CN110274896B

CN110274896B - ATP bioluminescence lgCB-lgIBMethod for evaluating fungus killing effect of liquid disinfectant by standard yeast method

Info

Publication number: CN110274896B
Application number: CN201910200132.1A
Authority: CN
Inventors: 李文杰; 闫冀焕; 崔宗岩; 钱云开; 郝凌云
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-03-15
Filing date: 2019-03-15
Publication date: 2021-11-30
Anticipated expiration: 2039-03-15
Also published as: CN110274896A

Abstract

The invention relates to a method for evaluating the fungus killing effect of a liquid disinfectant, which comprises the following steps: extracting a sample and determining the lowest effective concentration; selecting equipment types, and preparing reagents and culture media; strain preservation, activation and preparation of a strain suspension; viable bacteria content C of bacterial suspension after methylene blue dyeing_BBlood cell plate counting and inoculation bacterial liquid C_BCalibrating; lgC_BRelative fluorescence intensity log lgI_BEstablishing a standard curve; identifying a neutralizer and carrying out quantitative suspension test; recovery solution I_BDetermination of the viable bacteria content C_BAnd T_BCalculating; calculating the killing rate R and the sterilization index A; judging a result; it is characterized in that: and (3) applying an ATP fluorescence photometer to perform accurate quantitative evaluation on the killing effect of the disinfectant fungi characterized by R or A. The present invention specifies the test sample solution and the reference sample solution after different times of recovery, and determines I_BAnd in lgI_BCharacterizing and calculating R or A; and providing a result judgment basis. ATP bioluminescence lgC for evaluating fungus killing effect of disinfectant developed by the invention_B‑lgI_BThe standard yeast method can improve the related disinfection effect evaluation method system.

Description

ATP bioluminescence lgCB-lgIBMethod for evaluating fungus killing effect of liquid disinfectant by standard yeast method

Technical Field

The invention relates to a method for evaluating the fungus killing effect of a liquid disinfectant, in particular to ATP bioluminescence lgC for accurately and quantitatively testing the fungus killing effect of the liquid disinfectant characterized by a killing rate R or a sterilization index A by using an ATP fluorescence photometer_B-lgI_BA standard curve method belongs to the technical field of disinfectant sterilization effect evaluation.

Background

Along with the improvement of living standard and the enhancement of health consciousness of people, in order to cut off the transmission way of infectious diseases, liquid disinfectants such as peroxides, aldehydes, phenols, quaternary ammonium salts and the like are widely applied in the fields of medical treatment, food, education, catering, home and the like, and increasingly strict requirements on the killing action and broad spectrum of pathogenic microorganisms such as bacteria, fungi and spores thereof are increased; the industrial related process innovation and product research and development pressure are continuously rising, and the quality inspection technology and the test efficiency are promoted.

At present, the technical system for evaluating the antibacterial and disinfection effects at home and abroad mainly aims at bacteria and fungi, and the principle of the test method for the antibacterial and disinfection effects of various countries is mainly a plate counting method for carrying out isolated culture on candida albicans, and the few people related to the fungi exist; among them, one suitable test for Candida albicans is the dipping quantitative test method proposed by the Japanese Industrial Standard; secondly, bacteriostatic ring method from the textile industry of America; thirdly, an international general oscillating flask method; and fourthly, a dripping method originated from the American textile enterprises. Although relevant standards such as GB 15981-. In addition, international mildew-proof effect evaluation technical systems are basically derived from American standards, daily standards and European standards for many years, and the related standards mainly comprise a soil-buried culture method, an agar plate method, a wet-room culture method and the like; because the mycelium formed in the growth process of the mould spores cannot be accurately counted, only qualitative judgment can be carried out, and quantitative test cannot be carried out; and the growth period of the mould is long, and the culture time is at least 2-4 weeks, so that the test period is long, and the time and the economic cost are high. Whether the Candida albicans or the mould is aimed at, the experimental process of the existing standard method is complicated, and the technical difficulty is higher; the related operation is greatly influenced by human factors, so that the test error is large and the comparability is lacked. In recent years, the development of an ATP fluorescence analysis method in the technical field of international bacteria detection is mature day by day, and compared with the traditional plate method, the correlation of a detection result is 98%, the accuracy is high, and the rapid detection can be realized. Drawn by demand, aiming at the current trends of quantification, rapidity and simplification in the field of performance detection technology of foreign antibacterial materials, an ISO 20743:2007-2013 < Textiles-Determination of antibacterial activity of textile products and ISO 13629-1:2012 < Textiles-Determination of antibacterial activity of textile products < Part Lu1nescence are formulated by referring to the ATP fluorescence analysis principle, and a fluorescence Determination method for representing the antibacterial performance of textile products by the ATP content change after sample inoculation is specified, but the absorption method, the transfer method and the transfer method provided by the fluorescence Determination method are only suitable for textile materials or microporous materials with water absorption and the contrast sample bacterial/mold growth value > 0; the evaluation of the fungus killing effect of the liquid disinfectant needs to form a complete set of clear and specific methods aiming at key technologies such as neutralization effect evaluation, result calculation formulas and the like, and meanwhile, the evaluation of related measurement uncertainty needs to be provided. In addition, the standard method has single antibacterial performance characterization parameter, only relates to an antibacterial activity value A, and is generally used in China for killing rate R.

Therefore, in order to improve the disease prevention and control effect, standardize the domestic market order and promote the transformation and upgrading of the industry, a scientific, advanced, high-accuracy and high-reproducibility simple and easy liquid disinfectant fungus killing effect testing technology is urgently needed to be researched. The technical route of the patent is designed to meet international orbit, the detection method belongs to global initiatives, and can fill up the blank of related technical fields at home and abroad.

Disclosure of Invention

The technical problem to be solved by the invention is to provide ATP bioluminescence lgC for evaluating the fungus killing effect of a liquid disinfectant characterized by a killing rate R or a sterilization index A by using an ATP fluorescence photometer_B-lgI_BThe standard curve method can solve the problem of accurate and quantitative evaluation of the sterilization effect of liquid disinfectants and other medical and health fields.

In order to solve the technical problems, the invention adopts the technical scheme that:

a method of evaluating the effectiveness of a liquid disinfectant for fungus kill, comprising: (1) extracting a sample and determining the lowest effective concentration; (2) selecting equipment types, and preparing reagents and culture media; (3) strain preservation, activation and preparation of a strain suspension; (4) viable bacteria content C of bacterial suspension after methylene blue dyeing_BBlood cell plate counting and inoculation bacterial liquid C_BCalibrating; (5) log value of viable bacteria content lgC_BRelative fluorescence intensity log lgI_BEstablishing a standard curve; (6) identifying a neutralizer and carrying out quantitative suspension test; (7) relative fluorescence intensity value I of recovered liquid_BMeasuring; (8) viable bacteria content C of the recovered liquid_BAnd T_BCalculating the sterilization rate R and the sterilization index A; (9) judging a result; it is characterized by applying ATP fluorescence photometer pair to killATP bioluminescence lgC for accurately and quantitatively evaluating fungus killing effect of liquid disinfectant characterized by rate R or sterilization index A_B-lgI_BStandard curve method, in particular:

relative fluorescence intensity value I in the recovered liquid_BIn the measurement:

determining the extraction requirement of a sample, determining the minimum effective concentration of the inhibiting and killing indicator bacteria in 10min of the disinfectant, carrying out passage and activation on standard strains of fungus test strains, and taking fresh candida albicans or mould spore cultures to prepare a bacterial suspension; measuring viable bacteria content C of bacteria liquid by adopting a blood counting chamber after methylene blue staining_BAnd inoculating the inoculum C_BAnd (3) calibrating: 5.0X 10⁶CFU/mL～9.0×10⁶CFU/mL. Selecting viable bacteria content C_BIs 3.5 multiplied by 10³CFU/mL、3.5×10⁴CFU/mL、3.5×10⁵CFU/mL bacterial liquid is used as standard series bacterial suspension, and ATP fluorescence photometer is used for measuring relative fluorescence intensity value I of CFU/mL bacterial liquid_BCarrying out measurement; drawing lgC_B-lgI_BStandard curve, linear equation Y ═ a derived from curve_BX+b_BAnd the correlation coefficient R_B ². Then, carrying out a neutralizer identification test according to the minimum effective concentration of the disinfectant to be tested, and determining the type and concentration of the neutralizer; meanwhile, 4.41mL of test sample liquid and control sample liquid with various concentrations are placed in a water bath at the temperature of (20 +/-2) DEG C and mixed with 0.49mL of inoculation bacterial liquid; the effect to be sterilized lasts for t₁、t₂、t₃、t₄After four periods of different time, 0.49mL of the contaminated sample liquid is respectively mixed with 4.41mL of neutralizing agent solution, and after neutralization is carried out for 10min, the uniformly mixed solution is used as a recovery solution; measuring relative fluorescence intensity value of recovered solution by using ATP fluorescence photometer

And

and according to the standard curve equation of the indicated strainFormula Y ═ a_BX+b_BCalculating the content of corresponding viable bacteria

And

in the calculation of the killing rate R or the sterilization index A:

according to the standard curve lgC of the indicated strain_B-lgI_BLinear equation of (a)_BX+b_BFor t₁、t₂、t₃、t₄Four sections of different sterilization time, taking the relative fluorescence intensity measured values of the recovered live bacteria of the test sample liquid and the control sample liquid with each concentration in each quantitative suspension test as basic data; calculating the kill rate of the fungus

Or sterilization index

Taking the arithmetic mean value of the five tests as the kill rate of the liquid disinfectant sample with corresponding concentration to be tested on fungi in the same sterilization time

Or sterilization index

Simultaneously, related data reduction and measurement uncertainty requirements are defined;

in the result determination:

referring to the common practice in the health industry and the biological evaluation standard of related disinfection effect, if a liquid disinfectant sample liquid to be tested with a certain concentration is in a single quantitative suspension test, the liquid disinfectant sample liquid is subjected to ATP bioluminescence lgC_B─lgI_BThe fungus killing rate in a specific sterilization time measured by a standard curve method is more than or equal to 99.9 percent or the sterilization index is more than or equal to 3.0, and the sterilization effect of the fungus in the test is judged to be qualified. When the disinfection effect of the liquid disinfectant sample liquid to be measured in the concentration is qualified within the same disinfection time in 5 times of quantitative suspension tests, the minimum concentration and the minimum time for the liquid disinfectant sample liquid to be measured to have the disinfection effect on fungi can be determined (in practical application, the minimum concentration and the minimum time of the organic matter protection test are used as the concentration and the time required by the liquid disinfectant sample to be measured to achieve the practical disinfection effect).

Compared with the prior art, the invention adopting the technical scheme has the beneficial effects that:

(1) the advancement is as follows: the modern precise instrument, namely an ATP fluorescence photometer, is used as testing equipment, so that the quantity of live bacteria recovered after a disinfectant sample is sterilized for a specific time can be accurately and quickly determined, and the modernization of the evaluation of the fungus killing effect is achieved; the influence of artificial factors in the experimental process can be effectively reduced, and the limitation of complex operation and long period of the traditional plate culture method is broken through; the precision simplification of the evaluation process and the precision of the evaluation data are realized; the evaluation technology has certain advancement.

(2) Scientifically: according to the ATP fluorescence analysis method test principle, the viable bacteria ATP concentration logarithmic value lgC suitable for multiple strains is established_BRelative log of fluorescence intensity lgI_BA standard curve; a mathematical model of the ATP bioluminescence real-time quantitative analysis method for the fungus killing effect of the liquid disinfectant is constructed. Meanwhile, the cognitive habits of consumers in different countries are considered, the killing rate R and the sterilization index A are taken as relevant effect evaluation indexes, and the scientificity and the universality of the evaluation method are improved.

(3) The innovation is as follows: compared with the conventional general method, the ATP fluorescence photometer with higher automation and intelligence levels is introduced in the test process, so that the experimental steps are greatly simplified, the precision and quantification of the evaluation of the fungus killing effect of the disinfectant are realized, and the method has good reproducibility and comparability; meanwhile, the test period is greatly shortened, and the evaluation cost is reduced; can effectively fill the blank in the prior related test technical field.

(4) Prospective: establishes the communication with lgC_B、lgI_BThe standard curve quantitative analysis method based on the linear relation is innovative and enriches the viable bacteria concentration determination method of the candida albicans and the mould spore suspension and the disinfectant sample dilution mode, and definitely contrasts the technical contents of the sample, the standard solution concentration, the determination steps, the calculation formula, the uncertainty and the like; the relative fluorescence intensity value I of the recovered viable bacteria directly measured by an instrument is initiated_BThe killing rate R and the sterilization index A are calculated and judged as a result judgment form, and the uncertainty of measurement is investigated through the intra-group and inter-group variation coefficients C.V, so that the method has certain prospect technically.

