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Primer probe combination for identifying four components of canine animal origin, kit and multiple real-time fluorescence PCR (polymerase chain reaction) detection method

A technology of animal-derived ingredients and compositions, applied in the field of molecular biology, can solve problems such as inaccurate test results, achieve stable and easy operation, reduce the risk of interference, and achieve good accuracy

Inactive Publication Date: 2015-09-23
BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In addition, the current fluorescent quantitative PCR detection research of animal origin in feed and food lacks the positive amplification internal standard (IAC) to monitor the reaction system, which cannot avoid the generation of false negative results caused by reaction inhibitory factors, resulting in the detection results Inaccurate
At present, in the world, the multiplex fluorescent quantitative PCR detection method of animal origin in feed and food added with exogenous amplified internal standard is still blank.

Method used

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  • Primer probe combination for identifying four components of canine animal origin, kit and multiple real-time fluorescence PCR (polymerase chain reaction) detection method
  • Primer probe combination for identifying four components of canine animal origin, kit and multiple real-time fluorescence PCR (polymerase chain reaction) detection method
  • Primer probe combination for identifying four components of canine animal origin, kit and multiple real-time fluorescence PCR (polymerase chain reaction) detection method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] 1. Universal primer design: Download the mitochondrial 16S rRNA gene sequences of fox, mink, raccoon dog and dog from the NCBI database, and use the homology analysis tool Mega5.0 software for homology comparison, and use the primer design software Primer 5.0 according to A pair of universal primers were designed for similar sequences at both ends of the screened core fragment.

[0066] 2. Design and construct a recombinant plasmid containing internal standard DNA: use DNA random generation software to generate a DNA sequence, there is no homologous DNA fragment after BLAST in NCBI, connect fox and mink to the upstream and downstream of this random DNA sequence The universal primer sequences derived from 4 kinds of canines, raccoon and dog, thus forming a 107bp internal standard DNA sequence. This internal standard sequence was entrusted with artificial gene synthesis, the synthetic fragment was connected to the PMD18-T vector, transformed into competent DH5a, and the p...

Embodiment 2

[0073] Embodiment 2 detection method

[0074]1. DNA extraction: Grind 50g of the sample to be tested and mix thoroughly, take 50mg for DNA extraction, use the animal tissue extraction kit to extract DNA, or use the classic hand-held method (refer to the DNA extraction method of animal tissue in the Molecular Cloning Manual). The Nanodrop nucleic acid detector detects the concentration and purity of the extracted DNA, the required concentration is 1-20ng / μL, and the OD value is between 1.7-1.8.

[0075] 2. Real-time fluorescent PCR amplification of the sample DNA to be tested

[0076] Prepare the 20 μL reaction system in the PCR reaction tube according to the following table 1, put the prepared PCR reaction tube into the fluorescence quantitative PCR instrument, and complete the PCR amplification according to the following reaction conditions: Amplification program: pre-denaturation at 95°C for 10 minutes; 95°C for 10s, 58°C for 35s, collect fluorescence signal here, 45 cycles...

Embodiment 3

[0095] Embodiment 3 specificity test

[0096] In order to verify the specificity of primer and probe of the present invention, carry out following experiment:

[0097] Respectively from fox meat, mink meat, raccoon dog meat, dog meat, raw beef, raw sheep meat, raw goat meat, raw pork, raw rabbit meat, raw duck meat, raw chicken meat, raw goose meat, raw fish meat, corn, wheat Genomic DNA was extracted and used as a DNA template to perform fluorescent quantitative PCR detection according to the detection method of the above-mentioned Example 2, so as to test the specificity of primers and probes.

[0098] The experimental results are shown in the following table 4. As can be seen from the data in the table, fox meat, mink meat, raccoon dog meat and dog meat are all effectively amplified, and the Ct value<35, while other samples are not amplified. It can be seen that the primers of the present invention And the probe has strong species specificity.

[0099] Table 4

[0100] ...

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Abstract

The invention discloses a primer probe combination for identifying four components of canine animal origin. The primer probe combination comprises a pair of universal primers suitable for foxes, minks, raccoon dogs and dogs, four peculiar probes for the foxes, the minks, the raccoon dogs and the dogs, an interior label and an interior label probe. The invention further discloses a kit and a multiple real-time fluorescence PCR (polymerase chain reaction) detection method. The universal primers and the peculiar probes are high in sensitivity and accuracy, false-negative results can be indicated effectively during origin detection of the foxes, the minks, the raccoon dogs and the dogs, the primer probe combination has multiple advantages of accuracy, stability, simplicity in operation, high sensitivity, specificity and flux and the like, and a new approach for identification of components of animal origin in feed and food is explored.

Description

technical field [0001] The present invention relates to a primer probe composition and a kit for identifying four canine-derived components in feed and food, and also relates to a method for detecting the four canine-derived components using multiple real-time fluorescent PCR detection methods , belonging to the technical field of molecular biology. Background technique [0002] Feed and food safety hazards are closely related to animal production, environmental pollution, and human health, and have become a hot issue of global concern. In 2014, according to statistics from the Ministry of Agriculture, in Hebei, Shandong and other places in northern my country, the annual breeding volume of foxes, minks and raccoon dogs was about 100 million. The skins of these animals are used as fabrics for expensive clothes because of their warmth retention properties, but the whereabouts of the meat of these animals is unknown. According to reports, these meats are purchased by illegal...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2537/143C12Q2561/101C12Q2561/113
Inventor 张全芳步迅范阳阳刘艳艳
Owner BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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