Test strip for simultaneously detecting avian influenza virus and Newcastle disease virus as well as preparation method and application of test strip

Abstract

The invention discloses a test strip for simultaneously detecting avian influenza virus and Newcastle disease virus as well as a preparation method and application thereof, and belongs to the technical field of animal epidemic disease diagnosis and detection. According to the test strip prepared by the invention, mouse anti-FluA-NP-E and NDV-HN-8H2 are used as gold-labeled antibodies, and mouse anti-FluA-NP-F and NDV-HN-1C10 are used as capture antibodies. The test strip has good universality and specificity, has no cross reaction with other poultry viruses, has high sensitivity, has detection limits of 103.0 EID50 / mL and 102.6 EID50 / mL for AIV and NDV respectively, can be preserved for 6 months at 4 DEG C and normal temperature, and compared with RT-PCR, the total coincidence rate of AIV detection is 95.8%, and the total coincidence rate of NDV detection is 100%. The test strip provides technical support for basic clinical rapid identification of the two epidemic diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of animal epidemic disease diagnosis and detection, in particular to a test strip for simultaneously detecting avian influenza virus and Newcastle disease virus, and a preparation method and application thereof. BACKGROUND

[0002] Avian influenza virus (AIV) is a member of Orthomyxoviridae and Influenza virus A, which is an important zoonosis pathogen, mainly infecting wild birds and poultry. In addition, AIV can also infect many mammals, such as humans, foxes, seals, and even cows and cats. AIV is mainly transmitted through the fecal-oral route of wild birds and poultry, and is carried and transferred through bird migration, and finally spreads and exists in nature for a long time. The adaptability of AIV changes accordingly with continuous transmission, thereby affecting its pathogenicity, transmission ability and host range.

[0003] Newcastle disease virus (NDV) is a member of paramyxovirus and Avulavirus, and its serotype is Avian paramyxovirus-1 (APMV-1). NDV has a wide host range, mainly infecting poultry (chickens, ducks, geese, etc.), and in addition, more than 200 species of birds have been reported to be naturally infected with NDV, but the symptoms caused by infection with the same strain may differ depending on the host. Infected birds are the main source of infection, mainly transmitted through the respiratory and digestive tracts, and can cause severe damage to the respiratory, digestive and central nervous systems. AIV and NDV, as two high-risk avian respiratory infectious disease pathogens, have spread globally, not only causing huge economic losses to the poultry industry, but also seriously threatening human public health safety due to the recombination mutation of different strains. Therefore, early diagnosis of the two viruses and the adoption of rapid prevention and control measures are very important, and there is an urgent need for a simple, rapid, convenient, accurate and reliable detection method for clinical rapid diagnosis of poultry.

[0004] Avian influenza and Newcastle disease have similar clinical symptoms, and sometimes it is difficult to differentiate the two diseases based on clinical symptoms, which seriously affects the scientific prevention and control of the epidemic. The routine diagnosis methods of AIV and NDV are basically the same, including virus isolation and identification, HA-HI, PCR, etc. However, the above methods are limited by complicated operation techniques, professional laboratory technicians, long diagnosis process and special instruments and equipment, etc., and cannot meet the requirements of clinical rapid diagnosis and screening of a large number of samples.

[0005] Colloidal gold immunochromatography (GICA) is a new type of immunolabeling technology based on colloidal gold labeling technology applied to antigen-antibody reaction with colloidal gold as tracer. Its core technology is to use nitrocellulose membrane as a solid carrier, and the sample solution is moved on the chromatography strip due to capillary action, and the sample in the sample solution and the receptor (such as antigen or antibody) on the chromatography strip for the sample to be detected can occur high specificity and high affinity immune reaction in a short time. It has the advantages of simplicity, rapidness, strong specificity, high sensitivity and low cost. SUMMARY

[0006] The purpose of the present application is to provide a test strip for simultaneously detecting avian influenza virus and Newcastle disease virus and a preparation method and application thereof, so as to solve the problems existing in the prior art and provide important technical support for early clinical monitoring and differential diagnosis of AIV and NDV.

[0007] To achieve the above-mentioned purpose, the present application provides the following scheme:

[0008] In a first aspect, the present application provides a test strip for simultaneously detecting avian influenza virus and Newcastle disease virus, which comprises a base plate, a sample pad, a gold label combination pad, an NC membrane and a water absorption pad, wherein the sample pad, the gold label combination pad, the NC membrane and the water absorption pad are sequentially and sequentially bonded on the base plate in the chromatography direction.

[0009] The gold label combination pad is coated with colloidal gold labeled Flu A NP gold antibody and NDV HN gold antibody; the NC membrane is provided with two detection lines and a quality control line, the two detection lines are respectively coated with NDV-HN antibody and Flu A NP antibody, and the quality control line is coated with goat anti-mouse IgG protein.