(5) Operability: the ATP fluorescence photometer has the advantages of low price, simple operation and wide application, the established Meilan stained blood platelet viable count method and the ATP fluorescence test method for evaluating the disinfectant fungus killing effect are simple and easy to implement, the related technical explanation is clear and concrete, and the ATP fluorescence photometer is easy to understand and master; the method has strong operability in the implementation process, is suitable for microorganism experiment personnel with different professional levels, and is favorable for promoting the achievement transfer and transformation and popularization and application.

(6) Universality: because ATP is generally present in cells of a living body, the patent method can provide evaluation technical support with broad spectrum value for experimental strain amplification; the introduction of related instruments can greatly simplify the experimental steps and reduce the test cost; the method is favorable for expanding the popularization and application in inspection, study, research and production fields, can support the disinfectant fungus killing effect evaluation technology to realize universality, and can provide reference for the antibacterial effect evaluation technology research in the fields of daily chemicals, sanitary articles and the like.

Further, the preferred scheme of the invention is as follows:

the sample extraction and the determination of the minimum effective concentration are carried out according to the following steps:

(1) control sample: the Chao's culture solution is used as a control sample;

(2) test samples: randomly extracting 1 liquid disinfectant sample of the minimum sale package from 1 complete transport package of a certain production lot number for neutralizer identification and quantitative suspension test; for each set of test samples, a set of control samples was selected as a reference and effectively identified.

(3) Determination of the minimum effective concentration of disinfectant sample: diluting the disinfectant sample to be detected with sterile water into three proper concentrations, namely high concentration, medium concentration and low concentration, according to the effective concentration range marked by the use instruction of the disinfectant sample to be detected; 4.41mL of diluent with different concentrations is divided into three sterile test tubes by a sterilization pipette, and 0.49mL of inoculated bacteria liquid is respectively dripped into each test tube; after shaking the test tube at 3000r/min for 30s, it was placed in a water bath at (20. + -. 2 ℃ C.). Timing when the temperature of the solution in the test tube is balanced with the temperature of the water bath to ensure that the killing effect of the disinfectant on the fungi lasts for 10 min; then, the mixed solution is used as a recovery solution according to the relative fluorescence intensity value I in the patent_BAnd (3) measuring and recording the relative fluorescence intensity value of the recovered liquid. At the same time, the above-mentioned sterilization test and recovery solution I were repeated using 4.41mL of the Kircher's culture medium as a control sample_BA measuring process; calculating the killing rate of the diluent with different concentrations according to a killing rate R calculation formula in the patent; if the killing rate R of a certain concentration is 99.9%, setting the concentration as the minimum effective concentration of the disinfectant sample to be detected;

the equipment selection and reagent and culture medium preparation are carried out according to the following steps:

(1) the general requirements are as follows: the analytical reagent for the test and the tertiary water (distilled water or deionized water) meeting the requirements of GB/T6682-;

(2) the instrument equipment comprises: a secondary biological safety cabinet or an ultra-clean workbench with the cleanliness grade not lower than 100; an ATP bioluminescence rapid detection system containing an ATP fluorescence photometer, a special test tube and the like, wherein the wavelength range of the ATP fluorescence photometer is 300 nm-650 nm, and the total number detection range of fungal cells/spores is 10¹CFU/mL～10⁶CFU/mL; a biological optical microscope at a magnification of 40X to 400X; (25-37) a constant temperature incubator at +/-1 ℃; (10-50) DEG C +/-1 ℃ constant-temperature water bath tank; (0-50) DEG C +/-1 ℃ and (50-300) r/minA constant temperature oscillator; a centrifuge with the rotating speed more than or equal to 8000r/min and a matched centrifuge tube; a vortex oscillator with a rotating speed range (500-3000) r/min; a pressure steam sterilizer at (121 + -2) deg.C and (103 + -5) kPa; a low-temperature refrigerator at the temperature of minus 20 ℃ to minus 80 ℃; a freezer at 0-10 ℃; an electronic balance with a sensory volume of 0.001 g; an ultrasonic cleaner with a frequency range (30-50) kHz; a pH meter with the precision of +/-0.1 (25 ℃); an electric furnace;

(3) material utensil: a blood counting chamber and a special cover glass; 1mL and 10mL sterile graduated pipettes; 0.05mL, 0.1mL, 0.2mL, 1mL, 5mL, 10mL (metering error less than 1%) of single-channel variable range pipette and sterile pipette tip; sterile conical bottles and bottle stoppers with the capacities of 100mL, 250mL and 500 mL; a sterile culture dish; a glass funnel; a sterile cock test tube; an inoculating loop with the diameter not more than 4 mm; glass beads with a diameter of 5 mm; an alcohol lamp; sterilizing the tweezers; absorbent cotton and gauze for biochemical detection; sterile filter paper;

the thermometer of (1); a stopwatch with an accuracy of 0.01 s;

(4) reagent: 0.1% of Lv's basic methylene blue staining solution; calf serum; the following reagents are sterilized by high pressure for 30min at 121 ℃ and stored for 30d at 5-10 ℃: 85% of normal saline; one of N-methyl ethanesulfonic acid, Tween 80 and dioctyl sodium sulfosuccinate is selected to prepare a 0.05% wetting agent aqueous solution; 5g of sodium thiosulfate was dissolved in 1000mL of water (for chlorine type disinfectants); dissolving 1.36g of monopotassium phosphate, 2.83g of disodium hydrogen phosphate, 10g of lecithin, 10g of glycine and 30g of tween (80) in 1000mL of water or dissolving 1.36g of monopotassium phosphate, 2.83g of disodium hydrogen phosphate, 3g of lecithin and 20g of tween (80) in 1000mL of water (for a non-oxidizing disinfectant); dissolving 20g of tween (80) and 1g of sodium thiosulfate in 1000mL of phosphate buffer solution (used for oxygen type disinfectant) and the like (or selecting other applicable neutralizing agents according to the type of a liquid disinfectant sample to be detected);

(5) culture medium/liquid (commercially available culture medium/liquid): subpackaging the prepared culture medium/liquid, sterilizing at 121 ℃ for 30min under high pressure, and storing at 2-8 ℃ for 30 d;

sakeh medium/liquid (for candida albicans strain activation): 40g of glucose, 10g of peptone and 20g of agar were dissolved in 1000mL of water under heating (the culture medium was not agar), and the pH was adjusted to 5.6. + -. 0.2(25 ℃ C.);

the Chaudou culture solution (for preparing mould spore solution, diluting the bacterial suspension and eluting the sample) comprises the following components: heating and dissolving 2g of sodium nitrate, 1g of dipotassium phosphate, 0.5g of potassium chloride, 0.5g of magnesium sulfate, 0.01g of ferrous sulfate and 30g of cane sugar in 1000mL of aqueous solution containing 0.05% of wetting agent, and adjusting the pH value to 6.0-6.5 (25 ℃);

potato-glucose culture medium (for activating mold spore strain) is prepared by peeling fresh potato 300g, cutting into pieces, and boiling in 1000mL water for 20-30 min; filtering to obtain juice, adding 20g of glucose, 0.1g of chloramphenicol and 20g of agar into the filtrate, and diluting to 1000 mL;

(6) ATP fluorescence reaction reagent (or with commercially available reagent): the prepared ATP fluorescent reaction reagent is preserved at the temperature of-20 ℃ to-70 ℃ besides phosphate buffer solution and is used within 6 months;

diluting the buffer solution: 0.005mol/L disodium hydrogen phosphate solution containing 0.037% of sucrose, and adjusting the pH to 7.2 +/-0.2; sterilizing at 121 deg.C under high pressure for 15min, and storing at 2-8 deg.C for 30 d;

ATP fluorescent reagent buffer solution: heating and dissolving 1117mg of trihydroxymethyl aminomethane, 183mg of disodium ethylene diamine tetraacetate, 808mg of magnesium acetate, 6.7mg of dimercaptothreitol, 25000mg of beta-cyclodextrin and 925mg of glucose in 250mL of water, and adjusting the pH to 7.5 +/-0.2; the composition is used within 8 h;

ATP lysate: 4.6 International units/mL apyrase (EC:3.6.1.5) and 46 International units/mL adenosine deaminase (EC:3.5.4.6 or EC:3.5.4.17), 37mg sucrose and 20mg bovine serum albumin were dissolved in 10mL of 0.05 mol/L2-morpholinoethanesulfonic acid buffer solution, the pH was adjusted to 6.0 + -0.5, and the solution was used within 8h (1mL of lysate reduced ATP concentration to 10 in Sabouraud's medium within 15 min)^-11Less than mol/L);

ATP extracting solution: heating 45mg of tris (hydroxymethyl) aminomethane to dissolve in 9.8ml of water, uniformly mixing with 0.2ml of 10% benzalkonium chloride solution, and adjusting the pH to 12.0 +/-0.5 (the ATP extraction efficiency of fungal cells is not lower than 80%);

ATP fluorescent reagent: dissolving 0.7mg of luciferase, 12.6mg of D-luciferin and 56mg of bovine serum albumin in 30mL of ATP fluorescent reagent buffer solution, uniformly mixing, standing at room temperature for 15min, and using within 3 h;

the strain preservation, activation and preparation of the strain suspension are carried out according to the following steps:

(1) indicating strains: candida albicans ATCC 10231; aspergillus niger ATCC 16404; chaetomium globosum ATCC 6205; penicillium chrysogenum ATCC 9179 (or other species provided and traceable by the national collection of microbial cultures);

(2) and (3) strain preservation: candida albicans, opening a freeze-dried strain tube by aseptic operation, injecting a proper amount of Sabouraud's culture solution into the tube by using a capillary suction tube, blowing and sucking for several times to melt and disperse strains; dripping a little strain suspension into a test tube filled with 5-10 mL of Sabouraud's culture solution, and culturing for 18-24 h at 37 ℃. Mould-the mould test strain is inoculated on the slant of the potato-glucose culture medium by aseptic operation and the inoculation date is marked, and the mould is cultured at 28 ℃ to 30 ℃ until the slant is full of mould spores (7d to 14 d); preserving for 4 months at 3-10 ℃ as preserving bacteria;

(3) activating strains: candida albicans-typical colonies in the 1 st generation culture were scraped with an inoculating loop and streaked onto a Shaw's medium plate; after culturing for 18-24 h at 37 ℃, selecting a typical colony in the culture of the 2 nd generation to inoculate on a slope of a Sabouraud's medium; culturing at 37 deg.C for 18-24 hr, storing at 4 deg.C under sealed condition, and using within 6 weeks. The mould is that the preserved fungus spores are scraped by an inoculating loop, the potato-glucose culture medium slant is inoculated, and the culture is carried out for 7 to 14 days at the temperature of between 28 and 30 ℃ until a large amount of spores are generated. Before preparing spore suspension, a test tube plug of a mould strain cannot be pulled out, each test tube is opened and then only used for preparing spore liquid once, and newly cultured mould spores are used for preparing suspension each time;

(4) preparing a bacterial suspension: candida albicans test-selecting fresh strain culture from a slant with an inoculating loop, inoculating into a sterile conical flask filled with 50mL of Sabouraud's culture solution, placing in a constant temperature oscillator at (30 +/-1) ° C for culturing for 18 h-24 h at 150r/min, and storing in a sealed manner at 4 ℃ for use on the same day. Mold test-Add 10mL of sterile water to the strain tube, scrape the fresh mold spores on the surface of the medium with an inoculating loop, inject the washed spore stock solution into a sterile stoppered conical flask containing 15 glass beads and 45mL of Cnahs' culture solution, shake the tube at 3000r/min for 2min, break up the spore clusters, and mix the spore solution. Then, placing a glass funnel covered with sterile absorbent cotton or eight layers of gauze on a conical flask, and filtering the spore suspension to remove hyphae and culture medium fragments; transferring the filtrate into a sterilized centrifuge tube, separating at room temperature at 8000r/min for at least 10min, and removing supernatant; the spore precipitate was washed with 50mL of the Kirchner's medium and centrifuged, and after washing was repeated 3 times, the centrifuged spore precipitate was diluted with the Kirchner's medium. Preparing spore suspension for each test mould according to the method, and mixing spore liquid of each strain in equal volume; storing at 0-7 deg.c for use in the same day or 4 days;

the viable bacteria content C of the bacterial suspension after the methylene blue dyeing_BBlood cell plate counting and inoculation bacterial liquid C_BThe calibration is carried out according to the following steps:

(1) and (3) dyeing with methylene blue: sterile 50. mu.L dilution to 10^-2～10^-3Respectively transferring the candida albicans suspension or mold mixed spore liquid and 30 mu L of 0.05% Lv's alkaline methylene blue staining solution into the same sterile test tube (the dilution of the bacterial liquid is that each cell of a blood counting plate contains 4-5 candida albicans cells or mold spores), and shaking the test tube for 1min at 1000r/min to fully and uniformly mix the bacterial suspension and the staining solution;

