CN110272960A

CN110272960A - ATP bioluminescence lgCA-lgIAThe method of calibration curve method detection antibacterial rubber fungicidal properties

Info

Publication number: CN110272960A
Application number: CN201910199763.6A
Authority: CN
Inventors: 李文杰; 崔宗岩; 杨志伟; 葛娜; 刘晓茂
Original assignee: INSPECTION AND QUARANTINE TECHNOLOGY CENTER OF QINHUANGDAO ENTRY-EXIT INSPECTION AND QUARANTINE BUREAU
Current assignee: INSPECTION AND QUARANTINE TECHNOLOGY CENTER OF QINHUANGDAO ENTRY-EXIT INSPECTION AND QUARANTINE BUREAU
Priority date: 2019-03-15
Filing date: 2019-03-15
Publication date: 2019-09-24

Abstract

The present invention relates to a kind of detection method of antibacterial rubber fungicidal properties, detecting step includes: sample preparation and pretreatment；Lectotype selection and reagent, culture medium are prepared；Culture presevation, activation and spore liquid preparation；OD value-viable bacteria content logarithm lgC_BStandard curve is established；ATP log concentration value lgC_ARelative intensity of fluorescence logarithm lgI_AStandard curve is established and inoculation bacterium solution viable bacteria C_ACalibration；Sample inoculation, culture and elution recycling；Recovered liquid I_AMeasurement and its viable bacteria ATP concentration C_AAnd T_AIt calculates；Antibiotic rate R or antibacterial activity value A is calculated；Evaluation of result；Its special feature is that: accurate quantitative test is carried out to the rubber fungicidal properties with R or A characterization using ATP fluophotometer.Present invention provide that control sample and antimicrobial sample after elution recycling contact 0h and culture 48h, measure recovered liquid I_AAnd with lgI_ACharacterization and calculating R or A.The ATP bioluminescence lgC for the antibacterial rubber fungicidal properties detection that the present invention researches and develops_A‑lgI_ABent method is marked, will be promoted with the advanced detection technique support product quality of science.

Description

ATP bioluminescence lgCA-lgIACalibration curve method detects antibacterial rubber fungicidal properties Method

Technical field

The present invention relates to the fungicidal properties test method of antibacterial rubber, specifically a kind of application ATP fluophotometer to The antibacterial rubber fungicidal properties of antibiotic rate R or antibacterial activity value A characterization carries out the ATP bioluminescence lgC of accurate quantitative detection_A- lgI_ACalibration curve method belongs to antibacterial rubber anti-microbial property detection technique field.

Background technique

Antibacterial rubber product is widely used in industry and people's livelihood field, although the polymerization in antibacterial rubber material and its product Carbon source needed for object ingredient does not have fungus growth, has inhibiting effect to fungus growth；But since vulcanization used in process promotees It is infected into substances such as agent, reinforcing fillers for mould and provides necessary nutrient source, its appearance and physicochemical property is easily caused to decline； Transmission channel is constituted simultaneously.Therefore, the sterilization of antibacterial rubber material and product is for Improving The Quality of Products, raising disease control Level has a very important significance；Currently, antibacterial rubber floor, ground cushion and antibiotic property silicon/antibacterial rubber material are defended in medical treatment The numerous areas such as life, style education, tourist catering and household electrical appliance are used widely.Antimicrobial technology is in antibacterial rubber industry Using and popularize irresistible, antibacterial functions have become one very characteristic " attraction " of antibacterial rubber product；But existing correlation The missing of performance detection technology but seriously restricts industry development.

Currently, anti-biotic material performance detection technical system is mainly for bacterium and fungi both at home and abroad, wherein mould proof detection side Method is originated from American Standard, day mark and Europe superscript substantially, and involved standard mainly includes burying cultivation, agar plate method and moist chamber culture based on soil The ISO 846:1997 " the antibacterial culture of plastic products is assessed " of method, " the antimycotic measurement of antimicrobial macromolecule material of DIN 53793 Method " and GB/T 24128-2009 " plastic mould-proof method for testing performance ", QB/T 4199-2011 " Leather mildew-proof performance test Method ", HG/T 4301-2012 " antibacterial rubber fungicidal properties test method " etc..In general, existing fungicidal properties detection side There are following common problems in technology contents and practical application for method: first is that due to the mycelia formed in mycotic spore growth course Body can not accurate counting, can only be qualitatively judged, can not quantitative test；Second is that because the fungus growth period is longer, incubation time At least 2 weeks~4 weeks, cause test period longer, time and economic cost are high；Third is that experimentation is cumbersome, technical difficulty is higher； Relevant operation is affected by human factors greatly, so that test error is big, lacks comparativity.In recent years, in international Bacteria Detection technology Field ATP fluorescence analysis development it is increasingly mature, with traditional Plating compared with testing result correlation be 98%, accuracy height and Quickly detection can be achieved.By demand pull, external anti-biotic material performance detection technical field of research for current quantification, quickly Change and summary development trend have used for reference ATP fluorescence analysis principle and have formulated ISO 20743:2007-2013 " Textiles- Determination of antibacterial activity of textile products (spin by textile-antibiotic finish The antibacterium performance measurement of fabric) " and ISO 13629-1:2012 " Textiles-Determination of antifungal Activity of textile products.Part1 Luminescence (textile-antibiotic finish textile mildew resistance Can measurement) ", it is specified that with the fluorimetry of anti-thin/mould performance of ATP changes of contents characterization after sample inoculation, but it is only fitted For with water imbibition and control sample it is thin/weaving of mould increasing value > 0 or poromerics, have in viable bacteria way of recycling, test Be not suitable in terms of the key technologies such as effect condition the rubber with unwetted property hard surface and control sample mould increasing value < 0, Plastic or other material, while uncertainty of measurement assessment not being provided.In addition, the standard method dependent antimicrobial performance characterization parameter is more It is single, only relate to antibacterial activity value A, and China then usual antibiotic rate R.

Therefore, to resist foreign technology barrier, specification domestic market order pushes industrial transformation upgrading, it would be highly desirable to research section It emulates the advanced, accuracy and reproducibility are high, easy-to-use antibacterial rubber Performance Testing Technology.The art of this patent highway route design integrates with The world, detection method belong to the whole world and initiate, and can effectively fill up domestic and international correlative technology field blank.

