Rapid source-tracking method of lactobacillus fermenti, combined sequences used for method, and construction method of combined sequences

A technology of Lactobacillus fermentum and construction method, which is applied in the field of rapid traceability of Lactobacillus fermentum, and can solve problems such as low efficiency, inability to ferment Lactobacillus isolate gene fragments, and a large amount of sequence comparison

Active Publication Date: 2017-03-08
完美(广东)日用品有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, due to the different species and differences in the target gene fragments selected in the literature among different strains, this technical solution cannot be applied to Lactobacillus fermentum, and the corresponding gene fragments of Lactobacillus fermentum isolates cannot be obtained through this technical solution.
In addition, in order to realize the traceability of Streptococcus thermophilus, this technical scheme uses 10 single-copy alleles selected in a scattered manner for comparison, and the amount of sequence comparison is huge, because the implementation efficiency is low

Method used

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  • Rapid source-tracking method of lactobacillus fermenti, combined sequences used for method, and construction method of combined sequences
  • Rapid source-tracking method of lactobacillus fermenti, combined sequences used for method, and construction method of combined sequences
  • Rapid source-tracking method of lactobacillus fermenti, combined sequences used for method, and construction method of combined sequences

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Experimental program
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Effect test

Embodiment 1

[0052] Activation and DNA extraction of embodiment 1 bacterial strain

[0053] Selected from the western region of Inner Mongolia, Shuanghe Town, Tuoketuo County, Hohhot City, Inner Mongolia, Yousuo Town, Eryuan County, Yunnan Province, Arixiyatu Sumu, Bulgan Province, Mongolia, and Bayinaobao Sumu, Kent Province, Mongolia. 35 strains of Lactobacillus fermentum were isolated from the collected samples of sour porridge, kumiss, lactic acid whey and yogurt. See Table 2 for the strain isolation information.

[0054] Table 2 Source information of Lactobacillus fermentum isolates

[0055]

[0056] Activate the strains separately: put the freeze-dried bacterial powder stored in the ampoule tube into skim milk to activate and rejuvenate (37°C, 24h), and perform microscopic examination on the bacterial liquid smear, and the microscopic examination results are Gram-positive Pure cultures were subcultured to the third generation.

[0057] Use liquid nitrogen freeze-thaw-CTAB method...

Embodiment 2

[0058] The construction of embodiment 2 joint sequence

[0059] 2.1 Design primers

[0060] Using the Lactobacillus fermentum F-6 genome (genbank number CP005958) as a template, starting from a large number of known housekeeping genes of Lactobacillus fermentum, through comparative genomics analysis results and the selection basis of housekeeping genes, it is considered that housekeeping genes must be It is closely related to core metabolism, and the selected genes should cover the entire genome as much as possible to more accurately reflect the evolutionary relationship between strains, and the distance between two adjacent housekeeping genes should not be too close to avoid genetic linkage. Influenced by this, the inventor finally selected 6 housekeeping genes related to the core metabolism of Lactobacillus fermentum, including dnaA, pyrG, rpoB, recA, dnaK and murC, as targets after many experiments. Primer Premier 5.0 software was used to design specific amplification prim...

Embodiment 3

[0073] Embodiment 3 builds developmental tree

[0074] The MEGA6.0 software package was used to analyze the combined sequence, and compared with Clustal W, the combined sequence of the 35 strains of bacteria isolated in Example 1 was compared in pairs, the distance between the sequences was calculated, and the distance matrix was constructed to reflect the various relationship between sequences.

[0075] Then construct a phylogenetic tree by constructing the neighbor-joining method (Neighour-joining, N-J) (see Figure 7 ).

[0076] Figure 7 The phylogenetic tree constructed based on the joint gene sequence of danA-pyrG-rpoB-recA-dnaK-murC can be seen that the phylogenetic tree constructed by 35 Lactobacillus fermentum strains is clearly divided into two branches, which are isolated from China It was composed of Lactobacillus fermentum isolates from Mongolia, and the results obtained were completely consistent with the collection source, indicating that the strains in the s...

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Abstract

The invention provides applications of combined gene sequences comprising lactobacillus fermenti genomes in source tracking of lactobacillus fermenti. The combined gene sequences comprise six house-keeping genes, namely, dna A, pyrG, rpoB, recA, dnaK and murC. The invention further provides a rapid source-tracking method of lactobacillus fermenti. The rapid source-tracking method comprises the following steps: (1) extracting genome DNA of lactobacillus fermenti; (2) constructing the combined gene sequences of the bacterial strains; (3) constructing adjacent method phylogenetic trees for the combined gene sequences by adopting MEGA 6.0 software, thus obtaining the source-tracking analysis for the lactobacillus fermenti strains through the phylogenetic trees; and (4) judging the bacterial strains in the same branch in the phylogenetic trees as the same resource. With the method, the bacterial strains with different resources can be effectively and rapidly separated, so that the source tracking for lactobacillus fermenti is realized, and the phylogenetic tree relationship of the lactobacillus fermenti strains with different resources is determined.

Description

[0001] 【Technical field】 [0002] The invention relates to a rapid traceability method for Lactobacillus fermentum, in particular to a traceability method based on a phylogenetic tree constructed by a joint sequence composed of multiple housekeeping genes. [0003] 【Background technique】 [0004] Lactobacillus fermentum (Lactobacillus fermentum) belongs to the Lactobacillus genus in the Lactobacillus family, Gram-positive facultative anaerobic bacteria, and does not produce spores. It is the main heterofermentative Lactobacillus in the human intestinal tract. At present, a large number of reports indicate that Lactobacillus fermentum has the functions of hydrolyzing bile salts and lowering cholesterol. It is mostly used in soymilk fermentation and sausage fermentation, can prevent the growth of harmful bacteria, and has an important beneficial effect on the health of the host. [0005] In recent years, more and more molecular methods have been used to analyze the genetic divers...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/10
CPCC12Q1/6806C12Q1/686C12Q1/689C12Q2600/156C12Q2600/158C12Q2565/125
Inventor 刘文俊张和平丹彤陈永福孙志宏
Owner 完美(广东)日用品有限公司
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