CN-116794304-B - Quantum dot microsphere fluorescence immunochromatography test paper for combined detection of ASFV antibody and nucleic acid and application thereof

Abstract

The invention provides quantum dot microsphere fluorescence immunochromatography test paper for combined detection of ASFV antibodies and nucleic acids and application thereof, and belongs to the technical field of veterinary biological diagnostic products. The quantum dot microsphere fluorescence immunochromatographic test paper for combined detection of ASFV antibodies and nucleic acids comprises a bottom plate, wherein a sample pad, a binding pad, a nitrocellulose membrane and a water absorption pad are sequentially connected in an end-to-end mode on the bottom plate, an antibody detection line, a nucleic acid detection line and a quality control line are sequentially arranged on the nitrocellulose membrane from the binding pad to the water absorption pad, a goat anti-pig IgG-Fc antibody is coated on the antibody detection line, streptavidin is coated on the nucleic acid detection line, a rabbit anti-sheep IgG antibody is coated on the quality control line, and a recombinant protein P22 marked by quantum dot microspheres and a sheep anti-digoxin antibody marked by the quantum dot microspheres are coated on the binding pad. The test paper can be used for rapidly detecting ASFV antibodies and nucleic acids with high specificity and high sensitivity.

Inventors

• LIU FEI
• SHAN YANKE
• FENG ZHIXIN
• BAI YUN
• ZHAO YIRAN
• LI JIAHAO
• XIE QINGYUN
• YI WEIJIE
• WEI YANNA

Assignees

• 南京农业大学
• 江苏省农业科学院

Dates

Publication Date

20260508

Application Date

20230630

Claims (7)

1. 1. The quantum dot microsphere fluorescence immunochromatographic test paper for combined detection of ASFV antibodies and nucleic acids is characterized by comprising a bottom plate, wherein a sample pad, a binding pad, a nitrocellulose membrane and a water absorption pad are sequentially connected in an end-to-end mode on the bottom plate, an antibody detection line, a nucleic acid detection line and a quality control line are sequentially arranged on the nitrocellulose membrane in the direction from the binding pad to the water absorption pad, a goat anti-pig IgG-Fc antibody is coated on the antibody detection line, streptavidin is coated on the nucleic acid detection line, a rabbit anti-sheep IgG antibody is coated on the quality control line, a recombinant protein P22 with quantum dot microsphere marks and a sheep anti-digoxin antibody with quantum dot microsphere marks are coated on the binding pad, the amino acid sequence in the recombinant protein P22 is shown as SEQ ID NO:5, the sheep anti-digoxin antibody is purchased from Beijing biological technology Co., product number is 3210-0488, the goat anti-pig IgG-Fc antibody is purchased from Abmart Co, product number is 112748, and where the anti-sheep IgG antibody is purchased from Abcam, product number is 31480.
2. 2. The test paper according to claim 1, wherein the sample pad is obtained by immersing a glass cellulose membrane in a boric acid buffer solution containing Triton, BSA and glucose and drying.
3. 3. The test paper according to claim 2, wherein the glass cellulose membrane is soaked in Tris-HCl buffer solution containing BSA, sucrose, PEG1500 and Tween20, then the recombinant protein P22 marked by the quantum dot microsphere and the sheep anti-digoxin antibody marked by the quantum dot microsphere are sprayed in sequence, and the binding pad is obtained after drying.
4. 4. The test paper according to claim 3, wherein the quantum dot microsphere is coupled with recombinant protein P22 and sheep anti-digoxin antibody respectively to obtain quantum dot microsphere-labeled recombinant protein P22 and quantum dot microsphere-labeled sheep anti-digoxin antibody respectively.
5. 5. The test paper according to claim 4, wherein the mass ratio of the quantum dot microsphere to the recombinant protein P22 is 5-7:1, and the mass ratio of the quantum dot microsphere to the sheep anti-digoxin antibody is 6-10:1.
6. 6. A method for testing a blood sample using the test strip of claim 1 for non-diagnostic purposes, comprising the steps of: (1) Using genome DNA in a blood sample as a template, and adopting a primer F, a primer R and a probe to carry out isothermal nucleic acid amplification; (2) Diluting the nucleic acid amplification product, and mixing the diluted nucleic acid amplification product with serum of the blood sample to obtain a mixed product; (3) Dripping the recombinant protein P22 marked by the quantum dot microsphere and the sheep anti-digoxin antibody marked by the quantum dot microsphere into the mixed product, vibrating and uniformly mixing, diluting with a buffer solution containing Tween-20, dripping the diluted solution onto a sample pad of the test paper, and judging the result by using a fluorescence analyzer or an ultraviolet laser lamp after chromatography.
7. 7. The method according to claim 6, wherein the nucleotide sequence of the primer F is shown as SEQ ID NO. 1, the nucleotide sequence of the primer R is shown as SEQ ID NO. 2, the 5' end of the primer R is marked with biotin, the nucleotide sequence of the probe is shown as SEQ ID NO. 3, the 5' end of the probe is connected with digoxin, the 3' end is connected with C3-spacer, and a tetrahydrofuran is marked between the 30 th nucleotide and the 31 st nucleotide of the SEQ ID NO. 3.

