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Primer pair for detecting epizootic hemorrhagic disease viruses of deer and application thereof

A deer epidemic hemorrhage and primer pair technology, which is applied to the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of EHDV economic loss, export trade restrictions, etc., and achieve rapid and accurate detection. , high specificity and stability

Inactive Publication Date: 2014-05-14
江苏博爱生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no effective vaccine and treatment for this disease at present. It takes a lot of manpower and material resources to control and eliminate EHD, and the export trade is also limited, which brings great economic losses to the countries where EHDV is endemic.

Method used

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  • Primer pair for detecting epizootic hemorrhagic disease viruses of deer and application thereof
  • Primer pair for detecting epizootic hemorrhagic disease viruses of deer and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1: Preparation of the kit.

[0017] 1. Source and treatment of viruses and disease materials

[0018] The clinical data of deer epidemic hemorrhage were collected from the internal organs of Henan deer in October 2009. Take 0.6 g of visceral tissue in a sterile mortar, grind with saline, freeze and thaw 3 times, and then centrifuge at 6000 rpm for 4 min. Take the supernatant and store it at -20°C.

[0019] 2. Reagents

[0020] PCR pre-mix and proteinase K were purchased from Dalian Takara Company; DNA Marker and GoldView were purchased from Nanjing Shanggao Biological Company; primers were synthesized by Sigma Company. Other reagents are imported or domestic analytical reagents.

[0021] 1. Design and synthesis of primers

[0022] According to the full gene sequence of EHD in GenBank, a pair of detection primers was designed using the primer design software Primer5.0. EHD VP7 upstream primer P1 is: 5'-GGCAGCGCCAGAAGTTACC-3';

[0023] Downstream primer P2 (...

Embodiment 2

[0026] Embodiment 2: detection method

[0027] Follow the steps below to check:

[0028] (1) Sample processing: The clinical materials of deer epidemic hemorrhage were collected from the internal organs of Henan deer in October 2009. Take 0.6 g of visceral tissue in a sterile mortar, add physiological saline to grind, freeze and thaw 3 times, centrifuge at 6000 rpm for 4 min, and take the supernatant and store it at -20°C.

[0029] (2) Extraction of viral DNA: Freeze and thaw the tissue suspension of suspected disease material 3 times, centrifuge to obtain 450 μL of supernatant, add proteinase K to a final concentration of 500 μg / mL, add SDS to a final concentration of 10 g / L, and bathe in water at 55°C for 30 minutes. Extract once each with phenol, phenol-chloroform (volume ratio 1:1) mixture and chloroform, absorb the water phase, add 1 / 10 volume of 3mol / L NaAc (pH5.2) and 2.5 volumes of absolute ethanol For precipitation, centrifuge at 12000r / min at 4°C for 15min, suspend...

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Abstract

The invention discloses a primer pair for detecting epizootic hemorrhagic disease viruses of deer. Nucleotide sequences of the primer pair comprise SEQIDNO:1 and SEQIDNO:2. The invention further discloses a detection kit and method for the epizootic hemorrhagic disease viruses of the deer, and the detection kit includes the primer pair. According to the primer pair for detecting the epizootic hemorrhagic disease viruses of the deer, on basis of a specific primer pair, a PCR condition is optimized, the nucleotide sequences of the epizootic hemorrhagic disease viruses of the deer are expanded, detection conditions and a detection system are optimized, and the kit for detecting the viruses is manufactured. The kit has high specificity and stability, flexibility can be 6 pg, and the epizootic hemorrhagic disease viruses of the deer can be detected fast and accurately.

Description

technical field [0001] The invention relates to the field of virus detection, in particular to a primer pair for detecting deer epidemic hemorrhagic disease virus and its application. Background technique [0002] Epizootic haemorrhagic disease of deer (EHD) is a seasonal, non-contact viral infectious disease. Deer epidemic hemorrhagic disease virus (EHDV), also a member of the Orbivirus genus of the Reoviridae family, is a double-stranded RNA virus transmitted by arthropods, mainly Culicoides. Under natural conditions, deer epidemic hemorrhagic disease virus can cause cattle, sheep and other livestock as well as white-tailed deer, mink, eland antelope and other domesticated and wild ruminants infection. The clinical features were a transient rise in body temperature, symptoms of shock, mucosal and serosal hemorrhage, and death in a coma. Lesions showed extensive congestion, hemorrhage, severe edema, and vascular necrosis, and sometimes peritoneal edema. At present, eight...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/70C12Q1/686
Inventor 詹爱军
Owner 江苏博爱生物科技有限公司
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