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RDA method and kit for rapidly detecting canine coronaviruses (CCV)

A coronavirus and kit technology, applied in the field of molecular biology, can solve problems such as cost and application scope limitations, and achieve the effects of low detection cost, high-precision rapid molecular detection, and wide applicability

Pending Publication Date: 2021-02-02
GUANGZHOU PLUSLIFE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The detection of molecular biology is mostly based on PCR. The detection needs to rely on PCR machine or expensive real-time quantitative PCR machine, and other supporting equipment. It needs to be equipped with special PCR laboratory and professional operators. The cost and application range are limited limit

Method used

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  • RDA method and kit for rapidly detecting canine coronaviruses (CCV)
  • RDA method and kit for rapidly detecting canine coronaviruses (CCV)
  • RDA method and kit for rapidly detecting canine coronaviruses (CCV)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1 A canine coronavirus (CCV) RDA fluorescence detection kit

[0082] (1) Acquisition of recombinant enzyme KX and KY proteins

[0083] The reported recombinant enzyme UvsX has poor stability and is difficult to mass produce and store for a long time. In order to solve this problem, the research and development team used bioinformatics methods to analyze and simulate large quantities of protein structures, and finally found a new recombinant enzyme Enzyme KX and its accessory protein KY.

[0084]In this example, the R&D team extracted the key functional site information in the recombinase structure, such as DNA binding site, ATP hydrolysis site, etc., and mapped it to the three-dimensional structure of the protein to obtain secondary structure information and tertiary structure information , by integrating the functional residues of the primary structure sequence, secondary structure features and tertiary structure spatial distance, a data model for protein stru...

Embodiment 2

[0136] Example 2 RDA fluorescence method detection reagent sensitivity test

[0137] The positive control is the pUC57 plasmid containing the S gene of canine coronavirus (CCV), and the negative control is the empty vector pUC57 plasmid.

[0138] The specific operation is as follows:

[0139] Step 1: Dilute the positive control plasmid to 10^4c, and then dilute to 10^3c, 10^2c, and 10^1c by 10-fold serial dilution.

[0140] Step two, sample processing. Take 5 μL of each concentration of the plasmid in step 1 in an EP tube, and take 5 μL of the negative control in another EP tube, add 20 μL of Buffer A respectively, shake and mix, and let stand at room temperature for 10-15 minutes;

[0141] Step 3, system preparation and testing. Add 25 μL of Buffer B to each tube, shake and mix, add 50 μL of the mixed solution to the RDA fluorescence reaction module, cover the tube cap, shake and centrifuge, and detect immediately; the reaction program is: 39°C for 1 minute, 30 cycles, col...

Embodiment 3

[0153] Example 3 RDA fluorescence method detection reagent specificity test

[0154] 1 case of canine parvovirus (CPV), 1 case of canine distemper virus (CDV), 3 cases of canine coronavirus (CCV), and 1 case of canine parainfluenza virus were clinically collected. A total of 6 cases of 4 types were verified by fluorescent quantitative PCR. The corresponding pathogen-positive samples were tested to test the specificity of the kit.

[0155] The specific operation is as follows:

[0156] Step 1. Sample processing. Take 5 μL of each of the above 6 positive samples in EP tubes, and at the same time take 5 μL of the positive control and negative control of the kit in a new EP tube, add 20 μL Buffer A respectively, shake and mix, and let stand at room temperature for 10-15 minutes;

[0157] Step 3, system preparation and testing. Add 25 μL of Buffer B to each tube, shake and mix, add 50 μL of the mixed solution to the RDA fluorescence reaction module, cover the tube cap, shake and...

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Abstract

The invention discloses an RDA method and kit for rapidly detecting canine coronaviruses (CCV). The kit comprises a specific primer pair and an RDA fluorescence labeling probe, so as to realize safe,specific, sensitive, simple and convenient detection of the canine coronaviruses (CCV) and overcome the defects of the existing traditional detection technology. According to the kit provided by the invention, a nucleic acid extraction step can be omitted, the detection of the canine coronaviruses (CCV) is realized under a constant temperature condition within 20 minutes, and the specificity is 100%. Compared with a common PCR method, the RDA fluorescence method has the advantages of reaction at constant temperature, no need of temperature change and complex instruments, shorter reaction timeand suitability for on-site rapid detection. The method and the kit provided by the invention have the characteristics of simple and rapid operation, good specificity, high sensitivity, low cost and the like, can provide an effective technical means for on-site rapid detection and screening of the canine coronaviruses (CCV), and have a wide application prospect.

Description

technical field [0001] The invention belongs to the technical field of molecular biology. More specifically, it relates to a primer pair, probe and related kit for detecting canine coronavirus (canine coronavirus, CCV) nucleic acid based on RDA fluorescence detection technology. Background technique [0002] Canine coronavirus (CCV) is one of the important pathogens causing acute gastroenteritis in dogs. The disease is distributed worldwide, and it is popular in many areas of my country, causing serious losses to the dog industry. In recent years, a large number of researches on the molecular biology of CCV have been carried out abroad, and its protein composition and genome structure have been discussed in detail. In order to understand the molecular epidemiological characteristics of CCV in my country, clarify its genetic characteristics, and provide theoretical basis and molecular biology detection methods for the prevention and treatment of this disease, the present in...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2600/166C12Q2531/119C12Q2521/107C12Q2521/507C12Q2522/101C12Q2563/107C12Q2545/113C12Q2537/1376Y02A50/30
Inventor 季宇文荻琛刘华勇陈翀
Owner GUANGZHOU PLUSLIFE BIOTECH CO LTD
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