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High-throughput malt purity identification technology based on capillary electrophoresis and SSR marker

A capillary electrophoresis, high-throughput technology, applied in the biological field, can solve problems such as prone to errors, and achieve the effects of improved detection efficiency, high sensitivity, and efficient methods

Active Publication Date: 2017-05-24
TSINGTAO BREWERY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The samples detected by capillary electrophoresis all contain molecular weight internal standards, and DNA fragments can be directly compared with the molecular weight internal standards in their swimming lanes to obtain their size values ​​accurately, while the fragment sizes obtained by polyacrylamide gel electrophoresis can only be determined with the naked eye and DNA Marker It is estimated after comparison, especially the strips far away from the Marker are prone to errors in judgment
However, there are no reports on the high-throughput identification of malt purity based on capillary electrophoresis and SSR molecular markers.

Method used

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  • High-throughput malt purity identification technology based on capillary electrophoresis and SSR marker
  • High-throughput malt purity identification technology based on capillary electrophoresis and SSR marker
  • High-throughput malt purity identification technology based on capillary electrophoresis and SSR marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: Construction of malt fingerprint

[0035] Barley samples from 17 different origins were selected, germinated and dried to make malt.

[0036] Table 1. 17 kinds of malt samples

[0037]

[0038] (1) extract the test sample DNA

[0039] Randomly sample from the malt samples, and use the CTAB method to extract the DNA of the test samples: take a single-grain malt sample in a 1.5ml centrifuge tube, about 25-45mg, add 7-8mm steel balls, and use the Thmorgan CK1000D high-throughput tissue grinder. smash. Add 600ul of CTAB extraction buffer preheated at 65°C to the sample, vortex and mix to lyse the sample. Since malt is rich in proteins, polysaccharides, polyphenols and other substances, it is difficult to separate, so add 4ul β-mercaptoethanol and 1% PVP (polyvinylpyrrolidone) to prevent polyphenols from oxidative browning and DNA degradation, and effectively remove polyphenols and polysaccharides . Water bath at 65°C for 15 minutes, during which time s...

Embodiment 2

[0050] Example 2: Purity identification of commercial malt Copeland samples

[0051] The SSR primer set includes 3 pairs of SSR primers, which are respectively HVM68, Bmag0007 and EBmac755; the sequences of the 3 pairs of SSR primers are as follows:

[0052]

[0053] Utilize above-mentioned three pairs of SSR primers to carry out the method for the commercial malt Copeland sample of purchase to carry out purity identification, comprise the following steps:

[0054] (1) Randomly select 96 grains of malt from the Copeland malt sample, prepare malt DNA, fluorescent multiplex PCR amplification, capillary gel electrophoresis detection, and data analysis. The specific implementation steps are as in steps (1)-(4) of Example 1 ).

[0055] (2) Result analysis: compare the electrophoretic patterns amplified by the three pairs of fluorescent primers of each grain of malt DNA of the sample to be tested with the fingerprints of Copeland malt respectively. The results showed that the e...

Embodiment 3

[0056] Example 3: Purity Identification of Commercial Malt Commander Samples

[0057] Different from Example 2,

[0058] (1) Randomly select 96 grains of malt from the Commander malt sample, prepare malt DNA, fluorescent multiplex PCR amplification, capillary gel electrophoresis detection, and data analysis. The specific implementation steps are as in steps (1)-(4) of Example 1 ).

[0059] (2) Result analysis: compare the electrophoretic patterns amplified by three pairs of fluorescent primers of each grain of malt DNA of the sample to be tested with Commander malt fingerprints respectively. The results showed that the electrophoretic patterns of 90 of the 96 malts were consistent with the fingerprint data of Commander malt, and the SSR loci were 141bp, 224bp, and 267bp respectively. The peak characteristics of the other 6 malts were consistent, and the SSR loci were 146bp, 224bp and 257bp, indicating that other varieties were mixed, so the malt purity of this batch of Comma...

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Abstract

The invention provides a SSR primer pairs, which contain three pairs of SSR primers, namely HVM68, Bmag0007 and EBmac755. The invention also provides a method for high-throughput malt purity identification by the utilization of the SSR primer pairs. The method comprises the following steps: extracting DNA of test samples, carrying out multiplex PCR amplification by the use of SSR labeled with different fluorescent dyes, and carrying out capillary electrophoresis fluorescence detection and data analysis. By SSR band analysis after PCR amplification of the three pairs of SSR primers, a malt variety fingerprinting database can be established. The capillary electrophoresis and SSR marker combined high-throughput malt purity identification method is established. By the method, results are stable and reliable, detection efficiency is raised, lots of samples can be rapidly determined within a short period of time, and the results can be used as judgment basis of commercial arbitration. Therefore, adulteration and selling fakes of malt are eliminated, and product quality of beer brewing enterprises is guaranteed from the source.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a method for identifying malt purity, in particular to a method for identifying high-throughput malt purity based on capillary electrophoresis and SSR markers. Background technique [0002] Beer is a low-alcohol wine full of carbon dioxide brewed from malt, hops, and water through yeast fermentation. As the main raw material of beer, malt is obtained from barley through processes such as soaking, germinating, drying and roasting. The quality of malt directly determines the quality of beer. Compared with other malt indicators, malt purity identification is also an important indicator for evaluating malt quality. [0003] China's beer production has ranked first in the world for many years, but its domestic production of barley is insufficient and it needs to import a large amount from overseas, mainly from Canada, Australia and France. There is a large price difference be...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 尹花张志军岳杰余俊红王书谦房莉周月南祁明霞纪秀苹
Owner TSINGTAO BREWERY
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