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Dual-fluorescence PCR detection reagent, kit and detection method for classical swine fever virus and African swine fever virus

A technology of African swine fever virus and detection kit, which is applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., to achieve rapid diagnosis, good versatility, and high sensitivity

Pending Publication Date: 2020-02-07
HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] At present, the fluorescent RT-PCR and fluorescent PCR methods and patents for detecting single swine fever virus and African swine fever virus have been reported, and the double fluorescent PCR detection reagent capable of simultaneously detecting swine fever virus and African swine fever virus and its preparation method and application , also rarely reported

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  • Dual-fluorescence PCR detection reagent, kit and detection method for classical swine fever virus and African swine fever virus
  • Dual-fluorescence PCR detection reagent, kit and detection method for classical swine fever virus and African swine fever virus
  • Dual-fluorescence PCR detection reagent, kit and detection method for classical swine fever virus and African swine fever virus

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Embodiment 1

[0047] Development of a dual fluorescent PCR assay for the detection and identification of classical swine fever virus and African swine fever virus.

[0048] The first step is the design and synthesis of swine fever virus and African swine fever virus primers and probes:

[0049] On the basis of NCBI sequence comparison analysis, for the 5'-UTR gene of classical swine fever virus and the P72 gene of African swine fever virus, primers and probes that can cover all strains and are suitable for double detection were designed and screened, including two For specific primers and two specific probes, the lengths of the amplified target fragments are 94bp and 249bp respectively, and the sequences of the primers and probes are as follows:

[0050] Swine Fever Virus:

[0051] Upstream primer: F: 5'-TGCCCAYAGTAGGACTAG-3'SEQ ID NO.1

[0052] Downstream primer: R: 5'-CTACTGACGACTGTCCTG-3'SEQ ID NO.2

[0053] Probe: P: 5'-TGGCGAGCTCCCTGGGTGGTCTA-3'SEQ ID NO.3

[0054] The probe is lab...

Embodiment 2

[0089] Application of a dual fluorescent PCR assay for the detection and identification of classical swine fever virus and African swine fever virus.

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Abstract

The invention discloses a dual fluorescence PCR detection reagent, a kit and a detection method for swine fever virus and African swine fever virus, and belongs to the field of animal pathogen detection. Aiming at a 5'-UTR gene of the swine fever virus and a P72 gene of the African swine fever virus, primers and probes capable of covering all strains and suitable for double detection are separately designed and screened, including two pairs of specific primers and two specific probes; the invention also describes a kit containing the primers and the probes and a PCR detection method using theprimers and the probes; the invention, through the design of the primers and the adjustment of each component of PCR, realizes purposes of one-time analysis and simultaneous detection and differentiation of the swine fever virus and the African swine fever virus on the premise of no reduction in sensitivity and specificity, which not only reduces the workload and cost of detection, but also greatly saves the detection time, thus gaining valuable time for epidemic disease prevention and treatment.

Description

technical field [0001] The invention belongs to the field of detection of animal pathogens, and in particular relates to a double fluorescent PCR detection reagent, kit and detection method of swine fever virus and African swine fever virus. Background technique [0002] Classical Swine Fever Virus (CSFV) belongs to Flaviviridae, Pestivirus genus, is a single-stranded positive-sense RNA virus, is the pathogen of swine fever, harms domestic pigs and wild boars, and other animals do not get sick. Swine fever is an acute, febrile, highly contagious infectious disease characterized by high temperature and microvascular degeneration that can cause systemic hemorrhage, necrosis, and infarction. Swine fever is extremely harmful to pigs and will cause heavy losses in the pig industry. African swine fever (ASF) is a severe infectious disease of warthogs, porcupines, wild boars and domestic pigs caused by African swine fever virus (ASFV). The virus belongs to the African swine fever...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2600/166C12Q2537/143C12Q2563/107C12Q2545/113
Inventor 王金涛孙东波宋伟红郑家三李春秋邢晓旭陈国福刘光前师庆伟佟玲刁彩霞
Owner HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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