Molecular marker used for identification of Sanzan gum and synthesis bacteria thereof, and preparation and application of molecular marker

A technology of molecular markers and Sanzan glue, which is applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve the problems of cumbersome steps and achieve high accuracy and short detection time

Active Publication Date: 2020-07-24
HEBEI XINHE BIOCHEM
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The patent document with the application number 201811206214.9 discloses "A Primer and Molecular Marker for Identifying Sanzan Gum, Gellan Gum and Wenlun Gum", and the technology disclosed in this patent document can be used in Sanzan Gum However, in the compound glue system using Sanzan gum, gellan gum and Wenlun gum at the same time, since these three microbial glues are synthesized by different strains of Sphingomonas, the same The origin is high, and the differential primers will amplify three PCR products with similar sizes. The three colloids must be separated from each other by other operations such as cloning and sequencing, and the steps are cumbersome.

Method used

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  • Molecular marker used for identification of Sanzan gum and synthesis bacteria thereof, and preparation and application of molecular marker
  • Molecular marker used for identification of Sanzan gum and synthesis bacteria thereof, and preparation and application of molecular marker
  • Molecular marker used for identification of Sanzan gum and synthesis bacteria thereof, and preparation and application of molecular marker

Examples

Experimental program
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Effect test

Embodiment 1

[0033] A molecular marker for identifying sanzan gum and its synthetic bacteria. The sequence of the primer 1 of the molecular marker is SEQ5: 5'-TCAGGCCGTGTGGGGAA-3', and the sequence of the primer 2 is SEQ6: 5'-GATCCGATCCAGCTTTCGGG- 3'.

[0034] The above-mentioned method for obtaining primers for identifying Sanzan gum and its synthetic bacteria, the steps are as follows:

[0035]1. The method for obtaining primers for identifying molecular markers of Sanzan Gum and its synthetic bacteria, the steps are as follows: Synthesize Sanzan Gum Synthetic Bacteria—Sphingomonas sp.CGMCC No.1650 and gellan gum Bacteria—Sphingomonas sp.CGMCC No.14238, Wenlun glue-synthesizing bacteria—Sphingomonas sp.CGMCC No.1561, Xanthan gum-synthesizing bacteria—Xanthomonas campestris Xanthomonas campestris CGMCCNo.10122 were respectively inoculated into 250mL Erlenmeyer flasks with 100mL sterilized liquid medium, and the medium formula was as follows (g / L): glucose 12.0, malt extract powder 4.0, p...

Embodiment 2

[0046] Detection of Molecular Markers of Sanzan Glue-Synthesizing Bacteria

[0047] Sanzan gum synthetic bacteria Sphingomonas sp.CGMCCNo.1650 as described in Example 1, gellan gum synthetic bacteria, Wenlun gum synthetic bacteria, and xanthan gum synthetic bacteria were respectively inoculated into 100mL sterile In a 250mL Erlenmeyer flask of bacterial liquid culture medium, the medium formula is as follows (g / L): glucose 12.0, malt extract powder 4.0, peptone 2.5, yeast powder 1.5, pH 7.0. 150r / min, 30°C shaker culture for 48h Afterwards, centrifuge at 6000r / min for 15min, collect the bacterial cells, and use the bacterial genomic DNA extraction kit from Tiangen Biochemical Technology (Beijing) Co., Ltd. to extract the genomic DNA. Use primer 1 and primer 2 as primers for PCR amplification. The PCR amplification system is: 1 μL of template DNA of 10-30 ng / uL, 0.5 μL of upstream and downstream primers, 1.0 μL of 10 mM dNTP, 2.5 μL of 10× buffer, 5 U / μL Platinum Taq DNA poly...

Embodiment 3

[0049] Inoculate Sanzan gum synthetic bacteria Sphingomonas sp.CGMCC No.19480 into a 250mL Erlenmeyer flask containing 100mL sterilized liquid medium. The medium formula is as follows (g / L): glucose 12.0, malt infusion Powder 4.0, peptone 2.5, yeast powder 1.5, pH 7.0. 150r / min, 30°C shaker culture for 48h, 6000r / min, centrifuge for 15min, collect bacterial cells, use Tiangen Biochemical Technology (Beijing) Co., Ltd. Bacterial Genomic DNA Extraction Kit for genomic DNA extraction. Use primer 1 and primer 2 as primers for PCR amplification. The PCR amplification system is: 1 μL of 10-30ng / uL template DNA, 0.5 μL of upstream and downstream primers, 1.0 μL of 10 mM dNTP, 2.5 μL of 10× buffer, 5 U Add 0.5 μL of PlatinumTaq DNA polymerase / μL to 25 μL with water. The PCR reaction conditions are: 95°C pre-denaturation for 5 minutes; 94°C for 45s, 62°C for 45s, 72°C for 1 min, 30 cycles; 72°C for 10 minutes. Mix 5 μL of the PCR amplification product with 1 μL of loading buffer, spo...

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Abstract

The invention belongs to identification of microorganisms, in particular to a molecular marker used for identification of Sanzan gum and synthesis bacteria thereof, and preparation and application ofthe molecular marker. Sphingomonas sp.CGMCC No.1650 or genome DNA of CGMCC No.19480 is used as a PCR amplification formwork, a primer 1 having a sequence being SEQ No.5 and a primer 2 having a sequence being SEQ No.6 are used as primers, PCR amplification is performed, and the molecular marker is obtained. The problems that when a compounding glue system of the Sanzan gum, gellan gum and welan gumis used, only through other operations of cloning and sequencing and the like, the three gum bodies can be mutually separated, and the step is tedious, can be solved. The molecular marker has the advantages that the Sanzan gum can be identified from medium and light processed products containing the Sanzan gum, and is short in detection time and high in accuracy.

Description

technical field [0001] The invention belongs to the identification of microorganisms, in particular to a molecular marker for identification of sanzan gum and its synthetic bacteria, as well as its preparation and application. Background technique [0002] Food gum is composed of polysaccharides and their complexes, which can form viscous or gel-like polymer hydrosols after being fully hydrated, which can thicken, gel, and suspend in food processing, so that food can obtain the desired Various shapes and soft, hard, crisp, sticky and other textures. Food glue can be divided into vegetable glue, animal glue, microbial glue and chemically modified glue according to the source. Among them, microbial glue has superior performance, short production cycle, and is not affected by climate and geographical environment. It has been widely used in the food field, such as wild Xanthan gum synthesized by Xanthomonas rape and gellan gum synthesized by Sphingomonas paucimobilis, etc. [...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11
CPCC12Q1/689C12Q1/686C12Q2565/125C12Q2535/122C12Q2600/158Y02A50/30
Inventor 张禹张国沛张少华
Owner HEBEI XINHE BIOCHEM
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