SSR primer set and method for identifying malt varieties using the primer set

A variety identification and primer set technology, applied in biochemical equipment and methods, microbial determination/inspection, recombinant DNA technology, etc., can solve the problems affecting saccharification, filtration and brewing performance, no secondary germination, and difficult separation. , to prevent malt adulteration, maintain brand image, and achieve good results in amplification efficiency

Active Publication Date: 2018-09-14
TSINGTAO BREWERY
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

[0004] Compared with the relatively mature technology of barley variety identification, there is no clear detection method for malt variety identification. Conventional malt indicators are indicators that reflect the mixed state of malt and cannot reflect changes in malt varieties.
However, the existing methods for identification of barley varieties are not suitable for the identification of malt varieties.
(1) The appearance characteristics of barley have changed after germination and drying, and the protein begins to decompose under the action of protease, so the traditional barley morphology identification method and protein electrophoresis identification method are completely unsuitable for the identification of malt varieties
(2) In terms of molecular marker technology, barley DNA is usually extracted from germinated fresh seedling leaves. Fresh leaves contain less impurities and DNA extraction is easy; Made after the malting process, it cannot be germinated again, so DNA can only be extracted directly from the malt
Because malt is rich in proteins, polysaccharides, polyphenols and other substances, it is difficult to separate
(3) More critically, part of the DNA of the malt is broken after being roasted at 84-86°C for 3 hours, which directly affects the analysis of the results of PCR amplification fragments, resulting in many primers that play a decisive role in the identification of barley varieties being amplified by PCR. The shape and position of the electrophoresis bands on the polyacrylamide gel electrophoresis map of the increased products are basically the same, and the corresponding malt varieties cannot be distinguished
[0005] Due to the lack of technical means for identifying malt varieties, beer companies not only suffer huge economic losses when purchasing malt, but also the poor uniformity of adulterated malt directly affects the performance of saccharification, filtration and brewing, reduces the quality of beer, and ultimately affects the brand of beer companies image

Method used

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  • SSR primer set and method for identifying malt varieties using the primer set
  • SSR primer set and method for identifying malt varieties using the primer set
  • SSR primer set and method for identifying malt varieties using the primer set

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Select 21 barley samples from different origins, germinate and dry them to make malt.

[0036] Table 1. 21 kinds of malt samples

[0037]

[0038] (1) Extract the DNA of the test variety sample

[0039] The DNA of the tested variety sample was extracted by CTAB method: take a single grain malt sample in a 1.5ml centrifuge tube, about 25-45mg, add 7-8mm steel balls, and use Thmorgan CK1000D high-throughput tissue grinder to grind. Add 600ul of CTAB extraction buffer preheated at 65°C to the sample, vortex and mix to lyse the sample. Add 4ul β-mercaptoethanol and 1% PVP (polyvinylpyrrolidone) to prevent oxidative browning of polyphenols and DNA degradation, effectively remove polyphenols and polysaccharides, and vortex to mix. Water bath at 65°C for 15 minutes, during which time shake gently to mix. Add 600ul of chloroform / isoamyl alcohol (24:1), vortex and mix vigorously for 20s to form an emulsion. Centrifuge at 10,000 rpm for 10 minutes at room temperature, pipe...

Embodiment 2

[0052] The samples included 2 malt varieties to be tested, one was the control variety Copeland with 2 grains, and the other was the malt variety A to be identified with 6 grains, and it was determined whether the variety A was Copeland.

[0053] (1) Prepare malt DNA from the sample, perform PCR amplification using 6 pairs of SSR primers, polyacrylamide gel electrophoresis, silver staining, and map analysis. The specific implementation steps are as (1)-(4) in Example 1.

[0054] (2) Analysis of results: by Figure 7 It can be seen that lanes 1-8 are the first pair of primers scssr10148, lanes 1 and 2 are the control variety Copeland, lanes 3-8 are varieties to be identified, and the electrophoresis bands of lanes 3, 5, 6, and 7 are compared with lanes 1 and 2 The electrophoresis bands of the samples are completely consistent, but the electrophoresis bands of lanes 4 and 8 are different from the control sample Copeland, so it can be determined that lanes 4 and 8 are other varie...

Embodiment 3

[0056] The samples included 2 malt varieties to be tested, one was the control variety Metcalfe with 2 grains, and the other was the malt variety B to be identified with 6 grains.

[0057] (1) Prepare malt DNA from the sample, perform PCR amplification using 6 pairs of SSR primers, polyacrylamide gel electrophoresis, silver staining, and map analysis. The specific implementation steps are as in steps (1)-(4) of Example 1.

[0058] (2) Analysis of results: by Figure 8It can be known that swimming lanes 1-8 are the first pair of primers scssr10148, swimming lanes 1 and 2 are the reference variety Metcalfe, and swimming lanes 3-8 are varieties to be identified, wherein the electrophoresis bands of swimming lanes 4-8 are the same as those of the comparison samples in swimming lanes 1 and 2 The bands are exactly the same, but the electrophoresis band in lane 3 is different from that of the control sample Metcalfe, so it can be determined that lane 3 is another species. Swimming l...

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PUM

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Abstract

The invention provides an SSR primer group, which comprises 6 pairs of SSR primers, namely scssr07759, HVM68, Bmac0030, scssr10148, scssr03907 and Bmag0120. The invention also provides a method for malt variety identification by virtue of the SSR primers, wherein the method comprises the following steps: extraction of DNA of a sample of experimental variety, PCR amplification by virtue of the SSR primers, polyacrylamide gel electrophoresis as well as silver staining and SSR spectrum band analysis. The method, through SSR spectrum band analysis after the PCR amplification of the 6 pairs of SSR primers, can be used for identifying experimental malt variety and known malt variety and can be used for constructing a malt variety database. Through malt variety identification by virtue of the specific SSR primer group, the method can be used for rapidly identifying various malt varieties, and the method has the advantages of being rapid and accurate, and convenient in operation.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for identifying malt varieties, in particular to a technique for identifying malt varieties using SSR primers. 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. Variety identification of barley and malt is the most important indicator to evaluate the quality of barley and malt, both of which are important indicators to ensure the quality of beer. [0003] At present, barley variety identification technology has been relatively mature. There are not only traditional morphological identification methods, but also protein electrophoresis identification methods and DNA molecular markers...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/6895
Inventor 董建军张志军林艳余俊红岳杰房莉周月南祁明霞张翠
Owner TSINGTAO BREWERY
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