[0010] Preferably, the Flu A NP gold antibody is a mouse anti-Flu A-NP-E monoclonal antibody, the NDV HN gold antibody is a mouse anti-NDV-HN-8H2 monoclonal antibody, the NDV-HN antibody is a NDV-HN-1C10 mouse monoclonal antibody, and the Flu A NP antibody is a Flu A-NP-F mouse monoclonal antibody.

[0011] Preferably, the preparation method of the gold label combination pad is as follows: preparing a colloidal gold solution, adding 20-28 μg / mL of mouse anti-Flu A NP monoclonal antibody and 44-52 μg / mL of mouse anti-NDV HN monoclonal antibody into the colloidal gold solution for labeling reaction by adjusting the pH value of the colloidal gold solution, and after the reaction is completed, the gold label antibody is obtained by blocking, centrifugation and re-dissolving; the gold label antibody is uniformly sprayed on the gold label pad and dried to obtain the gold label combination pad.

[0012] Preferably, the colloidal gold solution is diluted to 40 OD colloidal gold solution with 0.01 M Tris-HCl buffer solution having a pH of 7.0-8.5.

[0013] In a second aspect, the application further provides a preparation method of the test strip, comprising the following steps:

[0014] Preparation of the gold-labeled conjugate pad;

[0015] Preparation of the nitrocellulose membrane;

[0016] Assembly of the test strip;

[0017] The preparation method of the gold-labeled conjugate pad is as follows: preparing a colloidal gold solution, adding 20-28 μg / mL of mouse anti-Flu A NP monoclonal antibody and 44-52 μg / mL of mouse anti-NDV HN monoclonal antibody into the colloidal gold solution respectively for a labeling reaction by adjusting the pH value of the colloidal gold solution, and obtaining the gold-labeled antibody after blocking, centrifugation and re-dissolving after the reaction; and the gold-labeled antibody is uniformly sprayed on the gold-labeled pad and dried to obtain the gold-labeled conjugate pad.

[0018] Preferably, the mouse anti-Flu A NP monoclonal antibody is added in an amount of 24 μg / mL, and the mouse anti-NDV HN monoclonal antibody is added in an amount of 48 μg / mL.

[0019] Preferably, the labeling reaction is performed at room temperature for 30-90 min; after the reaction, 1-9 μL of BSA solution is added for blocking reaction for 0.5-1.5 h, centrifugation is performed to discard the supernatant, and colloidal gold re-dissolving solution is added for re-dissolving to obtain the gold-labeled antibody.

[0020] In a third aspect, the application further provides application of the test strip in preparation of a product for simultaneously detecting avian influenza virus and Newcastle disease virus.

[0021] Preferably, the product is a detection kit.

[0022] In a fourth aspect, the application further provides a product for detecting avian influenza virus and Newcastle disease virus, wherein the product comprises the test strip.

[0023] The application discloses the following technical effects:

[0024] The GICA can realize the clinical rapid differential diagnosis of AIV and NDV due to the advantages of simple operation, rapid diagnosis, no need of professional equipment and personnel, high specificity and high sensitivity, and is suitable for screening of a large number of clinical samples. The application is based on the colloidal gold immunochromatography technology and the antigen-antibody immunodetection method, selects a pair of influenza A virus monoclonal antibodies and a pair of Newcastle disease virus monoclonal antibodies, and develops the AIV and NDV double colloidal gold differential diagnosis test strip based on the double-antibody sandwich principle, thereby providing important technical support for the early clinical monitoring and differential diagnosis of AIV and NDV.

[0025] The prepared test strip has good universality and specificity, and only produces specific bands with different subtypes of AIV and different genotypes of NDV, and has no cross reaction with other avian viruses; the sensitivity is relatively high, and the detection limits of AIV and NDV are 10 3.0 EID 50 / mL and 10 2.6 EID 50 / mL respectively; the repeatability is good, and the batch detection rate and the inter-batch detection rate are both 100%; the stability is good, and the test strip can be stored for 6 months at 4 DEG C and at normal temperature; the clinical test detects 167 clinical samples, and compared with RT-PCR, the overall coincidence rate of AIV detection is 95.8%, and the overall coincidence rate of NDV detection is 100%.

[0026] The prepared test strip for simultaneously detecting AIV and NDV has the advantages of good universality, strong specificity, high sensitivity, good stability, good accuracy, convenient operation, easy result reading, convenient storage and transportation, no need of professional personnel and special instrument, and provides technical support for the primary clinical rapid differential diagnosis of the two diseases. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments will be briefly introduced below. Obviously, the drawings in the following description only some embodiments of the present application, and for those skilled in the art, other drawings can be obtained without creative labor on the basis of these drawings.

