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Method and biological indicator for quickly determining sterilizing effect

A biological indicator, sterilization effect technology, applied in chemiluminescence/bioluminescence, analysis by chemical reaction of materials, measurement devices, etc., can solve the problems of low accuracy and long evaluation time, and achieve high accuracy degree of effect

Inactive Publication Date: 2017-12-22
XINTRUM PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a method for quickly and accurately determining the sterilization effect, especially a method for directly detecting the survival of spores after sterilization treatment, so as to solve the problems of long evaluation time or low accuracy in the prior art

Method used

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  • Method and biological indicator for quickly determining sterilizing effect
  • Method and biological indicator for quickly determining sterilizing effect
  • Method and biological indicator for quickly determining sterilizing effect

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Preparation of bacterial spore suspension

[0034]The preparation of the spore suspension refers to the preparation method of the bacterial spore suspension in "Disinfection Technical Specifications (2002 Edition)". (1) Take the freeze-dried strain tube, open it under aseptic operation, and add an appropriate amount of nutrient broth medium. Take a test tube containing nutrient broth, add a little strain suspension dropwise, and incubate at 37°C for 18-24h. Use an inoculation loop to take the bacterial suspension of the first generation culture, streak inoculate it on a nutrient agar medium plate, and incubate at 37°C for 18-24h. Pick typical colonies from the above-mentioned second-generation culture, inoculate them in nutrient broth medium, and cultivate them at 37°C for 18-24 hours to form the third-generation culture. (2) Take the 18-24h nutrient broth culture of the 3rd-5th generation, inoculate it on the surface of the nutrient agar medium in the Roche...

Embodiment 2

[0035] Example 2 Preparation of spore ATP detection reagent

[0036] Follow the steps below to prepare the spore ATP detection reagent:

[0037] (1) Use freshly prepared high-purity water to prepare 100mL 150-250mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) solution; add 0.15-1.20g chlorhexidine acetate, 1.5-2.5 mL Triton X-100 (Triton X-100), 0.3-0.5g magnesium chloride hexahydrate, 0.7-1.0g dihydrate ethylenediaminetetraacetic acid disodium salt (EDTA); use 1M sodium hydroxide to adjust the pH of the solution to After 7.5, use a 0.22 μm filter membrane to filter and sterilize; add 20-50 mg of luciferase (Sigma) and 100-200 mg of D-luciferin (Sigma), dissolve, and store at -20°C.

[0038] Or (2) Use freshly prepared high-purity water to prepare 100mL of 150-250mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) solution; add 0.06-0.10g hexadecyltrimethyl Ammonium bromide (CTAB), 0.15-1.20g chlorhexidine acetate, 1.5-2.5mL Triton X-100 (Triton X-1...

Embodiment 3

[0040] Example 3 Spore ATP detection reagent detects spores

[0041] Get the spores of Geobacillus stearothermophilus (ATCC 7953) prepared in Example 1, use sterile water to dilute into different concentrations, add different concentrations of spore suspensions in the 96-orifice plate, so that the spores in each hole are respectively 0, 1, 10, 100, 1000, 10 000, 100 000 and 1 000 000, and then add the spore ATP detection reagent in Example 2, and use a multifunctional plate reader (PerkinElmer) to detect the luminescence value. figure 1 Shown is the ATP luminescence of different amounts of spores. The S:N (signal-to-noise ratio) of the luminescence is positively correlated with the amount of spores. The more spores, the greater the S:N (signal-to-noise ratio). When the number of spores is 1, the S:N (signal-to-noise ratio) is still greater than 10, indicating that the spore ATP detection reagent of the present invention has ultra-high sensitivity for detecting spores.

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Abstract

The invention relates to a method and a biological indicator for quickly determining sterilizing effect. The method detects ATP of sterilized spores directly, the ATP of the spores is detected by means of a bioluminescent method, and the sterilizing effect can be determined within several minutes. The method detects ATP from survival spores directly. Compared with an indirect method of detecting related parameters of killed spores, the method has higher accuracy. The biological indicator is a detection agent containing the spores and the ATP of the spores based on the method, and can provide an evaluation result of the sterilizing process within about 5 minutes.

Description

Technical field: [0001] The invention relates to a method for quickly determining the sterilization effect. The invention also relates to a biological indicator for quickly determining the sterilization effect. Background technique: [0002] It is well known in the art that microbial spores (endospores, or spores) are more difficult to inactivate than most other types of microorganisms. Determining the survival of spores after sterilization is the most powerful evidence to determine whether the sterilization is complete. [0003] Biological indicators have been widely used to test and / or determine the effectiveness of sterilization methods. Exposure is typically determined by exposing biological indicators containing microbial spores to a selected sterilant or sterilization method, and then placing the exposed spores in an incubation environment capable of sustaining spore germination and microbial growth. Survival of treated spores. [0004] Currently commercially avail...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/76
CPCG01N21/763
Inventor 羌维兵童明庆张怡
Owner XINTRUM PHARMA
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