Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Fluorescent PCR (Polymerase Chain Reaction) detection kit for African swine fever virus, preparation method and application method

A technology of African swine fever virus and detection kit, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of large error in detection results, unsuitable early diagnosis, and susceptibility to environmental influences, etc., to reduce The effects of missed detection, good repeatability, excellent sensitivity and specificity

Active Publication Date: 2018-07-20
湖南国测生物科技有限公司 +1
View PDF11 Cites 36 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing detection methods have disadvantages such as complex operation, easy to be affected by the environment, easy to infect, unsuitable for early diagnosis, and large error in detection results.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Fluorescent PCR (Polymerase Chain Reaction) detection kit for African swine fever virus, preparation method and application method
  • Fluorescent PCR (Polymerase Chain Reaction) detection kit for African swine fever virus, preparation method and application method
  • Fluorescent PCR (Polymerase Chain Reaction) detection kit for African swine fever virus, preparation method and application method

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0072] The embodiment of the present invention also provides a preparation method of the above-mentioned African swine fever virus fluorescent PCR detection kit, comprising:

[0073] S110, select the sequence of the highly conserved region in the p72 gene of the ASFV genome, design multiple sets of primers and probes, respectively amplify the multiple sets of primers and probes, and screen the amplification efficiency, probe signal-to-noise ratio and amplification A group with the best curve shape as the primer and the probe of the ASFV-reaction solution;

[0074] S120, synthesize the recombinant cloning plasmid numbered pUC-p72, the recombinant cloning plasmid contains the target nucleotide sequence of the amplified product of the primer in the ASFV-reaction solution, and use a protein and nucleic acid analyzer to analyze the recombinant cloning plasmid The concentration is quantified, and the quantified recombinant cloning plasmid is diluted by TE buffer solution to obtain m...

Embodiment 1

[0100] The preparation method of embodiment 1 kit

[0101] Analyzed and compared the p72 gene sequence information of 23 ASFV strains registered in NCBI, and selected highly conserved region sequences to design three sets of specific primers and probe sequences. Please refer to Table 1 for the sequences and marker information of each set. see Figure 1 to Figure 3 Amplification tests were carried out on the above three sets of primers and probes respectively, and the indicators such as amplification efficiency, probe signal-to-noise ratio, and amplification curve shape of each group were obtained. The curve shape and sensitivity of the third group are the best, and there is no mismatch between the probe sequence and some current ASFV strain sequences, so it is selected as the best primer and probe set.

[0102] The cloned plasmid pUC-p72 was synthesized according to the amplified product of the target gene of the ASFV primer obtained by the above screening, and its sequence w...

Embodiment 2

[0129] The usage method of embodiment 2 kit

[0130] Sample collection: dissect sick pigs, collect lungs, spleen, liver, lymph nodes and other tissues; or collect blood and tonsils from living organisms.

[0131] Sample pretreatment: Serum and plasma samples do not need to be processed and can be directly used for detection; other tissue samples need to be processed, take about 1g and grind it in a grinding device, add 1.2mL normal saline to grind until homogenized, transfer to a 1.5mL centrifuge tube, and centrifuge at 8000g After 5 minutes, the supernatant was diluted 10 times with normal saline for detection.

[0132] Amplification reagent preparation: Take out all the components in the packaging box except the DNase mixture, place them at room temperature, shake and mix them after they are completely dissolved; 2μL / portion+ASFV-built-in reference 0.5µL / portion) Take the corresponding amount of reagents, mix well to form PCR-Mix, and centrifuge briefly.

[0133] Nucleic a...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a fluorescent PCR (Polymerase Chain Reaction) detection kit for African swine fever virus (ASFV). The fluorescent PCR detection kit comprises an ASFV-reaction solution, a DNA (Deoxyribonucleic Acid) enzyme mixed solution, an ASFV-internal standard, an ASFV-positive control, an ASFV-negative control, a nucleic acid releasing reagent and an ASFV-quantitative reference material, wherein the ASFV-reaction solution is prepared from primers: ASF03F03 and ASF03R03; internal standard primers: IPC05F01 and IPC05R01; a probe ASF03P and an internal standard probe IPC05P; the primers and the probes correspond to highly conserved fragments of an African swine fever virus p72 gene. The invention further discloses a method for preparing the fluorescent PCR detection kit for the African swine fever virus and a detection method utilizing the fluorescent PCR detection kit.

Description

technical field [0001] The invention relates to the technical field of molecular diagnosis of animal pathogens, in particular to a fluorescent PCR detection kit for African swine fever virus, a preparation method and a use method. Background technique [0002] African swine fever (ASF) is a highly contagious disease of pigs, with a fatality rate of up to 100%. It is caused and spread by African swine fever virus (ASFV). The disease develops very rapidly, and in less than a century, it has swept from Africa to almost the whole world. ASF is an animal disease that seriously endangers the "world economy", and it is also a class A animal disease stipulated by the World Organization for Animal Health (OIE). At present, although my country is not an ASF-infected area, the surrounding countries are constantly affected by the epidemic. Therefore, the prevention and control of ASF has been put on the urgent agenda by relevant departments. In April this year, the Ministry of Agricul...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12Q1/70C12Q1/6851
CPCC12Q1/6851C12Q1/701C12Q2531/113C12Q2563/107C12Q2545/101
Inventor 喻正军石建杨磊罗哲容杨忠苹李增强
Owner 湖南国测生物科技有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com