Real-time fluorescent quantitative PCR detection method of porcine-derived components in meat or meat products

A real-time fluorescence quantitative and detection method technology, applied in the field of biotechnology detection, can solve problems such as reaction system errors, PCR instrument failures, false negatives, etc., and achieve the effects of ensuring accuracy, improving supervision levels, maintaining social stability and economic development

Inactive Publication Date: 2019-04-26
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In the process of meat product processing, various raw materials are added and processed by various methods. During these processes, many substances that inhibit the PCR reaction will be produced, resulting in false negatives.
In addition, due to the presence of inhibitors in the reaction system, PCR instrument failure, reaction system error, polymerase inactivation and other reasons will also cause false negative results

Method used

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  • Real-time fluorescent quantitative PCR detection method of porcine-derived components in meat or meat products
  • Real-time fluorescent quantitative PCR detection method of porcine-derived components in meat or meat products
  • Real-time fluorescent quantitative PCR detection method of porcine-derived components in meat or meat products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Extraction of Genomic DNA in Meat and Meat Products

[0030] Collect pork samples, beef samples, mutton samples, chicken samples, duck samples, goose samples, rabbit meat samples, donkey meat samples, horse meat samples, venison samples, yak meat samples, dog meat samples, fish meat samples with known components sample. Genomic DNA in meat and meat products was extracted according to the blood / cell / tissue genomic DNA extraction kit (Tiangen Biochemical Technology Co., Ltd.).

Embodiment 2

[0031] Embodiment 2: the selection of pig-specific nucleic acid sequence and primer, probe design

[0032] Through comparative analysis of the gene sequences of various animals and a large number of PCR test screening verification, the specific primers and probes for detecting pig-derived components are determined as: SEQ ID NO 1 5′-cacacaatgggaataaattg-3′; SEQ ID NO 2 5 '-gtcagtcatggttctcta-3'; SEQ ID NO 3 JOE 5'-ccttcaagcagtgcagccttac-3'BHQ1. The results are attached figure 1 As shown, the designed primers and probes have high specificity.

[0033] Also determine that the universal primers and probes for detecting vertebrate-derived components are: SEQ ID NO 5 5'-ctgctaaacaatccaataaac-3'; SEQ ID NO 6 5'-gaggtctccattactaataga-3'; SEQ ID NO 7CY5-5'-taacctcttgtctcttcggctgatg-3 '-BHQ2. The results are attached figure 2 As shown, the designed primers and probes have high specificity.

Embodiment 3

[0034] Embodiment 3: Construction of amplification internal standard plasmid

[0035] Select canine distemper virus nucleocapsid protein gene as target gene, design upstream primer: CDV-F 5′-cattgttacaagatctcgac-3′, downstream primer: CDV-R 5′-agttaatttagggccgtt-3′, the primer pair can amplify Canine distemper virus nucleocapsid protein gene 79bp fragment, and then add porcine-derived components real-time PCR detection primers SEQ ID NO 1 and SEQ ID NO 2 to the outside of CDV-F and CDV-R, respectively, to form primers SEQ ID NO 1 - CDV-F 5'-cacacaatgggaataaattgcattgttac-3' and SEQ ID NO 2 - CDV-R 5'-gtcagtcatggttctcta_agttaatttagggccg -3' primers. Using the 79bp PCR product as a template, using SEQ ID NO 1-CDV-F and SEQ ID NO2-CDV-R as primers, the length of the amplified PCR product is 104bp, and the PCR product is subjected to agarose gel electrophoresis, and ordinary agarose is used Gel DNA Recovery Kit recovers DNA fragments, connects the DNA fragments to pMD18-T Simple V...

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Abstract

The invention provides a real-time fluorescent detection method of porcine-derived components in meat and meat products. The method is capable of detecting fluorescent signals in an amplification process; electrophoresis used in an ordinary PCR detection method is not needed; the detection time is reduced; the detection results can be observed in real time; meanwhile, internal amplification control and positive control are added in the detection process, so that false negative detection results can be avoided; negative control and blank control are added, so that false positive detection results can be avoided; the accuracy of the detection results can be guaranteed from multiple aspects; the method has positive effects of improving animal-derived foodsafety supervision level, ensuring food quality safety, guaranteeing body health and living quality of broad masses of the people and maintaining social stability and economic development.

Description

technical field [0001] The invention belongs to the field of biotechnology detection, and relates to a detection method for animal-derived components in meat or meat products, in particular to a detection method for pig-derived components in meat or meat products. Background technique [0002] With the rapid development of my country's economy, the consumption of animal-derived commodities is increasing, and unscrupulous businesses often encounter fraudulent or even adulterated animal-derived raw materials in the process of food processing in pursuit of economic interests. Illegal conduct. Therefore, establishing a fast and accurate method for identifying animal-derived ingredients is of great importance for improving the safety supervision level of animal-derived food, ensuring food quality and safety, protecting the health and quality of life of the general public, and maintaining social stability and economic development. positive effects. [0003] Various raw materials ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6851
CPCC12Q1/6851C12Q1/6888C12Q2600/166C12Q2531/113C12Q2563/107C12Q2545/101
Inventor 姚大伟杨德吉周光宏孙昭宇徐幸莲叶可萍
Owner NANJING AGRICULTURAL UNIVERSITY
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