Summary of the invention

The technical problem to be solved in the present invention is to provide a kind of application ATP fluophotometers to living with antibiotic rate R or antibacterial Property value A characterization the ATP bioluminescence lgC that is detected of antibacterial rubber fungicidal properties_A-lgI_ACalibration curve method is able to solve anti- Bacterium rubber or even other product scope anti-biotic materials and the accurate quantitative test problem of product fungicidal properties.

In order to solve the above technical problems, the technical solution adopted by the present invention is that:

A kind of detection method of antibacterial rubber fungicidal properties, comprising: (1) sample preparation and pretreatment；(2) lectotype selection and Reagent, culture medium are prepared；(3) culture presevation, activation and spore suspension preparation；(4) OD value-viable bacteria content logarithm lgC_BStandard Curve is established；(5) ATP log concentration value lgC_ARelative intensity of fluorescence logarithm lgI_AStandard curve is established and inoculation bacterium solution viable bacteria ATP concentration C_ACalibration；(6) sample inoculation, culture and elution recycling；(7) reclaim liquid phase is to fluorescence intensity level I_AMeasurement；(8) it recycles Liquid viable bacteria ATP concentration C_AAnd T_AIt calculates and antibiotic rate R and antibacterial activity value A is calculated；(9) evaluation of result；It is characterized in that, using ATP fluophotometer carries out accurate quantitative test to the antibacterial rubber fungicidal properties with antibiotic rate R or antibacterial activity value A characterization ATP bioluminescence lgC_A-lgI_ACalibration curve method, specific:

In reclaim liquid phase to fluorescence intensity level I_AIn measurement:

Preparation, size, quantity and the pre-treatment requirement for specifying control sample and antimicrobial sample, by the mark of mould test strain After quasi- bacterial strain is passed on, activated, fresh mycotic spore culture is taken to prepare spore suspension；It is established using MTT colorimetric analysis OD value-viable bacteria content logarithm lgC_BStandard curve is derived from the linear equation Y=a of curve₀X+b₀And coefficient R₀ ²； And viable bacteria content C is carried out to the spore suspension of different dilutions_BCalibration.Then, with ATP fluorescent reagent buffer solution by 1.0 × 10^-3The ATP standard stock solution of mol/L is diluted to high and low concentration standard serial solution: 7.0 × 10^-8mol/L、7.0×10^-7mol/ L、7.0×10^-6Mol/L and 2.1 × 10^-9mol/L、2.1×10^-8mol/L、2.1×10^-7It is strong to measure its relative fluorescence by mol/L Angle value I_A；Draw the lgC of two respective concentrations_A-lgI_AStandard curve is derived from fitting equation Y=a_A0X+b_A0It is (highly concentrated Degree), Y=a_AX+b_A(low concentration) and linearly dependent coefficient R_A0 ²(high concentration), R_A ²(low concentration).Meanwhile with Cha Shi culture solution pair Viable bacteria content C_BIt is 1.0 × 10⁸CFU/mL~5.0 × 10⁸It is dilute that the mould mixing spore liquid of CFU/mL carries out continuous 10 times of gradients It releases, measures its relative intensity of fluorescence value I_A, and according to high standard fitting equation Y=a_A0X+b_A0Calculate corresponding viable bacteria ATP concentration C_A；It is adjusted to obtain C_ARange is 5.0 × 10^-7Mol/L~9.0 × 10^-7The inoculation bacterium solution of mol/L.Then, to each 0.3mL inoculation bacterium solution is added dropwise in group sample surface to be measured respectively, is eluted immediately with 4.6mL eluent to 6 groups of 0h contact samples Recycling, measures the relative intensity of fluorescence value I of recovered liquid_AC0ij、I_AT0ij, according to low concentration calibration curve equation formula Y=a_AX+b_AIt pushes away Calculate its viable bacteria ATP concentration C_AOijAnd T_AOij.Meanwhile the control sample and antimicrobial sample that 6 groups are sealed in sterilized petri dishes are (28 ± 2) after DEG C, cultivating 48h ± 2h under conditions of (95 ± 2) %RH；Recycling table is eluted using mode identical with 0h contact sample Face residual bacterium and the relative intensity of fluorescence value I for measuring recovered liquid_ACtij、I_ATtij, calculate corresponding viable bacteria ATP concentration C_AtijWith T_Atij；

In antibiotic rate R and antibacterial activity value A is calculated:

According to ATP low concentration standard curve lgC_A-lgI_ALinear equation Y=a_AX+b_A, contacted with 0h and trained through 48h The relative intensity of fluorescence measured value of every control sample and antimicrobial sample recovered liquid after supportingWith As base Plinth data；In the case where testing condition for validity, its mould increasing value F after 48h is cultivated is calculated_ij、G_ijAnd antibiotic rate R_ijAnd antibacterial Activity value A_ij；To the R of every group of sample_ijAnd A_ijArithmetic mean of instantaneous value is taken to obtain corresponding R_iAnd A_i；Every batch of antibacterial rubber sample resists Bacterium rate R and antibacterial activity value A is its three groups of sample R_iAnd A_iArithmetic mean of instantaneous value；And the clear related data revision of the convention and measurement be not true Fixed degree requires；

In evaluation of result:

With reference to health industry common practice and the requirement of dependent antimicrobial product standard, determine that fungicidal properties is classified criterion； As the antibiotic rate R of certain group (part) antibacterial rubber sample_i(R_ij) or antibacterial activity value A_i(A_ij) with other two groups (four) samples When fungicidal properties is compared to a levels are at least differed, one group of (part) sample is extracted again and repeats to test；It is calculated through ATP biology Fluorescence lgC_A─lgI_AThe antibiotic rate or antibacterial activity value that calibration curve method measures.If two groups of front and back (part) antibacterial rubber sample Fungicidal properties level is identical, then abandons it；Take other two groups (four) remaining sample antibiotic rate R_i(R_ij) or antibacterial activity value A_i (A_ij) evaluation result of the arithmetic mean of instantaneous value as batch (group) the antibacterial rubber sample fungicidal properties.

The present invention by adopting the above technical scheme, compared with prior art, beneficial effect is:

(1) advanced: using modern precision instrument-ATP fluophotometer as test equipment, precisely can quickly to survey Determine the viable bacteria amount recycled after antibacterial rubber sample culturing specific time, reaches the modernization of antibacterial rubber fungicidal properties detection； Can effectively reduce human factor in experimentation influences, and breaches the qualitative analysis limitation of traditional plate culture；Realize inspection Result quantification is surveyed, and increases substantially detection data accuracy；Detection technique has certain advance.