(2) and (3) counting blood corpuscle plates: and (3) placing 5 mu L +/-0.5 mu L of the stained bacterial suspension on the edge of a cover glass by using a sterile pipette, enabling the bacterial suspension to slowly permeate into a blood cell counting plate along the gap of the glass slide, and preventing bubbles from being generated between the counting plate and the glass slide, otherwise, operating again. And (4) completely sucking the redundant bacteria liquid in the tank by using sterile filter paper, standing for 2min +/-20 s, and completely settling into a counting chamber. When a 16-cell counting plate is used, counting candida albicans cells or mold spores in 4 middle cells (namely 100 small squares) which are positioned at the upper left, the upper right, the lower left and the lower right in a diagonal direction; if a 25-middle-grid counting plate is used, in addition to the 4 diagonal orientations, the central 1 middle grid (namely 80 small grids) is counted; when the test strains are positioned on the double lines of the middle lattice, only the Candida albicans cells or the mold spores on the upper line and the right line or the lower line and the left line are counted;

(3) viable bacteria content C_BAnd (3) calculating: after the magnification of the biological optical microscope is adjusted from low to high to 400 multiplied, the Candida albicans cells or the mould spores at the corresponding space position of the blood counting plate are immediately counted; wherein the colorless Candida albicans cells are viable bacteria, and the blue or light blue ones are dead bacteria; the mold spores with dark blue edge, colorless or light blue and light red inside are live bacteria, while those with dark blue edge and inside are dead bacteria, so only the spores with different colors are counted. If the number of the aseptic hypha monospores in the mold spore suspension is less than 90%, the spore liquid is prepared again. The candida albicans cells or mold spores in each cell of the hemacytometer were counted in duplicate by microscopy three times, and the average was taken. When the specification of the blood counting chamber is 16X 25, the viable bacteria content (colony forming unit per milliliter, CFU/mL) C of 1mL of bacterial liquid_B＝N÷5×16×K×10⁴(ii) a When the specification of the blood counting chamber is 25X 16, C_B＝N÷5×25×K×10⁴(ii) a Wherein N is the total number of viable candida albicans cells or mold spores in five middle lattices of the blood counting plate, and K is the bacterial liquid dilution factor. Then, the known viable bacteria content C is treated by the Czochralski culture solution_BDiluting the Candida albicans suspension or the mold mixed spore liquid to obtain C_BIn the range of 5.0X 10⁶CFU/mL～9.0×10⁶CFU/mL inoculated bacterial liquid;

the log value of viable bacteria content lgC_BRelative fluorescence intensity log lgI_BEstablishing a standard curve according to the following steps:

using Chao's culture solution to treat known viable bacteria content C_BAfter the candida albicans suspension or the mold mixed spore liquid is subjected to continuous gradient dilution, a standard series of bacterial suspensions are obtained: 3.5X 10³CFU/mL、3.5×10⁴CFU/mL、3.5×10⁵CFU/mL and mix well. Then, according to the relative fluorescence intensity value I in this patent_BThe determination method is based on the viable bacteria content C_BThe relative fluorescence intensity values of the above standard solutions were measured and recorded in descending order. Then, lgI is calculated as the relative fluorescence intensity logarithm of the standard series bacterial suspension_BAs abscissa, corresponding viable bacteriaLogarithmic value of content lgC_BPlotting the ordinate; calibrating the mathematical relationship between the two, and deriving a curve equation Y as a by using a least square fitting method_BX+b_BAnd a linear correlation coefficient R_B ²(ii) a When R is_B ²When the confidence level is more than or equal to 0.98 and more than or equal to 0.95, the determination according to the method is effective;

the neutralizer identification and quantitative suspension test is carried out according to the following steps:

(1) and (3) identification test of a neutralizer: selecting the lowest effective concentration of a disinfectant with 99.9 percent of sterilization inhibiting indicator bacteria for 10min as the concentration of a sample solution of a neutralizer identification test, and mixing 1mL of the test sample solution with 9mL of neutralizer solution; after 10min of action to form a neutralized product, experimental groups were performed as follows: 0.49mL of inoculated broth (with lgC)_B-lgI_BThe curve calibration bacterial liquid is taken from the same test tube indicating strain stock solution, stored at 2 +/-0.2 ℃ and used within 2 h), and five test sample liquids (1) containing 4.41mL are separately packaged^#、2^#) 4.41mL of neutralized product solution (3)^#) 4.41mL of Chao's medium (4)^#) 4.41mL of neutralizer solution (5)^#) The tube was shaken at 1000r/min for 10min to mix the solution thoroughly. Then, 0.49mL of each mixed solution was dispensed into five portions containing 4.41mL of the Kirch's culture medium (1)^#、3^#、4^#、5^#) 4.41mL of neutralizer solution (2)^#) In the sterile test tube of (4.9 mL of the Chaudhuri's culture solution in another sterile test tube was used as the 6 th^#Test samples were prepared. Shaking each group of test tubes at 1000r/min for 10min, and collecting the mixed solution as the recovered solution of the test sample solution according to the relative fluorescence intensity value I_BMeasuring and recording the relative fluorescence intensity values of the 6 groups of recovery liquid.

(2) Evaluation of neutralizing effect: if it is 1 st^#、6^#Relative fluorescence intensity measurement value I of group-collected liquid_B1≥0、I_B60, 2 nd^#～5^#The relative fluorescence intensity measurements of the pooled solutions are related: i is_B2> 0 and I_B2＜I_B3、I_B2＜I_B4、I_B2＜I_B5(ii) a No. 3^#、4^#、5^#The relative fluorescence intensity measurements of the group-recovered solutions are close, i.e. I_B3≈I_B4≈I_B5The viable bacteria content C is calculated according to the formula (1)_B3、C_B4、C_B5The error rate delta between groups is less than or equal to 10 percent; the type of the selected neutralizer is used for a fungus killing effect test of a liquid disinfectant sample to be tested, and the neutralizer identification test is repeated after the concentration of the neutralizer is adjusted according to an equivalent neutralization principle, so that the concentration of the neutralizer corresponding to the test concentration of the disinfectant is determined.

In the formula:

delta-3 rd^#、4^#、5^#Viable bacteria content C of the composition recovery liquid_B3、C_B4、C_B5Inter-group error rate,%;

I_B3、I_B4、I_B5-No. 3^#、4^#、5^#Relative fluorescence intensity measurements, RLU, of the panel recovery fluids;

-No. 3^#、4^#、5^#The average viable bacteria content of the group recovery liquid is as follows

a_BStandard curve lgC_B-lgI_BA slope; b_BStandard curve lgC_B-lgI_BIntercept at the longitudinal axis;

(3) quantitative suspension test: diluting the disinfectant with sterile water to high, medium and low concentrations according to the effective concentration and action time range marked in the instructionThe appropriate concentration is: c₁、C₂、C₃(C₁Is the minimum use concentration specified in the product description), wherein C₂＝2C₁、C₃＝2C₂(ii) a Selecting four different sterilization times t simultaneously₁、t₂、t₃、t₄(t₂The minimum action time specified in the product description), where t₁＝0.5t₂、t₃＝1.5t₂、t₄＝2t₂. Then, 4.41mL of test sample liquid with different concentrations is subpackaged into three sterile test tubes by a sterilization pipette; and placing the mixture in a water bath at the temperature of 20 +/-2 ℃, and respectively dropwise adding 0.49mL of inoculated bacterial liquid after the temperature of the solution in the test tube is balanced with the temperature of the water bath. Starting to record after shaking the test tube for 30s at 3000r/min, and continuing the sterilization until the set t₁、t₂、t₃、t₄After four periods of different time, 0.49mL of the mixed solution with different concentrations is divided into three sterile test tubes containing 4.41mL of neutralizer solution by using a sterilization pipette, the test tubes are shaken at 3000r/min for 30s and begin to time, and after the neutralization lasts for 10min, the mixed solution is used as the recovery solution of the test sample solution with each concentration. Then, replacing the test sample liquid with the Chachi culture solution to repeat the test;

the experiment was repeated 5 times.

Then, calf serum was added to the bacterial suspension to give a final serum content of 10% and to ensure inoculation of bacterial suspension C_BIs 5.0X 10⁶CFU/mL～9.0×10⁶CFU/mL, repeat the quantitative suspension test 5 times; determining the effective concentration and time of a liquid disinfectant sample to be tested having a disinfection effect on fungi in the presence of organic matters;

the relative fluorescence intensity value I of the recovered liquid_BThe determination is carried out according to the following steps:

(1) calibrating relative fluorescence intensity background values of instruments and reagent groups: respectively adding 0.1mL of the Chaudou culture solution, 0.35mL of the physiological saline and 0.05mL of the ATP lysate into the same sterile test tube by using a sterilized pipette, and shaking the test tube for 30s at 3000 r/min; standing for 10-20 min to serve as a primary blank sample; then 0.1mL of the primary blank was transferred to another sterile test tube and 0.4mL of the crude sample was added dropwiseBrine was treated and mixed well as a secondary blank. Then, sequentially transferring 0.1mL of secondary blank samples into three special sterile test tubes of instruments by using a sterilization liquid-transferring gun to serve as blank test parallel samples; dripping 0.1mL of ATP extraction reagent into each of the three parallel samples, mixing uniformly, dripping 0.1mL of ATP fluorescence reagent, shaking the test tube at 3000r/min for 5s, and immediately measuring the relative fluorescence intensity value I of the test tube by using an ATP fluorescence photometer_BAnd recording (ensuring the operation time of each link to be consistent and avoiding cross contamination). Measuring each parallel sample for no more than 15s, and taking the arithmetic mean value of the relative fluorescence intensity values of the three blank test parallel samples as the background value of the instrument and reagent group (or calibrating the background according to the instruction of the instrument);

(2) relative fluorescence intensity value I of recovered liquid_BAnd (3) determination: if the background level of the blank reagent group meets the use requirement of the instrument, respectively adding 4.9mL of recovery solution and 0.1mL of ATP lysate into the same sterile test tube by using a sterile pipette, and shaking the test tube for 30s at 3000 r/min; standing at room temperature for 20min, adding 5.0mL of ATP extraction reagent dropwise, and mixing uniformly again; standing at room temperature for 10 min. 0.1mL of the above mixed solution was sequentially transferred to three instrument-dedicated sterile test tubes by a sterile pipette gun to serve as an ATP bioluminescence test replicate. Dripping 0.1mL of ATP fluorescent reagent into each of the three parallel samples, shaking the test tube for 5s at 3000r/min, and immediately measuring the relative fluorescence intensity value I of the three parallel samples by using an ATP fluorescence photometer_BAnd recording (ensuring the operation time of each link to be consistent and avoiding cross contamination). The determination time of each parallel sample is not more than 15s, and the arithmetic mean value of the relative fluorescence intensity values of the three ATP bioluminescence test parallel samples is taken as I of the recovery liquid of the sample to be detected_BA measured value;

the recovery liquid has viable bacteria content C_BAnd T_BCalculating the calculation of the killing rate R and the sterilization index A according to the following steps:

(1) viable bacteria content C of the recovered liquid_BAnd T_BCalculation of

Marking strain lgC according to indication_B-lgI_BLinear equation Y ═ a_BX+b_BCalculating viable bacteria content T of recovered liquid after four sections of sterilization/contamination time of test sample liquid and control sample liquid with different concentrations each time_BAnd C_B. The correlation calculation is shown in formulas (2) to (9):

in the formula:

the amount of viable bacteria recovered after four sterilization sessions for each test sample of different concentration, in colony forming units per milliliter (CFU/mL); the test times i are 1,2,3,4 and 5; concentration number j is 1,2, 3;

the relative fluorescence intensity measured value, RLU, of the recovered solution after four sterilization periods of the test sample solution with different concentrations each time; the test times i are 1,2,3,4 and 5; concentration number j is 1,2, 3;

the amount of viable bacteria recovered after four sessions of contamination of control sample solutions of different concentrations each time, in units of colony forming units per milliliter (CFU/mL); the test times i are 1,2,3,4 and 5; concentration number j is 1,2, 3;

after the control sample liquid with different concentrations is subjected to four times of bacterial contamination, the relative fluorescence intensity measured value, RLU, of the recovered liquid is obtained; the test times i are 1,2,3,4 and 5; concentration number j is 1,2, 3;

(2) calculation of kill Rate R

Under the condition that the identification result of the neutralizer is qualified, the test sample liquid with different concentrations kills fungi within four periods of sterilization time

And the killing rate of the liquid disinfectant sample to be tested with corresponding concentration in the same sterilization time

Calculated according to equations (10) to (17), respectively:

in the formula:

the killing rate of the fungus in the test sample liquid with different concentrations in four sterilization periods is percent; the test times i are 1,2,3,4 and 5; concentration number j is 1,2, 3;

