Specific primers for PCR rapid detection of lactobacillus helveticus and method

A technology based on specific primers and lactobacilli, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection. and other problems, to achieve the effect of fast detection speed, good specificity, and reduced detection cost

Inactive Publication Date: 2017-03-08
BAOLINGBAO BIOLOGY
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the detection method of Lactobacillus helveticus is mainly the traditional enrichment culture-pure separation culture-biochemical identification. The disadvantage of this method is that the operation is cumbersome, the detection time is long, and there are many items for biochemical identification.
With the improv

Method used

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  • Specific primers for PCR rapid detection of lactobacillus helveticus and method

Examples

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Example Embodiment

[0026] Example 1

[0027] First, aseptically take a sample of 1g of yogurt to be tested, add it to a sterile container containing 9ml of MRS broth, incubate at 36°C±1°C under anaerobic conditions for 48h. Take the cultured bacterial solution and add it to a centrifuge tube, centrifuge at 12000 rpm for 1 min, discard the supernatant, extract genomic DNA using bacterial pellet and dilute it for later use.

[0028] Then carry out the PCR reaction. The total volume of each PCR reaction is 25μl, including the extracted genomic DNA, 2μl; 10×PCR Buffer, 2.5μl; Mg 2+ 1.5mmol / L; Taq enzyme 1u; dNTP 0.1mmol / L; Lactobacillus helveticus specific primers each 10pmol; add ddH at the end 2 0 to 25μl; the program parameters for setting the PCR instrument are: denaturation at 95°C for 5min; 95°C for 30sec, 53°C for 30s, 72°C for 40sec, the reaction is carried out 35 cycles; 72°C for extension 5min, and stored at 4°C. The specific primers for Lactobacillus helveticus are:

[0029] L.helv-F: 5’-TTCGAA...

Example Embodiment

[0032] Example 2

[0033] First, aseptically take 1g of tomato sauce sample (containing Lactobacillus helveticus), add it to a sterile container containing 9ml of MRS broth medium, and culture at 36°C±1°C for 48h under anaerobic conditions. Take the cultured bacterial solution and add it to a centrifuge tube, centrifuge at 12000 rpm for 1 min, discard the supernatant, extract genomic DNA using bacterial pellet and dilute it for later use.

[0034] Then carry out the PCR reaction. The total volume of each PCR reaction is 25μl, including the extracted genomic DNA, 2μl; 10×PCR Buffer, 2.5μl; Mg 2+ Concentration 1.5mmol / L; Taq enzyme 1U; dNTP 0.1mmol / L; Lactobacillus helveticus specific primers each 10pmol; add ddH at the end 2 O to 25μl;

[0035] Set the program parameters of the PCR machine as follows: 95°C pre-denaturation 5min; 95°C denaturation 30sec, 53°C annealing 30s, 72°C extension 40sec, 35 cycles; 72°C final extension 5min, 4°C storage.

[0036] The specific primers for Lactoba...

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Abstract

The invention relates to specific primers for PCR rapid detection of lactobacillus helveticus and a method. The primers are in a pair, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO.1, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.2. The invention also relates to the method for PCR rapid detection of the lactobacillus helveticus. The specific primers and the method have the advantages that the specific primers for PCR rapid detection of the lactobacillus helveticus are obtained for the first time by adopting the non-coding sequence of the genome of the lactobacillus helveticus, and due to less selective pressure of the non-coding sequence in the genome evolution and poor conservation among close species, the specificity of amplified products is better and the accuracy is higher.

Description

technical field [0001] The invention relates to a specific primer and method for rapid detection of Lactobacillus helveticus by PCR, belonging to the technical field of biotechnology. Background technique [0002] Lactobacillus helveticus is a common lactic acid bacteria, mainly used in the production of cheese, yogurt, feed additives and other products. Lactobacillus helveticus is also an important probiotic. It is a kind of micro-ecological preparation strain that has risen rapidly at home and abroad in recent years. It is widely used in medical, health care, food, animal husbandry and aquatic products and other industries. Because of its advantages of regulating intestinal dysfunction, maintaining the balance of intestinal flora, improving the health of the body, and avoiding problems such as drug resistance and double infection caused by taking antibiotics, it has always attracted the attention of the world. In recent years, research on Lactobacillus helveticus in ferme...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/225
CPCC12Q1/689C12Q1/686C12Q2531/113C12Q2565/125
Inventor 刘云国应汉杰李克文崔志军尹郑张倩
Owner BAOLINGBAO BIOLOGY
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