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Kit and detection method capable of synchronously detecting African swine fever virus antigens and antibodies in swine blood

A technology for simultaneous detection of African swine fever virus, which is applied in the field of kits for the simultaneous detection of African swine fever virus antigens and antibodies in swine blood, which can solve the problems of inability to directly detect whole blood, increase biological risks, and inconvenient on-site detection operations. , to reduce biological risks, facilitate on-site operation, and achieve the effect of simple operation

Pending Publication Date: 2020-10-02
TROPICAL CORP STRAIN RESOURCE INST CHINESE ACAD OF TROPICAL AGRI SCI +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no product capable of simultaneously detecting African swine fever virus pathogens (nucleic acid or protein antigens) and African swine fever virus antibodies
In addition, the traditional method of detecting antibodies is to detect antibodies in serum, which requires blood collection and separation of serum, which is easy to form aerosols and increases biological risks
On the other hand, whole blood cannot be tested directly, and serum separation equipment is required, making on-site testing inconvenient

Method used

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  • Kit and detection method capable of synchronously detecting African swine fever virus antigens and antibodies in swine blood
  • Kit and detection method capable of synchronously detecting African swine fever virus antigens and antibodies in swine blood
  • Kit and detection method capable of synchronously detecting African swine fever virus antigens and antibodies in swine blood

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1: Recombinant expression of African swine fever virus P30 protein

[0062] The African swine fever virus P30 protein is a protein encoded by the African swine fever virus gene, its amino acid sequence is shown in SEQ ID NO: 1 in the sequence listing, and its accession number in GeneBank is ACJ61575.1. The purpose of this embodiment is to efficiently express and prepare this African swine fever virus P30 protein (hereinafter referred to as P30 recombinant protein), so as to be used in the following examples as the detection line marker protein on the colloidal gold two-linked card, specifically comprising the following steps:

[0063] 1.1. Construction and identification of recombinant bacteria

[0064] The gene encoding the P30 protein of African swine fever virus (GenBank: EU874316.1) was retrieved from the database, and its codons were optimized to enable its efficient expression. This step was entrusted to Zhongmeitaihe Biotechnology (Beijing) Co., Ltd. ...

Embodiment 2

[0086] Embodiment 2: A kit capable of synchronously detecting African swine fever virus antigen and antibody in pig blood

[0087] This embodiment combines the following two detections to obtain a kit capable of simultaneously detecting African swine fever virus antigen and antibody in pig blood: the P30 recombinant protein double-antigen sandwich method prepared in the above-mentioned embodiment 1 is used to detect the African swine fever virus P30 protein antibody, And the African swine fever virus P72 protein antigen was detected by the African swine fever virus P72 protein monoclonal antibody double antibody sandwich method. The construction of the kit is based on the colloidal gold immunochromatography technology, that is, the kit mainly includes colloidal gold composed of a glass fiber membrane sample pad, a conjugate release pad, a water-absorbing pad, and an analysis membrane (nitrocellulose membrane (NC membrane)). A test card (named colloidal gold dual card) made of ...

Embodiment 3

[0115] Example 3: Comparison of Antigen and Antibody Detection Methods

[0116] 3.1 Comparison of African swine fever pathogen detection methods

[0117] This experiment utilizes the kit provided in the above-mentioned embodiment 2 to compare with the existing fluorescent PCR reagents used for African swine fever pathogen detection, to illustrate the sensitivity and specificity of the detection kit provided by the invention to the African swine fever virus antigen , including the following steps:

[0118] 3.1.1. Samples to be tested: 20 blood samples of suspected African swine fever pigs (from a pig farm in Hebei) were collected; 20 blood samples of healthy pigs were collected.

[0119] 3.1.2. The above 40 samples to be tested were detected with the kit provided in Example 2 and the African swine fever fluorescent PCR reagent produced by Tangshan Yian Bioengineering Co., Ltd., wherein the fluorescent PCR method was used as a reference method.

[0120] The detection results o...

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Abstract

The invention discloses a kit and detection method capable of synchronously detecting African swine fever virus antigens and antibodies in swine blood, and belongs to the technical field of biology. According to the kit provided by the invention, a detection line for detecting African swine fever virus antigens and antibodies is arranged on an analysis membrane of a colloidal gold duplex card at the same time; preferably, the African swine fever virus P30 protein is used as an antibody for detection, the monoclonal antibody of the African swine fever virus P72 protein is used as an antigen fordetection, and the matched sample diluent is matched for use, so the African swine fever virus P30 protein antibody and the P72 protein antigen in the whole blood of the pig can be simultaneously detected through one-step operation.

Description

technical field [0001] The invention belongs to the field of antigen and antibody detection in the field of biotechnology, in particular to a kit and a detection method capable of synchronously detecting African swine fever virus antigen and antibody in pig blood. Background technique [0002] African swine fever (ASF) is an acute, febrile and highly contagious disease of domestic pigs and wild boars caused by African swine fever virus (ASFV). Since the African swine fever epidemic was first discovered in 1921, the disease has been prevalent in dozens of countries around the world. According to the analysis of gene phylogenetic tree, it was found that the strain belonged to genotype II and belonged to the same evolutionary branch as the Georgia 2007 / 1 strain. [0003] At present, there is no effective vaccine to prevent African swine fever virus, and biological protection can only be carried out through the "quarantine-elimination" model. There are currently two types of d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/558G01N33/543
CPCG01N33/56983G01N33/54313G01N33/558G01N2333/01
Inventor 荣光郑金来赵军明乌哈干图草都吉林巴雅尔鞠珑株吴园园周汉林侯冠彧孙卫平彭维祺胡海超
Owner TROPICAL CORP STRAIN RESOURCE INST CHINESE ACAD OF TROPICAL AGRI SCI
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