-the kill rate of the liquid disinfectant sample to be tested with corresponding concentration against fungi within four sterilization periods,%; the test times i are 1,2,3,4 and 5; concentration number j is 1,2, 3;

a_Bstandard curve lgC_B-lgI_BA slope;

(3) calculation of Sterilization index A

Under the condition that the identification result of the neutralizer is qualified, the sterilization index of the test sample liquid with different concentrations to the fungi in four periods of sterilization time

And sterilization index of liquid disinfectant sample to be tested with corresponding concentration in the same sterilization time

Calculated according to equations (18) to (25), respectively:

in the formula:

-sterilization index of the test sample liquid at different concentrations against fungi during four periods of sterilization time each time; the test times i are 1,2,3,4 and 5; concentration number j is 1,2, 3;

sterilization of fungi in four sterilization periods for liquid disinfectant samples to be tested of corresponding concentrationAn index; the test times i are 1,2,3,4 and 5; concentration number j is 1,2, 3;

a_Bstandard curve lgC_B-lgI_BA slope;

(4) data reduction requirements: calibration of viable bacteria content C of bacterial suspension by adopting blood counting plate_BWhen referred to the relevant provisions in GB 4789.2-2016, when C_BWhen the concentration is less than 100CFU/mL, the integer is taken as "rounding off"; when C is present_BWhen the number is not less than 100CFU/mL, taking the first 2 digits after the 3 rd digit is rounded off, and replacing the digits with 0; it can also be expressed in the form of an exponent of 10, with two significant digits taken after "rounding". After the test sample liquid and the comparison sample liquid are sterilized/contaminated for four periods of time,the measured value of the relative fluorescence intensity of the recovered solution is an integer, the calculation result of the sterilization rate of the fungus is three significant digits, and the calculation result of the sterilization index is two significant digits;

(5) measurement uncertainty: the method comprises the steps of calculating four different sterilization/infection times of test sample liquid and reference sample liquid with different concentrations in 5 times of quantitative suspension tests, and obtaining a coefficient of variation C.V ═ σ ÷ μ × 100% (mu, σ and C.V calculation results are reserved to two decimal points) of 15 parallel ATP fluorescence test samples relative to fluorescence intensity measurement values by recovering liquid; the reproducibility of applying the ATP bioluminescence analysis method to the fungus killing effect test of the liquid disinfectant is judged, and the specified coefficient of variation C.V is less than or equal to 10 percent. The correlation calculation is shown in formulas (26) to (33):

in the formula:

warp t₁、t₂、t₃、t₄After four sections of different sterilization time, the relative fluorescence intensity measured values of 15 ATP fluorescence test parallel samples in test sample liquid with the same concentration in 5 tests

The arithmetic mean of (a); the test times i are 1,2,3,4 and 5; concentration number j is 1,2, 3; the parallel sample number k is 1,2, 3;

warp t₁、t₂、t₃、t₄After four sections of different sterilization time, 15 ATP fluorescence test parallel sample relative fluorescence intensity measured values of recovery liquid of test sample liquid with the same concentration in 5 tests

A standard deviation of (d); the test times i are 1,2,3,4 and 5; concentration number j is 1,2, 3; the parallel sample number k is 1,2, 3;

warp t₁、t₂、t₃、t₄After four different times of contamination, the relative fluorescence intensity measured values of 15 ATP fluorescence test parallel samples of the control sample liquid with the same concentration in 5 times of tests

warp t₁、t₂、t₃、t₄After four different times of contamination, 15 recovered solutions of control sample solution with the same concentration in 5 times of experiments test are used for testing the relative fluorescence intensity measured values of parallel samples

the result judgment of the fungus killing effect of the liquid disinfectant sample is carried out according to the following steps:

referring to the common practice in the health industry and the biological evaluation standard of related disinfection effect, if a liquid disinfectant sample liquid to be tested with a certain concentration is in a single quantitative suspension test, the liquid disinfectant sample liquid is subjected to ATP bioluminescence lgC_B─lgI_BThe sterilization effect in the test is judged to be qualified if the fungus killing rate in the specific sterilization time measured by the standard curve method is more than or equal to 99.9 percent or the sterilization index is more than or equal to 3.0. When the disinfection effect of the liquid disinfectant sample liquid to be measured in the concentration is qualified within the same disinfection time in 5 times of quantitative suspension tests, the minimum concentration and the minimum time for the liquid disinfectant sample liquid to be measured to have the disinfection effect on fungi can be determined (in practical application, the minimum concentration and the minimum time of an organic matter protection test are taken as the concentration and the time required by the liquid disinfectant sample to be measured to achieve the practical disinfection effect);

Detailed Description

The present invention is described in detail below with reference to preferred embodiments so that the advantages and features of the present invention can be more easily understood by those skilled in the art, and thus the scope of the present invention can be clearly and clearly defined.

In this example, the killing effect of a 13 α - (N, N-dimethylamino) ethylamino matrine disinfectant sample with a concentration of 120g/L on fungi is examined as an example.

The specific evaluation method is carried out according to the following steps:

(1) sample extraction and minimum effective concentration determination

1.1 control sample: the control sample was obtained in Chaudhur's broth.

1.2 test samples: randomly extracting 1 liquid disinfectant sample of the minimum sale package from 1 complete transport package of a certain production lot number for neutralizer identification and quantitative suspension test; for each set of test samples, a set of control samples was selected as a reference and effectively identified.

1.3 determination of the minimum effective concentration of disinfectant sample: diluting the disinfectant sample to be detected with sterile water into three proper concentrations, namely high concentration, medium concentration and low concentration, according to the effective concentration range marked by the use instruction of the disinfectant sample to be detected; 4.41mL of diluent with different concentrations is divided into three sterile test tubes by a sterilization pipette, and 0.49mL of inoculated bacteria liquid is respectively dripped into each test tube; after shaking the test tube at 3000r/min for 30s, it was placed in a water bath at (20. + -. 2 ℃ C.). Timing when the temperature of the solution in the test tube is balanced with the temperature of the water bath to ensure that the killing effect of the disinfectant on the fungi lasts for 10 min; then, the mixed solution is used as a recovery solution according to the relative fluorescence intensity value I in the patent_BAnd (3) measuring and recording the relative fluorescence intensity value of the recovered liquid. At the same time, the above-mentioned sterilization test and recovery solution I were repeated using 4.41mL of the Kircher's culture medium as a control sample_BA measuring process; calculating the killing rate of the diluent with different concentrations according to a killing rate R calculation formula in the patent; if the killing rate R of a certain concentration is 99.9%, the concentration is set as the minimum effective concentration of the disinfectant sample to be detected.

(2) Equipment model selection and preparation of reagent and culture medium

2.1 general requirements: the analytical reagent for the test and the tertiary water (distilled water or deionized water) meeting the requirements of GB/T6682-.

2.2 instrumentation: a secondary biological safety cabinet or an ultra-clean workbench with the cleanliness grade not lower than 100; an ATP bioluminescence rapid detection system containing an ATP fluorescence photometer, a special test tube and the like, wherein the wavelength range of the ATP fluorescence photometer is 300 nm-650 nm, and the total number detection range of fungal cells/spores is 10¹CFU/mL～10⁶CFU/mL; magnification of 40X-400 × biological optical microscope; (25-37) a constant temperature incubator at +/-1 ℃; (10-50) DEG C +/-1 ℃ constant-temperature water bath tank; a constant temperature oscillator at (0-50) DEG C +/-1 ℃ and (50-300) r/min; a centrifuge with the rotating speed more than or equal to 8000r/min and a matched centrifuge tube; a vortex oscillator with a rotating speed range (500-3000) r/min; a pressure steam sterilizer at (121 + -2) deg.C and (103 + -5) kPa; a low-temperature refrigerator at the temperature of minus 20 ℃ to minus 80 ℃; a freezer at 0-10 ℃; an electronic balance with a sensory volume of 0.001 g; an ultrasonic cleaner with a frequency range (30-50) kHz; a pH meter with the precision of +/-0.1 (25 ℃); an electric furnace.

2.3 materials utensil: a blood counting chamber and a special cover glass; 1mL and 10mL sterile graduated pipettes; 0.05mL, 0.1mL, 0.2mL, 1mL, 5mL, 10mL (metering error less than 1%) of single-channel variable range pipette and sterile pipette tip; sterile conical bottles and bottle stoppers with the capacities of 100mL, 250mL and 500 mL; a sterile culture dish; a glass funnel; a sterile cock test tube; an inoculating loop with the diameter not more than 4 mm; glass beads with a diameter of 5 mm; an alcohol lamp; sterilizing the tweezers; absorbent cotton and gauze for biochemical detection; sterile filter paper;

the thermometer of (1); stopwatch with accuracy 0.01 s.

2.4 reagent: 0.1% of Lv's basic methylene blue staining solution; calf serum; the following reagents are sterilized by high pressure for 30min at 121 ℃ and stored for 30d at 5-10 ℃: 85% of normal saline; one of N-methyl ethanesulfonic acid, Tween 80 and dioctyl sodium sulfosuccinate is selected to prepare a 0.05% wetting agent aqueous solution; 5g of sodium thiosulfate was dissolved in 1000mL of water (for chlorine type disinfectants); dissolving 1.36g of monopotassium phosphate, 2.83g of disodium hydrogen phosphate, 10g of lecithin, 10g of glycine and 30g of tween (80) in 1000mL of water or dissolving 1.36g of monopotassium phosphate, 2.83g of disodium hydrogen phosphate, 3g of lecithin and 20g of tween (80) in 1000mL of water (for a non-oxidizing disinfectant); 20g of Tween (80), 1g of sodium thiosulfate were dissolved in 1000mL of phosphate buffer solution (for oxygen-type disinfectant), etc. (or other suitable neutralizing agents were selected for the type of liquid disinfectant sample to be tested).

2.5 Medium/liquid (commercially available Medium/liquid): subpackaging the prepared culture medium/liquid, sterilizing at 121 ℃ for 30min under high pressure, and storing at 2-8 ℃ for 30 d;

potato-glucose culture medium (for activating mold spore strain) is prepared by peeling fresh potato 300g, cutting into pieces, and boiling in 1000mL water for 20-30 min; filtering to obtain juice, adding 20g of glucose, 0.1g of chloramphenicol and 20g of agar into the filtrate, and diluting to 1000 mL.

2.6ATP fluorescence reaction reagent (or with commercially available reagent): the prepared ATP fluorescent reaction reagent is preserved at the temperature of-20 ℃ to-70 ℃ besides phosphate buffer solution and is used within 6 months;

ATP fluorescent reagent: 0.7mg of luciferase, 12.6mg of D-luciferin and 56mg of bovine serum albumin are dissolved in 30mL of ATP fluorescent reagent buffer solution, and the mixture is uniformly mixed and then is kept stand for 15min at room temperature for use within 3 h.

(3) Strain preservation, activation and preparation of bacterial suspension

3.1 indicating the strain: candida albicans ATCC 10231; aspergillus niger ATCC 16404; chaetomium globosum ATCC 6205; penicillium chrysogenum ATCC 9179 (or other species supplied and traceable by the national collections of microorganisms).

3.2 strain preservation: candida albicans, opening a freeze-dried strain tube by aseptic operation, injecting a proper amount of Sabouraud's culture solution into the tube by using a capillary suction tube, blowing and sucking for several times to melt and disperse strains; dripping a little strain suspension into a test tube filled with 5-10 mL of Sabouraud's culture solution, and culturing for 18-24 h at 37 ℃. Mould-the mould test strain is inoculated on the slant of the potato-glucose culture medium by aseptic operation and the inoculation date is marked, and the mould is cultured at 28 ℃ to 30 ℃ until the slant is full of mould spores (7d to 14 d); preserving at 3-10 deg.c for 4 months as preserving bacteria.

3.3 strain activation: candida albicans-typical colonies in the 1 st generation culture were scraped with an inoculating loop and streaked onto a Shaw's medium plate; after culturing for 18-24 h at 37 ℃, selecting a typical colony in the culture of the 2 nd generation to inoculate on a slope of a Sabouraud's medium; culturing at 37 deg.C for 18-24 hr, storing at 4 deg.C under sealed condition, and using within 6 weeks. The mould is that the preserved fungus spores are scraped by an inoculating loop, the potato-glucose culture medium slant is inoculated, and the culture is carried out for 7 to 14 days at the temperature of between 28 and 30 ℃ until a large amount of spores are generated. Before the spore suspension is prepared, the test tube plug of the mould strain cannot be pulled out, each test tube is opened and then only used for preparing the spore liquid once, and the suspension is prepared by using newly cultured mould spores each time.

