African swine fever virus rapid detection test strip and preparation method and application thereof

An African swine fever virus and detection test paper technology, which is applied in the field of virus disease diagnosis technology and animal quarantine, can solve problems such as lack of methods, and achieve the effects of good stability, fast detection speed and high sensitivity

Active Publication Date: 2020-07-31
杭州恒奥科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as the 3D fine structure of African swine fever virus has been deciphered [Liu et al. lacking

Method used

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  • African swine fever virus rapid detection test strip and preparation method and application thereof
  • African swine fever virus rapid detection test strip and preparation method and application thereof
  • African swine fever virus rapid detection test strip and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Preparation of Anti-African Swine Fever Virus Monoclonal Antibody

[0030]1. Preparation of African swine fever virus P30 protein

[0031] 1 material

[0032] 1.1 Escherichia coli BL21 competent cells were purchased from Biomed Technology Co., Ltd.; gene synthesis was synthesized by General Biosystems (Anhui) Co., Ltd. (providing plasmid PET-30a); DNA Marker was purchased from Thermo Company, and plasmid kit was purchased from From AXYGEN Company; other chemical reagents are domestic analytical grade.

[0033] 1.2 LB medium Weigh 5.0g of yeast extract, 10g of tryptone, and 10g of NaCl, dissolve in 1L of purified water, and sterilize by high-pressure steam at 121°C for 20 minutes.

[0034] 1.3 LB (Kan+) liquid medium Add kanamycin to the LB medium to make the final concentration 100 μg / mL, store at 2-8°C.

[0035] 1.4 LB (Kan+) solid medium Add 1.5g agar powder to 100mL LB medium, and sterilize under high pressure steam at 121°C for 20 minutes. Before use, ...

Embodiment 2

[0104] Embodiment 2 African swine fever virus rapid detection test strip preparation

[0105] 1. Preparation and testing of monoclonal antibodies

[0106] 1.1 Recovery of hybridoma cells Take the hybridoma cells (7D5 strain and 4C11 strain) out of the liquid nitrogen tank respectively, put them in water at 37°C to melt rapidly, centrifuge at 1000r / min for 10 minutes, discard the supernatant, and use 15% After the DMEM medium of fetal bovine serum was resuspended, it was transferred into a T75 Kirkner flask, and the DMEM medium containing 15% fetal bovine serum was added to 10 mL, and placed at 37°C, 5% CO 2 Culture in the incubator for 2-6 hours. After the cells adhere to the wall, gently shake the bottle body to make the dead cells fall off and pour it out. Add 15mL of DMEM medium containing 15% fetal bovine serum again, and mark the cell name on the cell bottle. , generation and time, set at 37°C, 5% CO 2 cultured in an incubator.

[0107] 1.2 Preparation of monoclonal an...

Embodiment 3

[0146] Embodiment 3 African swine fever virus rapid detection test strip performance evaluation

[0147] 1. Specificity test

[0148] 1 material

[0149] 1.1 Reagent strips African swine fever virus rapid detection test strips are prepared by referring to Example 2, and the batch numbers are 190301, 190302, and 190305 respectively.

[0150] 1.2 Specific quality control samples of porcine foot-and-mouth disease virus antigen come from the improved foot-and-mouth disease O-type liquid phase blocking ELISA antibody detection kit (purchased from Lanzhou Veterinary Research Institute of Chinese Academy of Agricultural Sciences, batch number 2018091013S) of foot-and-mouth disease type O virus antigen; swine fever virus (Live vaccine purchased from Pulaike Bioengineering Co., Ltd., batch number 12CW1801001), porcine reproductive and respiratory syndrome virus (live vaccine purchased from Zhejiang Shihua Nuobeiwei Biotechnology Co., Ltd., batch number 2018002), pig Pseudorabies viru...

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Abstract

The invention provides an African swine fever rapid detection test strip. The African swine fever rapid detection test strip is composed of a test strip and a sample treatment liquid. On the other hand, the invention also provides a method for preparing the test strip. The method comprises the following steps of 1) preparing an anti-African swine fever virus P30 protein monoclonal antibody and ananti-African swine fever virus P72 protein monoclonal antibody, 2) preparing a latex microsphere pad and a sample pad, 3) preparing a nitrocellulose membrane detection line and a quality control line,4) preparing and sub-packaging a sample treatment solution, and 5) assembling test strips. The African swine fever rapid detection test strip provided by the invention is suitable for detection of African swine fever viruses in porcine serum, porcine whole blood and porcine tissues, is strong in specificity, high in sensitivity, good in stability and high in detection speed, can be used for earlyscreening of the African swine fever virus, and is particularly suitable for on-site African swine fever infection diagnosis, epidemiological investigation, pig international trade quarantine inspection and the like.

Description

technical field [0001] The invention belongs to the field of virus epidemic disease diagnosis technology and animal quarantine, and in particular relates to a rapid detection test strip for detecting African swine fever virus, a preparation method and application thereof. Background technique [0002] African swine fever (English name: African Swine fever, referred to as ASF) is caused by African swine fever virus (English name: African Swine fever virus, referred to as ASFV) that infects domestic pigs and various wild boars (such as African wild boars, European wild boars, etc.) Cause an acute, hemorrhagic, severe infectious disease. The World Organization for Animal Health (OIE) listed it as a legally notifiable animal disease, and this disease is also a class of animal epidemics that my country focuses on preventing. It is characterized by a short course of disease, the most acute and acute infection mortality rate is as high as 100%, clinical manifestations are fever (u...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/58G01N33/577G01N33/569G01N33/558G01N33/532
CPCG01N33/585G01N33/577G01N33/56983G01N33/558G01N33/532G01N2333/01G01N2469/10Y02A50/30
Inventor 孙统威舒建洪查银河刘影周晶晶徐慧珍
Owner 杭州恒奥科技有限公司
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