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Primer pair for detecting AKV and application thereof

A technology of Akabane disease virus and primer pairs, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of low sensitivity, poor specificity, time-consuming and labor-intensive operation, and achieve high specificity Performance and stability, fast and accurate detection

Inactive Publication Date: 2014-05-28
江苏博爱生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Routine diagnostic methods are pathogen isolation and identification and serological methods, but these methods have limitations such as low sensitivity, poor specificity, and time-consuming and labor-intensive operations.

Method used

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  • Primer pair for detecting AKV and application thereof
  • Primer pair for detecting AKV 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 materials of Akabane disease were collected from internal organs of Henan cattle in October 2012. Take 0.5 g of visceral tissue in a sterile mortar, grind with saline, freeze and thaw 3 times, centrifuge at 5500 rpm for 3 min, and store the supernatant 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. AKV upstream primer P1 is: 5'-TAAGTATGGGCAGCAGCTCAAC-3';

[0023] Downstream primer P2 (5'-TCAGCAATCCAGCGAGCTAA-...

Embodiment 2

[0026] Embodiment 2: detection method

[0027] Follow the steps below to check:

[0028] (1) Sample processing: The clinical materials of Akabane disease were collected from the internal organs of Henan cattle in October 2012. 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) 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 , centrifuged at 12000r / min at 4°C for 15min, suspended in ultrapu...

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PUM

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Abstract

The invention discloses a primer pair for detecting AKV (Akabane virus). The nucleotide sequence of the primer pair is shown in SEQ ID NO.1 and SEQ ID NO.2, and the invention further discloses a detection kit including the primer pair and used for detecting AKV, and a detecting method thereof. The primer pair for detecting AKV has the advantages that: based on a specific primer pair, the PCR condition is optimized, the nucleotide sequence of AKV is amplified, the detection condition and system are optimized and the kit for detecting AKV is prepared; the kit is higher in specificity and stability, has the sensitivity up to 8 pg, and can be used for quickly and accurately detecting AKV.

Description

technical field [0001] The invention relates to the field of virus detection, in particular to a primer pair for detecting Akabane disease virus and its application. Background technique [0002] Akabane disease is an animal vector-borne infection caused by Akabane virus (AKV), which causes abortion, stillbirth, stillbirth, fetal malformation, and congenital arthrogryposis-hydrocephalus anencephaly in cattle, sheep and other animals. sick. In 1990, it was confirmed that the disease existed in our country. Akabane disease virus belongs to a subgroup of the serogroup of Simbu virus in the family Bunyaviridae (Bunyaviridae), and is a single-stranded negative-sense RNA virus. At present, the diagnosis of Akabane disease is mainly through virus isolation, agar immunodiffusion test (AGID), neutralization test and ELISA test and other methods. AKA has caused serious economic losses to animal husbandry, and is the key quarantine object in the prevention and control of animal dise...

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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