3.4 preparation of bacterial suspension: candida albicans test-selecting fresh strain culture from a slant with an inoculating loop, inoculating into a sterile conical flask filled with 50mL of Sabouraud's culture solution, placing in a constant temperature oscillator at (30 +/-1) ° C for culturing for 18 h-24 h at 150r/min, and storing in a sealed manner at 4 ℃ for use on the same day. Mold test-Add 10mL of sterile water to the strain tube, scrape the fresh mold spores on the surface of the medium with an inoculating loop, inject the washed spore stock solution into a sterile stoppered conical flask containing 15 glass beads and 45mL of Cnahs' culture solution, shake the tube at 3000r/min for 2min, break up the spore clusters, and mix the spore solution. Then, placing a glass funnel covered with sterile absorbent cotton or eight layers of gauze on a conical flask, and filtering the spore suspension to remove hyphae and culture medium fragments; transferring the filtrate into a sterilized centrifuge tube, separating at room temperature at 8000r/min for at least 10min, and removing supernatant; the spore precipitate was washed with 50mL of the Kirchner's medium and centrifuged, and after washing was repeated 3 times, the centrifuged spore precipitate was diluted with the Kirchner's medium. Preparing spore suspension for each test mould according to the method, and mixing spore liquid of each strain in equal volume; storing at 0-7 deg.c for use in the same day or 4 days.

(4) Viable bacteria content C of bacterial suspension after methylene blue dyeing_BBlood cell plate counting and inoculation bacterial liquid C_BCalibration

4.1 blue staining: sterile 50. mu.L dilution to 10^-2～10^-3The candida albicans suspension or the mold mixed spore solution and 30 mu L of 0.05 percent Lv's alkaline methylene blue staining solution are respectively transferred into the same sterile test tube (the dilution of the bacterial solution is that each cell of a blood counting plate contains 4-5 candida albicans cells or mold spores), and the test tube is shaken at 1000r/min for 1min, so that the bacterial suspension and the staining solution are fully and uniformly mixed.

4.2 blood cell plate count: and (3) placing 5 mu L +/-0.5 mu L of the stained bacterial suspension on the edge of a cover glass by using a sterile pipette, enabling the bacterial suspension to slowly permeate into a blood cell counting plate along the gap of the glass slide, and preventing bubbles from being generated between the counting plate and the glass slide, otherwise, operating again. And (4) completely sucking the redundant bacteria liquid in the tank by using sterile filter paper, standing for 2min +/-20 s, and completely settling into a counting chamber. When a 16-cell counting plate is used, counting candida albicans cells or mold spores in 4 middle cells (namely 100 small squares) which are positioned at the upper left, the upper right, the lower left and the lower right in a diagonal direction; if a 25-middle-grid counting plate is used, in addition to the 4 diagonal orientations, the central 1 middle grid (namely 80 small grids) is counted; when the test species are on the double lines of the middle lattice, only Candida albicans cells or mold spores are counted on the upper and right lines or on the lower and left lines.

4.3 viable bacteria content C_BAnd (3) calculating: after the magnification of the biological optical microscope is adjusted from low to high to 400 multiplied, the Candida albicans cells or the mould spores at the corresponding space position of the blood counting plate are immediately counted; wherein the colorless Candida albicans cells are viable bacteria, and the blue or light blue ones are dead bacteria; the mold spores with dark blue edge, colorless or light blue and light red inside are live bacteria, while those with dark blue edge and inside are dead bacteria, so only the spores with different colors are counted. If the number of the aseptic hypha monospores in the mold spore suspension is less than 90%, the spore liquid is prepared again. The candida albicans cells or mold spores in each cell of the hemacytometer were counted in duplicate by microscopy three times, and the average was taken. When the specification of the blood counting chamber is 16X 25, the viable bacteria content (colony forming unit per milliliter, CFU/mL) C of 1mL of bacterial liquid_B＝N÷5×16×K×10⁴(ii) a When the specification of the blood counting chamber is 25X 16, C_B＝N÷5×25×K×10⁴(ii) a Wherein N is the total number of viable candida albicans cells or mold spores in five middle lattices of the blood counting plate, and K is the bacterial liquid dilution factor. Then, the known viable bacteria content C is treated by the Czochralski culture solution_BDiluting the Candida albicans suspension or the mold mixed spore liquid to obtain C_BIn the range of 5.0X 10⁶CFU/mL～9.0×10⁶CFU/mL inoculated bacterial fluid.

(5) Log value of viable bacteria content lgC_BRelative fluorescence intensity log lgI_BStandard curve establishment

Using Chao's culture solution to treat known viable bacteria content C_BAfter the candida albicans suspension or the mold mixed spore liquid is subjected to continuous gradient dilution, a standard series of bacterial suspensions are obtained: 3.5X 10³CFU/mL、3.5×10⁴CFU/mL、3.5×10⁵CFU/mL and mix well. Then, according to the relative fluorescence intensity value I in this patent_BThe determination method is based on the viable bacteria content C_BThe relative fluorescence intensity values of the above standard solutions were measured and recorded in descending order. Then, lgI is calculated as the relative fluorescence intensity logarithm of the standard series bacterial suspension_BAs abscissa, log value lgC of corresponding viable bacteria content_BPlotting the ordinate; calibrating the mathematical relationship between the two, and deriving a curve equation Y as a by using a least square fitting method_BX+b_BAnd a linear correlation coefficient R_B ²(ii) a When R is_B ²When the confidence level is more than or equal to 0.98 and more than or equal to 0.95, the determination according to the method is effective.

(6) Neutralizer identification and quantitative suspension test

6.1 neutralizer identification test: selecting the lowest effective concentration of a disinfectant with 99.9 percent of sterilization inhibiting indicator bacteria for 10min as the concentration of a sample solution of a neutralizer identification test, and mixing 1mL of the test sample solution with 9mL of neutralizer solution; after 10min of action to form a neutralized product, experimental groups were performed as follows: 0.49mL of inoculated broth (with lgC)_B-lgI_BThe curve calibration bacterial liquid is taken from the same test tube indicating strain stock solution, stored at 2 +/-0.2 ℃ and used within 2 h), and five test sample liquids (1) containing 4.41mL are separately packaged^#、2^#) 4.41mL of neutralized product solution (3)^#) 4.41mL of Chao's medium (4)^#) 4.41mL of neutralizer solution (5)^#) The tube was shaken at 1000r/min for 10min to mix the solution thoroughly. Then, 0.49mL of each mixed solution was dispensed into five portions containing 4.41mL of the Kirch's culture medium (1)^#、3^#、4^#、5^#) 4.41mL of neutralizer solution (2)^#) In the sterile test tube of (4.9 mL of the Chaudhuri's culture solution in another sterile test tube was used as the 6 th^#Test samples were prepared. Shaking each group of test tubes at 1000r/min for 10min, and collecting the mixed solution as the recovered solution of the test sample solution according to the relative fluorescence intensity value I_BMeasuring and recording the relative fluorescence intensity values of the 6 groups of recovery liquid.

6.2 evaluation of neutralizing Effect: if it is 1 st^#、6^#Relative fluorescence intensity measurement value I of group-collected liquid_B1≥0、I_B60, 2 nd^#～5^#The relative fluorescence intensity measurements of the pooled solutions are related: i is_B2> 0 and I_B2＜I_B3、I_B2＜I_B4、I_B2＜I_B5(ii) a No. 3^#、4^#、5^#The relative fluorescence intensity measurements of the group-recovered solutions are close, i.e. I_B3≈I_B4≈I_B5The viable bacteria content C is calculated according to the formula (1)_B3、C_B4、C_B5The error rate delta between groups is less than or equal to 10 percent; the type of the selected neutralizer is used for a fungus killing effect test of a liquid disinfectant sample to be tested, and the neutralizer identification test is repeated after the concentration of the neutralizer is adjusted according to an equivalent neutralization principle, so that the concentration of the neutralizer corresponding to the test concentration of the disinfectant is determined.

In the formula:

a_BStandard curve lgC_B-lgI_BA slope; b_BStandard curve lgC_B-lgI_BIntercept at the longitudinal axis.

6.3 quantitative suspension test: diluting the disinfectant with sterile water to obtain the disinfectant with effective concentration and action time range marked in the instructionThree suitable concentrations are high, medium and low: c₁、C₂、C₃(C₁Is the minimum use concentration specified in the product description), wherein C₂＝2C₁、C₃＝2C₂(ii) a Selecting four different sterilization times t simultaneously₁、t₂、t₃、t₄(t₂The minimum action time specified in the product description), where t₁＝0.5t₂、t₃＝1.5t₂、t₄＝2t₂. Then, 4.41mL of test sample liquid with different concentrations is subpackaged into three sterile test tubes by a sterilization pipette; and placing the mixture in a water bath at the temperature of 20 +/-2 ℃, and respectively dropwise adding 0.49mL of inoculated bacterial liquid after the temperature of the solution in the test tube is balanced with the temperature of the water bath. Starting to record after shaking the test tube for 30s at 3000r/min, and continuing the sterilization until the set t₁、t₂、t₃、t₄After four periods of different time, 0.49mL of the mixed solution with different concentrations is divided into three sterile test tubes containing 4.41mL of neutralizer solution by using a sterilization pipette, the test tubes are shaken at 3000r/min for 30s and begin to time, and after the neutralization lasts for 10min, the mixed solution is used as the recovery solution of the test sample solution with each concentration. Then, the above test was repeated by replacing the test sample with the Kircher's culture solution.

The experiment was repeated 5 times.

Then, calf serum was added to the bacterial suspension to give a final serum content of 10% and to ensure inoculation of bacterial suspension C_BIs 5.0X 10⁶CFU/mL～9.0×10⁶CFU/mL, repeat the quantitative suspension test 5 times; determining the effective concentration and time of the liquid disinfectant sample to be tested having a disinfection effect on fungi in the presence of organic matters.

(7) Relative fluorescence intensity value I of recovered liquid_BMeasurement of

7.1 Instrument and reagent set relative fluorescence intensity background calibration: respectively adding 0.1mL of the Chaudou culture solution, 0.35mL of the physiological saline and 0.05mL of the ATP lysate into the same sterile test tube by using a sterilized pipette, and shaking the test tube for 30s at 3000 r/min; standing for 10-20 min to serve as a primary blank sample; then 0.1mL of the primary blank was transferred to another sterile test tube, 0.4mL of physiological saline was added dropwise and mixedAnd (6) homogenizing to obtain a secondary blank sample. Then, sequentially transferring 0.1mL of secondary blank samples into three special sterile test tubes of instruments by using a sterilization liquid-transferring gun to serve as blank test parallel samples; dripping 0.1mL of ATP extraction reagent into each of the three parallel samples, mixing uniformly, dripping 0.1mL of ATP fluorescence reagent, shaking the test tube at 3000r/min for 5s, and immediately measuring the relative fluorescence intensity value I of the test tube by using an ATP fluorescence photometer_BAnd recording (ensuring the operation time of each link to be consistent and avoiding cross contamination). The measurement time of each parallel sample does not exceed 15s, and the arithmetic mean of the relative fluorescence intensity values of three blank test parallel samples is used as the background value of the instrument and reagent group (or the background is calibrated according to the instruction of the instrument).

7.2 relative fluorescence intensity value I of the recovered liquid_BAnd (3) determination: if the background level of the blank reagent group meets the use requirement of the instrument, respectively adding 4.9mL of recovery solution and 0.1mL of ATP lysate into the same sterile test tube by using a sterile pipette, and shaking the test tube for 30s at 3000 r/min; standing at room temperature for 20min, adding 5.0mL of ATP extraction reagent dropwise, and mixing uniformly again; standing at room temperature for 10 min. 0.1mL of the above mixed solution was sequentially transferred to three instrument-dedicated sterile test tubes by a sterile pipette gun to serve as an ATP bioluminescence test replicate. Dripping 0.1mL of ATP fluorescent reagent into each of the three parallel samples, shaking the test tube for 5s at 3000r/min, and immediately measuring the relative fluorescence intensity value I of the three parallel samples by using an ATP fluorescence photometer_BAnd recording (ensuring the operation time of each link to be consistent and avoiding cross contamination). The determination time of each parallel sample is not more than 15s, and the arithmetic mean value of the relative fluorescence intensity values of the three ATP bioluminescence test parallel samples is taken as I of the recovery liquid of the sample to be detected_BAnd (d) measuring the value.

(8) Viable bacteria content C of the recovered liquid_BAnd T_BCalculating out the killing rate R and the sterilization index A

8.1 viable bacteria content C of the recovered liquid_BAnd T_BCalculation of

in the formula:

after the control sample liquid with different concentrations is subjected to four times of bacterial contamination, the relative fluorescence intensity measured value, RLU, of the recovered liquid is obtained; the test times i are 1,2,3,4 and 5; the concentration number j is 1,2, 3.

8.2 calculation of kill Rate R

Calculated according to equations (10) to (17), respectively:

in the formula:

control samples of different concentrations at a timeThe amount of viable bacteria recovered after the liquid is subjected to four times of bacterial contamination time is in the unit of colony forming unit per milliliter (CFU/mL); the test times i are 1,2,3,4 and 5; concentration number j is 1,2, 3;

a_Bstandard curve lgC_B-lgI_BThe slope.

