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RPA (recombinase polymerase amplification) primer for detecting fowl adenovirus serotype 4 and detection method of RPA primer

A poultry adenovirus and serum technology, applied in the field of molecular biology detection, can solve problems such as complicated operation, inability to guide epidemic diseases, and long time-consuming, and achieve the effects of accurate detection results, high sensitivity, and low cost

Active Publication Date: 2017-11-24
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The existing methods for detecting FAdV-4 are complex and time-consuming, and cannot provide timely guidance for clinical control of diseases caused by FAdV-4. Therefore, it is urgent to find a rapid on-site detection and diagnosis technology for FAdV-4

Method used

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  • RPA (recombinase polymerase amplification) primer for detecting fowl adenovirus serotype 4 and detection method of RPA primer
  • RPA (recombinase polymerase amplification) primer for detecting fowl adenovirus serotype 4 and detection method of RPA primer
  • RPA (recombinase polymerase amplification) primer for detecting fowl adenovirus serotype 4 and detection method of RPA primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: The synthesis of the primer that is used to detect poultry adenovirus serotype 4

[0038] According to the FAdV-4 gene sequence comparison results published in GenBank, the sequence at the 2355bp-2390bp position in the FAdV-4 genome nucleotide sequence (5'-FITC-TTACACCCCCTACCACGGACAACCAGCGGACAGCAGCC-3') was selected as the upstream primer for the RPA amplification reaction , when synthesizing the upstream primer, label fluorescein isothiocyanate (FITC) at its 5' end; use the reverse complementary sequence of the 2620bp-2652bp position sequence in the FAdV-4 genome nucleotide sequence as the downstream primer of the RPA amplification reaction, synthesize The downstream primer is labeled with biotin (Biotin) at its 5' end: (5'-Biotin-GATCTGTCAGTTCGCCCATGTACATAAAGTCGG-3').

[0039] Primer synthesis was completed by Sangon Bioengineering (Shanghai) Co., Ltd.

Embodiment 2

[0040] Embodiment 2: the preparation of colloidal gold lateral flow immunochromatography test strip

[0041] (1) Preparation of streptavidin-coated gold nanoparticles: Take 1 ml of gold nanoparticle solution (0.15 pmol / mL), add 200 mM borax solution, and adjust the pH value to 6.5. At the same time, in another test tube, 2 μl of streptavidin (2 mg / ml) was mixed with 398 μl of borax solution (2 mM); The amount is gradually added, stirring while adding. Place the mixture at room temperature for 45 minutes, add 155.6 μl of 2 mM borax solution containing 10% BSA, continue to place the solution at room temperature for 10 minutes, centrifuge at 4500 g for 15 minutes, discard the liquid, and wash with 1 ml of washing solution (containing 10 g / L BSA 2mM borax solution) to resuspend the precipitate, centrifuge at 4500g for 15 minutes, aspirate and discard the liquid, and resuspend the red precipitate in 250 μl buffer solution containing 5% BSA, 137mM NaCl and 0.025% Tween-20. Accordi...

Embodiment 3

[0043] Example 3: DNA Extraction

[0044] Extraction of avian adenovirus serotype 4 DNA: Total DNA was extracted using the TIANamp Genomic DNA Kit kit from TIANGEN Company, and the specific operations were as follows: ①Take 200 μl of FAdV-4 cytotoxicity in a 1.5ml eppendorf tube, add 20 μl of proteinase K (concentration 20mg / ml); ②Add 200μl of buffer GB to each tube, mix thoroughly by inversion, place at 70°C for 10min, centrifuge briefly after the solution becomes clear to remove water droplets in the tube; ③Add 200μl absolute ethanol, shake and mix well, briefly Centrifuge to remove water droplets in the tube; ④ Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3 (the adsorption column is placed in the collection tube), centrifuge at 12000rpm for 30s, pour off the waste liquid, and put the adsorption column CB3 Put it back into the collection tube; ⑤Add 500μl buffer GD to the adsorption column CB3, centrifuge at 12000rpm for ...

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Abstract

The invention discloses an RPA (recombinase polymerase amplification) primer for detecting a fowl adenovirus serotype 4. The nucleotide sequence of the RPA primer is as shown in SEQ ID No.1 and SEQ ID No.2 in the description. The invention further discloses a method for detecting the RPA of the fowl adenovirus serotype 4, and the method mainly comprises the steps of primer synthesis, extraction of DNA in a sample to be tested, RPA amplification reaction and analysis of an RPA amplification product. According to the RPA primer disclosed by the invention, the specificity is strong, the sensitivity is high, and the detection result is accurate. The detection method disclosed by the invention is simple in operation and good in stability, and a low-cost, rapid and specific on-site diagnosis method for effectively detecting and authenticating chicken cecum hepatitis-hydropericardium syndrome is provided.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, and in particular relates to an RPA primer for detecting poultry adenovirus serotype 4 and a detection method thereof. Background technique [0002] Chicken hepatitis-pericardial effusion syndrome caused by Fowl adenovirus serotype 4 (FAdV-4) is a highly contagious disease. The disease mainly occurs in broiler chickens aged 3-10 weeks and dies at the age of 4-5 weeks. In the peak period, the mortality rate can reach as high as 80%, which brings great economic losses to the poultry industry in my country. Rapid on-site diagnosis of the disease can buy valuable time for the effective prevention and control of the disease, and minimize losses and the risk of disease spread. [0003] At present, the methods for detecting poultry adenovirus mainly include virus isolation and identification, neutralization test, electron microscopy, agar diffusion test, ELISA, PCR and so on. Virus isolation ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6844C12Q1/701C12Q2521/507C12Q2522/101C12Q2527/125C12Q2531/119C12Q2565/625
Inventor 赵军王川庆万文妍刘延珂杨霞常洪涛王新卫陈陆高冬生王永生
Owner HENAN AGRICULTURAL UNIVERSITY
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