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Rapid Qualitative and Quantitative Determination of Saccharomyces cerevisiae in Additive Premix Samples

A technology for the qualitative determination of Saccharomyces cerevisiae, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, and material stimulation analysis. The effect of short cycle, good accuracy and high detection efficiency

Active Publication Date: 2011-12-28
BEIJING DABEINONG TECH GRP CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional methods are easily affected by external environmental factors and culture performance, and the test results lack stability and reliability, and are time-consuming and labor-intensive.

Method used

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  • Rapid Qualitative and Quantitative Determination of Saccharomyces cerevisiae in Additive Premix Samples
  • Rapid Qualitative and Quantitative Determination of Saccharomyces cerevisiae in Additive Premix Samples
  • Rapid Qualitative and Quantitative Determination of Saccharomyces cerevisiae in Additive Premix Samples

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Rapid qualitative detection of Saccharomyces cerevisiae in premix samples:

[0056] This embodiment uses the gel recovery product of the purpose amplified fragment as a positive control; Sterile water is used as a negative control; In addition, two parts of feed samples 1# and 2#, Enterococcus faecalis, and Pichia pastoris collected from the market are used as samples to carry out .

[0057] A. Preparation of Template DNA

[0058] The template DNA of the feed sample was prepared by the glass bead method, and the specific steps were as follows:

[0059] (1) Take 1 g feed sample containing Saccharomyces cerevisiae in a 1.5 mL centrifuge tube, add 500 μL PBS to suspend the sample, and centrifuge at 2500 × g for 3 min;

[0060] (2) Discard the supernatant, add an appropriate amount of acid-washed glass beads, and shake vigorously for 2 minutes;

[0061] (3) Add 400 μL TE and an equal volume of Tris-saturated phenol, invert and mix well, and centrifuge at 15,00...

Embodiment 2

[0075] Example 2: Quantitative determination of Saccharomyces cerevisiae in premix samples

[0076] Quantitative determination of Saccharomyces cerevisiae was carried out on the different samples mentioned in Example 1. Its determination steps are as follows:

[0077] A. Preparation of total sample DNA

[0078] Use enzymatic method to break the yeast cell wall and extract the total DNA of the sample with the high-purity total DNA rapid extraction kit (spin column type): add 0.5-1.0 g of the sample to a 1.5 mL centrifuge tube, add 600 μL of sorbitol buffer, add 50 U of Lyticase, and mix well Treat at 30°C for 30 minutes, centrifuge at 1500×g for 10 minutes, discard the supernatant, collect the precipitate, extract the sample DNA according to the instructions of the high-purity total DNA rapid extraction kit, measure the concentration with a UV spectrophotometer, and detect it by 1% agarose gel electrophoresis The integrity of the DNA was then stored at -20°C.

[0079] B. Flu...

Embodiment 3

[0089] Example 3: Quantitative determination of Saccharomyces cerevisiae in feed samples

[0090] Quantitative determination of Saccharomyces cerevisiae was carried out on the different samples mentioned in Example 1. Its determination steps are as follows:

[0091] A. Preparation of sample total RNA

[0092] Use enzymatic method to break the yeast cell wall and extract total RNA with high-purity total RNA rapid extraction kit (spin column type): add 0.5-1.0 g of sample to a 1.5 mL centrifuge tube, add 600 μL of sorbitol buffer, add 50 U of Lyticase, and mix well Treat at 30°C for 30 minutes, centrifuge at 1500×g for 10 minutes, discard the supernatant, collect the precipitate, extract the sample RNA according to the instructions of the high-purity total RNA rapid extraction kit, and then treat it twice with DNaseI to ensure that the DNA contamination in the RNA is completely eliminated. For the purified RNA sample, use a UV spectrophotometer to measure the concentration, us...

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Abstract

The invention belongs to the detection technical fields of feed science and feed additives, and more specifically relates to a rapid qualification and quantitation measurement method of saccharomyces cerevisiae in an additive premix sample. According the invention, cDNA gradient dilution is taken as an external standard substance used for distinguishing dead bacteria and live bacteria for accurately detecting the amount of the live bacteria in a sample to be measured. The invention is capable of measuring saccharomyces cerevisiae in premix sample with simple, rapid and accurate qualification and quantitation without pure culture of saacharomyces cereisiae, the whole process has the advantages of simple detection process, high detection efficiency, high accuracy and short detection period; and the invention provides a guarantee a long term development for a microecological preparation industry.

Description

technical field [0001] The invention belongs to the technical field of feed science and feed additive detection, and in particular relates to a rapid qualitative and quantitative determination method for Saccharomyces cerevisiae in additive premix samples. Background technique [0002] Micro-ecological feed additives have become a hot spot in the field of premixes because of their rich nutritional value, special probiotic effects, and environmental protection characteristics. There are various types of micro-ecological feed additives on the market, and they are increasing at a high rate every year. Although the application of micro-ecological feed additives in animal husbandry is becoming more and more widespread, unified quality standards and perfect management mechanisms have not yet been formed, and the supervision and certification of their quality, especially in the qualitative and quantitative testing, lacks scientific, reasonable and rapid methods. method, the reason...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/06C12Q1/04G01N21/64
Inventor 周娜刘滢刘鹏王安如彭子欣
Owner BEIJING DABEINONG TECH GRP CO LTD
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