8.3 Sterilization index A calculation

Calculated according to equations (18) to (25), respectively:

in the formula:

-sterilization index of a liquid disinfectant sample to be tested at a corresponding concentration against fungi within four periods of sterilization time; the test times i are 1,2,3,4 and 5; concentration number j is 1,2,3；

a_Bstandard curve lgC_B-lgI_BThe slope.

8.4 data reduction requirements: calibration of viable bacteria content C of bacterial suspension by adopting blood counting plate_BWhen referred to the relevant provisions in GB 4789.2-2016, when C_BWhen the concentration is less than 100CFU/mL, the integer is taken as "rounding off"; when C is present_BWhen the number is not less than 100CFU/mL, taking the first 2 digits after the 3 rd digit is rounded off, and replacing the digits with 0; it can also be expressed in the form of an exponent of 10, with two significant digits taken after "rounding". After the test sample liquid and the reference sample liquid are sterilized/infected for four periods of time, the measured values of the relative fluorescence intensity of the recovered liquid are taken as integers, and the integrated values are comparedThe calculation result of the sterilization rate of the fungus takes three significant figures, and the calculation result of the sterilization index takes two significant figures.

8.5 measurement uncertainty: the method comprises the steps of calculating four different sterilization/infection times of test sample liquid and reference sample liquid with different concentrations in 5 times of quantitative suspension tests, and obtaining a coefficient of variation C.V ═ σ ÷ μ × 100% (mu, σ and C.V calculation results are reserved to two decimal points) of 15 parallel ATP fluorescence test samples relative to fluorescence intensity measurement values by recovering liquid; the reproducibility of applying the ATP bioluminescence analysis method to the fungus killing effect test of the liquid disinfectant is judged, and the specified coefficient of variation C.V is less than or equal to 10 percent. The correlation calculation is shown in formulas (26) to (33):

in the formula:

A standard deviation of (d); the test times i are 1,2,3,4 and 5; concentration number j is 1,2, 3; the parallel number k is 1,2, 3.

(9) Determination of results

Referring to the common practice in the health industry and the biological evaluation standard of related disinfection effect, if a liquid disinfectant sample liquid to be tested with a certain concentration is in a single quantitative suspension test, the liquid disinfectant sample liquid is subjected to ATP bioluminescence lgC_B─lgI_BThe sterilization effect in the test is judged to be qualified if the fungus killing rate in the specific sterilization time measured by the standard curve method is more than or equal to 99.9 percent or the sterilization index is more than or equal to 3.0. When the disinfection effect of the liquid disinfectant sample liquid to be measured in the concentration is qualified within the same disinfection time in 5 times of quantitative suspension tests, the minimum concentration and the minimum time for the liquid disinfectant sample liquid to be measured to have the disinfection effect on fungi can be determined (in practical application, the minimum concentration and the minimum time of the organic matter protection test are used as the concentration and the time required by the liquid disinfectant sample to be measured to achieve the practical disinfection effect).

Laboratory biosafety qualifications, instrumentation, media/liquids, chemical reagents, standard strains used in this example:

(1) laboratory biosafety qualifications

In a secondary biological safety laboratory with the agency registration number CNAS BL0059, relevant experiments are completed by microorganism evaluation professionals with secondary advanced technical titles.

(2) Instrument and test device

2.1 second-level biosafety cabinet: thermo Scientific 1300 series second-level B2 type biosafety cabinet with 0.55m of surface area of workbench²Exhaust gas quantity 1130m³H, filtration efficiency 99.99% (0.3 μm).

2.2ATP Biofluorescence Rapid detection System: a portable system SURE ATP fluorescence detector of Hygiena company in America comprises a matched kit, a special plastic test tube and the like; ATP content detection lower limit of 4X 10^-18mol/ml, the detection limit of total microbial quantity of 1.0CFU/ml, the RLU reading range of 0-9999, the detection time of 10s, the sampling rate of 1000 times/s and the measurement error of +/-5 percent.

2.3 biological optical microscope: an Olympus BX43 three-mesh research grade biological microscope with a 10-time wide-field adjustable eyepiece and 4-time, 10-time, 20-time, 40-time and 100-time flat field achromatic objectives.

2.4 constant temperature and humidity incubator: a constant temperature and humidity incubator with model WS-380H of Guansen Biotechnology (Shanghai) Limited, volume 380L, temperature control range (0-50) DEG C +/-0.8 ℃, humidity control range (30-95)% RH +/-2% RH.

2.5 constant temperature water bath: the electrothermal constant-temperature water tank of Shanghai Renwei test instrument equipment Limited model DK-S420 has the volume of 15L, the temperature control range (5-99.9) DEG C +/-0.5 ℃ and the timing range of 1-999 min.

2.6 constant temperature oscillator: the Shanghai constant type WS-380H, the temperature control range (4-65) DEG C +/-0.1 ℃, the oscillation frequency (40-300) r/min and the timing range (1-99) H.

2.7 refrigerated centrifuge: the model DL7M-12L vertical freezing centrifuge of Beijing Xinnuo Li Hua apparatus Limited has the rotation speed of 8000r/min +/-20 r/min and the relative centrifugal force of 12300 Xg; the capacity is 14400ml, the timing range is 1 s-99 h59min99s, the temperature control range is (-20-40) DEG C +/-1 ℃, and the specification of the centrifugal tube is phi 74mm multiplied by 168 mm.

2.8 pressure steam sterilizer: an MLS-3780 autoclave manufactured by Sanyo corporation of Japan, with a volume of 75L, a sterilization temperature of 105-135℃ + -2 ℃, a maximum pressure of 0.235MPa, and a timer of 1-250 min.

2.9 low-temperature refrigerator: the ultra-low temperature freezing storage box of the Mike Mitsubishi low temperature science and technology corporation model DW-HL398 has an effective volume of 398L and a storage temperature of-10 ℃ to-86 ℃.

2.10 refrigerator: a cryobox ultra-low temperature refrigerator of HXL-25-250AD model of Haoxin instruments and equipment Limited in Dongguan city is characterized in that a refrigerator (2-10) DEG C +/-0.1 ℃ and a freezer (30-20) DEG C +/-0.1 ℃.

2.11 electronic balance: an electronic balance of Japan Shimadzu model AUX220, measuring range 220g, precision + -0.1 mg.

2.12 ultrasonic cleaner: the capacity of an ultrasonic cleaner of Shanghai Yijing ultrasonic instrument Limited type YQ-120C is 3.2L, the ultrasonic frequency is 40KHz/28KHz/25KHz, and the timing range is 1 min-30 min/99 min.

2.13 vortex oscillator: the rotation speed range (500-3000) r/min +/-3 r/min and the timing range (1-60 s) of a vortex oscillator of American brand Coleparmer Votex-Genie 2.

2.14pH meter: a precision pH meter of Shanghai precision instruments and meters, namely MP512-03, has a measuring range of (-2.000-19.999) pH +/-0.002 pH and a temperature range of (-10-110) DEG C +/-0.4 ℃.

2.15 electric furnace: a 1KW closed temperature-adjusting electric furnace of model DLD-1KW of Changzhou German instruments Co.

2.16 hemocytometer: shanghai refinement specification is a 25 × 16 blood count plate.

(3) Culture medium/liquid

Culture medium/liquid produced by Beijing Luqiao technology GmbH.

(4) Chemical reagent

4.10.1% of luwenshi alkaline methylene blue staining solution: purchased from Shanghai Heart Biotech, Inc.

4.2 adenosine disodium triphosphate standard and a series of biochemical reagents required for preparing ATP fluorescent reagent buffer solution, ATP lysate, ATP extract and ATP fluorescent reagent were purchased from America Amresco brand of Shanghai gold Pai Biotech Co., Ltd.

(5) Standard strains

Candida albicans ATCC 10231 was purchased from China medical fungi collection and management center; aspergillus niger ATCC 16404, Chaetomium globosum ATCC6205 and Penicillium chrysogenum ATCC 9179 are purchased from China general microbiological culture Collection center.

Evaluation data and result calculation of the present example:

using ATP fluorophotometer lgC_B-lgI_BDetecting the fungus killing effect of 13 alpha- (N, N-dimethylamino) ethylamino matrine disinfectant liquid sample by a standard curve method within 10minThe minimum effective concentrations of the compound for killing candida albicans and mold with the killing rate of 99.9 percent are 113g/L and 105g/L respectively. TPS solution containing 10g/L lecithin, 35g/L glycine and 100g/L tween (80) is selected as a neutralizer, so that the residual action of a to-be-detected disinfectant sample on an indication strain can be effectively neutralized; the identification of related neutralizer, the test data of quantitative suspension, the evaluation of disinfection effect and the calculation result of measurement uncertainty are respectively shown in tables 1-3.

TABLE 1 neutralizer identification test data and inter-group error rate calculation results

TABLE 2 quantitative suspension test data and kill rate R_ijtSterilization index A_ijtCalculation results

TABLE 3 evaluation of Disinfection Effect and calculation of the uncertainty of the measurement of the relative fluorescence intensity value

The above description is only an example of the present invention, and is not intended to limit the scope of the present invention, and all equivalent modifications made by the present invention in the specification or other related fields directly or indirectly are included in the scope of the present invention.

Claims

1. A method of evaluating the effectiveness of a liquid disinfectant for fungus kill, comprising: firstly, extracting a sample and determining the lowest effective concentration; selecting equipment, preparing reagents and culture media; thirdly, strain preservation, activation and preparation of bacterial suspension; fourthly, the viable bacteria content C of the bacterial suspension after the dyeing of the methylene blue_BBlood cell plate counting and inoculation bacterial liquid C_BCalibrating; log value of viable bacteria content lgC_BRelative fluorescence intensity log lgI_BEstablishing a standard curve; sixthly, identifying the neutralizer and carrying out quantitative suspension test; seventhly, recovering the relative fluorescence intensity value I of the liquid phase_BMeasuring; content of viable bacteria C in recovered liquid_BAnd T_BCalculating the sterilization rate R and the sterilization index A; ninthly, judging a result; the method is characterized in that ATP bioluminescence lgC for accurately and quantitatively evaluating the fungus killing effect of the liquid disinfectant characterized by a killing rate R or a sterilization index A by using an ATP fluorescence photometer_B-lgI_BStandard curve method, in particular:

determining the extraction requirement of a sample, determining the minimum effective concentration of the inhibiting and killing indicator bacteria in 10min of the disinfectant, carrying out passage and activation on standard strains of fungus test strains, and taking fresh candida albicans or mould spore cultures to prepare a bacterial suspension; measuring viable bacteria content C of bacteria liquid by adopting a blood counting chamber after methylene blue staining_BAnd inoculating the inoculum C_BAnd (3) calibrating: 5.0X 10⁶CFU/mL～9.0×10⁶CFU/mL; selecting viable bacteria content C_BIs 3.5 multiplied by 10³CFU/mL、3.5×10⁴CFU/mL、3.5×10⁵CFU/mL bacterial suspension is used as standard series bacterial suspension, and ATP fluorescence photometer is used for measuring relative fluorescence intensity value I_BCarrying out measurement; drawing lgC_B-lgI_BStandard curve, linear equation Y ═ a derived from curve_BX+b_BAnd the correlation coefficient R_B ²；a_BStandard curve lgC_B-lgI_BThe slope of the linear equation;b_Bstandard curve lgC_B-lgI_BLinear equation intercept at vertical axis; then, carrying out a neutralizer identification test according to the minimum effective concentration of the disinfectant to be tested, and determining the type and concentration of the neutralizer; simultaneously placing 4.41mL of test sample liquid and control sample liquid with various concentrations in a water bath with the temperature of 20 +/-2 ℃ and mixing the test sample liquid and the control sample liquid with 0.49mL of inoculation bacterial liquid; the effect to be sterilized lasts for t₁、t₂、t₃、t₄After four periods of different time, 0.49mL of the contaminated sample liquid is respectively mixed with 4.41mL of neutralizing agent solution, and after neutralization is carried out for 10min, the uniformly mixed solution is used as a recovery solution; measuring relative fluorescence intensity value of recovered solution by using ATP fluorescence photometer

And

and according to the linear equation Y ═ a of the standard curve of the indicated strain_BX+b_BCalculating the content of corresponding viable bacteria

And

the test times i are 1,2,3,4 and 5; concentration number j is 1,2, 3;

the amount of viable bacteria recovered after four periods of sterilization time of the test sample liquid with different concentrations at each time is in units of colony forming units per milliliter (CFU/mL);

the relative fluorescence intensity measured value, RLU, of the recovered solution after four sterilization periods of the test sample solution with different concentrations each time;

the amount of viable bacteria recovered after four times of contamination of control sample liquid with different concentrations each time is in the unit of colony forming unit per milliliter (CFU/mL);

after the control sample liquid with different concentrations is subjected to four times of bacterial contamination, the relative fluorescence intensity measured value, RLU, of the recovered liquid is obtained;

in the calculation of the killing rate R or the sterilization index A:

