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Specific primers, probes, kits and detection methods for detecting duck-derived components in meat products

A meat product, duck source technology, applied in the field of food inspection and biological detection, can solve the problems of prone to false positives and low efficiency of ordinary PCR amplification

Active Publication Date: 2018-11-02
苗丽
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the amplification efficiency of ordinary PCR is low, qPCR needs to establish a standard curve and is prone to false positives and other factors, so its quantitative system needs to be further improved

Method used

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  • Specific primers, probes, kits and detection methods for detecting duck-derived components in meat products
  • Specific primers, probes, kits and detection methods for detecting duck-derived components in meat products
  • Specific primers, probes, kits and detection methods for detecting duck-derived components in meat products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Design and synthesis of primers and probes

[0048] A total of 17 sequences of β-actin single-copy genes published in GenBank were compared by the nucleic acid comparison software DNAMAN, including 5 duck-derived β-actin single-copy genes (the accession numbers in GenBank are respectively EF667345.1, NM_001310421.1, GU564232.1, AY251275.1, DQ675572.1), 1 goose-derived β-actin single-copy gene (accession number in GenBank is M26111.1), 3 chicken-derived β-actin Single copy gene (accession numbers in GenBank are L08165.1, NM_205518.1, JF436880.1), 2 bovine β-actin single copy genes (accession numbers in GenBank are AY141970.1, BT030480.1) , 3 sheep-derived β-actin single-copy genes (the accession numbers in GenBank are NM_001009784.1, U39357.1, AF035422.1), and 3 pig-derived β-actin single-copy genes (the accession numbers in GenBank are respectively DQ452569.1, DQ845171.1, U07786.1), screen out the specific sequence of duck β-actin single-copy gene (its nucle...

Embodiment 2

[0054] Embodiment 2: the extraction of sample DNA

[0055] (1) Sample pretreatment: cut meat or meat products into about 1cm 2 -2cm 2 Diced meat of different sizes, washed with distilled water, soaked in water at 10°C overnight to remove excess salt and grease, minced with a tissue homogenizer and stirred evenly, and then placed in an oven at 80°C-85°C to dry Dry for 72 hours, then put the dried meat into a pulverizer for pulverization and homogenization treatment to obtain a pretreated meat sample.

[0056] (2) DNA extraction: the genomic DNA of the sample was extracted by the phenol / chloroform method, and the specific steps were as follows:

[0057] Take 50 mg of the meat sample treated in step (1), add 700 μL of tissue lysate and 20 μL of proteinase K (20 mg / mL) to it, vortex and oscillate, bathe in 56°C water for 2 hours, add an equal amount of Tris to balance phenol, and turn it upside down Mix well, and centrifuge at 10000rpm for 10min; take the supernatant, add 1 / 2 v...

Embodiment 3

[0058]Example 3: Establishment of a microdroplet digital PCR method for detecting duck-derived components in meat products using specific primers and probes of the present invention

[0059] (1) Optimization of the droplet digital PCR amplification reaction system: After optimizing the concentration of primers, probes and templates, the size of the droplet digital PCR amplification reaction system of the present invention was finally determined to be 20 μL, and its specific configuration is shown in Table 1.

[0060] (2) The reaction program of the microdroplet digital PCR amplification reaction is as follows: the annealing temperature is optimized in the reaction program, wherein, the annealing temperature is from 55°C to 63°C, and a total of 9 gradients are set for optimization, and each gradient has a difference of 1°C; Optimizing, the reaction program of the microdroplet digital PCR amplification reaction of the present invention is finally determined as follows: pre-denatu...

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Abstract

The invention discloses a combination of a specific primer and a probe for detecting duck-origin components in meat products. The nucleotide sequences of the primer and the probe are respectively represented by SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3. The invention further provides a detection method and a detection kit for quantitatively detecting the content of the duck-origin components in meat products. The detection method and the detection kit have the advantages of accuracy in detection, high sensitivity, strong specificity, simplicity, rapidness and the like.

Description

technical field [0001] The invention relates to the technical fields of food inspection and biological detection, in particular to a specific primer, probe, kit and detection method for quantitative detection of duck-derived components in meat products. Background technique [0002] In recent years, the problem of meat adulteration in my country's meat product market has become increasingly serious. Driven by economic interests, some criminals have adulterated and faked meat in order to make huge profits, and sold relatively cheap duck and pork as beef and mutton after deep processing Among them, compared with pork, the texture and color of duck meat is the closest to that of beef and mutton. Therefore, in the commercially available beef and mutton and its products, many of them are mixed with duck meat to pretend to be beef and mutton for sale. Add mutton powder, mutton extract and other additives to whitewash its adulteration. The adulteration of meat products has seriously...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6851C12Q1/6888C12Q2563/159C12Q2545/114C12Q2563/107
Inventor 苗丽陈静张秀平杨娜李轲徐超王永杰白杰李志娟王珊
Owner 苗丽
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