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Method for quick quantitative determination of active bifidobacteria

A technology for quantitative detection of bifidobacteria, used in the determination/inspection of microorganisms, biochemical equipment and methods, etc.

Inactive Publication Date: 2008-12-10
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

TaqMan technology has been used to quantitatively detect Bifidobacteria in fecal samples, and real-time PCR methods based on SYBR Green I have also been used for the detection of Bifidobacteria species or genus, but no molecular beacon method has been applied to detect Bifidobacteria bacillus report

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  • Method for quick quantitative determination of active bifidobacteria
  • Method for quick quantitative determination of active bifidobacteria
  • Method for quick quantitative determination of active bifidobacteria

Examples

Experimental program
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Embodiment 1

[0119] A method for rapid quantitative detection of living bifidobacteria. After sampling, the sample is diluted 10 times for EMA treatment, then the DNA of the sample is extracted, and the molecular beacon-real-time PCR detection is used to calculate the content of bifidobacteria according to the standard curve. The DNA extraction process described is: add 18% sodium citrate and 1M NaOH to the bifidobacterium-containing products after EMA treatment, centrifuge at 10000rpm for 10min, collect bacterial cells, wash with sterile ultrapure water, and suspend in sterile Take the bacterial suspension in ultrapure water, add it to the Tritox-100 solution with a concentration of 2%, heat it at 100°C for 10 minutes, and immediately place it in ice water for rapid cooling, and the obtained supernatant is directly used for molecular beacon-real-time PCR amplification, the parts by weight of the bifidobacterium-containing product is 1, the parts by weight of the sodium citrate is 0.15, the...

Embodiment 2

[0121] The method for rapid quantitative detection of living bifidobacteria described in embodiment 1, described sampling, aseptic operation will fully mix the sample containing bifidobacteria products into a sterilized Erlenmeyer flask containing PBS buffer Make a uniform dilution of 1:10, and let it stand at room temperature for 30 minutes to hydrate.

Embodiment 3

[0123] The method for rapid quantitative detection of living bifidobacteria described in embodiment 1 or 2, the described EMA treatment sample, get the sample diluent that contains bifidobacterium products that mixes homogeneously, add concentration and be 1mg / mLEMA stock solution, Mix well, react in the dark for 5 minutes, and irradiate with a 650W halogen lamp for 2 minutes. The sample is 20 cm away from the light source. The operation is carried out on ice. Each sample is made in two parallels. The weight part of the sample diluent of the bifidobacterium product The number is 1, and the parts by weight of the EMA stock solution is 0.0015.

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Abstract

The invention discloses a method for quickly and quantificationally detecting live bifidobacterium and relates to a quick and quantificational detection method for the live bifidobacterium in products containing bifidobacterium. A detection technology of the bifidobacterium is divided into the prior detection with plate bacterial colony counting as a base and various detections with molecular biology as a base. The method comprises the following steps that: a sample is taken, is diluted up to 10 times, and is subjected to EMA treatment, DNA of the sample is extracted, the molecular beacon-real-time PCR detection is performed, the content of the bifidobacterium is calculated according to a standard curve, wherein, the extraction process of the DNA is that: the sample which is subjected to the EMA treatment is added with 18 percent sodium citrate and 1M NaOH and is centrifugated for 10min at a rotating speed of 10,000rpm, somatic cells are collected and are washed by ultrapure water, a bacterial suspension is taken, is added into a Tritox-100 liquid with a concentration of 2 percent, is subjected to water bath treatment for 10min at a temperature of 100 DEG C, and then is cooled immediately, and supernate is used for molecular beacon-real-time PCR amplification. The method is used for quantitative detection of the live bifidobacterium in the products containing the bifidobacterium.

Description

Technical field: [0001] The invention relates to a rapid quantitative detection method for living bifidobacteria in products containing bifidobacteria. Background technique: [0002] Bifidobacterium (Bifidobacterium) is one of the most important physiological bacteria in the intestinal tract of humans and animals. Since the discovery of Bifidobacteria by Dr. Tissier of the Pasteur Institute in France in 1989, people have learned from the morphology of Bifidobacteria It has been widely studied in many aspects such as physiology, taxonomy and genetics, microecology, physiological functions and applications. Bifidobacterium is a kind of Gram-positive obligate anaerobic bacteria that exists in the digestive tract of humans and animals. It is the dominant intestinal flora and is of great significance to maintaining the health of the body. Based on the current literature reports, bifidobacteria can maintain the balance of intestinal flora, stimulate the immune function of the bod...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/06
Inventor 孟祥晨庞睿王超
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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