Or sterilization index

Or sterilization index

the recovery liquid has viable bacteria content C_BAnd T_BCalculating and killingCalculating the ratio R and the sterilization index A according to the following steps:

(1) viable bacteria content C of the recovered liquid_BAnd T_BCalculation of

According to the standard curve lgC of the indicated strain_B-lgI_BLinear equation Y ═ a_BX+b_BCalculating viable bacteria content T of recovered liquid after four sections of sterilization/contamination time of test sample liquid and control sample liquid with different concentrations each time_BAnd C_B(ii) a The correlation calculation is shown in formulas (1) to (8):

(2) calculation of kill Rate R

Calculated according to equations (9) to (16), respectively:

in the formula:

the killing rate of the fungus in the test sample liquid with different concentrations in four sterilization periods is percent;

-the kill rate of the liquid disinfectant sample to be tested with corresponding concentration against fungi within four sterilization periods,%;

(3) calculation of Sterilization index A

Calculated according to equations (17) to (24), respectively:

in the formula:

-sterilization index of the test sample liquid at different concentrations against fungi during four periods of sterilization time each time;

-sterilization index of a liquid disinfectant sample to be tested at a corresponding concentration against fungi within four periods of sterilization time;

(4) data reduction requirements: calibration of viable bacteria content C of bacterial suspension by adopting blood counting plate_BReference is made to GB 4789.2-2016 when C_BWhen the concentration is less than 100CFU/mL, the integer is taken as "rounding off"; when C is present_BAt not less than 100CFU/mL, the 3 rd digit is "rounded offTaking the first 2 digits and replacing the digits with 0 at the back; it can also be expressed in the form of an exponent of 10, "rounded" followed by two significant digits; after the test sample liquid and the reference sample liquid are sterilized/infected for four periods of time, taking an integer from the measured value of the relative fluorescence intensity of the recovered liquid, taking three effective numbers from the calculation result of the sterilization rate of the fungus, and taking two effective numbers from the calculation result of the sterilization index;

(5) measurement uncertainty: by calculating four different sterilization/infection times of test sample liquid and control sample liquid with different concentrations in 5 times of quantitative suspension tests, the variation coefficient C.V of 15 ATP fluorescence test parallel samples relative to fluorescence intensity measured values of the recovery liquid is sigma/mu multiplied by 100 percent, and the calculation results of mu, sigma and C.V are reserved to two decimal points; judging the reproducibility of applying the ATP bioluminescence analysis method to the fungus killing effect test of the liquid disinfectant, wherein the specified coefficient of variation C.V is less than or equal to 10 percent; the correlation calculation is shown in formulas (25) to (32):

in the formula:

warp t₁、t₂、t₃、t₄After four sections of different sterilization time, the recovery liquid of the test sample liquid with the same concentration in 5 tests measures the relative fluorescence intensity measured values of 15 ATP fluorescence test parallel samples

The arithmetic mean of (a); the parallel sample number k is 1,2, 3;

A standard deviation of (d);

warp t₁、t₂、t₃、t₄After four different times of contamination, the recovery liquid of the control sample liquid with the same concentration in 5 times of experiments measures the relative fluorescence intensity of 15 parallel samples of ATP fluorescence test

The arithmetic mean of (a);

A standard deviation of (d);

in the result determination:

referring to the common practice in the health industry and the biological evaluation standard of related disinfection effect, if a liquid disinfectant sample liquid to be tested with a certain concentration is in a single quantitative suspension test, the liquid disinfectant sample liquid is subjected to ATP bioluminescence lgC_B─lgI_BThe fungus killing rate in a specific sterilization time measured by a standard curve method is more than or equal to 99.9 percent or the sterilization index is more than or equal to 3.0, and the sterilization effect of the fungus in the test is judged to be qualified; when the disinfection effect of the liquid disinfectant sample liquid with the concentration to be detected in 5 times of quantitative suspension tests within the same disinfection time is qualified, the minimum concentration and the minimum time for the liquid disinfectant sample liquid to have the disinfection effect on fungi can be determined; in practical application, the minimum concentration and the minimum time of an organic matter protection test are taken as the concentration and the time required by the liquid disinfectant sample to be detected to achieve the practical disinfection effect.

2. A method of assessing the effectiveness of a liquid disinfectant for combating fungi according to claim 1, wherein said sample extraction and determination of minimum effective concentration is performed by the steps of:

(1) control sample: the Chao's culture solution is used as a control sample;

(2) test samples: randomly extracting 1 liquid disinfectant sample of the minimum sale package from 1 complete transport package of a certain production lot number for neutralizer identification and quantitative suspension test; selecting a group of control samples as reference substances for each group of test samples and effectively identifying the reference substances;

(3) determination of the minimum effective concentration of disinfectant sample: diluting the disinfectant sample to be detected with sterile water into three proper concentrations, namely high concentration, medium concentration and low concentration, according to the effective concentration range marked by the use instruction of the disinfectant sample to be detected; 4.41mL of diluent with different concentrations is divided into three sterile test tubes by a sterilization pipette, and 0.49mL of inoculated bacteria liquid is respectively dripped into each test tube; shaking the test tube at 3000r/min for 30s, and placing the test tube in a water bath at 20 +/-2 ℃; timing when the temperature of the solution in the test tube is balanced with the temperature of the water bath to ensure that the killing effect of the disinfectant on the fungi lasts for 10 min; then, the mixed solution is used as a recovery solution according to the relative fluorescence intensity value I_BA measuring method for measuring and recording the relative fluorescence intensity value of the recovered liquid; at the same time, the above-mentioned sterilization test and recovery solution I were repeated using 4.41mL of the Kircher's culture medium as a control sample_BA measuring process; calculating the killing rate of the diluent with different concentrations according to a killing rate R calculation formula; if the killing rate R of a certain concentration is 99.9%, the concentration is set as the minimum effective concentration of the disinfectant sample to be detected.

3. The method for evaluating the fungicidal effectiveness of a liquid disinfectant according to claim 1, wherein the selection of the apparatus, the preparation of the reagents and the preparation of the culture medium are carried out by the following steps:

(1) the general requirements are as follows: the analytical reagent for the test and the tertiary water which conforms to the GB/T6682-;

(2) the instrument equipment comprises: a secondary biological safety cabinet or an ultra-clean workbench with the cleanliness grade not lower than 100; an ATP bioluminescence rapid detection system containing an ATP fluorescence photometer and a special test tube, wherein the wavelength range of the ATP fluorescence photometer is 300 nm-650 nm, and the total number detection range of fungal cells/spores is 10¹CFU/mL～10⁶CFU/mL; a biological optical microscope at a magnification of 40X to 400X; a constant temperature incubator at 25 +/-1 ℃ to 37 +/-1 ℃; a constant temperature water bath tank with the temperature of 10 +/-1 ℃ to 50 +/-1 ℃; constant temperature oscillation at 0 +/-1-50 +/-1 ℃ and 50-300 r/minA machine; a centrifuge with the rotating speed more than or equal to 8000r/min and a matched centrifuge tube; a vortex oscillator with the rotating speed range of 500r/min to 3000 r/min; a steam sterilizer with the temperature of 121 +/-2 ℃ and the pressure of 103kPa +/-5 kPa; a low-temperature refrigerator at the temperature of minus 20 ℃ to minus 80 ℃; a freezer at 0-10 ℃; an electronic balance with a sensory volume of 0.001 g; an ultrasonic cleaner with a frequency range of 30 kHz-50 kHz; a pH meter with an accuracy of +/-0.1 at 25 ℃; an electric furnace;

(3) material utensil: a blood counting chamber and a special cover glass; 1mL and 10mL sterile graduated pipettes; 0.05mL, 0.1mL, 0.2mL, 1mL, 5mL, 10mL of single-channel variable-range pipetting gun and sterile pipetting gun head, wherein the metering error is less than 1%; sterile conical bottles and bottle stoppers with the capacities of 100mL, 250mL and 500 mL; a sterile culture dish; a glass funnel; a sterile cock test tube; an inoculating loop with the diameter not more than 4 mm; glass beads with a diameter of 5 mm; an alcohol lamp; sterilizing the tweezers; absorbent cotton and gauze for biochemical detection; sterile filter paper; 0 plus or minus 0.2-100 plus or minus 0.2 deg.C thermometer; a stopwatch with an accuracy of 0.01 s;

(4) reagent: 0.1% of Lv's basic methylene blue staining solution; calf serum; the following reagents are sterilized by high pressure for 30min at 121 ℃ and stored for 30d at 5-10 ℃: 85% of normal saline; one of N-methyl ethanesulfonic acid, Tween 80 and dioctyl sodium sulfosuccinate is selected to prepare a 0.05% wetting agent aqueous solution; 5g of sodium thiosulfate was dissolved in 1000mL of water (for chlorine type disinfectants); dissolving 1.36g of monopotassium phosphate, 2.83g of disodium hydrogen phosphate, 10g of lecithin, 10g of glycine and 30g of tween 80 in 1000mL of water or dissolving 1.36g of monopotassium phosphate, 2.83g of disodium hydrogen phosphate, 3g of lecithin and 20g of tween 80 in 1000mL of water (for a non-oxidizing disinfectant); 20g of Tween 80, 1g of sodium thiosulfate were dissolved in 1000mL of phosphate buffer solution (for oxygen disinfectant);

(5) as the medium/liquid, commercially available medium/liquid: subpackaging the prepared culture medium/liquid, sterilizing at 121 ℃ for 30min under high pressure, and storing at 2-8 ℃ for 30 d;

sakeh medium/liquid (for candida albicans strain activation): dissolving 40g of glucose, 10g of peptone and 20g of agar in 1000mL of water by heating; agar is not added into the culture solution; adjusting the pH to 5.6 +/-0.2 at 25 ℃;

the Chaudou culture solution (for preparing mould spore solution, diluting the bacterial suspension and eluting the sample) comprises the following components: heating and dissolving 2g of sodium nitrate, 1g of dipotassium phosphate, 0.5g of potassium chloride, 0.5g of magnesium sulfate, 0.01g of ferrous sulfate and 30g of cane sugar in 1000mL of aqueous solution containing 0.05% of wetting agent, and adjusting the pH value to 6.0-6.5 at 25 ℃;

(6) ATP fluorescence reaction reagent, or with commercially available reagents: the prepared ATP fluorescent reaction reagent is preserved at the temperature of-20 ℃ to-70 ℃ besides phosphate buffer solution and is used within 6 months;

ATP lysate: dissolving 4.6 international units/mL apyrase (EC:3.6.1.5) and 46 international units/mL adenosine deaminase (EC:3.5.4.6 or EC:3.5.4.17), 37mg sucrose and 20mg bovine serum albumin in 10mL 2-morpholinoethanesulfonic acid buffer solution with concentration of 0.05mol/L, adjusting pH to 6.0 +/-0.5, and using within 8 h; 1mL of lysate can reduce the ATP concentration in the Sasa culture solution to 10 within 15min^-11Less than mol/L;

ATP extracting solution: heating and dissolving 45mg of tris (hydroxymethyl) aminomethane in 9.8ml of water, uniformly mixing with 0.2ml of 10% benzalkonium chloride solution, and adjusting the pH value to 12.0 +/-0.5; the ATP extraction efficiency of the fungal cells is not lower than 80%;

4. The method of claim 1 wherein the steps of strain preservation, activation and suspension preparation are performed by:

(1) indicating strains: candida albicans ATCC 10231; aspergillus niger ATCC 16404; chaetomium globosum ATCC 6205; penicillium chrysogenum ATCC 9179;

(2) and (3) strain preservation: candida albicans, opening a freeze-dried strain tube by aseptic operation, injecting a proper amount of Sabouraud's culture solution into the tube by using a capillary suction tube, blowing and sucking for several times to melt and disperse strains; dripping a little strain suspension into a test tube filled with 5-10 mL of Sa's culture solution, and culturing for 18-24 h at 37 ℃; mould-a mould test strain is inoculated on the slant of a potato-glucose culture medium by aseptic operation, the inoculation date is marked, and the test strain is cultured at the temperature of between 28 and 30 ℃ until the slant is full of mould spores and the culture time is 7 to 14 days; preserving for 4 months at 3-10 ℃ as preserving bacteria;

(3) activating strains: candida albicans-typical colonies in the 1 st generation culture were scraped with an inoculating loop and streaked onto a Shaw's medium plate; after culturing for 18-24 h at 37 ℃, selecting a typical colony in the culture of the 2 nd generation to inoculate on a slope of a Sabouraud's medium; culturing at 37 ℃ for 18-24 h, and storing at 4 ℃ in a sealed manner for use within 6 weeks; mould, namely scraping preserved fungus spores by using an inoculating ring, inoculating a potato-glucose culture medium inclined plane, and culturing at 28-30 ℃ for 7-14 days until a large number of spores are generated; before preparing spore suspension, a test tube plug of a mould strain cannot be pulled out, each test tube is opened and then only used for preparing spore liquid once, and newly cultured mould spores are used for preparing suspension each time;

(4) preparing a bacterial suspension: candida albicans test, selecting fresh strain culture from an inclined plane by using an inoculating loop, inoculating the fresh strain culture into a sterile conical flask filled with 50mL of Sha's culture solution, placing the sterile conical flask in a constant temperature oscillator at 30 +/-1 ℃ for culturing for 18-24 h at 150r/min, and storing the culture in a sealed manner at 4 ℃ for use on the same day; mold test, namely adding 10mL of sterile water into a strain test tube, scraping fresh mold spores on the surface of a culture medium by using an inoculating loop, injecting the washed spore stock solution into a sterile stoppered conical flask filled with 15 glass beads and 45mL of Cnahs' culture solution, shaking the test tube for 2min at 3000r/min, breaking up spore clusters, and uniformly mixing spore solution; then, placing a glass funnel covered with sterile absorbent cotton or eight layers of gauze on a conical flask, and filtering the spore suspension to remove hyphae and culture medium fragments; transferring the filtrate into a sterilized centrifuge tube, separating at room temperature at 8000r/min for at least 10min, and removing supernatant; cleaning the spore precipitate with 50mL of Chao's culture solution, centrifuging, repeatedly cleaning for 3 times, and diluting the centrifuged spore precipitate with Chao's culture solution; preparing spore suspension for each test mould according to the method, and mixing spore liquid of each strain in equal volume; storing at 0-7 deg.c for 4 days.

