PCR (Polymerase Chain Reaction) rapid detection method for Bacillus coagulans

A Bacillus coagulans, rapid technology, applied in the directions of microorganism-based methods, microorganism determination/inspection, biochemical equipment and methods, etc., can solve the problems of long detection time, cumbersome operation, and many biochemical identification items, etc. Low, fast detection speed, the effect of reducing the detection cost

Inactive Publication Date: 2013-01-16
COFCO TUNHE
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  • Application Information

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Problems solved by technology

At present, the detection method of Bacillus coagulans is mainly the traditional enrichment culture-pure isolation 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 Bacillus coagulans 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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Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0013] First, take 1g of the sample to be tested by aseptic operation, add it into a sterilized container filled with 9ml of acidic broth (acid canned food) or bromcresol purple glucose broth (low-acid canned food and other samples to be tested), at 55 ° C ± 1 ℃, cultivated under aerobic conditions 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.

[0014] Then carry out PCR reaction, the total volume of each PCR reaction is 25 μl, including extracted genomic DNA, 2 μl; 10×PCRBuffer, 2.5 μl; Mg 2+ 1.5mmol / L; Tag enzyme 1u; dNTP 0.1mmol / L; Bacillus coagulans specific primers 10pmol each; finally add ddH 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 56°C, and 40sec at 72°C for 35 cycles; extension at 72°C for 5min, and storage a...

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Abstract

The invention relates to a PCR (Polymerase Chain Reaction) rapid detection method for Bacillus coagulans, which comprises the following steps of: first extracting the genome DNA of an enrichment liquid of a sample to be detected and diluting for later use; then designing a specific primer by using the conservative DNA sequence of the genome of the Bacillus coagulans; then carrying out PCR augmentation on the genome DNA of the enrichment liquid of the sample to be detected by using the primer and carrying out agarose gel electrophoresis detection on a PCR product. The loading parameters are that the total reaction volume of each PCR is 25 microlitres, including 2 microlitres of genome DNA of the enrichment liquid of the sample to be detected, 2.5 microlitres of 10*PCR Buffer, 1.5mmol / L of Mg2+, 1 microlitre of Tag enzyme, 0.1mmol / L of dNT, 10pmol of specific primer according to claim 1 and finally 25 microlitres of ddH2I; the PCR reaction program comprises degenerating at 95 DEG C for 5min, 95 DEG C for 30s, 56 DEG for 30s and 72 DEG C for 40s. The reaction comprises 35 circulations, extension at 72 DEG C for 5min and conservation at 4 DEG C. The invention has the advantages of rapid detection speed and low cost.

Description

technical field [0001] The invention relates to a PCR rapid detection method for bacillus coagulans. Background technique [0002] Microorganisms that cause canned food to become rancid but not fat (that is, produce acid but not gas) are called flat acid bacteria in the canning industry. It is a group of high-temperature types in the family of Aerobic Bacillus, with the characteristics of thermophile and heat resistance, and its suitable growth temperature is 45°C to 60°C. Bacillus coagulans is an important microorganism that causes the rancidity of canned food. Bacillus coagulans is also a probiotic. It is a kind of microecological preparation that has risen rapidly at home and abroad in recent years. It is widely used in medical treatment, 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 ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12R1/07
Inventor 刘云国丁生林徐榕蔓宋佳
Owner COFCO TUNHE
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