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Method for rapidly detecting pig source components in red meat through real-time fluorescence loop-mediated isothermal amplification (LAMP) method

A real-time fluorescence, beef and mutton technology, applied in the field of molecular biology, to achieve high sensitivity and high specificity

Inactive Publication Date: 2013-08-14
长沙市食品质量安全监督检测中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Through searching, it is found that there are no literatures at home and abroad that use loop-mediated constant temperature amplification technology combined with real-time fluorescence method to detect pig-specific cytochrome b gene in beef and mutton, so as to quickly identify meat products.

Method used

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  • Method for rapidly detecting pig source components in red meat through real-time fluorescence loop-mediated isothermal amplification (LAMP) method
  • Method for rapidly detecting pig source components in red meat through real-time fluorescence loop-mediated isothermal amplification (LAMP) method
  • Method for rapidly detecting pig source components in red meat through real-time fluorescence loop-mediated isothermal amplification (LAMP) method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: Ordinary PCR reaction and detection method sensitivity comparison of the present invention

[0037] (1) Extraction of genomic DNA of the sample to be tested: the CTAB method is used to extract and purify the DNA of the sample;

[0038] (2) Preparation reaction system:

[0039] In the described reaction system, include:

[0040] Fluorescent indicator SYBR GREEN I: 0.5 μL;

[0041] Buffer: 12.5μL;

[0042] Bst DNA Polymerase Large Fragment 8U: 1 μL.

[0043] The ratio of inner primer / loop primer / outer primer is 6:1:1, and the concentration of primer / loop primer / outer primer is 1.2 μM / 0.2 μM / 0.2 μM. The nucleotide sequence of the primer is:

[0044] Inner primer FIP:

[0045] 5′-TGAAGGCTGTTGCTATAACGGTTTTTCCTATTCATCCACGTAGGC-3′

[0046] Inner primer BIP:

[0047] 5′-CCTGCCCTGAGGACAAATATCATTTTCTCAGATTCATTCTACGAGGT C-3′

[0048] Outer primer F3: 5′-ACGGATGAGTTATTCGCTATC-3′

[0049] Outer primer B3: 5′-GGTAATGATGAATGGCAGGATA-3′

[0050] Loop primer LB: 5...

Embodiment 2

[0056] Embodiment 2: specificity test

[0057] (1) Extraction of genomic DNA of the sample to be tested: the CTAB method is used to extract and purify the DNA of the sample;

[0058] (2) Preparation reaction system:

[0059] In the described reaction system, include:

[0060] Fluorescent indicator SYBR GREEN I: 0.5 μL;

[0061] Buffer: 12.5μL;

[0062] Bst DNA polymerase large fragment 8U: 1 μL;

[0063] The ratio of inner primer / loop primer / outer primer is 6:1:1, the concentration of primer / loop primer / outer primer is 1.2 μM / 0.2 μM / 0.2 μM, and the nucleotide sequence of the primer is the same as that in Example 1.

[0064] The DNA is isolated and purified beef, mutton, and pork genomes, and the template amount is 10ng;

[0065] wxya 2 O supplemented to a total volume of 25 μL;

[0066] (3) Constant temperature amplification reaction: Mix the above reaction system, centrifuge it, and react it at 63°C for 45 minutes; set up a negative control, the template is sterilized ...

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Abstract

The invention discloses a method for rapidly detecting pig source components in red meat through a real-time fluorescence loop-mediated isothermal amplification (LAMP) method. According to the method, the pig specific cytochrome b genes in the red meat are detected by employing a method for combining the LAMP technology with real-time fluorescence. According to the detection method, the genome DNA of the meat to be detected is extracted, six specific primers and Bst DNA polymerase fragments with strand displacement activity are amplified at the temperature of 60-65 DEG C for 45-60 minutes, SYBR GREEN I is added into the reaction system, and the amplification conditions of the sample template can be detected in real time, so that whether pig source components are contained in beef and mutton samples can be judged. The method has the advantages of high sensitivity, high specificity, simplicity and rapidness, and the result is not required to be subjected to gel electrophoresis.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a method for rapidly detecting pig-derived components in beef and mutton by a real-time fluorescent LAMP method. Background technique [0002] Meat and its products are one of the main foods consumed by humans. Meat can provide the protein, fat, inorganic salts and vitamins needed by the human body, and contains sufficient essential amino acids for the human body. The nutrient composition of meat varies greatly due to animal species, age, part, and degree of fatness and leanness. Although in meat products, due to price, religion, health and other reasons, many countries have enacted laws and regulations requiring food labels to truly and clearly indicate the source of meat and prohibit adulteration to protect the interests of consumers, but meat varieties are confused in the market The phenomenon is still very common, mainly because high-priced meat is mixed with low-pr...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 杨丽霞付淑君彭新凯周明刘佩曾林李乐
Owner 长沙市食品质量安全监督检测中心