[0028] Figure 1 The mode diagram of the prepared test strip of the present application;

[0029] Figure 2Results of optimal labeling pH (A) and antibody labeling concentration (B) for Flu A-NP-E mouse monoclonal antibody; wherein, 1-8 in A represent the experimental results of 0.01 M Tris-HCl buffer system with pH values of 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, and 1-8 in B represent the experimental results with antibody labeling concentrations of 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, respectively;

[0030] Figure 3 Results of optimal labeling pH (A) and antibody labeling concentration (B) for NDV-HN-8H2 mouse monoclonal antibody; wherein, 1-8 in A represent the experimental results of 0.01 M Tris-HCl buffer system with pH values of 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, and 1-8 in B represent the experimental results with antibody labeling concentrations of 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, respectively;

[0031] Figure 4 Selection of sample diluent; wherein, A is the detection results of AIV with different sample diluents, and B is the detection results of NDV with different sample diluents;

[0032] Figure 5 Results of general detection experiment of test strip;

[0033] Figure 6 Results of specific detection experiment of test strip;

[0034] Figure 7 Results of sensitivity detection experiment of test strip; wherein, A is the sensitivity detection results of AIV, and B is the sensitivity detection results of NDV;

[0035] Figure 8 Results of repeatability detection experiment of test strip;

[0036] Figure 9 Results of stability detection experiment of test strip stored for 6 months. DETAILED DESCRIPTION

[0037] The various illustrative embodiments of the present application will now be described in detail in connection with the accompanying drawings. This description is intended to be illustrative, but not to be limiting, of the present application, and is understood that it is provided in the interest of promoting the understanding of the concepts of the present application.

[0038] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for the purposes of the present application, ranges provided herein are understood to be shorthand for describing each and every value that falls within the range. Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which the application pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. In case of conflict between the content of the specification and that of any document incorporated herein by reference, the content of the specification prevails.

[0039] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which the application pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. In case of conflict between the content of the specification and that of any document incorporated herein by reference, the content of the specification prevails.

[0040] Various modifications and variations of the described methods and materials of the application will be apparent to those skilled in the art from the foregoing disclosure. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. Additional embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope of the application being indicated by the following claims.

[0041] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," and the like are open-ended terms that are intended to mean including, but not limited to.

[0042] Example 1

[0043] 1. Materials and Methods

[0044] 1.1 Materials

[0045] 1.1.1 Avian Disease Pathogens and Antibodies

[0046] Avian influenza virus AIV (H1N1, H3N2, H4N2 and H9N2 subtypes of avian influenza virus were isolated from live poultry market by our laboratory; H12N2 subtype of avian influenza virus was isolated from Yun Cheng Ping Lu wetland by our laboratory (GISAID accession number: EPI3408601-EPI3408608); Newcastle disease virus NDV (SX04, GX1284, GX218 strains) and pigeon paramyxovirus PPMV-1 (SX01, SX02 strains) were isolated, identified and preserved by China Animal Health and Epidemiology Center; Infectious bronchitis virus IBV was isolated from a diseased chicken farm by our laboratory (GenBank accession number: OQ189490); Chicken infectious anemia virus CIAV was isolated from clinical samples by our laboratory; Pigeon circovirus PiCV was isolated from diseased pigeon tissue samples in Shanxi area by our laboratory (GenBank accession number: PP301888); Infectious bursal disease virus IBDV was isolated from clinical samples by our laboratory (GenBank accession number: OR493443); Mouse monoclonal antibodies Flu A-NP-F (item number: EKY0259F) and Flu A-NP-E (item number: EKY0259E) were purchased from Shanghai Xibao Biotechnology Co., Ltd.; Mouse monoclonal antibodies NDV-HN-1C10 (item number: 3ND5-1C10) and NDV-HN-8H2 (item number: 3ND5-8H2) were purchased from Haipai Biotechnology Co., Ltd.; Goat anti-mouse IgG was purchased from Changsha Youxite Biotechnology Co., Ltd.

[0047] 1.1.2 Main reagents and instruments

[0048] 60 nm ultra-high concentration colloidal gold was purchased from Zhengzhou Lingsi Biological Technology Co., Ltd.; bovine serum albumin (BSA), sucrose, trehalose, Tris-HCl, polyvinylpyrrolidone (PVP40000), Tween-20, anhydrous potassium carbonate (K2CO3) were purchased from Beijing Solabio Biological Technology Co., Ltd.; NC nitrocellulose membrane (Sartorius CN140), PVC bottom plate (DB-6), water absorption pad (H5072), gold-labeled combination pad (GL0194), sample pad (GL-b04), plastic card shell (A-9) were purchased from Shanghai Jieyi Biological Technology Co., Ltd.; analytical balance was purchased from Tianjin Jingtao Instrument Technology Co., Ltd.; table-top low-temperature centrifuge was purchased from Eppendorf Biological Technology Co., Ltd.; 3D rotary mixer was purchased from Hangzhou Miou Instrument Co., Ltd.; XYZ three-dimensional membrane drawing gold spraying instrument (HM3035), microcomputer automatic cutting machine (ZQ2002), numerical control high-speed cutting machine (CTS300) were purchased from Shanghai Jibiao Biological Technology Co., Ltd.

