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A lamp detection method for identifying mink meat from beef and mutton using mitochondrial DNA

A detection method, the technology of beef and mutton, is applied in the field of molecular biology detection, which can solve the problems of difficult popularization and application and long time-consuming, and achieve the effects of broad market prospect, simple operation and easy judgment of results

Inactive Publication Date: 2018-08-14
山东省兽药质量检验所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the methods used for the detection of animal-derived components mainly include conventional PCR and fluorescent quantitative PCR, etc., but the above-mentioned diagnostic methods take a long time, and the judgment results require the help of sophisticated instruments and equipment, which is difficult to popularize and apply in actual production

Method used

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  • A lamp detection method for identifying mink meat from beef and mutton using mitochondrial DNA
  • A lamp detection method for identifying mink meat from beef and mutton using mitochondrial DNA
  • A lamp detection method for identifying mink meat from beef and mutton using mitochondrial DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Design primers

[0032] The COXI sequence of mink was retrieved from GenBank, and software comparison analysis was performed to confirm the accuracy of the sequence; according to the above sequence, primers were designed using PrimerExplore software as follows:

[0033] DAF3: GCTATGGGCCTTAGGGTT (as shown in SEQ ID NO.1),

[0034] DAB3: CATTTAGGGTGTAGCCTGT (as shown in SEQ ID NO.2),

[0035] DAFIP: GTGAAGAACGATGTCTAGTGATGAGTTTTATTTACAGTGGGTGGC (shown in SEQ ID NO.3),

[0036] DABIP: GCACATTTTCACTACGTTCTTTCAAGGAACCAGTGAACGAATCC (shown in SEQ ID NO. 4).

Embodiment 2

[0037] Embodiment 2 specificity experiment

[0038] (1) Extract sample DNA as a template

[0039] The fresh muscle tissues of cattle, sheep, dogs, cats, foxes, and minks were strictly aseptically collected as samples, and then the DNA in the samples was extracted by phenol-chloroform extraction method. Use ultrapure water (negative control), positive plasmid containing mink meat COXⅠ gene (positive control), beef DNA, mutton DNA, dog meat DNA, cat meat DNA, fox meat DNA, and mink meat DNA as templates. A LAMP reaction system was established; the DNA template concentration was 20ng / uL.

[0040] (2) Establishment of LAMP reaction system

[0041] The LAMP reaction system established by exploring the concentration ratio of internal and external primers, dNTP concentration, reaction temperature, reaction time, etc.: 25 μL, its composition is: 0.5uL each of 10umol / L DAF3 and DAB3, 4uL each of 10umol / L DAFIP and DABIP, 2.5 mmol / L of dNTP 4uL, 50mmol / L of MgSO 4 2uL, 2.5uL of 5mo...

Embodiment 3

[0046] Collect 30 fresh beef samples and 30 fresh mutton samples from the slaughterhouse in Shandong Province; collect 10 fresh mink meat samples from Shandong Animal Hospital; use the method of Example 1 to extract the DNA of the samples as a template, set up a LAMP reaction system, and carry out LAMP amplification reaction, detection results are shown in Table 1:

[0047] Table 1

[0048]

beef

mutton

mink meat

beef and lamb

beef and mink

lamb and mink

Number of samples

30

30

10

30

10

10

number of positives

0

0

10

0

10

10

Positive ratio / %

0

0

100

0

100

100

[0049] It can be concluded from Examples 2 and 3 that the primers in Example 1 can specifically amplify mink DNA, and only when the template contains mink DNA, the reaction solution after adding the fluorescent dye SYBR GREEN Ⅰ to the LAMP reaction product turns green ; while the template contains any other no...

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Abstract

The invention relates to an LAMP detection method of utilizing mitochondrial DNA to identify sable meat in beef and mutton and belongs to the technical field of molecular biological detection. The LAMP detection method includes: taking DNA of a to-be-detected meat product as a template, and taking DAF3, DAB3, DAFIP and DABIP as primers to build an LAMP detection system; putting the LAMP detection system into a water bath at constant temperature of 64 DEG C for LAMP reaction for 40min, and adding 1uL of fluorescent dye SYBR GREEN I into reaction liquid after the reaction is finished; if the reaction liquid becomes green, determining that the to-be-detected meat product contains the sable meat; if the reaction liquid becomes orange, determining that the to-be-detected meat product does not contain the sable meat. Compared with conventional PCR, the LAMP detection method has the advantages that detection can be completed only through reaction for 40min in a common constant-temperature water bath, and results can be directly observed by naked eyes through a method of adding the dye into a final product, so that animal-origin components can be identified quickly and accurately; the LAMP detection method has the advantages of simplicity and convenience in operation, low requirement on experimental instrument and easiness in result determination.

Description

technical field [0001] The invention relates to a LAMP detection method for identifying mink meat from beef and mutton by using mitochondrial DNA, and belongs to the technical field of molecular biology detection. Background technique [0002] In recent years, as the market demand for beef and mutton has gradually increased and the price has risen rapidly, some illegal traders, driven by profit, have mixed other cheap meat to fake beef and mutton. Since 2012, there have been many incidents in China where pork, chicken, and duck meat have been passed off as beef and mutton. However, in some special economic animal breeding areas dominated by fur, the whereabouts of mink and fox meat has become a mystery. Many people speculate that it was mixed with beef and mutton and entered people's tables. This phenomenon not only disrupts the economic order, but also seriously endangers people's health. [0003] At present, the methods used for the detection of animal-derived components...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/6844
CPCC12Q1/6844C12Q1/6888C12Q2531/119C12Q2563/107
Inventor 刘少宁陈智高迎春吴家强邵兵杨志昆苏梅姚震王贵升程瑞红
Owner 山东省兽药质量检验所
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