5. The method of claim 1 wherein the viable bacteria content C of the liquid disinfectant suspension is measured after the methylene blue staining_BBlood cell plate counting and inoculation bacterial liquid C_BThe calibration is carried out according to the following steps:

(1) and (3) dyeing with methylene blue: sterile 50. mu.L dilution to 10^-2～10^-3Respectively transferring one of the candida albicans suspension or the mold mixed spore solution and 30 mu L of 0.05 percent Lv's alkaline methylene blue staining solution into the same sterile test tube, wherein the bacterial solution dilution is that each cell of a blood counting plate contains 4-5 candida albicans cells or mold spores, and shaking the test tube for 1min at 1000r/min to fully and uniformly mix the bacterial suspension and the staining solution;

(2) and (3) counting blood corpuscle plates: placing 5 microliter +/-0.5 microliter of the stained bacterial suspension on the edge of a cover glass by using a sterile pipette, enabling the bacterial suspension to slowly permeate into a blood cell counting plate along a slide gap, and not generating bubbles between the counting plate and the slide, or else, operating again; completely sucking the redundant bacteria liquid in the tank by using sterile filter paper, standing for 2min +/-20 s, and completely settling the bacteria liquid in a counting chamber; when a 16-cell counting plate is used, counting candida albicans cells or mold spores in 4 middle cells (namely 100 small squares) which are positioned at the upper left, the upper right, the lower left and the lower right in a diagonal direction; if a 25-middle-grid counting plate is used, in addition to the 4 diagonal orientations, the central 1 middle grid (namely 80 small grids) is counted; when the test strains are positioned on the double lines of the middle lattice, only the Candida albicans cells or the mold spores on the upper line and the right line or the lower line and the left line are counted;

(3) viable bacteria content C_BAnd (3) calculating: after the magnification of the biological optical microscope is adjusted from low to high to 400 multiplied, the Candida albicans cells or the mould spores at the corresponding space position of the blood counting plate are immediately counted; wherein the colorless Candida albicans cells are viable bacteria, and the blue or light blue ones are dead bacteria; the mould spores with dark blue edges and colorless or light blue or light red interiors are live bacteria, and the mould spores with dark blue edges and interiors are dead bacteria, so that only the spores with different colors of the edges and the interiors are counted; if the number of the single spores of the aseptic silks in the mould spore suspension is lower than 90%, preparing the spore liquid again; repeating microscopic counting for three times on candida albicans cells or mould spores in each small square of the blood counting plate, and taking an average value; when the specification of the blood counting chamber is 16X 25, the viable bacteria content (colony forming unit per milliliter, CFU/mL) C of 1mL of bacterial liquid_B＝N÷5×16×K×10⁴(ii) a When the specification of the blood counting chamber is 25X 16, C_B＝N÷5×25×K×10⁴(ii) a Wherein N is the total number of viable candida albicans cells or mold spores in five middle lattices of the blood counting plate, and K is the bacterial liquid dilution multiple; then, the known viable bacteria content C is treated by the Czochralski culture solution_BDiluting the Candida albicans suspension or the mold mixed spore liquid to obtain C_BIn the range of 5.0X 10⁶CFU/mL～9.0×10⁶CFU/mL inoculated bacterial fluid.

6. A method of assessing the fungicidal effectiveness of a liquid disinfectant as claimed in claim 1, wherein said log live bacterial content lgC is_BRelative fluorescence intensity log lgI_BEstablishing a standard curve according to the following steps:

using Chao's culture solution to treat known viable bacteria content C_BAfter the candida albicans suspension or the mold mixed spore liquid is subjected to continuous gradient dilution, a standard series of bacterial suspensions are obtained: 3.5X 10³CFU/mL、3.5×10⁴CFU/mL、3.5×10⁵CFU/mL and mixing; according to the relative fluorescence intensity value I_BThe determination method is based on the viable bacteria content C_BThe relative ratios of the above standard solutions were measured and recorded in descending orderA fluorescence intensity value; then, lgI is calculated as the relative fluorescence intensity logarithm of the standard series bacterial suspension_BAs abscissa, log value lgC of corresponding viable bacteria content_BPlotting the ordinate; calibrating the mathematical relationship between the two, and deriving a linear equation Y ═ a of the standard curve by using least square fitting method_BX+b_BAnd a linear correlation coefficient R_B ²(ii) a When R is_B ²The determination is effective when the confidence level is more than or equal to 0.98 and more than or equal to 0.95.

7. The method of claim 1 wherein the neutralizer assay and quantitative suspension test is conducted by the steps of:

(1) and (3) identification test of a neutralizer: selecting the lowest effective concentration of a disinfectant with 99.9 percent of sterilization inhibiting indicator bacteria for 10min as the concentration of a sample solution of a neutralizer identification test, and mixing 1mL of the test sample solution with 9mL of neutralizer solution; after 10min of action to form a neutralized product, experimental groups were performed as follows: 0.49mL of inoculated broth (with lgC)_B-lgI_BThe curve calibration bacterial liquid is taken from the same indicated strain stock solution test tube, stored at 2 +/-0.2 ℃ and used within 2 h) and is subpackaged with five test sample liquids 1 containing 4.41mL^#、2^#4.41mL of neutralized product solution 3^#4.41mL of Chao's culture solution 4^#4.41mL neutralizer solution 5^#Shaking the test tube at 1000r/min for 10min to make the solution mix thoroughly; then, 0.49mL of each mixed solution was dispensed into five bottles containing 4.41mL of Chaudhur culture solution 1 by using a sterile pipette^#、3^#、4^#、5^#4.41mL neutralizer solution 2^#In the sterile test tube of (4.9 mL of the Chaudhuri's culture solution in another sterile test tube was used as the 6 th^#Preparing a test sample liquid; shaking each group of test tubes at 1000r/min for 10min, and collecting the mixed solution as recovered solution of test sample solution according to relative fluorescence intensity value I_BA measuring method for measuring and recording the relative fluorescence intensity values of the 6 groups of recovered liquid;

(2) evaluation of neutralizing effect: if it is 1 st^#、6^#Relative fluorescence intensity of the pooled liquidMeasured value I_B1≥0、I_B60, 2 nd^#～5^#The relative fluorescence intensity measurements of the pooled solutions are related: i is_B2> 0 and I_B2＜I_B3、I_B2＜I_B4、I_B2＜I_B5(ii) a No. 3^#、4^#、5^#The relative fluorescence intensity measurements of the group-recovered solutions are close, i.e. I_B3≈I_B4≈I_B5The content of viable bacteria C calculated according to the formula (33)_B3、C_B4、C_B5The error rate delta between groups is less than or equal to 10 percent; the type of the selected neutralizer is used for a fungus killing effect test of a liquid disinfectant sample to be tested, and the neutralizer identification test is repeated after the concentration of the neutralizer is adjusted according to an equivalent neutralization principle, so that the concentration of the neutralizer corresponding to the test concentration of the disinfectant is determined;

in the formula:

CFU/mL；

(3) Quantitative suspension test: according to the effective concentration and action time range marked in the use instruction of the disinfectant to be detected, the disinfectant is diluted into three proper concentrations of high, medium and low by using sterile water: c₁、C₂、C₃，C₁Is the minimum use concentration specified in the product description, wherein C₂＝2C₁、C₃＝2C₂(ii) a Selecting four different sterilization times t simultaneously₁、t₂、t₃、t₄，t₂The minimum action time specified in the product description, where t₁＝0.5t₂、t₃＝1.5t₂、t₄＝2t₂(ii) a Then, 4.41mL of test sample liquid with different concentrations is divided into three sterile test tubes by a sterilization pipette; placing the mixture in a water bath at 20 +/-2 ℃, and respectively dropwise adding 0.49mL of inoculation bacterial liquid after the temperature of the solution in the test tube is balanced with the temperature of the water bath; starting to record after shaking the test tube for 30s at 3000r/min, and continuing the sterilization until the set t₁、t₂、t₃、t₄After four periods of different time, 0.49mL of the mixed solution with different concentrations is subpackaged into three sterile test tubes containing 4.41mL of neutralizer solution by using a sterilization pipette, the test tubes are shaken at 3000r/min for 30s and timing is started, and after the neutralization lasts for 10min, the mixed solution is used as the recovery solution of the test sample solution with each concentration; then, replacing the test sample liquid with the Chachi culture solution to repeat the test;

the test was repeated 5 times;

8. A method of assessing the fungicidal effectiveness of a liquid disinfectant according to claim 1, wherein the recovered liquid has a relative fluorescence intensity value I_BThe determination is carried out according to the following steps:

(1) calibrating relative fluorescence intensity background values of instruments and reagent groups: respectively adding 0.1mL of the Chaudou culture solution, 0.35mL of the physiological saline and 0.05mL of the ATP lysate into the same sterile test tube by using a sterilized pipette, and shaking the test tube for 30s at 3000 r/min; quietStanding for 10-20 min as a primary blank sample; transferring 0.1mL of the primary blank sample into another sterile test tube, dropwise adding 0.4mL of physiological saline, and uniformly mixing to obtain a secondary blank sample; then, sequentially transferring 0.1mL of secondary blank samples into three special sterile test tubes of instruments by using a sterilization liquid-transferring gun to serve as blank test parallel samples; dripping 0.1mL of ATP extraction reagent into each of the three blank test parallel samples, mixing uniformly, dripping 0.1mL of ATP fluorescence reagent, shaking the test tube at 3000r/min for 5s, and immediately measuring the relative fluorescence intensity value I of the test tube by using an ATP fluorescence photometer_BAnd recording; the operation time of each link is ensured to be consistent, and cross contamination is avoided; the determination time of each parallel sample is not more than 15s, and the arithmetic mean value of the relative fluorescence intensity values of the three blank test parallel samples is used as the background value of the instrument and the reagent group, or the background is calibrated according to the instruction of the instrument;

(2) relative fluorescence intensity value I of recovered liquid_BAnd (3) determination: if the background level of the blank reagent group meets the use requirement of the instrument, respectively adding 4.9mL of recovery solution and 0.1mL of ATP lysate into the same sterile test tube by using a sterile pipette, and shaking the test tube for 30s at 3000 r/min; standing at room temperature for 20min, adding 5.0mL of ATP extraction reagent dropwise, and mixing uniformly again; standing at room temperature for 10 min; sequentially transferring 0.1mL of the mixed solution into three special sterile test tubes of an instrument by using a sterilized liquid transferring gun to serve as ATP bioluminescence test parallel samples; dripping 0.1mL of ATP fluorescent reagent into each of the three fluorescence test parallel samples, shaking the test tube for 5s at 3000r/min, and immediately measuring the relative fluorescence intensity value I of the test tube by using an ATP fluorescence photometer_BAnd recording; the operation time of each link is ensured to be consistent, and cross contamination is avoided; the determination time of each fluorescence test parallel sample is not more than 15s, and the arithmetic mean value of the relative fluorescence intensity values of the three ATP bioluminescence test parallel samples is taken as I of the recovery liquid of the sample to be detected_BAnd (d) measuring the value.