[0049] 1.1.3 Preparation of main solutions

[0050] 0.01 M Tris-HCl solution: 1.576 g of Tris-HCl was weighed and dissolved in 1 L of ddH2O, filtered with a 0.22 μm filter, and stored at 4℃. Different pH values were adjusted by HC1 and K2CO3 solution for experiments.

[0051] 0.1 M K2CO3 solution: 1.382 g of anhydrous potassium carbonate was weighed and dissolved in 80 mL of ddH2O, and then diluted to 100 mL after complete dissolution. After filtration with a 0.22 μm filter, it was stored at 4℃.

[0052] 10% BSA solution: 10 g of BSA powder was weighed and dissolved in 80 mL of ddH2O, and then diluted to 100 mL after complete dissolution. After filtration with a 0.22 μm filter, it was stored at 4℃.

[0053] 10% NaCl solution: 10 g of NaCl was weighed and dissolved in 80 mL of ddH2O, and then diluted to 100 mL after complete dissolution. After filtration with a 0.22 μm filter, it was stored at 4℃.

[0054] Colloidal gold reconstitution solution: 1 g of BSA and 10 g of sucrose were weighed, 1 mL of Tween-20 and 50 μL of preservative ProClean300 were measured, and all were dissolved in 80 mL of 0.01 M Tris-HCl (pH: 8.5). After complete dissolution, it was diluted to 100 mL, filtered with a 0.22 μm filter, and stored at 4℃.

[0055] Gold label pad treatment solution: 0.5 g BSA and 4 g sucrose were weighed, 1 mL Tween-20 and 50 μL preservative ProClean300 were measured, and all were dissolved in 80 mL 0.01 M Tris-HCl (pH = 8.5). After complete dissolution, the volume was made up to 100 mL, and after filtration with a 0.22 μm filter, it was stored at 4°C.

[0056] Sample pad treatment solution: 0.5 g BSA and 0.5 g PVP40000 were weighed, 1 mL Tween-20 and 50 μL preservative ProClean300 were measured, and all were dissolved in 80 mL 0.01 M Tris-HCl (pH = 8.5). After complete dissolution, the volume was made up to 100 mL, and after filtration with a 0.22 μm filter, it was stored at 4°C.

[0057] 1.2 Method

[0058] 1.2.1 Determination of optimal labeling pH and antibody labeling concentration

[0059] 0.01 M Tris-HCl solution was adjusted to pH values of 6, 6.5, 7, 7.5, 8, 8.5, 9, and 9.5 with 0.1 M K2CO3 solution and 0.1 M HCl solution, respectively. 40 μL of colloidal gold with a concentration of 100 OD was diluted to 40 OD by taking up 60 μL of 0.01 M Tris-HCl buffer with different pH values. To 100 μL of colloidal gold solution with a concentration of 40 OD in each group of different pH values, the same mass of Flu A-NP-E and NDV-HN-8H2 mouse monoclonal antibodies was added to a final concentration of 50 μg / mL, and shaken to mix, and reacted at room temperature for 60 min (reaction system 100 μL). After the reaction was completed, 25 μL of labeled antibody colloidal gold was taken, 975 μL of ddH2O was added to dilute it to 1 OD, and 100 μL of 10% NaCl solution was added to each group of 1 OD labeled gold to mix, stand for 5 min, and observe the color change of the colloidal gold. This step is salt destruction, which is used to test the stability of the gold-labeled antibody, and the color is stable or the least aggregation is the optimal labeling pH of the antibody.

[0060] Take 40 μL of colloidal gold with a concentration of 100 OD, and take 60 μL of 0.01 M Tris-HCl buffer with the optimal pH value to dilute it to 40 OD. Add different amounts of Flu A-NP-E and NDV-HN-8H2 mouse monoclonal antibodies to the colloidal gold solution to a final concentration of 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, and 80 μg / mL, respectively, shake and mix, and react at room temperature for 60 min (reaction system: 100 μL). After the reaction is completed, follow the salt destruction procedure in "1.2.1" to test the stability of the gold-labeled antibodies. The optimal labeling concentration is the one with the best color and the least amount of antibodies, and 20% more antibodies are added based on this.

[0061] Take colloidal gold with a concentration of 1 OD as a control.

