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CN-122017234-A - Near infrared fluorescence immunochromatography test strip for detecting African swine fever virus and application thereof

CN122017234ACN 122017234 ACN122017234 ACN 122017234ACN-122017234-A

Abstract

The invention provides a near infrared fluorescence immunochromatography test strip for detecting African swine fever virus and application thereof, belonging to the technical field of biological detection. The test strip comprises a sample pad, a binding pad, an antibody coating NC film and a water absorption pad, wherein a fluorescent conjugate is coated on the binding pad, the fluorescent conjugate is obtained by coupling an African swine fever virus p30 protein monoclonal antibody 027B with a near infrared fluorescent dye through biotin and streptavidin, the antibody coating NC film is provided with a detection line and a quality control line, the African swine fever virus p30 protein monoclonal antibody 2-12B is fixed on the detection line, the monoclonal antibody 2-12B is secreted by hybridoma cell lines 2-12B, the preservation number is CCTCC NO: C2022116, the monoclonal antibody 027B is purchased from Wohan primary valley biotechnology Co., ltd, and the product number is 027B. The test strip can realize the on-site, rapid, high-sensitivity and high-specificity detection of ASFV.

Inventors

  • XIAO QI
  • Lai Hongqiao
  • SONG JINGZE
  • PAN RONGHUA
  • ZHU HENGZHI
  • HE KONGWANG
  • YU JING
  • WEN LIBIN
  • MA QINGXIA
  • LI NA
  • Xiang Zenghao

Assignees

  • 江苏省农业科学院
  • 佰孚生物科技(泰州)有限公司

Dates

Publication Date
20260512
Application Date
20260215

Claims (9)

  1. 1. A near infrared fluorescence immunochromatography test strip for detecting African swine fever virus comprises a sample pad, a binding pad, an antibody coating NC film and a water absorption pad which are sequentially arranged on a supporting pad, and is characterized in that the binding pad is coated with a fluorescence conjugate, the fluorescence conjugate is obtained by coupling an African swine fever virus p30 protein monoclonal antibody 027B with a near infrared fluorescence dye through biotin and streptavidin, the antibody coating NC film is provided with a detection line and a quality control line, the detection line is coated with an African swine fever virus p30 protein monoclonal antibody 2-12B, the monoclonal antibody 2-12B is secreted by a hybridoma cell line 2-12B, the preservation number is CCTCC NO: C2022116, and the monoclonal antibody 027B is purchased from the Wuhan primary cereal biotechnology Limited liability company, and the product number 027B.
  2. 2. The test strip of claim 1, wherein the fluorescent conjugate is prepared by labeling an african swine fever virus p30 protein monoclonal antibody 027B with biotin, labeling streptavidin with DyLight 800 dye, and coupling the biotin-labeled monoclonal antibody 027B with DyLight 800 dye-labeled streptavidin.
  3. 3. The test strip of claim 1 or 2, wherein the molar ratio of the biotin-labeled monoclonal antibody 027B to the DyLight 800 dye-labeled streptavidin is 1-4:1, preferably 4:1.
  4. 4. The test strip of claim 3, wherein the control line is coated with a rabbit anti-streptavidin polyclonal antibody.
  5. 5. The test strip of claim 4, wherein the nitrocellulose membrane is coated with a fluorescent conjugate at a concentration of 0.05-0.2mg/mL to obtain a conjugate pad, the nitrocellulose membrane is coated with 0.8-2mg/mL of p30 protein monoclonal antibody 2-12B to obtain a detection line, and 0.02-0.04mg/mL of rabbit anti-streptavidin polyclonal antibody to obtain a quality control line.
  6. 6. The test strip of claim 5, wherein the nitrocellulose membrane is treated with a pretreatment confining liquid to obtain the sample pad, the pretreatment confining liquid being an aqueous solution containing 0.1-0.2g/L boric acid, 45-55g/L trehalose, 1.8-2.2g/L polyethylene glycol, 2.3-2.8g/L bovine serum albumin, and 4-6 g/L tween 20.
  7. 7. A method for detecting african swine fever virus using the test strip of claim 1 for non-diagnostic purposes, comprising the steps of: (1) Adding a sample to be tested into the lysis solution, after the lysis, sampling and dripping the sample into a sample pad of the test strip, and performing chromatography; (2) Detecting the fluorescence signal intensity C of the C line and the fluorescence signal intensity T of the T line of the chromatographic test strip by adopting a dry fluorescence immunoassay instrument under the condition of 800 nm wavelength; (3) The result is judged that the ratio of the T/C value of the sample to be tested to the T/C value of the negative control is greater than or equal to 2, the sample to be tested contains African swine fever virus, the sample to be tested does not contain African swine fever virus if the sample to be tested only has a C line with obvious fluorescence signal peak and has no fluorescence signal peak, and the sample to be tested does not contain African swine fever virus if the sample to be tested only has a C line with obvious fluorescence signal peak and the ratio of the T/C value of the sample to be tested to the T/C value of the negative control is less than 2.
  8. 8. The method of claim 7, wherein the lysate is phosphate buffer containing 2.3-2.8 g/L tween 20.
  9. 9. The method of claim 8, wherein the negative control is african swine serum negative for swine fever virus.

