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A specific molecular marker dna sequence of plant lactobacillus and its application

A technology of Lactobacillus plantarum and molecular markers, which is applied in the field of specific molecular marker DNA sequences of Lactobacillus plantarum, can solve the problems of inconvenient molecular detection of Lactobacillus plantarum, and achieve the effects of low cost, strong specificity and simple operation

Active Publication Date: 2016-09-21
BRIGHT DAIRY & FOOD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is to provide a specific molecular marker DNA sequence of Lactobacillus plantarum and its use aiming at the inconvenient current situation of carrying out molecular detection of Lactobacillus plantarum.

Method used

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  • A specific molecular marker dna sequence of plant lactobacillus and its application
  • A specific molecular marker dna sequence of plant lactobacillus and its application
  • A specific molecular marker dna sequence of plant lactobacillus and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The screening of the specificity PCR band of embodiment 1 different lactobacillus plantarum strains

[0043] (1) Template preparation

[0044] 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.).

[0045] (2) PCR amplification

[0046] 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.

[0047] The PCR amplification program is: ①95°C, 5min; ②95°C, 30s; ③50°C, 30s; ④72°C, 2min;

[0048] (3) Acquisition of specific ban...

Embodiment 2

[0051] The acquisition of the specificity PCR band of embodiment 2 different lactobacillus plantarum strains

[0052] (1) Template preparation

[0053] 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.).

[0054] (2) PCR amplification

[0055] 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.

[0056] The PCR amplification program is: ① 95°C, 4min; ② 95°C, 20s; ③ 50°C, 20s; ④ 72°C, 60s;

[0057] (3) Acquisition of specific bands

[0058] The PCR product w...

Embodiment 3

[0060] The acquisition of the specific PCR band of embodiment 3 Lactobacillus plantarum strains

[0061] (1) Template preparation

[0062] 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.).

[0063] (2) PCR amplification

[0064] 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.

[0065] The PCR amplification program is: ① 96°C, 4min; ② 93°C, 40s; ③ 45°C, 40s; ④ 70°C, 120s;

[0066] (3) Acquisition of specific bands

[0067] The PCR product was...

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Abstract

The invention discloses a specific molecular marker DNA sequence of lactobacillus plantarum, application of the specific molecular marker DNA sequence and a detection method of the lactobacillus plantarum. The specific molecular marker DNA sequence is shown in the SEQ ID NO.6. The detection method includes the following steps that (1) genomic DNA of a sample to be detected is extracted; (2) single-primer PCR amplification is conducted by taking the genomic DNA obtained in the step (1) as a template and taking a semi-random primer shown in the SEQ ID NO.1 as an amplification primer; (3) clone sequencing is conducted on amplification products obtained in the step (2), the sequencing result is compared with the sequence shown in the SEQ ID NO.6, and if the homologous degree is larger than 99%, it is indicated that the sample to be detected contains the lactobacillus plantarum.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a specific molecular marker DNA sequence of Lactobacillus plantarum and its application. Background technique [0002] To detect a bacterium at the molecular level, it is necessary to know the specific sequence of the bacterium, and to obtain the specific sequence of a bacterium often requires analysis and comparison of a large number of known sequences on the Internet; Less, it is very difficult to analyze, and it is difficult to obtain its 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 is expensive. [0003] Lactobacillus plantarum (Lactobacillus plantarum) is a kind of probiotics, which is widely used in various functional foods, especially in the development and research of dairy products, and has been paid more and more attention. At present, there are few sequence...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/25
CPCC12Q1/689
Inventor 张红发任婧郭本恒刘振民
Owner BRIGHT DAIRY & FOOD CO LTD
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