[0062] 1.2.2 Preparation of gold-labeled antibodies

[0063] Take two 1.5 mL EP tubes, and add 40 μL of colloidal gold with a concentration of 100 OD to each. Take 60 μL of 0.01 M Tris-HCl buffer with the optimal labeling pH value to dilute it to 40 OD. Add a certain amount of Flu A-NP-E and NDV-HN-8H2 mouse monoclonal antibodies to the 40 OD colloidal gold solution to a final concentration of the optimal labeling concentration, shake and mix, and react at room temperature for 60 min (reaction system: 100 μL). Add 5 μL of 10% BSA solution to the gold-labeled antibody solution after the labeling is completed, shake and mix, and block at room temperature for 1 h. After the blocking is completed, centrifuge the gold-labeled antibodies (4°C, 8000 rpm, 5 min), discard the supernatant, add 100 μL of colloidal gold resuspension solution to resuspend it to 40 OD, mix well, and store it at 4°C or directly spray gold.

[0064] 1.2.3 Gold spraying and line drawing

[0065] Mix the gold-labeled antibodies Flu A-NP-E and NDV-HN-8H2 well, and use the XYZ three-dimensional membrane-scribing gold-spraying instrument to uniformly spray the gold-labeled pad, which has been cut and treated with the gold-labeled pad treatment solution, in an amount of 3 μL / cm, and place it in a 45°C oven to dry for 6 h. Dilute the antibodies Flu A-NP-F and NDV-HN-1C10 with PBS to 0.5 mg / mL, respectively, and add 2 μL of methanol and 2 μL of 20% trehalose solution (m / v) to each 40 μL system. On the NC nitrocellulose membrane, coat 1 μL / cm of Flu A-NP-F (0.5 mg / mL) and NDV-HN-1C10 (0.5 mg / mL) mouse monoclonal antibodies as the detection lines T1 (AIV) and T2 (NDV) at an interval of 0.5 cm, and coat 1 μL / cm of goat anti-mouse IgG (0.75 mg / mL) as the quality control line (C) at a distance of 0.5 cm from the detection line T1. Place the antibody-coated NC nitrocellulose membrane in a 37°C incubator to dry for 4 h.

[0066] 1.2.4 Assembly of test strips and result determination

[0067] Exposing the adhesive surface of the PVC base plate by removing the paper covering it, and attaching the NC nitrocellulose membrane to the middle region; attaching the gold-labeled pad below the T2 line, overlapping the NC membrane by 2 mm; attaching the sample pad below the gold-labeled pad, overlapping the gold-labeled pad by 2 mm; and finally attaching the water-absorbing pad above the C line, to complete the preparation of the AIV and NDV colloidal gold differential diagnosis large plate (6 cm x 30 cm). As shown in FIG. 1, cut the AIV and NDV colloidal gold differential diagnosis large plate into AIV and NDV differential diagnosis test strips (3.9 mm x 60 mm) using a numerical control high-speed cutting machine, and assemble the test strips with the plastic card shell to complete the assembly of the AIV and NDV differential diagnosis test card. Figure 1

[0068] Result interpretation: negative (C line coloration, T1 and T2 lines no coloration), AIV positive (C line and T1 line coloration, T2 line no coloration), NDV positive (C line and T2 line coloration, T1 line no coloration), AIV and NDV mixed positive (C line, T1 and T2 lines coloration), and invalid (C line no coloration, interpreted as invalid).

[0069] 1.2.5 Determination of the optimal sample diluent of the test strip

[0070] ​To verify the difference of different sample diluents on the detection effect of AIV and NDV differential diagnosis test strip, the best sample diluent was screened. Five kinds of sample diluents were set: PBS (pH = 7.4), PBST (pH = 7.4), Tris-HCl (pH = 8.5), 0.9% NaCl, ddH2O, and AIV and NDV differential diagnosis test strips were assembled under the optimal preparation conditions. H9N2 strain and Lasota vaccine strain were diluted to the same concentration and detected, and the test results were observed and recorded.

[0071] 1.2.6 Test strip detection performance evaluation

[0072] Under the optimal simultaneous detection of AIV and NDV differential diagnosis test strip assembly conditions, a large number of test strips of the same batch and different batches were prepared to evaluate their detection performance.

[0073] 1.2.6.1 General experiment

[0074] To evaluate the universality of the test strip, the simultaneous detection of AIV and NDV differential diagnosis test strip was prepared under the optimal conditions. Several different subtypes of avian influenza virus (H1N1, H3N2, H4N2, H9N2 and H12N2), Lasota vaccine strain, different chicken Newcastle disease virus (SX04, GX1284, GX218), pigeon Newcastle disease virus (SX01, SX02) and H9N2 strain and Lasota vaccine strain mixed virus were detected to evaluate the universality of virus detection.

