Nano PCR method for detecting fowl adenovirus type 4, kit and application thereof

The technology of a poultry adenovirus and a kit, applied in the field of molecular biology, can solve the problems that PCR amplification efficiency, amplification sensitivity and specificity improvement effect need to be further improved, so as to make up for the lack of specificity of primers, improve heat conduction, improve The effect of PCR specificity

Active Publication Date: 2020-08-18
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing various nanoparticles have to be further improved on PCR amplification efficiency, amplification sensitivity and specificity.

Method used

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  • Nano PCR method for detecting fowl adenovirus type 4, kit and application thereof
  • Nano PCR method for detecting fowl adenovirus type 4, kit and application thereof
  • Nano PCR method for detecting fowl adenovirus type 4, kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1, Ag / Au / Fe3O4 preparation

[0024] The preparation process of Fe3O4: 4.64g FeCl3·6H2O and 1.71g FeSO4·7H2O were added to 250mL of deionized water, and then 2mL of 0.2mol / L sulfuric acid was added to prevent the oxidation of Fe2+ in the solution. Then add 25% ammonia water until the pH reaches 9.0-9.5, stir at room temperature for 30 min, after amination, heat to 80° C. and continue stirring for 30 min, form a black suspension, and then sonicate for 10 min. Separate with a magnet, collect the precipitate, and wash the precipitate repeatedly with hot water until the supernatant becomes clear. Fe3O4 nanoparticles were resuspended in 100 mL of deionized water and kept in a volumetric flask for the preparation of Ag / Au / Fe3O4 nanoparticles.

[0025] Dissolve 0.229g of sodium citrate in 100mL of deionized water, heat to 95°C under magnetic stirring, then add 50mL of Fe3O4 prepared above, then add 10mL of 10mmol / L tetrachloroauric acid and 10mL of 10mmol / L AgNO3, a...

Embodiment 2

[0026] Embodiment 2, establishment of nano-PCR method

[0027] 1. Preparation of PCR template

[0028] Take 200 μL of FAdV-4GX0625 (isolated and preserved by Veterinary Research Institute of Guangxi Zhuang Autonomous Region) chicken embryo culture supernatant and extract the DNA in the sample according to the operation manual of EasyPure Genomic DNA Kit (purchased from Beijing Quanshijin Biotechnology Co., Ltd.), Store at -70°C for later use.

[0029] 2. PCR primer design

[0030]FAdV-4 belongs to group I of avian adenoviruses and has a 12-sided symmetrical structure, and the size of virus particles is 70-90nm. FAdV-4 virions have 252 capsid particles, including 240 hexons (Hexon) and 12 pentons (apex capsid particles). Studies have shown that the Hexon gene is a specific antigenic determinant, which can trigger a strong neutralization reaction. When the hexon mutates, it will lead to the loss of neutralization immunity, and it can be used as the first protein for diagnosin...

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Abstract

The invention discloses a nano PCR method for detecting fowl adenovirus type 4 and a kit, Ag / Au / Fe3O4 nanoparticles and primers shown as SEQ ID No.1-2 are added into a reaction system of the nano PCR,and the reaction system and reaction conditions are optimized. By adopting the Ag / Au / Fe3O4 nano composite material, the amplification efficiency of PCR is improved, the sensitivity is improved by 10times, non-specific amplification is effectively inhibited, and the defect of insufficient primer specificity is overcome.

Description

technical field [0001] This application relates to the technical field of molecular biology, in particular, to a nano-PCR method, kit and application for detecting avian adenovirus type 4 Background technique [0002] Hepatitis-pericardial effusion syndrome is an infectious disease characterized by avian pericardial effusion, liver jaundice, swelling and hemorrhage caused by a new genotype of avian adenovirus type 4 (FAdV-4). Since 2013, the disease has been prevalent in Shandong, Henan, Hebei, Jiangsu, Shaanxi and other places in my country, causing huge economic losses to the poultry breeding industry. Currently reported FAdV-4 detection methods include virus isolation and identification, ELISA, virus neutralization test and so on. [0003] In the nano-PCR detection kits or reported nano-PCR detection methods currently on the market, the commonly used nano-materials are gold nanoparticles, and the conventional detection kits on the market are also used in patent applicati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2563/155Y02A50/30
Inventor 谢芝勋黄娇玲谢丽基谢志勤邓显文罗思思范晴曾婷婷张艳芳王盛张民秀李孟李小凤万丽军李丹韦悠阮志华
Owner GUANGXI VETERINARY RES INST
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