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Method for obtaining specific sequence of Lactobacillus plantarum and semi-random primers used therefor

A semi-random primer, Lactobacillus plantarum technology, applied in DNA preparation, DNA/RNA fragments, recombinant DNA technology, etc. Low cost and high versatility

Active Publication Date: 2017-06-06
BRIGHT DAIRY & FOOD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The technical problem to be solved by the present invention is that it is difficult to obtain the specific sequence of Lactobacillus plantarum through sequence comparison at present, and it is impossible to directly obtain the specific sequence of the existing Lactobacillus plantarum strain that has not undergone whole genome sequencing. present situation, but provide a kind of method that can obtain the specific sequence of Lactobacillus plantarum conveniently and semi-random primers used without sequencing unsequenced Lactobacillus plantarum strains

Method used

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  • Method for obtaining specific sequence of Lactobacillus plantarum and semi-random primers used therefor
  • Method for obtaining specific sequence of Lactobacillus plantarum and semi-random primers used therefor
  • Method for obtaining specific sequence of Lactobacillus plantarum and semi-random primers used therefor

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] The screening of the specificity PCR band of embodiment 1 different lactobacillus plantarum strains

[0046] (1) Template preparation

[0047] Each strain was activated, isolated and cultivated, and a single colony was selected from the obtained colonies, inoculated in 1ml of MRS medium, cultured anaerobically at 37°C for 24 hours, and centrifuged to obtain bacterial cells. The bacterial genome was extracted using a kit: TaKaRaminibest bacterial genomic DNA extraction kit ver.2.0 (Takara Biotechnology (Dalian) Co., Ltd.).

[0048] (2) PCR amplification

[0049] The system composition of PCR amplification is: 1 μ mol / L semi-random primer (its sequence is shown in SEQ ID NO.1), 0.5mmol / L dNTP, the Mg of 1.5mmol / L 2+ , 0.05U / μL Taq DNA polymerase, and 500ng (please express in concentration) genomic DNA template, the total volume is 50μL.

[0050] The PCR amplification program is: ①95°C, 5min; ②95°C, 30s; ③50°C, 30s; ④72°C, 2min;

[0051] (3) Acquisition of specific ban...

Embodiment 2

[0054] The acquisition of the specificity PCR band of embodiment 2 different lactobacillus plantarum strains

[0055] (1) Template preparation

[0056] Each strain was activated, isolated and cultivated, and a single colony was selected from the obtained colonies, inoculated in 1ml of MRS medium, cultured anaerobically at 37°C for 24 hours, and centrifuged to obtain bacterial cells. The bacterial genome was extracted using a kit: TaKaRaminibest bacterial genomic DNA extraction kit ver.2.0 (Takara Biotechnology (Dalian) Co., Ltd.).

[0057] (2) PCR amplification

[0058] The system composition of PCR amplification is: 2 μmol / L semi-random primer (its sequence is shown in SEQ ID NO.1), 1mmol / L dNTP, the Mg of 2.5mmol / L 2+ , 0.10U / μL Taq DNA polymerase, and 2ng / μL genomic DNA template in a total volume of 50 μL.

[0059] The PCR amplification program is: ① 95°C, 4min; ② 95°C, 20s; ③ 50°C, 20s; ④ 72°C, 60s;

[0060] (3) Acquisition of specific bands

[0061] The PCR product w...

Embodiment 3

[0063] The acquisition of the specific PCR band of embodiment 3 Lactobacillus plantarum strains

[0064] (1) Template preparation

[0065] The strains were activated, isolated and cultivated, and a single colony was selected from the obtained colonies, inoculated in 1ml of MRS medium, cultured anaerobically at 37°C for 24 hours, and centrifuged to obtain bacterial cells. The bacterial genome was extracted using a kit: TaKaRaminibest bacterial genomic DNA extraction kit ver.2.0 (Takara Biotechnology (Dalian) Co., Ltd.).

[0066] (2) PCR amplification

[0067] The system composition of PCR amplification is: 0.5 μ mol / L semi-random primer (its sequence is shown in SEQ ID NO.1), 0.2 mmol / L dNTP, the Mg of 1.0 mmol / L 2+ , 0.02U / μL Taq DNA polymerase, and 0.5ng / μL genomic DNA template in a total volume of 50 μL.

[0068] The PCR amplification program is: ① 96°C, 4min; ② 93°C, 40s; ③ 45°C, 40s; ④ 70°C, 120s;

[0069] (3) Acquisition of specific bands

[0070] The PCR product was...

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Abstract

The invention discloses a method for obtaining a lactobacillus plantarum specific sequence and a semi-random primer adopted in the method. The primer is shown by SEQ ID NO.1. The method comprises the steps that 1, the genomic DNA of two or more lactobacillus plantarum strains is adopted as a template, and the primer shown by the SEQ ID NO.1 is used for carrying out single-primer PCR amplification; 2, a common DNA fragment is sequenced, sequence comparison is carried out, and if the result indicates that the gene homology of the sequence of the common DNA fragment and lactobacillus plantarum with a known sequence is larger than 99 percent, the sequence is adopted as the specific sequence of the lactobacillus plantarum. The method is easy and fast to operate and high in specificity, the nucleotide sequence of target bacteria does not need to be known, and a large amount of gene data analysis does not need to be depended on. The method is high in universality, low in cost and short in experimental period and has the very wide application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for obtaining the specific sequence of Lactobacillus plantarum and semi-random primers used therefor. Background technique [0002] To obtain the specific sequence of a bacterium, it is often necessary to analyze and compare a large number of known sequences on the Internet; however, some bacteria have fewer sequences, which is very difficult to analyze, and it is difficult to obtain the specific sequence. Of course, it is also possible to collect all strains of a certain bacterium for sequencing comparison, but this method takes a long time and costs a lot. At present, there are few sequenced sequences of Lactobacillus plantarum available online, so it is difficult to analyze and obtain the specific sequence of Lactobacillus plantarum. Contents of the invention [0003] The technical problem to be solved by the present invention is that it is difficult to obtain the spec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/10
Inventor 张红发任婧郭本恒刘振民
Owner BRIGHT DAIRY & FOOD CO LTD