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Method for rapidly detecting African swine fever virus by using fluorescence in-situ detection technology

An African swine fever virus and detection technology, which is applied in the field of rapid detection of African swine fever virus by using fluorescence in situ detection technology, can solve the problem of no antiviral drugs and vaccines being developed, and reduce the experimental cost and operation difficulty. High degree and universal effect

Pending Publication Date: 2022-03-18
HUAZHONG AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The mortality rate of ASF can reach 100%, but so far no effective antiviral drugs and vaccines have been developed to prevent and control the disease. Therefore, rapid detection of ASFV infection has become a key measure to prevent, control and eliminate the disease

Method used

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  • Method for rapidly detecting African swine fever virus by using fluorescence in-situ detection technology
  • Method for rapidly detecting African swine fever virus by using fluorescence in-situ detection technology
  • Method for rapidly detecting African swine fever virus by using fluorescence in-situ detection technology

Examples

Experimental program
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Effect test

preparation example Construction

[0066] 1) Preparation of fluorescent probes:

[0067] i. Based on the whole genome of African swine fever virus, design n primer probes with a length of 120 to 150 bp, which is the primer probe pool; wherein, in the primer probe pool, the GC of each primer probe The content is 45-55% and the T content is less than 45-55%; n is 5000-9000;

[0068] ii. Add a pair of universal primers to the front and rear ends of each of the above primer probes to amplify and synthesize synthetic primer probes to form a synthetic primer probe pool containing n synthetic primer probes; wherein, the universal The primers are the T7 promoter sequence at the 5' end is 5'-TAATACGACTCACTATAGGG-3';

[0069] The reverse transcription primer sequence at the 3' end is 5'-GCGTGAATAGTCCGATCTGG-3'.

[0070] iii. Using the synthetic primer probe pool as a template, using T7-ASFV amplification primers to carry out PCR amplification to obtain ASFV genome fragments; wherein,

[0071] T7-ASFV-F: 5'-TAATACGACTC...

Embodiment 1

[0101] Utilize the method for rapid detection of African swine fever virus by fluorescence in situ detection technology, comprising the following steps:

[0102] 1) Preparation of fluorescent probes

[0103] i. Based on the whole genome of African swine fever virus, according to the design requirements: the GC content is 45-55% and the T content is less than 45-55%. 5000 primer probes with a length of 120-150bp are synthesized by the gene company to form primers probe pool;

[0104]ii. Add a pair of universal primers to the front and rear ends of each of the above primer probes to amplify and synthesize synthetic primer probes to form a synthetic primer probe pool containing 5000 synthetic primer probes; wherein, the universal The primers are the T7 promoter sequence at the 5' end: 5'-TAATACGACTCACTATAGGG-3' and the reverse transcription primer sequence at the 3' end: 5'-GCGTGAATAGTCCGATCTGG-3';

[0105] iii.PCR amplified DNA library utilizes T7-ASFV primer amplification to ...

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Abstract

The invention discloses a method for rapidly detecting African swine fever virus by using a fluorescence in-situ detection technology. The method comprises the following steps: preparing a fluorescent probe, collecting and fixing cells of a sample to be detected, and hybridizing and dyeing the fluorescent probe and the cells, observing the dyed hybrid cells, and identifying whether the sample to be detected is infected by the African swine fever virus; signals are amplified through a primer pool method, a signal point is composed of thousands of DNA fragments, compared with a traditional in-situ hybridization method, the signals are stronger and easy to capture, a super-resolution imaging system is not needed, a signal cluster can be analyzed through a common fluorescence microscope, and the experiment cost and the operation difficulty are reduced.

Description

technical field [0001] The invention relates to the application field of molecular biology technology, in particular to a method for rapidly detecting African swine fever virus using a fluorescent in-situ detection technology. Background technique [0002] The purpose of in situ hybridization is to determine the presence of target DNA or RNA sequences and to map these sequences to specific cellular or chromosomal loci. The combination of probes and target DNA sequences is used to identify specific sequences in cells. The ability to detect nucleic acids by in situ hybridization (ISH) was first reported in 1969. making this method an important tool in scientific and clinical research. In situ hybridization is a technique in which the complementary strand of a nucleotide probe is hybridized to a specific sequence. Depending on the probe used, this can be visualized with autoradiography, fluorescence microscopy or immunohistochemistry, respectively. The technology can locate...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6841C12Q1/6806C12Q1/02C12R1/93
CPCC12Q1/701C12Q1/6841C12Q1/6806G01N33/5005C12Q2563/107C12Q2565/601C12Q2565/40C12Q2525/143
Inventor 邢玲玉曹罡林达王金月肖琴
Owner HUAZHONG AGRI UNIV
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