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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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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 improvement of people's living standards, the demand for food safety is getting higher and higher. At the same time, the development of Lactobacillus helveticus microecological preparations and the expansion of production scale, the low efficiency of traditional detection methods can no longer meet the current batch detection tasks

Method used

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

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Experimental program
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Effect test

Embodiment 1

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

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

[0029] L.helv-F: 5'-TTCGAA...

Embodiment 2

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

[0034] Then perform PCR reaction, the total volume of each PCR reaction is 25 μl, including extracted genomic DNA, 2 μl; 10×PCR Buffer, 2.5 μl; Mg 2+ Concentration 1.5mmol / L; Taq enzyme 1U; dNTP 0.1mmol / L; Lactobacillus helveticus-specific primers each 10pmol; finally add ddH 2 0 to 25 μl;

[0035] The program parameters of the PCR instrument were set as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 sec, annealing at 53°C for 30 s, extension at 72°C for 40 sec, 35 cycles; final extension at 72°C for...

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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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