Description

Quantum dot microsphere fluorescence immunochromatography test paper for combined detection of ASFV antibody and nucleic acid and application thereof Technical Field The invention belongs to the technical field of veterinary biological diagnostic products, and particularly relates to quantum dot microsphere fluorescence immunochromatography test paper for combined detection of ASFV antibodies and nucleic acids and application thereof. Background African swine fever (AFRICAN SWINE FEVER, ASF) is a devastating infectious disease caused by African Swine Fever Virus (ASFV) that affects pigs of various breeds and ages. Animal epidemic diseases which are listed by the world animal health organization (World Organisation ForAnimal Health, WOAH) as animal epidemic diseases which need to be reported in time are listed as a class of animal epidemic diseases in China. Healthy pigs and diseased pigs are the main routes for transmitting ASFV by direct contact with ASFV pollutants or by insect bites with chlorpyrifos and other vectors. At present, no specific medicine and vaccine for ASFV exist, and the main prevention and control strategy relies on strict biosafety measures and the killing of infected or exposed animals, so that the detection and early diagnosis of ASFV are important for epidemic situation confirmation and control. Currently, ASF field detection still mainly depends on clinical manifestations and pathological lesions. In the field diagnosis, veterinary personnel perform differential diagnosis according to certain characteristic symptoms or lesions of the swinery, and provide a rapid swinery prevention and control direction for the field epidemic prevention and control. However, african swine fever has various manifestations such as most acute, subacute, chronic, and the like, clinical symptoms are various, and specific symptoms are lacking, so that laboratory detection is needed in differential diagnosis of african swine fever. African swine fever virus and antibodies were detected by a variety of effective detection methods. In clinical applications, the sensitivity of ASFV antibody detection is significantly lower than that of nucleic acid detection, and sometimes the ASFV infection is negative because acute onset of ASF pigs is likely to die without producing antibodies, and in vivo diagnosis requires conventional polymerase chain reaction (Polymerase chain reaction, PCR) of tissues, as well as serological detection to detect antibodies. Currently, the laboratory detection methods for African swine fever virus suggested by WOAH include virus isolation, fluorescent antibody detection and conventional polymerase chain reaction detection. The virus separation has high requirements on the operation environment and long time, and the fluorescent antibody detection and PCR have good specificity and high sensitivity, but have high requirements on test conditions and instrument equipment, and the on-site rapid detection is difficult to realize. In addition, the detection method also comprises Enzyme-linked immunosorbent assay (ELISA) kit and colloidal gold immunochromatography test paper. The ELISA kit has good specificity, high sensitivity, relatively simple operation, high requirements on environmental factors such as temperature, pH value and the like, is interfered by autoantibodies, xenophils and the like, is easy to generate false positive, and does not meet the requirements of on-site rapid detection. Colloidal gold immunochromatography test strips for rapidly detecting ASFV antibodies have low sensitivity. Thus, the current market lacks in-situ detection technology for ASFV nucleic acids and antibodies that achieve both rapid, high specificity, and high sensitivity. Disclosure of Invention Aiming at the defects of the prior art, the first aim of the invention is to provide quantum dot microsphere fluorescence immunochromatography test paper for combined detection of ASFV antibodies and nucleic acids, which is used for rapid, high-specificity and high-sensitivity detection of the ASFV antibodies and the nucleic acids. It is a second object of the present invention to provide a method for testing blood samples using the test strip for non-diagnostic purposes. The invention adopts the following technical scheme: The quantum dot microsphere fluorescence immunochromatographic test paper for combined detection of ASFV antibodies and nucleic acids comprises a bottom plate, wherein a sample pad, a binding pad, a nitrocellulose membrane and a water absorption pad are sequentially connected in an end-to-end mode on the bottom plate, an antibody detection line, a nucleic acid detection line and a quality control line are sequentially arranged on the nitrocellulose membrane from the binding pad to the water absorption pad, a goat anti-pig IgG-Fc antibody is coated on the antibody detection line, streptavidin is coated on the nucleic acid detection line, a rabbit anti-sheep IgG antibody is coated on the quality c