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Metering method for rapid measurement of mould in stored grain through ATP bioluminescence method and applications

A bioluminescence, measurement method technology, applied in the direction of chemiluminescence/bioluminescence, analysis by chemical reaction of materials, etc., can solve the problem of grain storage mold without ATP bioluminescence method, so as to ensure food safety and high sensitivity , the effect of simple operation

Inactive Publication Date: 2013-10-02
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no research report on the application of ATP bioluminescence method to the detection of mold in stored grain

Method used

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  • Metering method for rapid measurement of mould in stored grain through ATP bioluminescence method and applications
  • Metering method for rapid measurement of mould in stored grain through ATP bioluminescence method and applications
  • Metering method for rapid measurement of mould in stored grain through ATP bioluminescence method and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 different ATP releasing agent releases the method and effect of ATP to mold

[0042] The methods for releasing ATP from molds by different ATP release agents and their corresponding effects will be described below.

[0043] Take 25g of corn, add it to an Erlenmeyer flask filled with 225mL of sterile water and 10 glass beads, oscillate on a shaker for 30 minutes at a speed of 140 rpm, and obtain a bacterial suspension for later use.

[0044] 1. BAC extraction method

[0045] Take 100mL of bacterial suspension for suction filtration, the filter membrane used for suction filtration is a filter membrane with a pore size of 0.22um, then transfer the filter membrane to a test tube containing 10mL of 0.15% BAC solution, and place it in a water bath at 30°C at 140 rpm. Min shaking and extracting for 3 min, the reaction solution was obtained, which was stored at 4°C until testing. 2. TCA method

[0046] Take 100mL of bacterial suspension for suction filtration, t...

Embodiment 2B

[0071] In summary, the use of surfactant BAC has the best effect on mold release of ATP. Embodiment 2 BAC is to the optimal condition of the influence of luciferin-luciferase reaction system and mold ATP extraction

[0072] 1. The effect of BAC on the luciferin-luciferase reaction system

[0073] Standard ATP (10 -7 mol / L) were diluted to 10 -8 、10 -9 、10 -10 、10 -11 mol / L ATP working solution. Prepare two reagents: Reagent A (75 μL sterile water,

[0074] as a control); Reagent B (25 μL 0.1% BAC+50 μL sterile water). 25 respectively μ Add 1 L of ATP working solution of different concentrations to 75 μL of the two reagents, take 100 μL of the mixed solution, add to 100 x In the LrL / L reagent, the luminescence value was measured after mixing, and the results are shown in Table 2.

[0075] The effect of table 20.1% BAC reagent on mold ATP bioluminescent reaction

[0076]

[0077] It can be seen from Table 2 that compared with the control group, the luminescence val...

Embodiment 3

[0088] Example 3 Correlation analysis between ATP luminescence method and traditional plate counting method and application in corn storage experiment

[0089] 1. ATP luminescence method to measure the number of molds

[0090] (1) Take 25g of corn, add it into a Erlenmeyer flask filled with 225mL of sterile water and 10 glass beads, shake it on a shaker for 30min at a speed of 140rpm, and obtain a bacterial suspension for later use.

[0091] (2) Take 100mL of bacterial suspension for suction filtration. The filter membrane used for suction filtration is a filter membrane with a pore size of 0.22um. Rotate / min shake and extract for 3 minutes to obtain the reaction solution, which is to be tested;

[0092] (3) Add 100 μL of the reaction solution in step (2) to 100 μL rL / L reagent, use the bioluminescence module of the multi-functional microplate reader TECANINFINITE200, set the measurement temperature to 30°C, and measure the luminescence value.

[0093] 2. Correlation analysi...

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Abstract

The invention relates to a metering method for rapid measurement of mould in stored grain through the ATP bioluminescence method. The metering method comprises the following steps. (1) 25 g grain and 10-20 glass beads are put into 225 mL sterile water and oscillated to obtain a bacterial suspension, standby. (2) 50-200 mL of the bacterial suspension is subjected to suction filtration through filter membranes with a pore size of 0.22 mum, the filter membranes are then transferred to a BAC solution which is 0.1 times the bacterial suspension in volume, the BAC solution is oscillated to extract ATP, and a reaction solution to be measured is obtained. (3) 100 muL of the reaction solution from step (2) is added into 100 muL of the rL / L reagent and mixed evenly, and luminescence values are measured. The metering method does not need cultivation processes to measure mould quantity through the ATP bioluminescence method and has simple operations and high sensitivity. Measurement results can be obtained in short time, and the metering method has obvious advantages compared with other measurement methods for microorganisms.

Description

technical field [0001] The invention relates to the technical field of mold detection, and relates to a metering method for rapidly detecting mold in stored grain by an ATP bioluminescence method and an application thereof. Background technique [0002] ATP is an abbreviation for the chemical substance adenosine triphosphate, which is present in all living organisms (from microorganisms to higher animals and plants). The main function of ATP is to provide energy in biological cells. At present, a large number of experiments have clearly confirmed that there is a correlation between the concentration of ATP and the concentration of microorganisms, and the actual situation of the concentration of microorganisms can be reflected by measuring the concentration of ATP. [0003] From a quantitative point of view, the detection of mold in stored grains refers to the determination of the number of molds in stored grains. From a qualitative point of view, it monitors the activity st...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/76
Inventor 李平兰丛苑卢彦廷杜奕君韩玉竹梁志宏
Owner CHINA AGRI UNIV
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