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DNA sequence of specific molecular marker of streptococcus thermophilus and use thereof

A Streptococcus thermophilus, molecular marker technology, applied in recombinant DNA technology, DNA/RNA fragments, determination/inspection of microorganisms, etc., can solve the problem of inconvenient molecular detection of Streptococcus thermophilus, and achieve low cost and experimental time. short, specific effects

Active Publication Date: 2015-04-08
BRIGHT DAIRY & FOOD
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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 Streptococcus thermophilus and its application aiming at the inconvenient current situation of molecular detection of Streptococcus thermophilus at present.

Method used

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  • DNA sequence of specific molecular marker of streptococcus thermophilus and use thereof
  • DNA sequence of specific molecular marker of streptococcus thermophilus and use thereof
  • DNA sequence of specific molecular marker of streptococcus thermophilus and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] The screening of the specific PCR band of different Streptococcus thermophilus bacterial strains of embodiment 1

[0040] (1) Template preparation

[0041] 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.).

[0042] (2) PCR amplification

[0043] 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 1ng / μL genomic DNA template, in a total volume of 50 μL.

[0044] The PCR amplification program is: ①95°C, 5min; ②95°C, 30s; ③50°C, 30s; ④72°C, 2min;

[0045] (3) Acquisition of specific bands

[0046] The P...

Embodiment 2

[0048] The acquisition of specific PCR bands of different Streptococcus thermophilus bacterial strains of embodiment 2

[0049] (1) Template preparation

[0050] 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.).

[0051] (2) PCR amplification

[0052] 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.

[0053] The PCR amplification program is: ① 95°C, 4min; ② 95°C, 20s; ③ 50°C, 20s; ④ 72°C, 60s;

[0054] (3) Acquisition of specific bands

[0055] The PCR...

Embodiment 3

[0057] The acquisition of the specific PCR band of embodiment 3 Streptococcus thermophilus bacterial strain

[0058] (1) Template preparation

[0059] 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.).

[0060] (2) PCR amplification

[0061] 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.

[0062] The PCR amplification program is: ① 96°C, 4min; ② 93°C, 40s; ③ 45°C, 40s; ④ 70°C, 120s;

[0063] (3) Acquisition of specific bands

[0064] The PCR...

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Abstract

The invention discloses a DNA sequence of a specific molecular marker of streptococcus thermophilus and use thereof as well as a detection method of streptococcus thermophilus. The DNA sequence of the specific molecular marker is shown in SEQ ID NO. 6. The detection method comprises the following steps: (1) extracting the genome DNA of a to-be-detected sample; (2) carrying out single primer PCR amplification by taking the genome DNA obtained in the step (1) as a template and a semi-random primer shown in the SEQ ID NO. 1 as an amplification primer; (3) clone-sequencing an amplification product obtained the step (2) and comparing the sequencing result with the sequence shown in the SEQ ID NO. 6; if the homologous degree is greater than 99%, showing that the to-be-detected sample contains streptococcus thermophilus.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a specific molecular marker DNA sequence of Streptococcus thermophilus 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] Streptococcus thermophilus 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 of Strep...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68C12Q1/14
Inventor 张红发任婧郭本恒刘振民
Owner BRIGHT DAIRY & FOOD
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