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RPA primer pair, probe, kit and detection method for detection of canine adenoviruses

A technology of canine adenovirus and primer pair, which is applied in the field of molecular biology detection, to achieve the effect of high efficiency amplification, short time consumption and high sensitivity

Active Publication Date: 2020-06-26
GUANGDONG LAB ANIMALS MONITORING INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no report on the detection of CAV by real-time fluorescent RPA at home and abroad, so it is necessary to establish a rapid and accurate RPA method for the detection of canine adenovirus CAV

Method used

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  • RPA primer pair, probe, kit and detection method for detection of canine adenoviruses
  • RPA primer pair, probe, kit and detection method for detection of canine adenoviruses
  • RPA primer pair, probe, kit and detection method for detection of canine adenoviruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1. Design and screening of RPA primer pairs and probes for detecting canine adenovirus

[0036] 1. Extraction of viral DNA

[0037] Follow the instructions of TIANGEN Company TIANamp Genomic DNAKit Blood / Cell / Tissue Genomic DNA Extraction Kit for DNA extraction from CAV. Add 200 μL of virus solution to a 1.5 mL centrifuge tube, add 20 μL of proteinase K and 200 μL of buffer GB, shake the mixture and mix. After 15 minutes in a 56°C water bath, add 200 μL of absolute ethanol, transfer to a tube, and centrifuge at 8000 rpm for 1 minute. Wash the washing solution twice, then add TE, leave it at room temperature for 5 minutes and centrifuge to obtain DNA.

[0038] 2. Cloning of the target gene

[0039] The PCR method was used to amplify the E3 gene. The amplification system was 95°C / 15min pre-denaturation, 94°C / 30Sec, 55°C / 30Sec and 72°C / 90Sec 35 cycles, 72°C extension for 10 minutes. The PCR target product was recovered by agarose gel electrophoresis, according to OMEGA ...

Embodiment 2

[0049] Example 2. Establishment of RPA method for detecting canine adenovirus

[0050] Use TwistAmp TM The kit (TwistDX, Cambridge, United Kingdom) performed the RPA reaction in a volume of 50 uL. Including 420nM forward primer, 420nM reverse primer, 120nM fluorescent probe, 1uL viral DNA and 280mM magnesium ion. The RPA reaction was amplified in a real-time thermostatic fluorescence detector (DEAOU Biotechnology, China) at 39°C for 30 minutes.

[0051] From figure 1 It can be seen that the RPA method of the invention has a good amplification effect.

Embodiment 3

[0052] Example 3. Specificity test and sensitivity test of RPA method for detecting canine adenovirus of the present invention

[0053] 3.1 Method

[0054] Specificity test: Determine the specificity of the constructed real-time RPA method by testing CAV containing positive nucleic acids and other nucleic acids from other pathogens, including canine coronavirus CCV, canine parvovirus CPV, and canine distemper virus CDV. Sensitivity test: use plasmid diluted standard, use easy dilution to dilute to 4 concentrations by 10 times, the template is 1*10 5 copies / μL to 1*10 2 Copies / μL for sensitivity analysis of the method.

[0055] 3.2 Results

[0056] Detect the specificity of the constructed real-time RPA method by detecting CAV positive nucleic acid and other pathogenic viruses (CDV, CCV, CPV), ddH 2 O is a blank control. From figure 2 It was shown that, except for CAV amplification, other negative nucleic acids were not amplified, proving that the method has good specificity and does...

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Abstract

The invention discloses a RPA primer pair, probe, kit and detection method for detection of canine adenoviruses, and belongs to the technical field of molecular biological detection. The real-time fluorescence RPA detection method for detection of canine adenoviruses has high amplification specificity and high sensitivity, and the sensitivity can reach 10<2> copies. The primer pair and probe are obtained through design and screening of gene sequences in E3 of the canine adenoviruses (CAV), so that amplification is more efficient, and the specificity is higher. The RPA method is simple, has lowrequirements for temperature and machines and a short consumption time, and is suitable for rapid detection in the laboratory or on-site detection, and a technical reference is provided for rapid detection, diagnosis and prevention of the canine adenoviruses.

Description

Technical field [0001] The invention relates to an RPA primer pair, probe, kit and detection method for detecting canine adenovirus, and belongs to the technical field of molecular biology detection. Background technique [0002] Canine adenovirus can be divided into Canine adenovirus type 1 (CAV-1) and Canine adenovirus type 2 (CAV-2). Canine adenovirus type 1 is also known as infectious hepatitis, fox Encephalitis virus and Robus disease virus are acute septic infectious diseases caused by canine adenovirus type 1 in dogs and other animals. Occurs mainly in dogs, but can also be seen in other canines, weasels, and bears. Hepatitis and circulatory disorders are mainly manifested in dogs, and encephalitis in foxes and bears. Canine adenoviruses belong to the Adenoviridae family (Adenoviridae) and Mastadenovirus in classification. The incubation period of natural canine adenovirus infection is 6-9 days. In the most acute case, death occurs within a few hours after symptoms such...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2521/507C12Q2561/113C12Q2531/119C12Q2522/101C12Q2563/107Y02A50/30
Inventor 练月晓丛锋黄韧朱才毅
Owner GUANGDONG LAB ANIMALS MONITORING INST
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