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RPA primer for detecting duck-derived component, kit and detection method

A kit and duck-derived technology, applied in the field of RPA primers for the detection of duck-derived components, can solve the problems of not being able to further satisfy the rapid detection of livestock products, achieve accurate and reliable detection results, reduce the formation of primer-dimers, and shorten the experiment The effect of the process

Inactive Publication Date: 2017-07-21
上海瑞丰农业科技有限公司 +1
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, among the reported detection methods of duck-derived components, the PCR instrument is mainly used for routine detection in the laboratory, and these methods cannot further meet the rapid detection of livestock products.
At present, there is no species-specific identification of duck-derived components using RPA technology at home and abroad

Method used

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  • RPA primer for detecting duck-derived component, kit and detection method
  • RPA primer for detecting duck-derived component, kit and detection method

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1 Obtaining RPA primers for detecting duck-derived components

[0036]By consulting the literature and analyzing with BLAST software, 50ng / μL duck total DNA was used as a template for optimal screening, and the gene nucleic acid sequence on duck mitochondria was screened out, and primers were designed and screened.

[0037] Consider the following points when designing RPA primers: (1) The length of RPA primers is generally 30 to 35 nucleotides; (2) The 3-5 nucleotides at the 5' end of RPA primers should avoid polyguanine, The GC content is between 40% and 60%, the bases are randomly distributed, and repeated sequences are avoided; (3) When designing RPA primers, try to avoid primer-primer interaction, secondary structure, and hairpin structure in the primer to reduce primer dimerization body formation.

[0038] The specific sequences of the screened RPA primers are as follows:

[0039] RPA-duck-F: 5'-TGAATCCTGTTGCCGGTCTTGCGATGATTATC-3';

[0040] RPA-duck-R: 5...

Embodiment 2

[0041] Example 2 Verification of an RPA detection method for detecting species specificity of duck-derived components

[0042] Taking common pigs, cattle, sheep, chickens and ducks as objects, the amplification reaction was carried out, and the test strips were used for detection, and the electrophoresis method was used as a comparison for verification.

[0043] 1) Extract the DNA of the samples to be tested (pigs, cattle, sheep, chickens and ducks);

[0044] RPA amplification

[0045] The RPA primers in Example 1 were subjected to polymerase recombinase isothermal amplification using the RPA kit developed by TwistDx Company.

[0046] Amplification reaction system: the total volume is 50 μl, add 2.4 μl of the forward and reverse primers of the RPA primer with a concentration of 10 μmol / L, add 2.0 μl of the DNA template with a concentration of 20 ng / μl, boil and then cool pure water to prepare phosphate buffer Solution (0.2M, PH7.05) 29.5μl, 250mmol / L magnesium phosphate solu...

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Abstract

The invention discloses an RPA primer for detecting a duck-derived component, a kit and a detection method. The RPA primer comprises a forward primer RPA-duck-F and a backward primer RPA-duck-R, and the forward primer RPA-duck-F and the backward primer RPA-duck-R respectively have the specific sequences as follows: 5'-TGAATCCTGTTGCCGGTCTTGCGATGATTATC-3' and 5'-TCTATCGCGTATTAAATGTATAATTGCGGGAC-3'. By the use of the RPA primer, whether a detected product contains the duck-derived component can be accurately, quickly and easily detected, and the RPA primer has the characteristics of high sensitivity, high amplification speed and high detection result intuitiveness and can realize quick and simple detection for animal-origin meat and meat products at normal temperature and meet the requirements of the government for market supervision and routine monitoring.

Description

technical field [0001] The invention belongs to the field of livestock product safety biotechnology, and in particular relates to an RPA primer, a kit and a detection method for detecting duck-derived components. Background technique [0002] Food is the first priority for the people, and safety is the first priority. Food is the first material basis for the survival of human society. With the improvement of people's living standards, the consumption of animal-derived food is increasing year by year. Foodborne diseases and the potential danger of meat products have not been effectively controlled in both developed and developing countries, and the quality and safety of livestock and poultry meat products have become a global problem. [0003] Species identification, species origin and diversity assessment of animals at the nucleic acid molecular level, as well as inspection and research on animal-derived foods, are currently the most effective means for animal-derived compo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/6888C12Q2521/507C12Q2521/101
Inventor 王金斌唐雪明王荣谈李文孙斌白蓝蒋玮武国干吴潇吕贝贝潘爱虎李鹏贾军伟
Owner 上海瑞丰农业科技有限公司
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