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Method for rapidly detecting viable count

A fast technology for the amount of viable bacteria, which can be used in measuring devices, biochemical equipment and methods, and determination/inspection of microorganisms. , the effect of accurate detection

Inactive Publication Date: 2021-08-13
厦门承葛医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a method for quickly detecting the amount of live bacteria, which solves the problems of cumbersome detection of live bacteria and inaccurate detection by simple detection methods. In order to achieve the above purpose, the present invention provides the following technical solutions :

Method used

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  • Method for rapidly detecting viable count

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Experimental program
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Effect test

Embodiment 1

[0024] (1) Accurately weighed 0.1-0.15g of volunteer A’s feces and diluted it to 10% with normal saline. 3 times, take 1.5ml samples respectively in 2ml centrifuge tubes, add 0.3U / ml apurase enzyme (9μL), and bathe in 30℃ water for 10min to enzymatically hydrolyze free ATP, and then inactivate the enzyme in 100℃waterbath for 1min (every The test samples were repeated three times, and the average value was taken).

[0025] (2) Fluorescence value measurement: take the sample in step (1) and use the ATP fluorescence detector to measure the luminous intensity, repeat (2), until the fluorescence value of the test does not change, measure the fluorescence value of the sample and record it, through the above fluorescence Value / viable bacteria standard working curve to get the viable bacteria.

Embodiment 2

[0027] (1) Accurately weighed 0.1-0.15g of volunteer B’s feces and diluted it with normal saline to 10 3 Take 1.5ml samples respectively in 2ml centrifuge tubes, add 0.3U / ml apurase enzyme (9μL), and bathe in 30℃ water for 10min to enzymatically hydrolyze free ATP, then inactivate the enzyme in 100℃waterbath for 1min. (Each test sample was repeated three times, and the average value was taken)

[0028] (2) Fluorescence value measurement: take the sample in step (1) and use the ATP fluorescence detector to measure the luminous intensity, repeat (1), until the fluorescence value of the test does not change, measure the fluorescence value of the sample and record it, through the above fluorescence Value / viable bacteria standard working curve to get the viable bacteria.

Embodiment 3

[0030] (1) Accurately weigh 0.1-0.15g of healthy feces and dilute to 10% with normal saline 3 Take 1.5ml samples respectively in 2ml centrifuge tubes, add 0.3U / ml apurase enzyme (9μL), and bathe in 30℃ water for 10min to enzymatically hydrolyze free ATP, then inactivate the enzyme in 100℃waterbath for 1min. (Each test sample was repeated three times, and the average value was taken)

[0031](2) Fluorescence value measurement: take the sample in step (1) and use the ATP fluorescence detector to measure the luminous intensity, repeat (1), until the fluorescence value of the test does not change, measure the fluorescence value of the sample and record it, through the above fluorescence Value / viable bacteria standard working curve to get the viable bacteria.

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Abstract

The invention discloses a method for rapidly detecting the viable count. The method is characterized by comprising the following steps of S1, acquiring a standard sample, and diluting a to-be-detected sample to 10<2> times, 10<3> times, 10<4> times, 10<5> times, 10<6> times, 10<7> times, 10<8> times and 10<9> times by using a buffer solution for later use; and S2, calculating bacterial colonies of the diluted standard samples of 10<2> times, 10<3> times, 10<4> times, 10<5> times, 10<6> times, 10<7> times, 10<8> times and 10<9> times by using a plate counting method. According to the method disclosed by the invention, the sample is treated by utilizing apurase enzyme, so that the measured fluorescence value can relatively and comprehensively reflect the viable bacteria amount in the flora. An ATP bioluminescence method is applied to viable bacteria detection, specifically, fecal bacteria are detected, flora in the flora transplantation process is simply, rapidly and accurately detected, quality normalization control over transplanted flora can be achieved, and a flora transplantation donor can be preliminarily screened, so that unnecessary waste of equipment and personnel funds caused by unqualified donor quality is avoided.

Description

technical field [0001] The invention relates to the field of live bacteria detection, in particular to a method for rapidly detecting the amount of live bacteria. Background technique [0002] Conventional methods for the detection of viable bacteria include culture medium method, PCR molecular detection method, PMA-qPCR method, flow cytometry method and ATP bioluminescence method, etc. The traditional medium method is to use the medium to cultivate bacteria, and then use the plate count method to count and determine the content of viable bacteria in the intestinal flora. The traditional medium method has a large linear range, can better reflect the dilution degree of the colony, and has good repeatability, but the operation is cumbersome, requires skilled operators, and takes too long; the PCR molecular detection method requires special instruments and Equipment requires special extraction of DNA from the sample, the sample pretreatment steps are cumbersome, and the operat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/06G01N21/64
CPCC12Q1/06G01N21/6486
Inventor 张帮周肖传兴袁文功李源涛陈章然林爱强何剑全
Owner 厦门承葛医学检验实验室有限公司