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Method for quantitatively detecting quail-derived components based on ddPCR technology

A technology derived from quail, applied in the field of molecular biology analysis, can solve problems such as inaccurate quantification and regulatory obstacles, and achieve the effects of convenient and fast determination process, good specificity and sensitivity, good repeatability and stability

Active Publication Date: 2021-06-18
南京市食品药品监督检验院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current standard methods for identifying various animal-derived components are usually based on ordinary PCR technology or fluorescent PCR technology, which can only qualitatively detect the target animal-derived components in the sample, but cannot be accurately quantified. The limitations of this technology have implications for market supervision. formed a certain obstacle

Method used

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  • Method for quantitatively detecting quail-derived components based on ddPCR technology
  • Method for quantitatively detecting quail-derived components based on ddPCR technology
  • Method for quantitatively detecting quail-derived components based on ddPCR technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1: the design of primer probe

[0039] Primers and probes were synthesized by Nanjing GenScript Company.

[0040] For the single-copy gene design in the quail nuclear genome, the specific probe and primer sequences are as follows:

[0041] Probe: 5'-AGCAGCCAACACAGCACCCA-3', SEQ ID NO.1;

[0042] Upstream primer: 5'-ACAGCAACGTTTCTTCAGCTC-3', SEQ ID NO.2;

[0043] Downstream primer: 5'-CTGGGAGCGCTTGCTTTATT-3', SEQ ID NO.3;

[0044] The 5' end of the probe is modified with FAM, and the 3' end is modified with BHQ1; wherein, FAM represents a fluorescent reporter group, and BHQ1 represents a quencher group.

[0045] The present invention adopts the fluorescent probe method, and its detection principle is to identify templates by using fluorescently labeled specific probes. Compared with the SYBR dye method in the prior art, the fluorescence-labeled specific probe of the present invention has stronger specificity and lower background interference.

Embodiment 2

[0046]Example 2: Specific verification of primer probes

[0047] (1) Samples: The samples of quail meat, chicken, duck, goose, pigeon, lamb, beef and pork came from local farmers' markets in Nanjing.

[0048] (2) Reagents and instruments: DNA extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. Premix ExTaq (Takara, Japan); American Bio-Rad and CFX96 Touch fluorescent PCR instrument.

[0049] (3) Specificity verification: DNA from quail, chicken, duck, goose, mutton, beef, and pork was extracted as templates, and the primers and probes in Example 1 were used for QPCR amplification, and three samples were made for each sample. Repeat while ddH 2 O replaced the DNA template as a blank control group.

[0050] Amplification reaction system: Premix Ex Taq 12.5 μL, upstream and downstream primers 0.5 μL each, probe 1 μL, template 1 μL, ddH 2 O 9.5 μL.

[0051] The reaction conditions were: 95°C for 5 min; 95°C for 15 s, 60°C for 1 min, 40 cycles....

Embodiment 3

[0053] Example 3: Quail-derived ddPCR quantitative detection standard curve design and linear range verification

[0054] (1) Samples: The quail meat samples come from the local farmer's market in Nanjing. Divide the quail meat sample into small pieces of about 1 cm×1 cm×1 cm. Using vacuum freeze-drying technology, dry the sample at a vacuum degree of 0.100 mbar or less at -60°C for 48-72 h to reduce the moisture content in the sample. Effect of concentration of quail-derived components. After freeze-drying, the sample is ground into a powder sample by a tissue grinder to ensure the uniformity of the sample and avoid errors in quantitative results caused by uneven samples.

[0055] (2) Reagents and instruments: DNA extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. ddPCR amplification master mix ddPCR Super mix for Probes (Bio-Rad, USA); QX200 droplet digital PCR system and CFX96 Touch fluorescent PCR instrument of Bio-Rad in the United Stat...

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Abstract

The invention discloses a method for quantitatively detecting a quail-derived component based on a ddPCR technology, and belongs to the field of molecular biological analysis. A sample to be detected is subjected to vacuum freeze drying to remove moisture and then ground into powder, a dried and uniform powder sample is obtained, DNA of the powder sample is extracted to serve as a template, the ddPCR primer pair and the probe of quails are added into an amplification reaction system, a microdroplet generator is used for generating microdroplets, a ddPCR instrument is used for amplification, after amplification is finished, the ddPCR instrument reads signals and analyzes data, and a quantitative detection result is obtained by contrasting a standard curve. According to the method, the sample freeze-drying treatment method can improve the uniformity of the sample, and meanwhile, the interference of the moisture content on quail-derived component quantification is removed; the ddPCR method is high in specificity and sensitivity, the quantitation limit is 5 mu g / mg, the detection limit is 1 mu g / mg, and quantitative detection work of quail-derived components in complex samples can be met.

Description

technical field [0001] The invention relates to a method for quantitatively detecting quail-derived components based on ddPCR technology, belonging to the field of molecular biology analysis. Background technique [0002] Quail, also known as domestic quail and domestic geese, is one of the common domesticated poultry. Quail meat is delicious and has high nutritional value. As a high-quality full-price protein, it has a low fat content and is rich in various amino acids necessary for human growth and development. It is easily digested and absorbed by the human body. Quail is known as "animal ginseng". Li Shizhen's "Compendium of Materia Medica" once pointed out that quail meat and eggs are beneficial to nourishing Qi, nourishing five internal organs, shape muscles and bones, resist cold and heat, and reduce heat knots, so they are very popular among consumers. . The phenomenon of using skinned chickens, sparrows or squabs as quail meat occurs from time to time in the marke...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6851
CPCC12Q1/6851C12Q1/6888C12Q2531/113C12Q2563/159C12Q2545/114C12Q2563/107
Inventor 刘新梅程逸宇刘延冯秋实吴海晶沈威周海波蒋卉
Owner 南京市食品药品监督检验院
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