Method for detecting and identifying Lactobacillus acidophilus NCFM

A technology of Lactobacillus acidophilus and bacteria, applied in the field of detection and identification of Lactobacillus acidophilus NCFM, can solve the problem of low product mismatch rate, and achieve the effect of low product mismatch rate and comprehensive sequencing results

Inactive Publication Date: 2018-01-26
AGRI PROD PROCESSING INST GUANGXI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with Taq DNA polymerase, the mismatch rate of products amplified by high-fidelity DNA polymerase is very low, but the products amplified by high-fidelity DNA polymerase are blunt-ended and cannot be directly used for TA cloning

Method used

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  • Method for detecting and identifying Lactobacillus acidophilus NCFM
  • Method for detecting and identifying Lactobacillus acidophilus NCFM
  • Method for detecting and identifying Lactobacillus acidophilus NCFM

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The 16S rDNA of the bacteria to be tested was amplified by high-fidelity DNA polymerase PCR and TA cloned. After sequencing, the Lactobacillus acidophilus NCFM was compared and identified.

[0035] (1) The primer sequence is:

[0036] plb16A:ACGA GACTTTGAGTC TGGCTCAG (the underline is the AhdI restriction site),

[0037] mlb16A:ACC GACGCTGCGTC CACGTAGTTAG (the underline is the AhdI restriction site),

[0038] plb16B:TTA ACTGGGAGAGT TTGATCCTGGCTCAG (the underline is the restriction site of BmrI),

[0039] mlb16B:GTA ACTGGGCGGCT GCTGGCACGTAGTTAG (the underline is the BmrI restriction site),

[0040] plb16X:ACG CCAGTTTTTATCCTGG CTCAG (the underline is the XcmI restriction site),

[0041] mlb16X:TAT CCACGCGTCTGCTGG CACGTAGTTAG (the underline is the XcmI restriction site);

[0042] Primers were entrusted to GenScript Biotechnology Company to synthesize.

[0043] (2) Extraction of genomic DNA of bacteria to be tested:

[0044] MRS broth culture: peptone 10.0g,...

Embodiment 2

[0059] The operation steps are the same as in Example 1, the difference is to use Ultra-Fidelity DNA Polymerase (NEB) Replacement Ultra-fidelity DNA polymerase. Test result is identical with embodiment 1.

Embodiment 3

[0061] The operation steps are the same as in Example 1, the difference is to use HS DNA polymerase (TAKARA) replacement Ultra-fidelity DNA polymerase. Test result is identical with embodiment 1.

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Abstract

The invention discloses a method for detecting and identifying Lactobacillus acidophilus NCFM. Fragments of to-be-detected strain 16SrDNA are subjected to PCR amplification by means of primers plb16A,mlb16A, plb16B, mlb16B, plb16X, mlb16X and high-fidelity DNA polymerase; an amplification product is digested by restriction endonucleases AhdI, BmrI and XcmI, purified and connected with a T vector;a connecting product is transformed into Escherichia coli, connexons are screened, plasmids are extracted for sequencing and comparison, and whether the detected strain is the Lactobacillus acidophilus NCFM is identified preliminarily. The method has the following advantages: the Lactobacillus acidophilus can be identified by means of the length of the amplification product; the Lactobacillus acidophilus can be identified by means of the length of the amplification product after being digested by AhdI, BmrI and XcmI; the amplification product is obtained through amplification by high-fidelityDNA polymerase, so that the mispairing rate of the amplification product is low, sequencing and comparison are performed after the amplification product is connected with the T vector, and the Lactobacillus acidophilus NCFM can be identified preliminarily by means of a result.

Description

【Technical field】 [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for detecting and identifying Lactobacillus acidophilus NCFM. 【Background technique】 [0002] Lactobacillus acidophilus (Lactobacillus acidophilus) is one of the more widely used strains of Lactobacillus, Gram-positive bacillus, the end of the rod is round, micro-aerobic, chemoorganotrophic type, complex nutritional requirements, needs growth factor. Its obvious feature is that it has high acid resistance, releases lactic acid, acetic acid and some antibiotics that act on harmful bacteria during growth, and has an antagonistic effect on pathogenic microorganisms. The optimum pH value is 5.5-6.2, and it can still grow below pH 5.0. Obligate to metabolize sugar compounds and produce more than 50% lactic acid. It is mostly distributed in dairy products, fermented plant products such as pickles, sauerkraut, silage and human intestines, especially in the intestin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869C12Q1/04C12R1/23
Inventor 廖东庆孙健李丽李昌宝零东宁周主贵何雪梅郑凤锦盛金凤刘国明李杰民辛明李志春唐雅园易萍
Owner AGRI PROD PROCESSING INST GUANGXI ACADEMY OF AGRI SCI
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