Affinis sheep meat detection method based on species specificity STR-SS

A detection method and specific technology, which is applied in biochemical equipment and methods, microbial measurement/inspection, genetic engineering, etc., can solve the problems of long detection time, difficult identification, high cost, etc., and achieve simple and easy operation , time-consuming effect

Pending Publication Date: 2019-07-12
NINGXIA UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

It can solve the problems of difficult identification, high cost, long detection time, large error and other problems in the prior art.

Method used

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  • Affinis sheep meat detection method based on species specificity STR-SS
  • Affinis sheep meat detection method based on species specificity STR-SS
  • Affinis sheep meat detection method based on species specificity STR-SS

Examples

Experimental program
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Effect test

Embodiment 1

[0044] The first step: DNA extraction of standard mutton and mutton samples to be tested: take the muscle tissues of 25 Tan sheep, small-tailed Han sheep and Tanhan hybrid sheep, grind them into powder with liquid nitrogen, add them to a 1.5 μL centrifuge tube, and add 400 μL BufferDigestion , shake to mix. Water bath at 65°C for 1 hour until the cells are completely lysed; add 200 μL Buffer PA, mix thoroughly by inversion, and place in a refrigerator at -20°C for 5 minutes; centrifuge at 10,000 rpm at room temperature for 5 minutes, transfer 500 μL of the supernatant to a new 1.5 μL centrifuge tube Put it into a centrifuge and centrifuge at 12,000rpm to take the supernatant; add an equal volume of isopropanol, invert it 5 times to make it fully mixed, and let it stand at room temperature for 2min; centrifuge at 10,000rpm at room temperature for 5min, discard the supernatant; Rinse upside down for 1 min, centrifuge at 10,000 rpm for 2 min, discard the supernatant; open the lid...

Embodiment 2

[0069] The first step: DNA extraction of standard mutton and mutton samples to be tested: take the muscle tissues of 40 Tan sheep, small-tailed Han sheep and Tanhan hybrid sheep, grind them into powder with liquid nitrogen, add them to a 1.5 μL centrifuge tube, and add 400 μL BufferDigestion , shake to mix. Water bath at 65°C for 1 hour until the cells were completely lysed; add 200 μL Buffer PA, mix thoroughly by inversion, and place in a refrigerator at -20°C for 5 minutes; centrifuge at 10,000 rpm at room temperature for 5 minutes, transfer 525 μL of the supernatant to a new 1.5 μL centrifuge tube Put it into a centrifuge and centrifuge at 12,000rpm to take the supernatant; add an equal volume of isopropanol, invert it 7 times to make it fully mixed, and let it stand at room temperature for 2.5min; centrifuge at 10,000rpm at room temperature for 5min, discard the supernatant; Ethanol, rinse upside down for 2 minutes, centrifuge at 10,000rpm for 2 minutes, discard the supern...

Embodiment 3

[0077] The first step: DNA extraction of standard mutton and mutton samples to be tested: take the muscle tissues of 50 Tan sheep, small-tailed Han sheep and Tanhan hybrid sheep, grind them into powder with liquid nitrogen, add them to a 1.5 μL centrifuge tube, and add 400 μL BufferDigestion , shake to mix. Bath at 65°C for 1 hour until the cells are completely lysed; add 200 μL Buffer PA, mix thoroughly by inversion, place in a refrigerator at -20°C for 5 minutes; centrifuge at 10,000 rpm at room temperature for 5 minutes, transfer 550 μL of the supernatant to a new 1.5 μL centrifuge tube Put it into a centrifuge and centrifuge at 12,000rpm to take the supernatant; add an equal volume of isopropanol, invert it 8 times to make it fully mixed, and let it stand at room temperature for 3min; centrifuge at room temperature for 5min at 10,000rpm, discard the supernatant; Rinse upside down for 3 minutes, centrifuge at 10,000rpm for 2 minutes, discard the supernatant; open the lid an...

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Abstract

The invention belongs to the technical field of food detection. The detection method comprises the following specific steps that DNA of a standard sheep meat sample and DNA of a to-be-detected sheep meat sample are extracted, the sheep meat sample DNA concentration of the standard sample DNA and the sheep meat sample DNA concentration of the to-be-detected sample DNA are detected respectively, theSSR gene sequence of the standard sample DNA and the SSR gene sequence of the to-be-detected sample DNA are subjected to fluorescence labeling, PCR amplification is conducted by using primers designed by the SSR sequences, the PCR amplification result is detected through agarose gel electrophoresis, the SSR gene sequence type of the standard sample and the SSR gene sequence type of the to-be-detected sample are detected through capillary electrophoresis, an STR-SSR standard type peak spectrogram is built. The detection method is s used for specifically identifying Tan sheep, small tailed Hansheep and Tan-Han crossbred sheep, the method takes short time, the operation is simple, and the specificity is high. The detection results are compared by using the peak spectrogram, the detection results can be mutually identified, the method can be used for precisely identifying the three kinds of affinis sheep species which cannot be identified morphologically, and sustainable and healthy development of the Tan sheep industry and consumer rights and interests are facilitated.

Description

[0001] Technical field: [0002] The invention belongs to the technical field of food detection, and in particular relates to a detection method for closely related sheep meat based on species-specific STR-SSR. [0003] Background technique: [0004] SSR is the abbreviation of Simple Sequence Repeat, which means simple repeat sequence marker. SSR is a DNA molecular marker technology with PCR technology as the core, or microsatellite sequence marker (Microsatellite sequence, MS), or short tandem repeat marker (Short Tandem Repeat , STR). [0005] In 1982, Hamade et al. discovered the simple repeat sequence marker (SSR) technology, or microsatellite sequence marker (Microsatellite sequence, MS), or short tandem repeat marker (Short Tandem Repeat, STR). There are a large number of tandem repeat sequences with extremely high repetition frequency and extremely low sequence complexity in eukaryotic genomes. According to the length of the repeat motif, the tandem repeat sequence can...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12Q1/6888
CPCC12Q1/6858C12Q1/6888C12Q2600/124C12Q2600/156C12N15/8261C12Q2525/151C12Q2563/107
Inventor 罗瑞明耿多王丽娟陈辉宋学丽
Owner NINGXIA UNIVERSITY
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