(2) scientific: according to ATP fluorescence analysis test philosophy, to establish the viable bacteria ATP concentration pair suitable for multi-cultur es Numerical value lgC_A- relative intensity of fluorescence logarithm lgI_AStandard curve；It is real-time to construct antibacterial rubber fungicidal properties ATP bioluminescence The mathematical model of quantitative analysis method.Meanwhile country variant consumer perceptions habit is taken into account, take antibiotic rate R or antibacterial activity Value A is correlated performance evaluation index, improves the science and versatility of detection method.

(3) innovative: with existing operation is numerous, the period is long, compared with the conventional method of non-quantitation, this patent method was being tested Automation and the higher ATP fluophotometer of intelligent level are introduced in journey, are greatly simplified experimental procedure, are realized antibacterial The precision of rubber fungicidal properties testing result and quantification simultaneously have good reproducibility and comparativity；It is greatly shortened simultaneously Test period reduces testing cost；Presently relevant the field of test technology blank can effectively be filled up.

(4) perspective: to establish with lgC_A、lgI_AStandard curve quantitative analysis method based on linear relationship, innovation is simultaneously Mycotic spore liquid viable bacteria content measuring method and antibacterial rubber sample pre-treatments mode are enriched, it is dense to specify control sample, standard liquid The technology contents such as degree, determination step, calculation formula, uncertainty；The pioneering recovered liquid viable bacteria directly measured with instrument is relatively glimmering Luminous intensity logarithm lgI_AEvaluation form calculates and determines antibiotic rate R or antibacterial activity value A as a result, and by group and Between-group variation coefficient CV investigates uncertainty of measurement, technically has certain perspective.

(5) operability: ATP fluophotometer is cheap, it is easy to operate, be widely used, the OD value-that this patent is established Viable bacteria content logarithm lgC_BMethod is simple for calibration curve method and antibacterial rubber fungicidal properties ATP fluorometric investigation, related skill It is clear and specific that art illustrates, should be readily appreciated that and grasps；Have stronger operability in implementation process, is suitable for different majors Horizontal Experiment on Microbiology personnel may advantageously facilitate achievement transfer conversion and promote and apply.

(6) universality: because ATP is prevalent in, life entity is intracellular, and patented method can expand for experimental strain and provide tool The detection technique support for thering is wide spectrum to be worth；The introducing of pertinent instruments can greatly simplify experimental procedure, reduce testing cost；Be conducive to Expand inspection, learn, grind, produces all circles promote and apply, can support antibacterial rubber fungicidal properties detection technique realization generalization, simultaneously Reference can be provided to the product scopes fungicidal properties detection technique research such as plastics, glass.

Further, preferred embodiment of the invention is:

The sample preparation and pretreatment carries out in the steps below:

(1) control sample: being injection moulded by hygienic level high-density polyethylene, quantity, size and thickness and antibacterial to be measured Rubber sample is identical；

(2) antibacterial rubber material to be measured or product (for cured sizing material) antimicrobial sample: are placed in (23 ± 2) DEG C, (50 ± 5) the no less than air conditioning of 16h is carried out in the environment of %RH, it is ensured that room air free flow simultaneously touches its each surface. Having a size of (50 ± 2) mm × (50 ± 2) mm, with a thickness of 1.0mm ± 0.1mm, 2.0mm ± 0.2mm or 4.0mm ± 0.2mm；Work as sample Product thickness be greater than 5mm when, need to be cut with leather slicer or similar functions cutting machine, and with flexibility sanding belt to sample not Smooth place polishes.Each strain test uses 6 groups of samples；Wherein 0h contact and 48h culture experiment respectively use 3 groups, every group 5 Sample selects 1 control sample as object of reference respectively and effectively identifies；

(3) sample pre-treatments: impregnating 1min in 70% ethanol solution for control sample and antimicrobial sample before experiment, and With the abundant cleaning sample of sterile water, to remove ethyl alcohol, (person that is unsuitable for ethanol disinfection can directly use sterile water on superclean bench It rinses)；Sample surface to be measured is put into upwards after natural drying spare in sterilized petri dishes；

The lectotype selection and reagent, culture medium are prepared, and are carried out in the steps below:

(1) General Requirement: test analytical reagents and meet tertiary effluent as defined in GB/T 6682-2008 (distilled water or Deionized water), laboratory has the safe qualification of two stage biological, and personnel have regular Microbiological Lab's working experience；

(2) instrument and equipment: the superclean bench of two stage biological safety cabinet or lustration class not less than 100；The light of fluorescence containing ATP The ATP bioluminescence rapid detection system of degree meter, Special test tube etc., ATP fluophotometer wavelength range 300nm~650nm, ATP Concentration Testing range 10^-11Mol/L~10^-6mol/L；Wave-length coverage 400nm~760nm, range of readings 0.0Abs~ The microplate reader of 4.0Abs；Amplification factor 40 ×~400 × Photobiology microscope；(25~30) DEG C ± 1 DEG C, (20~95) % The constant temperature and humidity incubator of RH ± 2%RH；46 DEG C ± 1 DEG C of constant water bath box；Revolving speed >=8000r/min centrifuge and mating Centrifuge tube；The vortex oscillator of the range of speeds (500~3000) r/min；The steam of (121 ± 2) DEG C, (103 ± 5) kPa Sterilizer；- 20 DEG C~-80 DEG C of low temperature refrigerator；0 DEG C~10 DEG C of refrigerating box；The electronic balance of sensibility reciprocal 0.001g；Frequency range The ultrasonic cleaner of (30~50) kHz；The pH meter of precision ± 0.1 (25 DEG C)；Electric furnace；Have counteredge, changeable blade Or the cutting machine of rotation cut-off knife and feed roller and cleavable different-thickness film；Surface abrasive is the electricity of aluminium oxide or silicon carbide Dynamic/mechanically flexible sanding belt；