Description

Near infrared fluorescence immunochromatography test strip for detecting African swine fever virus and application thereof Technical Field The invention belongs to the technical field of biological detection, and particularly relates to a near infrared fluorescence immunochromatography test strip for detecting African swine fever virus and application thereof. Background African swine fever (AFRICAN SWINE FEVER, ASF) is an acute, febrile, contagious animal infectious disease caused by African Swine Fever Virus (ASFV), has extremely high infection rate and mortality rate to domestic pigs and wild pigs, is an animal epidemic disease which is listed as a necessary report by the world animal health organization (WOAH), and constitutes a devastating threat to the global pig industry. Because the propagation paths are various, the incubation period is long, no effective commercial vaccine can be used for prevention, and the establishment of a rapid, accurate and sensitive early diagnosis method is important for prevention and control, extinguishment and eradication of epidemic situations. Currently, the detection methods of ASFV mainly include nucleic acid detection techniques represented by virus isolation and identification, fluorescent quantitative polymerase chain reaction (qPCR), and immunological detection techniques such as enzyme-linked immunosorbent assay (ELISA) and immunochromatographic assay (Immunochromatographic Assay, ICA). The virus separation takes too long to be suitable for large-scale rapid screening. Although the qPCR method has high sensitivity and specificity, the qPCR method is complex to operate, depends on expensive equipment and professionals, and is difficult to popularize in a basic layer and on site. In the immunological detection method, ASFV p30 protein (coded by the viral CP204L gene) is an important early expressed structural protein, and has excellent immunogenicity and conservation. It is expressed in large quantities in early stages of viral infection, i.e., in the cytoplasm of infected mononuclear-macrophages, and is one of the ideal targets for the development of ASFV detection reagents. The traditional colloidal gold immunochromatographic test strip (GICA) is simple and convenient to operate and low in cost, but has relatively low sensitivity, and the result interpretation depends on naked eyes and is high in subjectivity. In order to improve the detection sensitivity, fluorescent immunochromatography (Fluorescence Immunochromatographic Assay, FICA) has been developed. The FICA can remarkably improve the detection sensitivity and realize quantitative analysis by using fluorescent markers to replace colloidal gold and matching with a special reader. However, conventional FICAs may still suffer from insufficient lower detection limits in the face of low viral load african swine fever virus early infection samples. Disclosure of Invention The invention aims to overcome the defects of the prior art, and provides a near infrared fluorescence immunochromatography test strip for detecting African swine fever virus, which can realize on-site, rapid, high-sensitivity and high-specificity detection of ASFV. The invention also aims at providing a preparation method and a using method of the test strip. In order to achieve the above purpose, the invention adopts the following technical scheme: A near infrared fluorescence immunochromatography test strip for detecting African swine fever virus comprises a sample pad, a binding pad, an antibody coating NC film and a water absorption pad which are sequentially arranged on a supporting pad, and is characterized in that the binding pad is coated with a fluorescence conjugate, the fluorescence conjugate is obtained by coupling an African swine fever virus p30 protein monoclonal antibody 027B with a near infrared fluorescence dye through biotin and streptavidin, the antibody coating NC film is provided with a detection line and a quality control line, the detection line is coated with an African swine fever virus p30 protein monoclonal antibody 2-12B, the monoclonal antibody 2-12B is secreted by a hybridoma cell line 2-12B, the preservation number is CCTCC NO: C2022116, and the monoclonal antibody 027B is purchased from the Wuhan primary cereal biotechnology Limited liability company, and the product number 027B. In the invention, the fluorescent conjugate is prepared by labeling an African swine fever virus p30 protein monoclonal antibody 027B with biotin, labeling streptavidin with Dylight 800 dye, and coupling the biotin-labeled monoclonal antibody 027B with the Dylight 800 dye-labeled streptavidin to obtain the fluorescent conjugate. In the invention, the molar ratio of the biotin-labeled monoclonal antibody 027B to the Dylight 800 dye-labeled streptavidin is 1-4:1, preferably 4:1. In the invention, the quality control line is coated with rabbit anti-streptavidin polyclonal antibody. In the invention, a nitrocellulose membrane is coated b