 antigen, NDV antigen, IBDV antigen, IBV antigen , ILTV antigens were purchased from the National Animal Engineering Center of Harbin Veterinary Research Institute.

1. Using the H9N2 subtype AI test antigen as the test sample to determine the sensitivity of the H9 subtype avian influenza virus detection card prepared in Example 2.

The avian influenza virus H9N2 subtype AI test antigen is prepared into a series of concentrations (64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.063, 0.031, 0.016 , OHA unit), respectively added in the H9 subtype avian influenza virus fluorescent detection card prepared in Example 1 and equipped with a fluorescent detector to interpret the results. Detection steps: return the sample to be tested to room temperature (25°C) before the test, take 65 μl of the sample to be tested with a precise pipette and drop it vertically and slowly into the sample loading end of the H9 subtype avian influenza virus test card, and interpret the result after 10 minutes. The results are shown in Table 4 below.

Table 4 Detection results of H9 subtype avian influenza virus fluorescent detection card with different sample concentrations

From the detection results, it can be concluded that the sensitivity of the standard H9N2 subtype influenza virus detected by the H9 subtype avian influenza virus fluorescent detection card is 0.031HA unit.

2 Specificity evaluation test of fluorescent detection card for H9 avian influenza virus

H9 subtype avian influenza virus and other common respiratory viruses, including poultry respiratory viruses (Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV)), human respiratory viruses ( Parainfluenza virus, respiratory syncytial virus, measles virus, mumps virus, coronavirus) and other viruses of the same syndrome, prepared at a higher concentration with 0.02M PBS buffer solution with a pH of 7.4, and took 65 μl vertical Slowly drip into the sample loading end of the H9 subtype avian influenza virus detection card prepared in Example 2, and interpret the results in 10 minutes.

The established H9 subtype avian influenza virus fluorescent detection card was used to detect the viruses that cause human respiratory syndrome and are similar to influenza, and the detection results were all negative. Tests for viruses that are similar to influenza and cause poultry respiratory syndrome were also all negative. Detect negative matrix, including normal human nasopharyngeal swabs, sputum, cotton swabs of normal poultry, and normal poultry tissues. The test results are all negative, indicating that the established method is not affected by the matrix, and the detection results are highly specific . The test results are shown in Table 6 below. The H9 subtype avian influenza virus fluorescent detection card prepared according to this method can be used to detect H9 subtype avian influenza virus, and there is no cross-reaction with common respiratory viruses IBV, reovirus, and NDV, and the detection card cross-reaction detection result See Table 5 for details.

Table 5 Cross-reaction detection results of H9 subtype avian influenza virus fluorescence detection card

3. Small-scale field test of fluorescent detection card for H9 avian influenza virus

Field test samples were collected, including 179 poultry cotton swab samples and 21 clinical samples from hospitals. Fluorescence RT-PCR detection of avian influenza virus was performed on the samples simultaneously to determine the practicability of the established method.

For 200 field test samples, all of them were detected by the fluorescent detection card of H9 subtype avian influenza virus and the fluorescent RT-PCR detection at the same time. shown.

Table 6 Comparison results of H9 subtype avian influenza virus detection card detection method and RT-PCR detection method

The above is only a preferred embodiment of the present invention, and it should be pointed out that for those of ordinary skill in the art, some improvements and modifications can be made without departing from the principle of the present invention. It should be regarded as the protection scope of the present invention.

Claims (10)

1. A H9 subtype avian influenza virus fluorescent chromatography test strip, comprising backing (1), absorbent paper pad (7), cellulose film (2), sample pad (4), and fluorescent microspheres sprayed A fluorescent labeling pad labeled with H9 subtype avian influenza virus monoclonal antibody (2G4), the cellulose membrane (2) is on the backing (1), the right side of the fluorescent labeling pad (3) is on the upper surface of the cellulose membrane (2) on the left side of the fluorescent marker pad (3), the left side of the fluorescent marker pad (3) protrudes out of the cellulose membrane (2), the left side of the sample pad (4) is placed on the fluorescent marker pad (3), and the right side of the sample pad (4) protrudes Out of the fluorescent marker pad (3), the left side of the absorbent paper pad (7) is located on the right side of the upper surface of the cellulose membrane (2), and the right side of the absorbent paper pad (7) protrudes to the cellulose membrane (2) outside; Wherein, the upper surface of the cellulose membrane (2) is provided with another strain H9 subtype avian influenza virus monoclonal antibody (1C3) layer and goat anti-Balb/c mouse immunoglobulin layer successively from left to right, wherein the H9 subtype A detection line (5) is arranged on the monoclonal antibody (1C3) layer of type bird flu virus, and a control line (6) is arranged on the goat anti-Balb/c mouse immunoglobulin layer. 2. H9 subtype avian influenza virus fluorescent chromatography test strip according to claim 1, characterized in that, the cellulose membrane (2) is a nitrocellulose membrane. 3. The H9 subtype avian influenza virus fluorescence chromatography test strip according to claim 2, wherein the model of the nitrocellulose membrane is nitrocellulose membrane 140. 4. the H9 subtype avian influenza virus fluorescence chromatography test strip according to claim 2, is characterized in that, the thickness of described cellulose membrane (2) is 0.2mm, and width is 3.5mm. 5. H9 subtype avian influenza virus fluorescence chromatography test paper strip according to claim 1, is characterized in that, fluorescent microsphere is average Carboxyl-modified fluorescent microspheres with a diameter of 110nm. 6. H9 subtype avian influenza virus fluorescent chromatography test strip according to claim 1, is characterized in that, the mass ratio of described fluorescent microsphere and H9 subtype avian influenza virus hemagglutinin monoclonal antibody (2G4) It was 5:0.37. 7. H9 subtype avian influenza virus fluorescence chromatography test paper strip according to claim 1, is characterized in that, the coating amount of the avian influenza virus monoclonal antibody (1C3) that described detection line is used for delineation is 0.5 μl/cm～1.5μl/cm; the coating amount of goat anti-mouse secondary antibody used for quality control line is 0.5μl/cm～1.5μl/cm. 8. H9 subtype avian influenza virus fluorescence chromatography test paper strip according to claim 1, is characterized in that, the coating concentration of the avian influenza virus monoclonal antibody (1C3) that described detection line is used for delimiting film is 1.0 mg/ml～2.5mg/ml; the spray volume of fluorescent microspheres and H9 subtype avian influenza virus hemagglutinin monoclonal antibody (2G4) conjugate is 2.0μl/cm～3.0μl/cm. 9. the H9 subtype avian influenza virus fluorescence chromatography test paper strip according to claim 1, is characterized in that, the concentration of the goat anti-mouse secondary antibody that the quality control line is used for delineation is 0.5mg/ml～2mg /ml. 10. The application of a H9 subtype avian influenza virus fluorescent chromatography test strip in the detection of H9 subtype avian influenza virus, characterized in that the H9 subtype avian influenza virus described in any one of claims 1 to 9 is used Fluorescence chromatography test strips are used to detect samples: If there is no band in the control line (6), the detection is invalid; if there is a band in the control line (6) and no band appears in the detection line (5), then it is negative; if a band appears in the control line (6) , a band appears on the detection line (5), then it is positive.