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

A technology for detecting the number of kits and microorganisms, applied in the biological field, can solve the problems of high detection cost and unapplied application.

Inactive Publication Date: 2006-12-13
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 hi

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 bioluminescence rapid detection kit (luminescent 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 through a 0.22μm sterile microporous membrane to sterilize.

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

[0032] Acellular ATP remover AP: its specific composition and production process is to use containing 50mmol / L Tris-HCl, 10mmol / LMgSO 4 , 1mmol / L EDTA, 1mg / mL BSA, pH6.8 buffer 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, 2.0gDMSO, 0.05g EDTA, 0.05g MgSO per liter ...

Embodiment 2

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

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

[0042] It can be seen from Table 1 that: on the SHG-C luminometer for luminescence detection, when the ATP concentration is 10 -10 ~10 -6 When mol / L, the linearity is better, and the regression curve in the 0.5mL luminescence system is Lg[ATP]=-12.4792+1.0374LgΔCPM, R=0.9977, n=5; on the HKM-NRD (I) luminometer, when ATP concentration at 10 -10 ~10 -6 The linearity is better at mol / L, and the regression curve is Lg[ATP]=-10.8382+0.9547LgΔCP6S 1 , R=0....

Embodiment 3

[0044] Example 3: Luminescent detection of yeast artificial samples (SHG-C)

[0045] (1) Luminescence detection on SHG-C

[0046] Saccharomyces cerevisiae samples were detected on SHG-C in a 0.5mL system, and the result of artificial yeast sample plate culture was 1.44×10 7 cfu / mL (stock solution is abbreviated as Y0), detection system: 0.1mL FL+0.1mL standard ATP or H 2 O sample + 0.3mL GB, see Table 2 for the luminescence detection results.

[0047] sample

Luminescence detection

CPM

ΔCPM

LgΔ

CPM

Lg[ATP]

[ATP]mol /

ml

conversion cell

Number of cells / ml

h 2 o

Standard ATP

Y0

10 -1 Y0

10 -2 Y0

10 -3 Y0

10 -4 Y0

3472

241452

1096208

144266

19182

5020

3753

1092736

140794

15710

1548

281

6.0385

5.1486

4.196

3.190

2.4487

-6.2328

-7.1380

...

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Abstract

The invention relates to a rapid test method for microbial biomass using ATP bioluminescence and detection reagent cell, in which the said detection reagent cell consists of luciferase and its protecting agents, D-fluorescein, standard ATP, light-emitting detection buffer solution, non-cellular ATP remover, microbial cell ATP extractant and other agents. The rapid test standard method for microbial biomass using the detection reagent cell includes doing ATP normal curve in selected biological light-emitting device and selected system, obtaining linear regression equation after taking the logarithm, performing ATP biological light-emitting detection to specimen liquid by non-purpose ATP removal and micro-organism ATP extraction with the same enzyme in the same device and system, simultaneously doing blank control, then obtaining the specimen and blank CPM or RLU value, after taking the logarithm of the de-margined net relative luminescence value, and obtaining the mother ATP concentration with built regression equation. The ATP concentrations of bacteria, yeast, fungus and unicellular microalgae can be converted into the cell numbers of the said bacteria, yeast, fungus and unicellular microalgae, or homologous to the bacteria quantity.

Description

[technical field] [0001] The invention relates to a method for rapidly detecting the number of microorganisms by using an ATP bioluminescence method and a detection kit, which belong to the field of biotechnology. [Background technique] [0002] The bioluminescence method has the advantage of being fast and simple to detect the number of microbial cells, and the whole process only takes more than ten minutes, while the conventional method of counting the total number of bacteria, yeast and mold adopts the plate culture counting method, and it takes several days from the beginning of culture to the colonies visible to the naked eye, so It has a strong advantage in the rapid detection of the number of microorganisms. The microbial count determination technology by ATP bioluminescence method has high sensitivity because it does not require a cultivation process, and can be used in the factory for online detection of the total microbial count during the production process, provi...

Claims

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Application Information

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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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