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Adenosine triphosphate (ATP) bioluminescent reagent for detecting hygienic quality of drinking water and surface sanitation of GMP factory, method and kit

A technology for bioluminescent reagents and drinking water, which is applied in biochemical equipment and methods, and the determination/inspection of microorganisms, which can solve the problems of complex operation, limited application, and high cost.

Inactive Publication Date: 2014-04-30
GUANGDONG INST OF MICROORGANISM +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A clean surface needs to be sterile, but if it is contaminated by any biological material (indicating the presence of ATP), it provides the basis for rapid microbial reproduction, this source of contamination can be easily detected by ATP detection methods, but traditional microbial learning methods often fail to
Since the 1970s, Shape and his collaborators have applied ATP bioluminescence to detect the number of microorganisms in food. The research on the use of ATP bioluminescence to evaluate bacterial contamination has increased. Some commercial ATP bioluminescence systems have been used as a sanitation detector. Successfully used for in situ control of HACCP systems, however most commercial kits for removal of non-cellular ATP interference are expensive and complex to operate, thus limiting their use in the food industry
The detection sensitivity of general ATP bioluminescence technology is 10 4-5 Bacterial cells / mL, there is a certain gap with the evaluation index of effective surface sanitation and disinfection effect

Method used

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  • Adenosine triphosphate (ATP) bioluminescent reagent for detecting hygienic quality of drinking water and surface sanitation of GMP factory, method and kit
  • Adenosine triphosphate (ATP) bioluminescent reagent for detecting hygienic quality of drinking water and surface sanitation of GMP factory, method and kit
  • Adenosine triphosphate (ATP) bioluminescent reagent for detecting hygienic quality of drinking water and surface sanitation of GMP factory, method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1: Preparation of ATP bioluminescence reagent

[0039] A 10mg / ml concentrated sample was prepared by de-ATP and nitrogen-filled luminescent detection buffer containing 25mmol / L glycylglycine, 1.0mmol / L EDTA, 1.0mmol / L DTT, pH7.8, and passed through a 0.22μm filter membrane, and then Dilute to 1mg / ml with the same buffer to obtain D-luciferin. The purity of D-luciferin was over 99% (Area%, HPLC).

[0040] Luciferase must be purified. For the preparation method, refer to the invention patent ZL200610034384.4. The only difference is that the NTE buffer in the purification process is treated to remove ATP. The specific preparation method is as follows: take natural firefly insect tail, add enzyme extract to grind, centrifuge and ammonium sulfate precipitation to obtain crude enzyme preparation; The NTE buffer is treated to remove ATP, and further undergoes a molecular exclusion chromatography or affinity chromatography and a secondary molecular exclusion chroma...

Embodiment 2

[0044] Embodiment 2: Preparation of ATP bioluminescence reagent

[0045] A 10mg / ml concentrated sample was prepared by de-ATP and nitrogen-filled luminescent detection buffer containing 25mmol / L glycylglycine, 1.0mmol / L EDTA, 1.0mmol / L DTT, pH7.8, and passed through a 0.22μm filter membrane, and then Dilute to 1mg / ml with the same buffer to obtain D-luciferin. The purity of D-luciferin was over 99% (Area%, HPLC).

[0046]Luciferase must be purified. For the preparation method, refer to the invention patent ZL200610034384.4. The only difference is that the NTE buffer in the purification process is treated to remove ATP. The specific preparation method is as follows: take natural firefly insect tail, add enzyme extract to grind, centrifuge and ammonium sulfate precipitation to obtain crude enzyme preparation; The NTE buffer is treated to remove ATP, and further undergoes a molecular exclusion chromatography or affinity chromatography and a secondary molecular exclusion chromat...

Embodiment 3

[0050] Embodiment 3: ATP bioluminescence detection and plate culture method detection of the number of viable bacteria in artificial samples

[0051] In this example, the ATP bioluminescent reagent prepared in Example 1 is used for detection.

[0052] First compound the ATP bioluminescent reagent: ATP bioluminescent reagent is added to the luminescent stable buffer system that removes ATP aseptic treatment, so that the luminescent specific activity of the ATP bioluminescent reagent is in a 0.4mL luminescent system, and use 1×10 -7 The relative luminescence logarithmic value in / L ATP measurement reaches 6.5-7.5.

[0053] After culturing Escherichia coli 80991d in nutrient broth (NB) at 37°C, centrifuge at 10,000r / min for 2 minutes to obtain the sludge, then dilute it with sterile ultrapure water, and detect the total number of viable bacteria in the sample by ATP bioluminescence detection method. The NA medium was compared and tested by plate culture methods, and the test res...

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Abstract

The invention discloses an adenosine triphosphate (ATP) bioluminescent reagent for detecting hygienic quality of drinking water and surface sanitation of a GMP factory, a method and a kit. The ATP bioluminescent reagent disclosed by the invention comprises luciferase, fluorescein and a luminous stability buffer system for sterile treatment to remove ATP; the luciferase is necessary to be purified; the dynamic background ratio of the purified luciferase can be up to 50-3,000; the lighting specific activity of the ATP bioluminescent reagent is in 0.4mL of luminescent system; the relative lighting log value can be up to 6.5-7.5 in ATP measurement; the luciferase, the fluorescein and the luminous stability buffer system for sterile treatment to remove ATP are frozen after being mixed, so as to obtain freeze-dried powder. Rapid detection on the hygienic quality of the drinking water and the surface sanitation of the GMP factory is carried out by adopting the ATP bioluminescent reagent with the sensitivity and a stable lighting function; sampling and rapid detection can be finished within half an hour.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and in particular relates to an ATP bioluminescent reagent, a method and a kit for detecting the sanitation quality of drinking water and the surface sanitation of a GMP factory. Background technique: [0002] Water is the source of life, and water safety involves thousands of households and everyone's life and health. Drinking water safety includes two factors, physical chemistry and microbiology, and these two factors influence each other in some aspects. In the current national standard for drinking water, the total number of bacteria is a mandatory inspection item. Even though the total number of bacteria in the national standard for mineral water is not a mandatory inspection item due to the problem of disinfection by-products in drinking water, three new detections for pathogenic bacteria have been added. , and mineral water manufacturers implement the HACCP quality management system in the p...

Claims

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Application Information

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IPC IPC(8): C12Q1/66C12Q1/06
Inventor 吴慧清吴清平李程思张菊梅郭伟鹏
Owner GUANGDONG INST OF MICROORGANISM
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