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Kit for detecting wolf-derived components and application thereof

A technology of wolf-derived ingredients and kits, applied in the field of kits capable of detecting wolf-derived ingredients, can solve the problems of unreported methods for wolf-derived ingredients, and achieve market order maintenance, good specificity, and rapid detection methods simple effect

Active Publication Date: 2021-09-28
二连海关技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there have been many reports on the design of species-specific primers based on the differences in mitochondrial genomic DNA sequences, and the establishment of PCR and real-time fluorescent PCR methods for the detection of animal-derived components. The method of sexual composition has not been reported

Method used

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  • Kit for detecting wolf-derived components and application thereof
  • Kit for detecting wolf-derived components and application thereof
  • Kit for detecting wolf-derived components and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The design of embodiment 1 primer

[0039] Search for targets from nuclear genomic DNA, and search for sequence differences between dogs and wolves through repeated alignment and screening of wolf and dog nuclear genome sequences. Design primers by bacon designer 7.0 software, design wolf-specific upstream and downstream primers and Taqman probes, the lengths of the upstream and downstream primers are 19bp and 18bp nucleotides, and the probe length is 24bp nucleotides, and they are labeled at the 5' end On the FAM fluorescent group, the 3' end is labeled with the TAMRA quencher group, the sequence is:

[0040] Upstream primer: 5'-CCG TAC TCT AGC TTA CAT A-3'(SEQ ID NO.1)

[0041] Downstream primer: 5'-GAG ACC TTC CAG ATG AAA-3'(SEQ ID NO.2)

[0042] Fluorescent probe: 5'-FAM-TAC CTC ACA TTA GCC AAG AAG CCA-TAMRA-3' (SEQ ID NO.3).

Embodiment 2

[0043] Embodiment 2 Establishment of PCR detection method

[0044] 1. Extract sample DNA (CTAB method)

[0045] Take 0.5-1g sample and add 1.5-3.5mL CTAB extract, then add 12-20μL proteinase K (adjusted according to the amount of CTAB), make 2 tubes for each sample, and use blank water as the extraction control, mix well and incubate at 65°C for at least 1h (overnight incubation is best), if the sample swells, add more CATB, 1mL more each time, up to 10mL; centrifuge at 4000-5000rpm for 10min; add the supernatant (about 1mL) to a new test tube (2mL tube), add 700μL Chloroform, vortex for 30s, then centrifuge at 12000rpm for 10min (take 2 tubes each); take 600-650μL of the supernatant and add it to a new test tube, add 2 times the volume of CTAB precipitation solution, vortex to mix, and place at room temperature for 1h; centrifuge at 12000rpm for 10min , Remove the supernatant, dissolve the precipitate in 350 μL of 1.2mol / L NaCl solution, (2 tubes combined to share 350 μL sol...

Embodiment 3

[0053] Embodiment 3 Establishment of Fluorescent Quantitative PCR Detection Method

[0054] 1. Extract sample DNA with reference to the method in Example 2.

[0055] 2. Using the primers and probes of Example 1, the above-mentioned DNA is used as a template, and the reaction system of fluorescent quantitative PCR is shown in Table 2.

[0056] Table 2

[0057] Element concentration volume 2×Premix Ex Taq TM master mix

-- 12.5μL upstream primer 10mmol / L 1μL downstream primer 10mmol / L 1μL probe 10mmol / L 1μL template 1μL wxya 2 o

-- Make up to 25μL

[0058] The reaction conditions are: pre-denaturation: 94°C for 3min

[0059] Denaturation: 94°C 10s

[0060] Annealing extension: 60°C for 10s, read the absorbance value.

[0061] A total of 35 cycles.

[0062] After the amplification was completed, the same threshold was taken to analyze the data after deducting the background fluorescence signal, and ...

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Abstract

The invention provides a kit for detecting wolf-derived components and application of the kit, and belongs to the technical field of food and feed detection or species identification. The kit provided by the invention adopts primers and / or fluorescent probes to carry out PCR detection or fluorescent quantitative PCR detection on a to-be-detected sample, and whether the food contains wolf-derived components is judged according to whether specific fragments are amplified or not or according to a Ct value. The kit has the advantages of being accurate in detection, high in sensitivity, high in specificity, simple, convenient and rapid, the lowest detection limit is 15 micrograms per milliliter, the wolf-derived components in a sample to be detected can be accurately discriminated, adulteration and smuggling of meat products are effectively attacked, the market order is maintained, wild animal resources are protected, and powerful technical guarantee is provided.

Description

technical field [0001] The invention relates to the technical field of food and feed detection or species identification, in particular to a kit capable of detecting wolf-derived components and its application. Background technique [0002] Prairie wolves are national second-class protected wild animals. Prairie wolves are prone to carry viruses such as foot-and-mouth disease and Peste des petits ruminants. The entry of wolf-derived products increases the risk of disease transmission and threatens public health security at all times. It is necessary to establish a rapid and accurate detection method for coyote-derived components, accurately identify animal-derived components in imported and exported animals and their products, ensure the protection of wild animals and migratory wild animals in the world, maintain the public biosecurity system, and start from the source Crack down on smuggling and trafficking. The identification technology of animal-derived ingredients is a...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/686C12Q1/6851C12N15/11
CPCC12Q1/6888C12Q1/686C12Q1/6851C12Q2600/166C12Q2565/125C12Q2545/113C12Q2531/113C12Q2563/107
Inventor 王伊琴陈少博杨帆包勇敢荆文魁张志峰常鸿
Owner 二连海关技术中心
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