(3) material utensil: blood counting chamber and dedicated coverslip；The sterile measuring pipette of 1mL, 10mL；0.05mL, The single track changeable fluid liquid-transfering gun and sterile liquid transfer gun head of 0.1mL, 0.2mL, 1mL, 5mL, 10mL (measurement error is less than 1%)； Aperture is not more than 0.45 μm of syringe-driven filter；The sterile conical flask and bottle stopper of capacity 100mL, 250mL, 500mL；96 holes are flat Bottom culture plate；The sterile petri dish of diameter 90mm；Glass funnel；Sterile cock test tube；Diameter is not more than the oese of 4mm；L Stick；The bead of diameter 5mm；Alcolhol burner；Sterilize tweezers；Medical adhesive tape；Absorbent cotton and gauze for biochemistry detection；Sterile filter Paper；Thermometer；The stopwatch of precision 0.01s；

(4) reagent: 70% ethanol solution；(0.45 μm of solution of MTT (tetramethyl azo azoles salt) of 5mg/mL, pH value 7.4 Membrane filtration degerming, 4 DEG C~6 DEG C are kept in dark place 15d)；121 DEG C of high pressure sterilization 30min after the packing of following reagent, 5 DEG C~10 DEG C Store the physiological saline of 30d:85%；N monomethyl ethanesulfonic acid, Tween 80, two pungent sulfonation sodium succinates are chosen any one kind of them, and are configured to 0.05% wetting agent solution；

(5) medium/liquid (commercially available medium/liquid can be used): 121 DEG C of high pressure sterilizations after matched medium/liquid packing 30min, 2 DEG C~8 DEG C storage 30d；

Cha Shi culture solution (preparation of mycotic spore liquid, bacteria suspension dilution and sample elution use): by 2g sodium nitrate, 1g phosphoric acid hydrogen Dipotassium, 0.5g potassium chloride, 0.5g magnesium sulfate, 0.01g ferrous sulfate, 30g sucrose are dissolved by heating in 1000mL containing 0.05% wetting In the aqueous solution of agent, adjust pH to 6.0~6.5 (25 DEG C)；

One dextrose culture-medium of potato (mycotic spore actication of culture and its total plate count plate count use): 300g is new Fresh potato removes the peel stripping and slicing, boils 20min~30min in 1000mL water；Filter to take juice, into filtrate be added 20g glucose, 1000mL is settled to after 0.1g chloramphenicol, 20g agar；

(6) ATP fluorescence reaction reagent (or with commercial reagent): in addition to phosphate buffer solution, matched ATP fluorescence reaction examination - 20 DEG C~-70 DEG C of agent preservations, use in 6 months；

Dilution buffer: 0.005mol/L and the disodium phosphate soln for containing 0.037% sucrose, adjusting pH to 7.2 ± 0.2；121 DEG C of high pressure sterilizations 15min, 2 DEG C~8 DEG C storage 30d；

ATP fluorescent reagent buffer solution: by 1117mg trishydroxymethylaminomethane, 183mg disodium ethylene diamine tetraacetate, 808mg magnesium acetate, 6.7mg dithiothreitol, the beta-cyclodextrin of 25000mg and 925mg glucose are dissolved by heating in 250mL water In, adjust pH to 7.5 ± 0.2；It is used in 8h；

ATP lysate: 4.6 international units/ml apyrase (EC:3.6.1.5) and 46 is international single Adenosine phosphate deaminase (EC:3.5.4.6 or EC:3.5.4.17), 37mg sucrose, the 20mg bovine serum albumin of position/ml is dissolved in 10mL concentration is to adjust in pH to 6.0 ± 0.5,8h in the 2-morpholine ethane sulfonic acid buffer solution of 0.05mol/L and use (1mL cracking ATP concentration in Sharpe culture solution can be down to 10 in 15min by liquid^-11Mol/L or less)；

ATP extracting solution: 45mg trishydroxymethylaminomethane is dissolved by heating in 9.8ml water, is 10% with 0.2ml concentration Benza mix after, adjust pH to 12.0 ± 0.5 (fungal cell ATP extraction efficiency be not less than 80%)；

ATP fluorescent reagent: 0.7mg luciferase, the D- fluorescein of 12.6mg, 56mg bovine serum albumin are dissolved in 30mL ATP fluorescent reagent buffer solution, be stored at room temperature 15min after mixing, used in 3h；

Culture presevation, activation and the spore suspension preparation, carries out in the steps below:

(1) test strain: aspergillus niger ATCC 16404；Chaetomium globosum ATCC 6205；Penicillium chrysogenum ATCC 9179 (or by Other strains that national Culture Collection is provided and can be traced to the source)；

(2) mould test strain culture presevation: is inoculated in simultaneously by potato-dextrose culture-medium inclined-plane with sterile working Indicate the date, 28 DEG C~30 DEG C culture to inclined-planes cover with mycotic spore (7d~14d)；3 DEG C~10 DEG C preservation 4 months；

(3) actication of culture: with oese scrape preservation bacterium spore, be inoculated with potato-dextrose culture-medium inclined-plane, 28 DEG C ~30 DEG C of culture 7d~14d are until generate a large amount of spores.Mold species test tube plug must not be extracted before spore suspension by preparing, and every Only for spore liquid of preparation after test tube opening, suspension is prepared using the mycotic spore newly cultivated every time；

(4) prepared by spore suspension: the sterile water of 10mL being added into strain test tube, gently scrapes media surface with oese The spore stoste injection of wash-off is equipped with the sterile taper of jumping a queue of 15 beades and 45mL Cha Shi culture solution by fresh mycotic spore In bottle, 3000r/min shakes test tube 2min, breaks up spore ball, mixes spore liquid.Then, sterile absorbent cotton or eight layers of yarn will be covered with The glass funnel of cloth is placed on conical flask, and filtering spore suspension removes mycelia and culture medium fragment；By filtrate move to sterilizing from In heart pipe, 8000r/min separating treatment at least 10min, removes supernatant at room temperature；It is heavy that spore is cleaned with 50mL Cha Shi culture solution again Starch is simultaneously centrifuged, after repeated washing 3 times, with the spore sediment after the dilution centrifugation of Cha Shi culture solution.Every kind of test mould is pressed Spore suspension is prepared according to the above method, the spore liquid of each test strain is mixed in equal volume；0 DEG C~7 DEG C storages, 4d is interior to be used；

The OD value-viable bacteria content logarithm lgC_BStandard curve is established and inoculating spores liquid C_BCalibration, in the steps below It carries out:

