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A kind of African swine fever virus fluorescent PCR detection kit, preparation method and use method

An African swine fever virus, detection kit technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of large error in detection results, easy to be affected by the environment, unsuitable for early diagnosis, etc., to reduce The effect of missed detection, excellent sensitivity and specificity, and good reproducibility

Active Publication Date: 2020-05-29
湖南国测生物科技有限公司 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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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.

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  • A kind of African swine fever virus fluorescent PCR detection kit, preparation method and use method
  • A kind of African swine fever virus fluorescent PCR detection kit, preparation method and use method
  • A kind of African swine fever virus fluorescent PCR detection kit, preparation method and use method

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preparation example Construction

[0071] The embodiment of the present invention also provides a preparation method of the above-mentioned African swine fever virus fluorescent PCR detection kit, including:

[0072] 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 The curve shape is the best set as the primer and the probe of the ASFV-reaction solution;

[0073] S120, synthesize a recombinant cloning plasmid numbered pUC-p72, the recombinant cloning plasmid containing the target nucleotide sequence of the amplified product of the primer in the ASFV-reaction solution, and using a protein nucleic acid analyzer to analyze the recombinant cloning plasmid The concentration is quantified, and the quantified recombinant cloning plasmid is diluted with TE buffer solution to obtain multiple...

Embodiment 1

[0099] Example 1 Preparation method of kit

[0100] Analyze and compare the p72 gene sequence information of 23 ASFV genomes registered in NCBI, select the sequence of the highly conserved region to design three sets of specific primer and probe sequences. For the sequence and label information of each group, please refer to Table 1. See Figure 1 to Figure 3 , Perform amplification experiments on the above three sets of primers and probes to obtain the amplification efficiency, probe signal-to-noise ratio and amplification curve shape of each group. The curve shape and sensitivity of the third group are the best, and there is no mismatch between the probe sequence and the sequence of some current ASFV strains, so it is selected as the best primer and probe set.

[0101] 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 was GATGATGATTACCTTTGCTTTGAAGCCACGGGAGGAATATCAAC...

Embodiment 2

[0128] Example 2 Method of using the kit

[0129] Sample collection: dissecting sick pigs, collecting lung, spleen, liver, lymph nodes and other tissues; or collecting blood and tonsils in vivo.

[0130] Sample pretreatment: Serum and plasma samples do not need to be processed and used directly for testing; other tissue samples need to be processed, take about 1g and grind in a grinding tool, add 1.2mL saline to grind to homogenate, transfer to a 1.5mL centrifuge tube, and centrifuge at 8000g After 5 minutes, take the supernatant and dilute 10 times with normal saline and use it for detection.

[0131] Amplification reagent preparation: Take out the components except the DNA enzyme mixture in the packaging box and place them at room temperature. After they are completely dissolved, shake and mix well for later use; according to the proportion (ASFV-reaction solution 38μL / head + DNA enzyme mixture 2μL / head portion + ASFV-built-in reference 0.5μL / head portion) Take the corresponding a...

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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 infectious disease of swine, with a fatality rate of 100%. It is caused and spread by African swine fever virus (ASFV). The disease develops very rapidly, and within less than a century, it has spread from Africa to almost the entire world. ASF is an animal disease that seriously harms the "world economy", and it is also a Category A animal disease specified by the World Organization for Animal Health (OIE). Although my country is not an ASF-affected area at present, there are continuous epidemics in neighboring countries. Therefore, the prevention and control of ASF has been put on the urgent agenda by relevant departments. In April of this year, the Ministry of Agricultur...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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
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