Method for quantifying viable bacteria of flora and application of method

A technology of flora and live bacteria, applied in the field of microbial detection, can solve the problem of inaccurate distinction between dead and live bacteria

Active Publication Date: 2021-06-29
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

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Problems solved by technology

[0003] The purpose of the present invention is to provide a method for quantifying live bacteria used in human int

Method used

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  • Method for quantifying viable bacteria of flora and application of method
  • Method for quantifying viable bacteria of flora and application of method
  • Method for quantifying viable bacteria of flora and application of method

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Experimental program
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preparation example Construction

[0024] Preparation of the flora

[0025] Dissolve 100-200g of feces collected from healthy donors in 750mL-1L of 0.9% NaCl solution, and process them through the feces analysis pretreatment instrument TG-01 (patent number CN201930312740.2) and supporting consumables produced by Chengge Biotechnology Co., Ltd. Remove the residue to obtain 30-40g of fecal bacteria;

[0026] Dilute 10 times, 100 times, 500 times, 1000 times with PBS buffer containing 0.05% L-cysteine ​​hydrochloride, and sterilize at 115°C for 10 minutes to prepare heat-inactivated fecal bacteria suspension, respectively Pipette 500 μL into a 1.5ml centrifuge tube, perform PMA treatment according to step 1.2.1, and use no PMA treatment as a control, then use the kit method (QIAamp Fast DNAStool Mini Kit) to extract genomic DNA, perform qPCR detection, and use the Ct value as indicators to analyze the results.

[0027] See the experimental results figure 1 ,Depend on figure 1 It can be seen that in the undilut...

Embodiment 1

[0034] The live bacteria quantitative method of flora comprises the following steps:

[0035] Prepare the flora, dissolve the collected samples in 0.9% NaCl solution, and remove the residue through the stool analysis pretreatment instrument TG-01 (patent number CN201930312740.2) produced by Chengge Biotechnology Co., Ltd. and supporting consumables to obtain the flora , samples include feces, soil, silt, etc.;

[0036] Preparation of PMA stock solution: Dissolve PMA in ddH 2 O, the PMA mother solution with a solubility of 2mM was obtained, and the PMA mother solution was stored in the dark at -20°C;

[0037] Preparation of PMA bacterial suspension: Take part of the bacterial population, dilute it 100 times with PBS buffer containing 0.05% L-cysteine ​​hydrochloride, add PMA to make the final concentration of the system 50 μM, and mix well to obtain the PMA bacterial suspension ;

[0038]Treatment of PMA bacterial suspension: incubate the obtained PMA bacterial suspension at...

Embodiment 2

[0040] Use the excrement bacterium that embodiment 1 obtains;

[0041] Preparation of PMA stock solution: Dissolve PMA in ddH 2 O, the PMA mother solution with a solubility of 2mM was obtained, and the PMA mother solution was stored in the dark at -20°C;

[0042] Preparation of PMA bacterial suspension: Take part of the bacterial population, dilute it 100 times with PBS buffer containing 0.05% L-cysteine ​​hydrochloride, add PMA to make the final concentration of the system 50 μM, and mix well to obtain the PMA bacterial suspension ;

[0043] PMA bacterial suspension treatment: incubate the obtained PMA bacterial suspension at room temperature in the dark for 30 minutes, then place the sample on ice, use a 500w halogen lamp to illuminate for 20 minutes at a distance of 20cm, and rotate at 5000r / min at 4°C after illumination Centrifuge for 3-5min, collect the cells for DNA extraction, and perform qPCR detection and analysis.

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Abstract

The invention aims to provide a method for quantifying viable bacteria of a flora and application of the method. The method comprises the following steps: flora preparation: dissolving the flora in a 0.9% NaCl solution, and treating by an analysis pretreatment instrument and matched consumables to remove residues to obtain the flora; and flora PMA treatment: diluting the obtained coprophilous fungi with a PBS buffer solution containing 0.05% L-cysteine hydrochloride, then adding PMA to enable the final concentration of the system to be 30-80 [mu]M, then carrying out a dark reaction for 30 min, then carrying out illumination for 20 min, then extracting genome DNA, and carrying out qPCR detection analysis. According to the method provided by the invention, the accuracy of distinguishing dead bacteria and viable bacteria in the PMA-qPCR method is ensured by limiting the turbidity of coprophilous bacteria, the solubility of PMA and the illumination time, the problem that the dead bacteria and viable bacteria are not accurately distinguished is solved, and the method is used for quantifying the viable bacteria of human intestinal flora.

Description

technical field [0001] The invention relates to the field of microorganism detection, in particular to a method for quantifying live bacteria of flora and its application. Background technique [0002] PMA (propidium monoazide) is a photosensitive reactive dye with high affinity for nucleic acids, which can enter dead bacterial cells with incomplete cell walls or membranes and selectively cross-link dead bacterial DNA. Combining it with DNA amplification detection technology can effectively inhibit the amplification of dead cell DNA, thereby realizing the detection of live bacteria. At present, this method is mostly used for the quantification of live bacteria of certain bacteria in single bacteria or mixed microorganisms, as disclosed in the patent CN103509860A a kind of quantitative detection method for Escherichia coli O157:H7 live bacteria in food; disclosed in the patent CN102816850B A method for simultaneous detection of Salmonella typhimurium, Escherichia coli O157:H...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686
CPCC12Q1/689C12Q1/686C12Q2561/113C12Q2563/107C12Q2545/114Y02A50/30
Inventor 张帮周肖传兴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
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