(1) bacterium solution OD value measures: carrying out primary dcreening operation using spore total amount of the blood counting chamber to mixing spore suspension, uses Cha Shi Culture solution is 6.0 × 10 to concentration⁸The mixing spore liquid of CFU/mL carries out 1:10,1:15,1:20 and is serially diluted, and with culture solution As blank control sample liquid, zeroing correction is carried out to microplate reader.Then, the spore liquid of the above-mentioned different dilutions of 100 μ L is distinguished 96 hole flat-bottomed plates are injected, each dilution does 3 multiple holes；It is added dropwise the MTT solution of 20 μ L to every hole, (28 ± 1) DEG C, (95 ± 2) after %RH cultivates 30min；Maximum absorption band is scanned in 560nm~610nm wave-length coverage, determines maximum absorption wavelength； The viable bacteria OD value of each hole mixed solution is measured, the viable bacteria OD value of different dilution spore suspensions is its 3 multiple holes OD measured values Arithmetic mean of instantaneous value.Meanwhile the method referring to as defined in national standard GB 4789.15-2016, it is appropriate to carry out to above-mentioned mixing spore suspension After dilution, in (28 ± 1) DEG C culture 3d, and bacterium colony counting is carried out to plate, demarcate its viable bacteria content C_B；

(2) OD value-viable bacteria content logarithm lgC_BStandard curve is established and inoculating spores liquid C_BCalibration: with different dilutions The viable bacteria OD value of spore suspension is as ordinate, with its viable bacteria content logarithm lgC_BFor abscissa, OD value-lgC is drawn_BStandard Curve；The linear equation Y=a of curve is derived from using least square fitting method₀X+b₀And coefficient R₀ ².Work as R₀ ²≥ 0.98, when confidence level >=0.95, the spore liquid viable bacteria content C that is measured according to this patent method_BEffectively；

The ATP log concentration value lgC_ARelative intensity of fluorescence logarithm lgI_AStandard curve is established and inoculation bacterium solution is living Bacterium ATP concentration C_ACalibration carries out in the steps below:

(1) determination of ATP standard serial solution and its test specimens preparation: ATP fluorescent reagent buffer solution is by 1.0 × 10^- ³The ATP standard stock solution of mol/L is diluted to high and low concentration standard serial solution: 7.0 × 10^-8mol/L、7.0×10^-7mol/L、 7.0×10^-6Mol/L and 2.1 × 10^-9mol/L、2.1×10^-8mol/L、2.1×10^-7mol/L.It then, will with sterilizing liquid-transfering gun The above-mentioned ATP standard solution of 0.1mL is moved to respectively in three sterile test tubes, and 0.05mL sterile water and 0.35mL physiology salt is successively added dropwise Aqueous solution simultaneously mixes, as level-one ATP standard solution test specimens；0.1mL level-one ATP standard solution test specimens are moved to respectively again It is each that 0.4mL physiological saline is added dropwise and mixes in three sterile test tubes, as second level ATP standard solution test specimens；And it is moved with sterilizing Liquid rifle moves to the second level ATP standard solution test specimens of 0.1mL various concentration in three instrument sterile test tubes respectively, as ATP standard solution tests Duplicate Samples；

(2) blank background value calibration: the aqueous solution, 0.35mL with sterilizing liquid-transfering gun by 0.1mL containing 0.05% wetting agent are raw The ATP lysate of reason salt water and 0.05mL are separately added into same branch sterile test tube, and 3000r/min shakes test tube 30s；It stands Level-one blank sample is used as after 10min~20min；0.1mL level-one blank sample is moved in an other sterile test tube again, is added dropwise 0.4mL physiological saline simultaneously mixes, as second level blank sample.Then, 0.1mL second level blank sample is moved to respectively with sterilizing liquid-transfering gun In three instrument sterile test tubes, as skip test Duplicate Samples；The ATP that 0.1mL is successively added dropwise into three Duplicate Samples is mentioned Reagent is taken, instills the ATP fluorescent reagent of 0.1mL after mixing again.3000r/min shakes test tube 5s, uses ATP fluophotometer immediately Measure its relative intensity of fluorescence value I_AAnd it records and (ensures that each link operating time is consistent, avoid cross contamination).Each Duplicate Samples Minute is no more than 15s, using the arithmetic mean of instantaneous value of three skip test Duplicate Samples relative intensity of fluorescence values as instrument and examination Agent group background values (or calibrating background according to instrument operation instruction)；

(3) ATP standard solution relative intensity of fluorescence value I_AMeasurement: from low to high according to concentration, to various concentration ATP The ATP that 0.1mL is added dropwise in three test Duplicate Samples of standard solution respectively extracts reagent, and the ATP for instilling 0.1mL after mixing again is glimmering Light reagent；3000r/min shakes test tube 5s, uses its relative intensity of fluorescence value of ATP fluorescent spectrophotometer measuring I immediately_AAnd it records (really It is consistent to protect each link operating time, avoids cross contamination).Each Duplicate Samples minute is no more than 15s, with each concentration ATP mark The arithmetic mean of instantaneous value of three Duplicate Samples relative intensity of fluorescence values of quasi- solution is its I_AMeasured value；

(4) ATP log concentration value lgC_ARelative intensity of fluorescence logarithm lgI_AStandard curve is established: with various concentration ATP The relative intensity of fluorescence logarithm lgI of standard solution_AAs abscissa, with its ATP log concentration value lgC_AFor ordinate mapping； Calibration curve is carried out to mathematical relationship between the two, draws the lgC of two respective concentrations_A-lgI_AStandard curve；And application is most Small two multiply the linear equation Y=a that fitting process is derived from curve_A0X+b_A0(high concentration), Y=a_AX+b_AIt is (low concentration) and related Coefficients R_A0 ²(high concentration), R_A ²(low concentration).Work as R_A0 ²And R_A ²>=0.98, it when confidence level >=0.95, is done according to this patent Measurement is effective；

(5) it is inoculated with bacterium solution viable bacteria ATP concentration C_ACalibration: with Cha Shi culture solution to viable bacteria content C_BIt is 1.0 × 10⁸CFU/mL ~5.0 × 10⁸The mould mixing spore liquid of CFU/mL carries out continuous 10 times of gradient dilutions, measures its relative intensity of fluorescence value I_A；Root According to high standard fitting equation Y=a_A0X+b_A0It calculates and demarcates corresponding viable bacteria ATP concentration C_A, through Cha Shi culture solution tune It is whole, obtain C_ARange is 5.0 × 10^-7Mol/L~9.0 × 10^-7The inoculation bacterium solution of mol/L measures and records 0.3mL inoculation bacterium solution Relative intensity of fluorescence value I after the dilution of 4.6mL eluent in 1min_A0；

