Degenerate primer for quickly screening citrulline degrading bacteria and application of degenerate primer

A technology of degenerate primers and citrulline, which is applied in the field of microorganisms, can solve problems such as potential safety hazards of citrulline fermented food, achieve good screening and identification functions, high work efficiency, and good versatility

Pending Publication Date: 2021-11-02
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, with the deepening of research in recent years, citrulline has been found to bring serious safety hazards to fermented foods

Method used

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  • Degenerate primer for quickly screening citrulline degrading bacteria and application of degenerate primer
  • Degenerate primer for quickly screening citrulline degrading bacteria and application of degenerate primer
  • Degenerate primer for quickly screening citrulline degrading bacteria and application of degenerate primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Design of degenerate primer pairs

[0037] Due to the degeneracy of amino acid codons, different codons may correspond to the same amino acid.

[0038] The present invention adopts the Lactobacillus sakei The PTP gene sequence in CTC494 was imported into the GeneBank database [National Center for Biotechnology Information (NCBI), website: https: / / www.ncbi.nlm.nih.gov] for blast analysis, and a batch of Bacteria having a gene sequence with a high degree of similarity to the PTP gene sequence and its amino acid sequence. The amino acid sequences of PTP genes of different strains were compared by software MEGA7 to find out the conserved amino acid sequence Motif, thus obtaining suitable degenerate primers:

[0039] PTP1-F:5'-NACHACHGGCCAHACRAA-3'

[0040] PTP2-R:5'-GTHGAYATHATGGTMTTYAT-3'

[0041] Among them, the degenerate base code Y=C / T, R=A / G, H=A / C / T, B=G / T / C, N=A / G / C / T.

Embodiment 2

[0042] Example 2 Using the degenerate primers obtained in Example 1 to screen citrulline-degrading bacteria

[0043] Isolate single colonies to be detected by streaking from fermented products sausage, miso, soy sauce and kimchi, and use the above degenerate primers to perform colony PCR on PTP1-F and PTP2-R. The colony PCR reaction system is 50 μl, specifically: DNA template <50 ng, each 1 μl of upstream primer and downstream primer, 25 μl of 2*Es Taq MasterMix (Dye) DNA Polymerase, and make up to 50 μl with sterilized ultrapure water.

[0044] The PCR reaction conditions were: pre-denaturation at 98°C for 3 min, denaturation at 95°C for 30 s, annealing at 48°C for 30 s, extension at 72°C for 1 min, 35 cycles, and extension at 72°C for 2 min. After colony PCR amplification, 5 μL was taken for 1% agarose gel electrophoresis.

[0045] According to whether the amplified product contains a DNA fragment of about 1000bp to determine whether the strain to be tested contains the PTP...

Embodiment 3

[0046] Embodiment 3 Carry out classification identification to the bacterial classification that embodiment 2 screens out

[0047] Sequence Alignment and Phylogenetic Tree Construction

[0048]Activation of strains: After amplified by degenerate primers PTP1-F and PTP2-R, the amplified products contain 4 strains of DNA fragments of about 1000bp, namely strains LC-1, LC-2, JY-3 and PC -4 was inoculated in MRS medium and activated under the culture condition of 180rpm at 30°C.

[0049] Bacterial genomic DNA extraction: According to the instructions of the bacterial DNA extraction kit, the genomic DNA was extracted.

[0050] PCR amplification of 16S rDNA: 16S rDNA PCR was carried out with the extracted bacterial genomic DNA as a template and universal primers 27F and 1492R as primers. The reaction system was prepared as 50 μl, specifically: 2*pfu PCR MasterMix high-fidelity enzyme 25 μl, template DNA<50 ng, primer 27F 1μl, primer 1492R 1μl, sterilized ultrapure water to make up...

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Abstract

The invention discloses a degenerate primer for quickly screening citrulline degrading bacteria. The sequence of the degenerate primer is as follows: PTP1-F: 5 '-NACHACHGGCCAHACRAA-3'; and PTP2-R: 5 '-GTHGAYATHATGGTMTTYAT-3'; wherein the degenerate base code Y is equal to C / T, R is equal to A / G, H is equal to A / C / T, B is equal to G / T / C, and N is equal to A / G / C / T. The invention also discloses an application of the designed degenerate primer in screening citrulline degrading bacteria. A pair of degenerate primers is designed according to the PTP gene with citrulline utilization capacity for the first time, the degenerate primers are universal primers with a screening function, a specific probe primer does not need to be designed for a certain strain, and the degenerate primers are convenient and fast to use, good in universality and high in working efficiency and have good screening and recognition functions; a strain guarantee is provided for subsequently exploring the difference of citrulline utilization capacity of PTP genes of different strain sources and comparing the influence of environmental factors, and a new solution idea and method are provided for reducing or eliminating ethyl carbamate generated in the fermentation process.

Description

technical field [0001] The invention relates to the technical field of microorganisms, in particular to a method for rapidly screening melon [0002] The degenerate primer of the citrulline-degrading bacteria, and the invention also relates to a method for screening citrulline-utilizing bacteria using the degenerate primer. Background technique [0003] Citrulline (Citrulline, CIT) is a non-coded α-amino acid, which has an extremely important impact on human health. Citrulline is involved in a series of physiological metabolic processes of human beings, so it has received extensive attention from the fields of nutrition and medicine. However, with the deepening of research in recent years, citrulline has been found to bring serious safety hazards to fermented foods. [0004] The spontaneous reaction of citrulline and ethanol is considered to be the main way to form ethyl carbamate in a variety of alcoholic beverages (such as wine, etc.) and fermented condiments (such as so...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/686C12Q1/04C12N15/11
CPCC12Q1/686C12Q2565/125
Inventor 张继冉王文玉吴坤毛兆敏李肖杏郭兆鑫徐淑霞张世敏
Owner HENAN AGRICULTURAL UNIVERSITY
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