[0075] 1.2.6.2 Specificity experiment

[0076] To evaluate the specificity of the test strip, AIV+NDV mixed virus (Lasota+H9N2 group, referred to as Lasota+H9 group), AIV-H9N2 (referred to as AIV-H9 group), NDV-Lasota vaccine strain group, IBV, CIAV, PiCV, IBDV and other avian viruses were detected by the differential diagnosis test strip. Negative allantoic fluid (corresponding to NAF group) and ddH2O were set as negative controls. The samples were diluted to the same concentration, 90 μL was taken by pipette and added to the sample pad for chromatographic reaction, and the test results were observed and recorded.

[0077] 1.2.6.3 Sensitivity experiment

[0078] To evaluate the sensitivity of the test strip, the sample diluent was used to dilute H9N2 strain and 10 7.0 EID 50 / mL of H9N2 strain and 10 5.5 EID 50 / mL of Lasota vaccine strain were diluted by 2-fold and detected, and the detection sensitivity was observed and recorded.

[0079] 1.2.6.4 Reproducibility experiment

[0080] To evaluate the batch stability of the test strip, i.e. reproducibility, different batches of test strips were used to detect H9N2 strain, Lasota vaccine strain and negative samples, and each sample was detected 5 times using different batches of test strips, and the test results were observed and analyzed. ddH2O was used as a control group.

[0081] 1.2.6.5 Stability experiment

[0082] To evaluate the stability of the differential diagnosis test strip for simultaneous detection of AIV and NDV, the test strips assembled in the same batch were placed in a sealed tin foil paper, divided into two groups, and the same amount of desiccant was placed inside. After sealing and packaging, they were stored at 4°C and room temperature (RT) respectively, and detected every 15 days using H9N2 strain and Lasota vaccine strain to determine the optimal storage condition and longest shelf life.

[0083] 1.2.7 Preliminary clinical application and accuracy evaluation of test strip

[0084] A total of 167 clinical samples were collected from different regions of Shanxi Province, including live poultry markets, farms, and wild bird habitats. The sample types included throat swabs, anal swabs, tissue grinds, and allantoic fluid, etc. to verify the detection applicability of the test strip in different scenarios. At the same time, the test strip and RT-PCR detection methods were used to detect all clinical samples, and the detection results of the two methods were compared, and the overall coincidence rate was calculated.

[0085] 2. Results

[0086] 2.1 Determination of optimal marker pH and antibody marker concentration

[0087] The pH value of the buffer system is one of the important factors affecting the binding of antibodies and colloidal gold particles. Low pH makes the antibody protein carry more positive charge, leading to cross-linking of gold markers to form "dead gold"; high pH will increase the charge repulsion between antibody protein and colloidal gold, with the risk of falling off. Antibody marker concentration affects the color development effect and sensitivity of the test strip. Since the color stability state will change accordingly with longer storage time during pH screening, therefore, based on the standard of color stability or least aggregation, the optimal marker pH value of Flu A-NP-E mouse monoclonal antibody is 8.5 ( Figure 2 medium A), and the optimal antibody marker concentration is 24 μg / mL ( Figure 2 medium B); the optimal marker pH value of NDV-HN-8H2 mouse monoclonal antibody is 8.5 ( Figure 3(A), the optimal antibody labeling concentration is 48 μg / mL ( Figure 3 (B)

[0088] 2.2 Determination of the optimal sample diluent for the test strip

[0089] like Figure 4 As shown, five different sample diluents were used to dilute H9N2 avian influenza virus and Lasota vaccine strain to the same concentration, and test strips from the same batch and under the same assembly conditions were used for detection. The results showed that when using PBST to detect AIV, a non-specific band appeared at test line T2; when using 0.9% NaCl to detect NDV, a non-specific band appeared at test line T1. Furthermore, repeated experiments revealed that PBS and Tris-HCl also produced a certain proportion of non-specific bands, easily leading to false positives. When using ddH2O as the sample diluent to detect both viruses, the test bands were complete, clear, and uniformly colored with no significant color difference. Moreover, because ddH2O is pure and free of salt ions, it does not affect the reactions of various substances on the test strip, minimizing the occurrence of non-specific reactions and providing more reliable support for the detection and differential diagnosis of AIV and NDV. Therefore, ddH2O was chosen as the sample diluent for the AIV and NDV differential diagnostic test strips.

[0090] 2.3 Generality Experiment Results

[0091] Figure 5 The results showed that the diagnostic strip for the simultaneous detection of AIV and NDV could identify five subtypes of AIV strains: H1N1, H3N2, H4N2, H9N2, and H12N2. It could also identify the Lasota vaccine strain, three different genotypes of chicken Newcastle disease virus (SX04, GX1284, GX218), two different genotypes of pigeon Newcastle disease virus (SX01, SX02), and a mixture of H9N2 and Lasota vaccine strains. This indicates that the strip has good universality.

