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A specific molecular marker dna sequence of Lactobacillus casei and its use

A Lactobacillus casei, molecular marker technology, applied in the directions of recombinant DNA technology, DNA/RNA fragments, microorganism-based methods, etc., can solve the problems of inconvenient molecular detection of Lactobacillus casei, and achieve low cost, short experimental time, and specificity. strong effect

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

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

Method used

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  • A specific molecular marker dna sequence of Lactobacillus casei and its use
  • A specific molecular marker dna sequence of Lactobacillus casei and its use
  • A specific molecular marker dna sequence of Lactobacillus casei and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The screening of the specific PCR band of different Lactobacillus casei strains of embodiment 1

[0041] (1) Template preparation

[0042] 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: TaKaRa minibestbacterial genomic DNA extraction kit ver.2.0 (Takara Biotechnology (Dalian) Co., Ltd.).

[0043] (2) PCR amplification

[0044] 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 2ng / μL genomic DNA template in a total volume of 50 μL.

[0045] The PCR amplification program is: ①95°C, 5min; ②95°C, 30s; ③50°C, 30s; ④72°C, 2min;

[0046] (3) Acquisition of specific bands

[0047] The PCR product was ele...

Embodiment 2

[0049] The acquisition of the specificity PCR band of embodiment 2 different Lactobacillus casei bacterial strains

[0050] (1) Template preparation

[0051] 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.).

[0052] (2) PCR amplification

[0053] 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.

[0054] The PCR amplification program is: ① 95°C, 4min; ② 95°C, 20s; ③ 50°C, 20s; ④ 72°C, 60s;

[0055] (3) Acquisition of specific bands

[0056] The PCR pro...

Embodiment 3

[0058] The acquisition of the specific PCR band of embodiment 3 Lactobacillus casei bacterial strains

[0059] (1) Template preparation

[0060] 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.).

[0061] (2) PCR amplification

[0062] 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.

[0063] The PCR amplification program is: ① 96°C, 4min; ② 93°C, 40s; ③ 45°C, 40s; ④ 70°C, 120s;

[0064] (3) Acquisition of specific bands

[0065] The PCR produ...

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PUM

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Abstract

The invention discloses a specific molecular marker DNA sequence of lactobacillus casei, application of the specific molecular marker DNA sequence and a detection method of the lactobacillus casei. The specific molecular marker DNA sequence is shown as SEQ ID NO.6. the detection method comprises the steps of 1 extracting a genome DNA of a sample to be detected, 2 using the genome DNA obtained in the step 1 as a template and using a semi-random primer represented I the SEQ ID NO.1 as an amplification primer to perform single primer PCR amplification, 3 conducting clone sequencing on an amplification product and comparing a sequencing result with the sequence represented by the SEQ ID NO.6, wherein it is shown that the sample to be detected contains the lactobacillus casei if the homology degree is higher than 99%.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a specific molecular marker DNA sequence of Lactobacillus casei 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 casei (Lactobacillus casei) 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 sequenced sequences ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/68C12Q1/04C12R1/245
Inventor 张红发任婧郭本恒刘振民
Owner BRIGHT DAIRY & FOOD CO LTD
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