Sample inoculation, culture and the elution recycling, carries out in the steps below:

(1) inoculated and cultured: antibacterial rubber sample carries out antibacterial tests after completing air conditioning in 1h, with sterilizing liquid-transfering gun 0.3mL inoculation bacterium solution is added dropwise respectively (with lgC to each group control sample and antimicrobial sample surface to be measured_A-lgI_ACalibration curve spore Sub- liquid is derived from same branch mixing spore liquid test tube, and 0 DEG C of ± 1 DEG C of preservation, 2h is interior to be used), (adhered to inoculation bacterium solution with L stick but do not hung Drop) bacterium solution is smeared uniformly, so that it is covered sample whole surface.Ware lid is covered, sample will be contacted equipped with 6 groups of 48h with medical adhesive tape The plate of product seals；(28 ± 2) DEG C, (95 ± 2) %RH cultivate 48h ± 2h；

(2) elution recycling: after 6 groups of 0h contact sample inoculation moulds, 4.6mL eluent is drawn with sterilizing liquid-transfering gun immediately (i.e. Cha Shi culture solution), each sample inoculation surface of repeated flushing at least 4 times in plate, sufficiently elution are simultaneously anti-with liquid transfer gun head Mould washing lotion in multiple pressure-vaccum plate, until lawn again moves into washing lotion in sterile test tube after being broken up；3000r/min shakes test tube 2min, as the recovered liquid of sample to be tested (if recovered liquid adds eluent to 4.9mL) less than 4.9mL after mixing.Each group Sample after 48h is cultivated uses type of elution identical with 0h contact sample to recycle mould；

The reclaim liquid phase is to fluorescence intensity level I_AMeasurement carries out in the steps below:

(1) instrument and reagent set relative intensity of fluorescence background value calibration: according to lgC_A-lgI_ABlank when standard curve is established Background calibration methods, with the wetting agent solution of Cha Shi culture solution substitution 0.05%, determining instrument and reagent set background values；

(2) reclaim liquid phase is to fluorescence intensity level I_AMeasurement: if background level meets instrument requirement, with sterilizing liquid-transfering gun The ATP lysate of 4.9mL recovered liquid and 0.1mL is separately added into same branch sterile test tube, 3000r/min shakes test tube 30s； After being stored at room temperature 20min, then the ATP extraction reagent of 5.0mL is added dropwise and mixes；It is stored at room temperature 10min.It will with sterilizing liquid-transfering gun The above-mentioned mixed solution of 0.1mL is moved to respectively in three instrument sterile test tubes, tests Duplicate Samples as ATP bioluminescence.To The ATP fluorescent reagent of 0.1mL is respectively added dropwise in three Duplicate Samples, 3000r/min shakes test tube 5s, uses ATP fluophotometer immediately Measure its relative intensity of fluorescence value I_AAnd it records and (ensures that each link operating time is consistent, avoid cross contamination).Each Duplicate Samples Minute is no more than 15s, using the arithmetic mean of instantaneous value of three ATP bioluminescences test Duplicate Samples relative intensity of fluorescence value as to The I of sample recovered liquid_AMeasured value；

The recovered liquid viable bacteria ATP concentration C_AAnd T_AIt calculates and antibiotic rate R and antibacterial activity value A is calculated, by following steps It is rapid to carry out:

(1) recovered liquid viable bacteria ATP concentration C_AAnd T_AIt calculates

According to ATP low concentration standard curve lgC_A-lgI_ALinear equation Y=a_AX+b_A, calculate that each group sample connects through 0h Touch the viable bacteria ATP concentration C of recovered liquid after 48h is cultivated_AAnd T_A.Relevant calculation is shown in formula (1)~(12):

In formula:

C_A0And C_AtThe average ATP concentration that -3 groups of 0h are contacted and control sample recycles viable bacteria after 48h is cultivated, unit are to rub You are every liter (mol/L)；

C_A0iAnd C_AtiThe average ATP concentration that-every group 0h is contacted and control sample recycles viable bacteria after 48h is cultivated, unit are Mole every liter (mol/L)；Sample group i=1,2,3；

C_A0ijAnd C_AtijThe ATP concentration that-every 0h is contacted and control sample recycles viable bacteria after 48h is cultivated, unit are to rub You are every liter (mol/L)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

With- every 0h contact and after 48h is cultivated control sample recovered liquid relative intensity of fluorescence measured value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

a_A- low concentration standard curve lgC_A-lgI_ASlope；b_A- low concentration standard curve lgC_A-lgI_AIn vertical axis intercept；

T_A0And T_AtThe average ATP concentration that -3 groups of 0h are contacted and antimicrobial sample recycles viable bacteria after 48h is cultivated, unit are to rub You are every liter (mol/L)；

T_A0iAnd T_AtiThe average ATP concentration that-every group 0h is contacted and antimicrobial sample recycles viable bacteria after 48h is cultivated, unit are Mole every liter (mol/L)；Sample group i=1,2,3；

T_A0ijAnd T_AtijThe ATP concentration that-every 0h is contacted and antimicrobial sample recycles viable bacteria after 48h is cultivated, unit are to rub You are every liter (mol/L)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

With- every 0h contact and after 48h is cultivated antimicrobial sample recovered liquid relative intensity of fluorescence measured value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

(2) condition for validity is tested

If recovered liquid and 0.3mL inoculation the bacterium solution 1min after the dilution of 4.6mL eluent of every 0h contact control sample Interior relative intensity of fluorescence measured value is close, i.e.,Every after 48h is cultivated control sample recovered liquid relative fluorescence it is strong Spend the logarithm of measured valueThat is C_Atij≥0.1×C_A0ij.Then when 3 groups of 0h contact control sample reclaim liquid phase To fluorescent strength determining valueGroup in and when between-group variation coefficient CV≤10% (involved calculation formula is shown in this patent correlation Uncertainty of measurement requirement), the measurement carried out according to this patent method is effective；

(3) mould increasing value F_ij、G_ijIt calculates

Every control sample and antimicrobial sample are after 48h is cultivated, mould increasing value F_ij、G_ijRespectively according to formula (13), (14) it calculates:

