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Method for detecting ingredients of beef source by virtue of cross primers and dual-probe isothermal amplification

A constant temperature amplification detection and cross-primer technology, which is applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve the problems of slow detection speed and complicated detection methods, and achieve fast detection speed, Strong specificity and improved detection efficiency

Inactive Publication Date: 2015-03-04
CHINA JILIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Aiming at the problems existing in the current detection of animal-derived components, such as the need for specialized equipment and specialized technical personnel, complex detection methods, and slow detection speed, the purpose of the present invention is to provide cross-primers and double-probe constant temperature amplification nucleic acid sequences for detection of bovine-derived components. Component Primer Sets

Method used

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  • Method for detecting ingredients of beef source by virtue of cross primers and dual-probe isothermal amplification
  • Method for detecting ingredients of beef source by virtue of cross primers and dual-probe isothermal amplification
  • Method for detecting ingredients of beef source by virtue of cross primers and dual-probe isothermal amplification

Examples

Experimental program
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Effect test

Embodiment 1

[0055] Example 1 Determination of specific gene segments of beef source components

[0056] Select fresh beef samples, use the beef genomic DNA extracted from the animal tissue DNA kit (Hangzhou Xinjing Biological Reagent Development Co., Ltd., the same below) as the template, and use PCR to detect the beef mitochondrial ATP8 gene detection pair of forward and reverse primers (ie primers) BOS3F / 3R: SEQ ID No: 7 / 8)) PCR amplification, PCR reaction system (Taq enzyme 0.25μL, Taq Buffer 2μL, dNTPs 2μL, BOS3F 1μL, BOS3R 1μL, template 3μL, add double distilled water 10.75μL) , PCR products are detected by agarose gel electrophoresis (see figure 1 , Lane 1). The PCR product was sent to Shanghai Bioengineering Technology Co., Ltd. for sequencing verification, and the sequencing result was consistent with the expected 271bp sequence.

[0057] The genomic DNA of mouse, lamb, chicken, and duck was extracted with the animal tissue DNA kit as a template, and the BOS3F / 3R primers (SEQ ID No: 7...

Embodiment 2

[0058] Example 2 PCR amplification detection of beef mitochondrial ATP8 gene in different processed beef samples

[0059] Select samples of fresh beef, smoked beef, and beef tenderloin with peppers and grind them into beef floss. Take 0.25 mg of each sample; use the animal tissue DNA kit for DNA extraction; the PCR reaction system is the same as in Example 1 (Taq enzyme 0.25 μL , Taq Buffer 2μL, dNTPs 2μL, BOS 3F 1μL, BOS3R 1μL, template 3μL, add double distilled water 10.75μL), after PCR amplification, electrophoresis detection of different sources of beef samples obtained a consistent 271bp fragment (see figure 1 ). It shows that regardless of the processing method, the 271bp gene fragment of the beef mitochondrial ATP8 gene can be used for specific detection of beef.

Embodiment 3

[0060] Example 3 Design and screening of cross primers in the constant temperature amplification reaction system of the present invention

[0061] Experimental design: Refer to CPA reaction (Fang RD et al. Journal of C linical Microbiology, 2009, 47(3): 847; Xu GL et al. Scientific Reports, 2012, 2:246. doi:10.1038 / srep00246) and LAMP online software ( http: / / primerexplorer.jp / e / ) Design primer system, use oligo 7, primer 5 and other software to analyze primer parameters, a total of 7 sets of cross primers were designed in the PCR amplification 271bp sequence (6 of which were eliminated The primers are not listed), the target fragment is between 150bp-250bp. When designing the primers, it is required that the G and C bases in the primers are high; dimers should be avoided between the primers; the distance between the amplified fragments; Avoid forming a hairpin structure. Based on the above factors, we have screened for many times, and the seventh set of primers are cross primer...

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Abstract

The invention discloses a primer group for detecting ingredients of a beef source by virtue of cross primers and dual-probe isothermal amplification and belongs to the field of detection methods for food quality control. The primer group comprises a pair of peripheral primers, a pair of cross primers and a pair of detection probe primers and specially comprises nucleotide sequences as shown in SEQ ID No: 1-SEQ ID No: 6. The invention also discloses a detection kit containing the primer group and the method for detecting the ingredients of the beef source by virtue of the cross primers and dual-probe isothermal amplification. The primer group has high specificity and high sensitivity and can only be used to detect the ingredients of the beef source and has no cross-reactivity with genes of meats such as mutton, rat meat and chicken; secondly, the method has the advantages of high detection speed and high detection efficiency; no complex devices are needed, only water bath is needed and thus the method is convenient and practical; the detected results of amplification products are read by test strips and the method is especially suitable for base layer on-site detection.

Description

Technical field [0001] The invention belongs to the field of food quality control detection methods, and specifically relates to a primer set used for cross primer and dual probe constant temperature amplification and detection of beef source components, and a detection kit containing the primer set, and also relates to the use of cross primer and dual probe Constant temperature amplification method for detecting beef source components. Background technique [0002] Meat products are an important source of human protein. There are big differences in the quality and price of meat from different animal sources. Due to profit-driven, illegal merchants' meat adulteration occurs from time to time, which seriously damages the interests of consumers and also brings food safety problems. On-site testing of animal-derived ingredients is an effective way to solve the problem of meat adulteration market supervision. [0003] Beef is more expensive than rat and rabbit meat, and is one of the...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888
Inventor 李素芳杨韩潘家荣张雪妍冯涛朱月城董圣禄何巧
Owner CHINA JILIANG UNIV
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