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ssr primer set and method for constructing malt fingerprint by using the primer set

A technology of fingerprints and standard fingerprints, which is applied in the field of creation, identification of malt varieties, construction of malt fingerprints, and construction of malt fingerprints by using SSR primers. It can solve the problems of high cost and achieve quality assurance, quality assurance and brand image maintenance. Effect

Active Publication Date: 2018-04-13
TSINGTAO BREWERY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, compared with the cost of only 30-50 yuan for ordinary primers, the current cost of 1OD fluorescent primers is too high, about 600-1000 yuan
However, there is no report on the application of SSR molecular markers based on capillary electrophoresis technology for identification of malt varieties.

Method used

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  • ssr primer set and method for constructing malt fingerprint by using the primer set
  • ssr primer set and method for constructing malt fingerprint by using the primer set
  • ssr primer set and method for constructing malt fingerprint by using the primer set

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: Construction of malt SSR fingerprint

[0036] 21 barley samples from different origins were selected, germinated and dried to make malt, see Table 1.

[0037] Table 1. 21 kinds of malt samples

[0038]

[0039] The SSR primer set includes 4 pairs of core SSR primers, namely Scssr10148, HVM68, HVWAXYG and EBmac755; the 5' end of the forward primer is connected to the M13 primer to synthesize a primer with an M13 tail, namely TP-M13 primer. The 4 pairs of core SSR primer sequences are as follows:

[0040]

[0041] Utilize above-mentioned SSR primer set to construct the method for malt variety fingerprint, comprise the following steps:

[0042] (1) extract the test sample DNA

[0043] 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. ...

Embodiment 2

[0058] Example 2: Identification of Copland Malt Varieties

[0059] Randomly check the company's purchased malt samples for malt variety identification to determine whether the sample is Copeland specified in the purchase contract, and randomly sample 8 grains of malt for variety identification.

[0060] The SSR primer set includes 4 pairs of core SSR primers, namely Scssr10148, HVM68, HVWAXYG and EBmac755; the 5' end of the forward primer is connected to the M13 primer to synthesize a primer with an M13 tail, namely TP-M13 primer. The 4 pairs of core SSR primer sequences are as follows:

[0061]

[0062] The method for identifying malt varieties using the above SSR primer set comprises the following steps:

[0063] (1) Prepare malt DNA, TP-M13-SSR marker PCR amplification, capillary electrophoresis detection, and data analysis on the sample. The specific implementation steps are as in steps (1)-(4) of Example 1.

[0064] (2) Analysis of results: by Figure 1-4 It can be...

Embodiment 3

[0065] Example 3: Gairdner Malt Variety Identification

[0066] Different from Example 2,

[0067] Sample The purchased malt sample is sampled by our company for malt variety identification to determine whether the sample is Gairdner specified in the purchase contract, and 8 grains of malt are randomly sampled for variety identification.

[0068] (1) Malt DNA preparation, TP-M13-SSR marker PCR amplification, capillary electrophoresis detection, and data analysis were carried out on the sample. The specific implementation steps were as in steps (1)-(4) of Example 1.

[0069] (2) Analysis of results: by Figure 5-8 It can be seen that the sizes of the amplified fragments on the 4 pairs of primers of the 8 malts to be identified are the same. Among them, the size of the amplified fragment of primer scssr10148 is 241bp, the size of the amplified fragment of HVM68 is 202 / 277, the size of the amplified fragment of HVWAXYG is 201bp, the size of the amplified fragment of EBmac755 is...

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Abstract

The present invention provides an SSR primer set, including 4 pairs of core SSR primers, namely Scssr10148, HVM68, HVWAXYG and EBmac755; wherein the 5' end of the forward primer is connected to the M13 primer to synthesize a primer with an M13 tail, namely TP‑M13 primer . The present invention also provides a method for constructing malt variety fingerprints by using the above-mentioned SSR primer set, comprising the following steps: extracting the DNA of the test sample, using three primers for PCR amplification, capillary electrophoresis fluorescence detection, data analysis and map construction . The identification of malt varieties can be carried out by using the above-mentioned standard malt fingerprints. The present invention establishes a high-channel malt purity identification method combining capillary electrophoresis technology and SSR fluorescent labeling, the result is accurate and reliable, the detection efficiency is improved, malt adulteration and counterfeiting behaviors are eliminated, and the product quality of beer production 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 constructing a malt fingerprint, in particular to a method for constructing a malt fingerprint by using SSR primers, and further identifying malt varieties. 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. Malt variety identification is the most important index to evaluate barley quality and malt quality, both of which are important indicators to ensure beer quality. Due to the lack of technical means for malt variety identification, beer companies not only suffer huge economic losses when purchasing malt, but also because of poor uniformity of adulterated malt, it will directly...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/6895C12Q1/6858
Inventor 尹花余俊红张志军岳杰林艳房莉周月南祁明霞杨梅张翠
Owner TSINGTAO BREWERY
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