[0092] 2.4 Specificity Experiment Results

[0093] like Figure 6 As shown, the test strip for the simultaneous detection of AIV and NDV was used to detect AIV+NDV mixed virus solution (Lasota+H9N2 group), AIV virus, NDV virus, and other avian pathogens. The results showed that the test strip prepared in this invention can specifically identify the H9N2 (AIV) strain and the Lasota vaccine strain, but shows no cross-reactivity with many other avian pathogens such as IBV, PiCV, and IBDV, indicating that the test strip has good specificity.

[0094] 2.5 Sensitivity Test Results

[0095] The H9N2 and Lasota vaccine strains with known virus titers were diluted by 2-fold dilution with the sample diluent respectively to detect the sensitivity. The results are shown in Table 2. Figure 7 As shown in Table 2, when the dilution of AIV virus was 2 13 , the T1 band was hardly seen; when the dilution of NDV virus was 2 11 , the T2 band was hardly seen. Therefore, the detection limit of the differential diagnosis test strip for AIV and NDV was about 10 3.0 EID 50 / mL and 10 2.6 EID 50 / mL respectively.

[0096] 2.6 Repetitive experiment results

[0097] Different batches of test strips prepared using the same assembly process were used for repetitive experiments on the H9N2 strain and the Lasota vaccine strain, and each sample was detected 5 times repeatedly. The detection results are shown in Table 3. Figure 8 As shown in Table 3, the detection rate of the test strip within the batch and between the batches was 100%, indicating that the simultaneous detection AIV and NDV differential diagnosis test strip prepared by the present application had good repeatability.

[0098] 2.7 Stability experiment results

[0099] In order to determine the optimal storage condition and the longest shelf life of the test strip, the test strip was placed in 4℃ and room temperature (RT) respectively, and was taken out every 15 days, and was detected using the H9N2 strain and the Lasota vaccine strain respectively at the same concentration. The results are shown in Table 4. Figure 9 As shown in Table 4, under the storage conditions of 4℃ and RT for 6 months, the detection results of the simultaneous detection AIV and NDV differential diagnosis test strip were stable, and there was no obvious difference.

[0100] 2.8 Preliminary clinical application and accuracy evaluation of the test strip

[0101] The simultaneous detection AIV and NDV differential diagnosis test strip was preliminarily applied clinically, and a total of 167 clinical samples were collected from different regions of Shanxi Province, live poultry markets, breeding farms, and wild bird habitats, and the two methods of test strip and RT-PCR were used for detection simultaneously. The results are shown in Table 1 and Table 2. Compared with RT-PCR, the test strip had 36 positive results of AIV, 124 negative results of AIV, the total coincidence rate was (36+124) / 167, and the result was 95.8%; the test strip had 10 positive results of NDV, 157 negative results of NDV, the total coincidence rate was (10+157) / 167, and the result was 100%. The results showed that the simultaneous detection AIV and NDV differential diagnosis test strip prepared by the present application was suitable for the clinical rapid diagnosis of AIV and NDV.

[0102] Table 1 AIV clinical diagnosis experiment results

[0103]

[0104] Table 2 NDV clinical diagnosis experiment results

[0105]

[0106] The present application selects 0.01 M Tris-HCL buffer with pH = 8.5 as the gold-labeled antibody buffer system, the isoelectric point of most monoclonal antibodies is 8.2, and the pH value of 8.5 can ensure that the labeled antibody carries a small amount of negative charge to maintain the overall stability of the gold-labeled antibody. Various components in the buffer system also determine the specificity and sensitivity of the test strip. BSA as a high-concentration inert protein can protect the activity of the labeled antibody protein; by pre-saturating the adsorption sites of the gold pad, it ensures the effective release of the gold-labeled complex; it can dynamically block in the chromatography process, neutralize the surface charge and hydrophobic force of the NC membrane, and reduce non-specific adsorption; at the same time, it can block non-specific proteins and reduce false positive phenomena. Sucrose and trehalose as a drying protector can prevent protein denaturation during drying and provide a relatively hydrophilic environment to avoid the gold label from being firmly combined with the gold pad through hydrophobic interaction and affecting chromatography release. Surfactant (Tween-20) has the effect of promoting the release of gold-labeled antibody, but if the concentration of surfactant in the buffer system is too high, the gold-labeled antibody has the risk of falling off, so the gold-labeled antibody solution should be sprayed and dried as soon as possible to reduce the storage time in the liquid phase. High molecular polymer (PVP40000) has the effect of promoting the reaction in addition to promoting the release, but it has the risk of causing non-specific reactions, so it cannot be added too much in the buffer system. Finally, the related consumables of the test strip, such as the sample pad, the gold-labeled pad, the NC membrane, and the water-absorbing pad, are closely related to the sensitivity and specificity of the diagnosis, such as the pore size and flow rate of the gold-labeled pad and the NC membrane, which will affect the release of the gold-labeled antibody.

