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Kit for anti-interference quick detection of microbe quantity by bioluminescence method

A detection kit and technology for the number of microorganisms, applied in the biological field, can solve the problems of high detection cost and unapplied use

Inactive Publication Date: 2009-08-26
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are sensitive bioluminescence instruments and high-purity detection kits in a few developed countries abroad. However, no matter whether the high-sensitivity luminescence detection instrument or the high-purity luminescence detection reagent, the price is very high, and the simple instrument is generally 5 ~100,000 yuan / unit, the slightly better price reaches 100,000-400,000 yuan / unit, and the price of the kit matching the instrument is 30-50 yuan / time. This is too high for the situation in our country, so Not used at all in the country

Method used

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  • Kit for anti-interference quick detection of microbe quantity by bioluminescence method
  • Kit for anti-interference quick detection of microbe quantity by bioluminescence method
  • Kit for anti-interference quick detection of microbe quantity by bioluminescence method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Construction of a rapid detection kit for bioluminescence method (the luminescence reagent is FL04113)

[0029] (1) Pre-preparation of reagents

[0030] Luciferase protective agent: Take 500mg trehalose, 500mg PEG6000 and 15mg BSA, dissolve in 10mL sterile distilled water, shake well and filter and sterilize through a 0.22μm sterile microporous membrane.

[0031] Standard ATP solution: 55.1mg of disodium salt A-2383 from Sigma, dissolved in 10mL of sterile distilled water to make a concentration of 1×10 -2 mol / L, packed in 1.5ml sterile centrifuge tube, 0.1ml per tube.

[0032] Non-cellular ATP remover AP: Its specific ingredients and production process are used with 50mmol / L Tris-HCl, 10mmol / LMgSO 4 , 1mmol / L EDTA, 1mg / mL BSA, pH6.8 buffer solution to prepare pyrophosphatase to a concentration of 5mg / ml, and filter through a 0.22μm sterile microporous membrane.

[0033] Microbial cell ATP extract E c : Add 20g TritonX-100, 2.0g CTAB, 2.0g DMSO, 0.05g EDTA, 0.05g...

Embodiment 2

[0040] Example 2: FL04113 ATP bioluminescence standard ATP curve

[0041] In the Shanghai Shangli Bioluminescence Apparatus SHG-C and the luminescence Apparatus HKM-NRD(1) developed by Guangdong Kai Microbiology Technology Co., Ltd., the 0.5mL system on-board, the detection standard concentration range is 10 -12 ~10 -6 ATP luminescence value of mol / L, the detection system is 0.1mL FL+0.1mL standard ATP+0.3mL GB, and a logarithmic curve and linear regression equation are made. The results are shown in Table 1 and attached. figure 1 .

[0042] It can be seen from Table 1: Perform luminescence detection on SHG-C luminometer, when ATP concentration is 10 -10 ~10 -6 The linearity is better at mol / L, the regression curve in the 0.5mL luminescence system is Lg[ATP] = -12.4792 + 1.0374Lg△CPM, R = 0.9977, n = 5; on the HKM-NRD (I) luminescence instrument, When the ATP concentration is 10 -10 ~10 -6 The linearity is better at mol / L, and the regression curve is Lg[ATP] = —10.8382+0.9547Lg△CP...

Embodiment 3

[0045] Example 3: Luminescence detection of artificial yeast samples (SHG-C)

[0046] (1) Luminescence detection on SHG-C

[0047] Saccharomyces cerevisiae samples were detected on SHG-C in a 0.5mL system, and the artificial yeast sample plate culture result was 1.44×10 7 cfu / mL (original solution abbreviated as YO), detection system: 0.1mL FL + 0.1mL standard ATP or H2O sample + 0.3mL GB, the luminescence detection results are shown in Table 2.

[0048] Table 2 Luminescence detection results of artificial yeast samples on SHG-C

[0049]

[0050] Note: The ATP content of yeast is 3×10 -14 g / cell meter. Standard ATP uses 1×10 -7 mol / L.

[0051] The results show that: when the concentration of the yeast sample is 10 3 cells / m~10 7 In cells / mL, the ATP bioluminescence method can better reflect the true level.

[0052] (2) Luminescence detection on HKM-NRD (1) machine

[0053] In the 0.5ml system, the artificial yeast sample was tested on the Guangdong Huankai HKM-NRD(1) machine. Th...

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Abstract

The invention relates to a method and a detection kit for rapidly detecting the number of microorganisms using ATP bioluminescence method. The rapid detection kit includes luciferase and its protective agent, D-luciferin, standard ATP, luminescent detection buffer, acellular ATP remover, microbial cell ATP extractant and other reagents. The standard method for using this detection kit to quickly determine the number of microorganisms is to make an ATP standard curve in a selected bioluminescence instrument and a selected system, and obtain a linear regression equation after taking the logarithm. After removing non-target ATP and microbial The sample solution extracted by ATP is detected in the same instrument and system with the same batch of enzymes for ATP bioluminescence detection, and a blank control is made at the same time to obtain the CPM or RLU value of the sample and the blank, and the net relative luminescence value after removing the blank is taken as logarithm Finally, the ATP concentration of the sample is obtained through the established regression equation. According to the ATP content of microorganisms such as bacteria, yeast, mold and unicellular microalgae, it can be converted into the number of cells of bacteria, yeast, mold spores or unicellular microalgae, or only calculated Equivalent to the number of bacteria.

Description

[Technical Field] [0001] The invention relates to a method and a detection kit for rapid detection of the number of microorganisms by using an ATP bioluminescence method, and belongs to the field of biotechnology. [Background technique] [0002] The bioluminescence method has the advantages of rapid and simple detection of the number of microbial cells. The whole process is only ten minutes. The conventional determination of the total number of bacteria, yeast and mold uses the plate culture counting method. It takes several days from the beginning of the culture to the visible colony. It has a strong advantage in rapid detection of the number of microorganisms. The microbial quantity determination technology by ATP bioluminescence method does not require a culture process and has high sensitivity. It can be used for online detection of the total number of microbes in the production process of a factory, providing a rapid detection method for product quality control. At present, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/04C12Q1/25G01N21/76
Inventor 吴清平吴慧清张菊梅李程思
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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