In formula:

F_ijAnd G_ijThe mould increasing value of-every control sample and antimicrobial sample after 48h is cultivated, sample group i=1, 2,3；Every group of sample number into spectrum j=1,2,3,4,5；

C_A0ijAnd C_AtijThe average ATP concentration that-every 0h is contacted and control sample recycles viable bacteria after 48h is cultivated, unit It is mole every liter (mol/L)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

With- every 0h contact and after 48h is cultivated control sample recovered liquid relative intensity of fluorescence measured value, RLU；Group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

T_A0ijAnd T_AtijThe average ATP concentration values that-every 0h is contacted and antimicrobial sample recycles viable bacteria after 48h is cultivated, Unit is mole every liter (mol/L)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

a_A- low concentration standard curve lgC_A-lgI_ASlope；

(4) antibiotic rate R is calculated

It tests under condition for validity, the antibiotic rate R of every antibacterial rubber sample_ij, every group and every batch of sample antibiotic rate R_iAnd R It is calculated respectively according to formula (15), (16), (17):

In formula:

R_ijThe antibiotic rate of-every antibacterial rubber sample, %；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2, 3,4,5；

R_iThe antibiotic rate of-every group antibacterial rubber sample, %；Sample group i=1,2,3；

R-every batch of antibacterial rubber sample antibiotic rate, %；

C_AtijAnd T_Atij- every antimicrobial sample and control sample recycle the average ATP concentration of viable bacteria after 48h is cultivated, single Position is mole every liter (mol/L)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

WithAfter 48h is cultivated, the relative intensity of fluorescence of recovered liquid is measured for-every antimicrobial sample and control sample Value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

a_A- low concentration standard curve lgC_A-lgI_ASlope；

(5) antibacterial activity value A is calculated

It tests under condition for validity, the antibacterial activity value A of every antibacterial rubber sample_ij, the antibacterial of every group and every batch of sample it is living Property value A_iIt is calculated respectively according to formula (18), (19), (20) with A:

In formula:

A_ijThe antibacterial activity value of-every antibacterial rubber sample；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2, 3,4,5；

A_iThe antibacterial activity value of-every group antibacterial rubber sample, sample group i=1,2,3；

A-every batch of antibacterial rubber sample antibacterial activity value；

a_A- low concentration standard curve lgC_A-lgI_ASlope；

(6) data revision of the convention requirement: calibration mycotic spore liquid viable bacteria content C_BAnd ATP concentration of standard solution and inoculation bacterium Liquid, sample recovered liquid viable bacteria ATP concentration C_AWhen, it is provided with reference to the data revision of the convention in GB 4789.2-2016 in relation to clump count, when C_BLess than 100CFU/mL or C_AWhen less than 100mol/L, " rounding up " round numbers；Work as C_BNot less than 100CFU/mL or C_AIt is not small When 100mol/L, take preceding 2 bit digital after the 3rd bit digital " rounding up ", behind with 0 replace digit；Can also with 10 index Form indicates that " rounding up " uses two effective digitals afterwards.Control sample and antimicrobial sample through 0h contact and 48h culture after, The relative intensity of fluorescence measured value round numbers of recovered liquid, antibiotic rate R_ij、R_i, R calculated result take three effective digitals；Mould increases Value F_ij、G_ijWith antibacterial activity value A_ij、A_i, A calculated result take two effective digitals；

(7) uncertainty of measurement: this patent method, which mainly passes through, calculates control sample and antimicrobial sample through 0h contact and 48h After culture, reclaim liquid phase is in the group of fluorescent strength determining value and between-group variation coefficient CV=σ ÷ μ × 100% (μ, σ and C V calculated result remains into 2 significant digits), judge for ATP fluorimetric assay for biological materials method to be applied to the test of rubber fungicidal properties Reproducibility；In regulation group and between-group variation coefficient CV≤10%.

5 control samples, 5 antimicrobial samples and 5 control samples after 48h is cultivated of every group of 0h contact, 5 it is anti- Bacterium sample, arithmetic mean of instantaneous value of the reclaim liquid phase to fluorescent strength determining valueAnd Respectively according to formula (21), (22) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k= 1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are contacted through 0h and 48h is cultivated Afterwards, the relative intensity of fluorescence measured value of each Duplicate Samples):

15 control samples, 15 antimicrobial samples and 15 control samples, 15 after 48h is cultivated that 3 groups of 0h are contacted Part antimicrobial sample, arithmetic mean of instantaneous value of the reclaim liquid phase to fluorescent strength determining valueAndRespectively according to public affairs Formula (23), (24) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k =1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are through 0h contact and 48h After culture, the relative intensity of fluorescence measured value of each Duplicate Samples):

5 control samples, 5 antimicrobial samples and 5 control samples after 48h is cultivated of every group of 0h contact, 5 it is anti- Bacterium sample, the standard deviation of the relative intensity of fluorescence measured value of recovered liquidAndRespectively according to public affairs Formula (25) and (26) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k =1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are contacted through 0h and 48h is trained After supporting, the relative intensity of fluorescence measured value of each Duplicate Samples):

15 control samples, 15 antimicrobial samples and 15 control samples, 15 after 48h is cultivated that 3 groups of 0h are contacted Part antimicrobial sample, the standard deviation of the relative intensity of fluorescence measured value of recovered liquidAndRespectively according to Formula (27) and (28) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples and compiles Number k=1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample contacted through 0h and After 48h culture, the relative intensity of fluorescence measured value of each Duplicate Samples):

The evaluation of result of the antibacterial rubber sample fungicidal properties carries out in the steps below:

(1) with reference to health industry common practice and the requirement of dependent antimicrobial product standard, following classification criterion is taken:

Such as using antibiotic rate R as correlated performance evaluation index: working as R_ij/R_iWhen/R < 80%, sample is without mildew-proof function；When 80%≤R_ij/R_iWhen/R < 90%, sample has mildew-proof function；As 90%≤R_ij/R_iWhen/R < 99%, sample mildew-proof function is suitable In；As 99%≤R_ij/R_iWhen/R < 99.9%, sample mildew-proof function is stronger；Work as R_ij/R_iWhen/R >=99.9%, the mould proof work of sample With extremely strong；

