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Real-time fluorescent quantitative PCR primer probe set, kit and detection method for detecting African swine fever virus

A real-time fluorescent quantitative, African swine fever virus technology, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of pollution, uneven quality of testing personnel, etc., to achieve the effect of reducing the possibility

Pending Publication Date: 2021-12-17
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the diagnosis of African swine fever is not limited to professional laboratories, and the detection of the disease has sunk to grass-roots farms. The traditional single-factor detection can no longer meet the clinical needs, and a set of scientific and reasonable quality control system must be established to monitor whether the sample collection is standardized, whether the nucleic acid extraction and PCR amplification are successful, so as to maximize the accuracy of the detection

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  • Real-time fluorescent quantitative PCR primer probe set, kit and detection method for detecting African swine fever virus
  • Real-time fluorescent quantitative PCR primer probe set, kit and detection method for detecting African swine fever virus
  • Real-time fluorescent quantitative PCR primer probe set, kit and detection method for detecting African swine fever virus

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Effect test

Embodiment 1

[0036] Design and screening of embodiment 1 primers and probes

[0037] According to the ASFV B646L gene sequence published by GeneBank and the porcine RNA polymerase II (PRP II) gene sequence, multiple sets of primers and probes were designed in the conserved region of the gene through sequence comparison, and a set of target genes based on ASFV B646L was selected after optimization experiments. Combined with specific primers and Taqman probes of the porcine PRP II gene, the sequence is shown in the table below, synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0038] Table 1 Primer and Probe Sequences

[0039]

Embodiment 2

[0040] Embodiment 2 detects the kit composition and detection method of African swine fever virus

[0041] I. Kit composition

[0042] 1) Real-time fluorescent quantitative PCR reaction solution: it includes: 0.02U / μL Taq DNA polymerase, 0.2mM dNTP, 1×PCR buffer, 0.2μM sense primer, 0.2μM antisense primer and 0.2μM antisense primer for detecting African swine fever virus B646L gene 0.4 μM fluorescent probe, 0.2 μM sense primer, 0.2 μM antisense primer and 0.4 μM fluorescent probe for detecting porcine RNA polymerase II gene, 0.5×ROX reference dye, 0.005% sodium thimerosal;

[0043] Optionally, the primers and probes can be packaged separately and mixed with other components in the reaction solution when used, or directly mixed in the reaction solution according to the above dosage.

[0044] Further, the Taq DNA polymerase is a TaKaRa Ex Taq hot-start version (article number RR006), comprising PCR buffer and dNTP, and the fluorescent quantitative PCR reference dye is ROX Refer...

Embodiment 3

[0067] The establishment of embodiment 3 kit amplification standard curve

[0068] The concentration of the above-mentioned recombinant plasmid DNA (pUC57-B646L) and PK-15 cell genomic DNA measured, using the formula: (6.02×10 23 copy number / mole) x (concentration g / ml) x10 -3 / (MW g / mol)=copy / μL, calculate the copy number of pUC57-B646L plasmid and PK-15 cell genomic DNA respectively, make continuous 10-fold gradient dilution with PCR-grade water, take 5 μL each as a template, use this The kit performs fluorescent PCR reaction, establishes kinetic amplification curve and standard curve. Such as figure 1 , figure 2 with image 3 shown.

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Abstract

The invention discloses a real-time fluorescent quantitative PCR primer probe set, a kit and a detection method for detecting African swine fever virus, and relates to the technical field of virus detection. The real-time fluorescent quantitative PCR primer probe set comprises a primer and a probe for detecting an African swine fever virus B646L gene and a primer and a probe for detecting a swine RNA polymerase II gene. The invention also provides the real-time fluorescent quantitative PCR kit and the detection method for detecting the African swine fever virus. According to the kit, a fluorescent PCR technology is adopted, and quantitative detection of the African swine fever virus is achieved through a TaqMan probe marked by a fluorescent reporter group; in addition, a reaction system also comprises a primer for detecting a pig RNA polymerase II gene conserved region and a fluorescent probe marked by different fluorescent reporter groups, which are used as internal quality control and are used for monitoring whether sample collection is standard or not and whether nucleic acid extraction and PCR amplification are successful or not.

Description

technical field [0001] The invention relates to the technical field of virus detection, more specifically, to a real-time fluorescence quantitative PCR primer probe set, a kit and a detection method for detecting African swine fever virus. Background technique [0002] African swine fever (ASF) is an acute, hemorrhagic, severe infectious disease caused by African swine fever virus (ASFV) infecting domestic pigs and various wild boars (such as African wild boar, European wild boar, etc.). African swine fever was discovered in Kenya in 1921 and spread from Africa to Europe in the 1950s. After being eliminated in Europe in the 1990s, it spread again from East Africa to the Eastern European country Georgia in 2007. Since then, the disease has spread widely in Eastern Europe. It was introduced to the Irkutsk region of Russia in 2017. Currently, there are ASF distribution or outbreaks in more than 50 countries around the world. my country is the largest pig raising and pork cons...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6851C12Q2600/16C12Q2600/166C12Q2531/113C12Q2563/107C12Q2537/143C12Q2561/101
Inventor 余斌袁秀芳徐丽华李军星苏菲叶十一
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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