[0107] The present application optimizes various conditions and successfully prepares a colloidal gold differential diagnosis test strip for simultaneously detecting AIV and NDV, realizing the synchronous rapid differential diagnosis of AIV and NDV. The test strip has good universality and specificity, can realize the rapid clinical diagnosis of different subtypes of avian influenza virus and different genotypes of Newcastle disease virus, and has no cross reaction with other avian pathogens; the detection limits of AIV and NDV are 10 3.0 EID 50 / mL and 10 2.6 EID 50 / mL; the repeatability is good, the batch detection rate and the batch detection rate are both 100%; the stability is good, and the detection effect is not affected after being stored at 4℃ or normal temperature for 6 months. Through comparison with RT-PCR in clinical trials, the overall coincidence rate of AIV detection is 95.8%, and the overall coincidence rate of NDV detection is 100%, which proves that the method has good detection performance and provides a reliable technical means for clinical rapid differential diagnosis of AIV and NDV.

[0108] The above-described embodiments are only used to describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements of the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A test strip for simultaneously detecting avian influenza virus and Newcastle disease virus, characterized in that, The test strip includes a base plate, a sample pad, a gold-labeled binding pad, an NC membrane, and an absorbent pad. The sample pad, the gold-labeled binding pad, the NC membrane, and the absorbent pad are sequentially overlapped and pasted onto the base plate in the direction of chromatography. The gold-labeled binding pad is coated with colloidal gold-labeled Flu A NP gold-labeled antibody and NDV HN gold-labeled antibody; the NC membrane is provided with two detection lines and one control line, the two detection lines are coated with NDV-HN antibody and Flu A NP antibody respectively, and the control line is coated with goat anti-mouse IgG protein.

2. The test strip according to claim 1, characterized in that, The Flu A NP gold-labeled antibody is a mouse-derived anti-Flu A-NP-E monoclonal antibody, the NDV HN gold-labeled antibody is a mouse-derived anti-NDV-HN-8H2 monoclonal antibody, the NDV-HN antibody is an NDV-HN-1C10 mouse monoclonal antibody, and the Flu A NP antibody is a Flu A-NP-F mouse monoclonal antibody.

3. The test strip according to claim 1, characterized in that, The preparation method of the gold-labeled conjugate pad is as follows: a colloidal gold solution is prepared, and 20-28 μg / mL of mouse anti-Flu A NP monoclonal antibody and 44-52 μg / mL of mouse anti-NDV HN monoclonal antibody are added to the colloidal gold solution by adjusting the pH value of the colloidal gold solution to carry out the labeling reaction. After the reaction is completed, the mixture is blocked, centrifuged, and reconstituted to obtain the gold-labeled antibody. The gold-labeled antibody is then uniformly sprayed onto the gold-labeled pad and dried to obtain the final product.

4. The test strip according to claim 3, characterized in that, The colloidal gold solution was diluted to a 40 OD colloidal gold solution with a pH of 7.0-8.5 and a 0.01 M Tris-HCl buffer solution.

5. A method for preparing a test strip as described in any one of claims 1-4, characterized in that, Includes the following steps: Preparation of gold-labeled conjugate pads; Preparation of nitrocellulose membranes; Assembly of test strips; The preparation method of the gold-labeled conjugate pad is as follows: a colloidal gold solution is prepared, and 20-28 μg / mL of mouse anti-Flu A NP monoclonal antibody and 44-52 μg / mL of mouse anti-NDV HN monoclonal antibody are added to the colloidal gold solution by adjusting the pH value of the colloidal gold solution to carry out the labeling reaction. After the reaction is completed, the mixture is blocked, centrifuged, and reconstituted to obtain the gold-labeled antibody. The gold-labeled antibody is then uniformly sprayed onto the gold-labeled pad and dried to obtain the final product.

6. The preparation method according to claim 5, characterized in that, The amount of the mouse anti-Flu A NP monoclonal antibody added was 24 μg / mL, and the amount of the mouse anti-NDV HN monoclonal antibody added was 48 μg / mL.

7. The preparation method according to claim 5, characterized in that, The labeling reaction is carried out at room temperature for 30-90 min; after the reaction is completed, 1-9 μL of BSA solution is added, the reaction is blocked for 0.5-1.5 h, the supernatant is discarded by centrifugation, and the gold-labeled antibody is obtained by reconstitution with colloidal gold solution.

8. The use of the test strip as described in any one of claims 1-4 in the preparation of a product for the simultaneous detection of avian influenza virus and Newcastle disease virus.

9. The application according to claim 8, characterized in that, The product in question is a test kit.

10. A product for detecting avian influenza virus and Newcastle disease virus, characterized in that, The product includes the test strip as described in any one of claims 1-4.

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