Method for screening and/or identifying lactobacillus and application of method

A Lactobacillus and subspecies technology, applied in the field of microorganisms, can solve the problems of low resolution of 16S rRNA gene, affect the industrial application of Lactobacillus, cannot meet the level of Lactobacillus species and subspecies, etc., and achieve the effect of high resolution and accuracy.

Inactive Publication Date: 2019-07-05
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] However, the 16S rRNA gene still has the limitation of low resolution in distinguishing different species of Lactobacillus. Therefore, this method cannot satisfy the detection of

Method used

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  • Method for screening and/or identifying lactobacillus and application of method
  • Method for screening and/or identifying lactobacillus and application of method
  • Method for screening and/or identifying lactobacillus and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: Construction of a kind of screening and / or the method for identification lactobacillus

[0038] Specific steps are as follows:

[0039] (1) Obtain the nucleotide sequence (total 118) of Lactobacillus groEL gene from gene databases such as NCBI (National Center for Biotechnology Information) and EMBL (European Molecular Biology Laboratory), construct the phylogenetic tree by MEGA software (such as figure 1 shown), by figure 1 It can be seen that the Lactobacillus groEL gene can well distinguish different species of Lactobacillus, therefore, this gene can be used for identification and detection of different species of Lactobacillus;

[0040](2) Download the nucleotide sequences of all known Lactobacillus groEL genes from gene databases such as NCBI (National Center for Biotechnology Information) and EMBL (European Molecular Biology Laboratory) (118 in total, including SEQ ID NO:1, SEQ ID NO:1, ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, S...

Embodiment 2

[0047] Example 2: Verification of a method for screening and / or identifying lactobacilli

[0048] In order to verify the accuracy of a method for screening and / or identifying lactobacilli obtained in Example 1, the present embodiment designed the following experiments:

[0049] Lactobacillus bulgaricus, Lactobacillus fermentum, Lactobacillus casei, Lactobacillus gasseri, Lactobacillus crispatus, Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus rhamnosus and Lactobacillus plantarum, a total of 10 kinds of milk Bacilli are mixed according to the live bacteria ratio of 0.01:0.05:0.1:0.5:1:2:5:10:20:50 to obtain a sample to be screened or identified; according to a screening and / or identification of Lactobacillus obtained in Example 1 The method for testing the sample to be screened or identified obtained in (1), the test results are as follows: figure 2 .

[0050] Depend on figure 2 It can be seen that the content of the lactobacillus...

Embodiment 3

[0051] Example 3: Detection of different species of Lactobacillus in human feces samples

[0052] Specific steps are as follows:

[0053] Step 1: Take 6 parts of human feces samples, extract the microbial genome DNA in the human feces samples (the DNA extraction kit of U.S. MPBiomedicals company is carried out), as shown in SEQ ID NO.11, SEQ ID NO.12 with nucleotide sequence The sequencing primers used were PCR primers, and the extracted microbial genomic DNA was subjected to PCR amplification, and the amplified products were collected, and the amplified products were purified using a QIAquick gel recovery kit to obtain gene fragments (gene fragments) with a size of about 400 to 600 bp. For electrophoresis results, see image 3 , this electrophoresis uses a 10Kbp DNA ladder purchased from Thermo Fisher as a Marker);

[0054] Step 2: Refer to the operating instructions of the Illumina Miseq sequencing platform, use the Varioskan LUX multi-functional microplate reader from The...

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Abstract

The invention discloses a method for screening and/or identifying lactobacillus and application of the method and belongs to the technical field of microorganisms. Compared with a traditional 16S rRNAmethod, the method has the advantages that the method is based on the specific primer of a lactobacillus groEL gene, so that the method can identify strains to the lactobacillus strain and subspecieslevel and is high in resolution and accuracy, and the accuracy of the method can reach up to 100%; by the method coordinated with a 16S rRNAV3-V4 region species diversity analysis method, the percentage of the number of each species and subspecies of lactobacillus in a complex sample accounting for the number of the total lactobacillus in the complex sample and the percentage of the number of each species and subspecies of the lactobacillus in the complex sample accounting for the number of the total bacteria in the complex sample can be measured accurately, and the accuracy reaches up to 100%.

Description

technical field [0001] The invention relates to a method for screening and / or identifying lactobacilli and an application thereof, belonging to the technical field of microorganisms. Background technique [0002] Lactobacillus, as an important probiotic that exists in the intestinal tract of humans and some other animals, can not only inhibit the growth of pathogenic bacteria in the intestinal tract to regulate and improve the intestinal flora, but also improve lactose intolerance, lower serum cholesterol, and promote immunity. and antioxidant effects. In addition, a large number of microecological studies at home and abroad have shown that Lactobacillus has various functions such as antibacterial, anti-cancer, and nutritional supplementation. Therefore, Lactobacillus has very important applications in food, medicine and other fields, and has attracted the attention of experts and scholars in industry and academia. [0003] As the application of Lactobacillus in various fi...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/04C12N15/11C12R1/225
CPCC12Q1/689
Inventor 陆文伟陈卫翟齐啸潘明罗谢妙琦崔树茂赵建新张灏
Owner JIANGNAN UNIV
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