Such as using antibacterial activity value A as correlated performance evaluation index: working as A_ij/A_iWhen/A < 0.5, sample is without mould proof work With；As 0.5≤A_ij/A_iWhen/A < 1.0, sample has mildew-proof function；As 1.0≤A_ij/A_iWhen/A < 2.0, sample mildew-proof function It is moderate；As 2.0≤A_ij/A_iWhen/A < 3.0, sample mildew-proof function is stronger；Work as A_ij/A_iWhen/A >=3.0, sample mildew-proof function pole By force；

(2) as the antibiotic rate R of certain group (part) antibacterial rubber sample_i(R_ij) or antibacterial activity value A_i(A_ij) and other two groups When the fungicidal properties of (four) sample is compared to a levels are at least differed, one group of (part) sample is extracted again and repeats to test；Meter It is calculated through ATP bioluminescence lgC_A─lgI_AThe antibiotic rate or antibacterial activity value that calibration curve method measures.If two groups of front and back antibacterial Rubber sample fungicidal properties level is identical, then abandons it；Take other two groups (four) remaining sample antibiotic rate R_i(R_ij) or antibacterial work Property value A_i(A_ij) evaluation result of the arithmetic mean of instantaneous value as batch (group) the antibacterial rubber sample fungicidal properties；

Specific embodiment

Below with reference to preferred embodiment, the present invention will be described in detail, so that advantages and features of the invention can be easier to It is readily appreciated by one skilled in the art, so as to make a clearer definition of the protection scope of the present invention.

The present embodiment by taking the detection of the fungicidal properties of the antibacterial rubber sample that added organic antibacterial agent is process as an example into Row explanation.

Specific detection method carries out in the steps below:

(1) sample preparation and pretreatment

1.1 control samples: being injection moulded by hygienic level high-density polyethylene, quantity, size and thickness and antibacterial to be measured Rubber sample is identical.

1.2 antimicrobial samples: antibacterial rubber material to be measured or product (for cured sizing material) are placed in (23 ± 2) DEG C, (50 ± 5) the no less than air conditioning of 16h is carried out in the environment of %RH, it is ensured that room air free flow simultaneously touches its each surface. Having a size of (50 ± 2) mm × (50 ± 2) mm, with a thickness of 1.0mm ± 0.1mm, 2.0mm ± 0.2mm or 4.0mm ± 0.2mm；Work as sample Product thickness be greater than 5mm when, need to be cut with leather slicer or similar functions cutting machine, and with flexibility sanding belt to sample not Smooth place polishes.Each strain test uses 6 groups of samples；Wherein 0h contact and 48h culture experiment respectively use 3 groups, every group 5 Sample selects 1 control sample as object of reference respectively and effectively identifies.

1.3 sample pre-treatments: impregnating 1min in 70% ethanol solution for control sample and antimicrobial sample before experiment, and With the abundant cleaning sample of sterile water, to remove ethyl alcohol, (person that is unsuitable for ethanol disinfection can directly use sterile water on superclean bench It rinses)；Sample surface to be measured is put into upwards after natural drying spare in sterilized petri dishes.

(2) lectotype selection and reagent, culture medium are prepared

2.1 General Requirements: test analytical reagents and meet tertiary effluent as defined in GB/T 6682-2008 (distilled water or Deionized water), laboratory has the safe qualification of two stage biological, and personnel have regular Microbiological Lab's working experience.

2.2 instrument and equipments: the superclean bench of two stage biological safety cabinet or lustration class not less than 100；The light of fluorescence containing ATP The ATP bioluminescence rapid detection system of degree meter, Special test tube etc., ATP fluophotometer wavelength range 300nm~650nm, ATP Concentration Testing range 10^-11Mol/L~10^-6mol/L；Wave-length coverage 400nm~760nm, range of readings 0.0Abs~ The microplate reader of 4.0Abs；Amplification factor 40 ×~400 × Photobiology microscope；(25~30) DEG C ± 1 DEG C, (20~95) % The constant temperature and humidity incubator of RH ± 2%RH；46 DEG C ± 1 DEG C of constant water bath box；Revolving speed >=8000r/min centrifuge and mating Centrifuge tube；The vortex oscillator of the range of speeds (500~3000) r/min；The steam of (121 ± 2) DEG C, (103 ± 5) kPa Sterilizer；- 20 DEG C~-80 DEG C of low temperature refrigerator；0 DEG C~10 DEG C of refrigerating box；The electronic balance of sensibility reciprocal 0.001g；Frequency range The ultrasonic cleaner of (30~50) kHz；The pH meter of precision ± 0.1 (25 DEG C)；Electric furnace；Have counteredge, changeable blade Or the cutting machine of rotation cut-off knife and feed roller and cleavable different-thickness film；Surface abrasive is the electricity of aluminium oxide or silicon carbide Dynamic/mechanically flexible sanding belt.

2.3 material utensils: blood counting chamber and dedicated coverslip；The sterile measuring pipette of 1mL, 10mL；0.05mL, The single track changeable fluid liquid-transfering gun and sterile liquid transfer gun head of 0.1mL, 0.2mL, 1mL, 5mL, 10mL (measurement error is less than 1%)； Aperture is not more than 0.45 μm of syringe-driven filter；The sterile conical flask and bottle stopper of capacity 100mL, 250mL, 500mL；96 holes are flat Bottom culture plate；The sterile petri dish of diameter 90mm；Glass funnel；Sterile cock test tube；Diameter is not more than the oese of 4mm；L Stick；The bead of diameter 5mm；Alcolhol burner；Sterilize tweezers；Medical adhesive tape；Absorbent cotton and gauze for biochemistry detection；Sterile filter Paper；Thermometer；The stopwatch of precision 0.01s.

2.4 reagents: 70% ethanol solution；(0.45 μm of solution of MTT (tetramethyl azo azoles salt) of 5mg/mL, pH value 7.4 Membrane filtration degerming, 4 DEG C~6 DEG C are kept in dark place 15d)；121 DEG C of high pressure sterilization 30min after the packing of following reagent, 5 DEG C~10 DEG C Store the physiological saline of 30d:85%；N monomethyl ethanesulfonic acid, Tween 80, two pungent sulfonation sodium succinates are chosen any one kind of them, and are configured to 0.05% wetting agent solution.

2.5 medium/liquids (can use commercially available medium/liquid): 121 DEG C of high pressure sterilizations after matched medium/liquid packing 30min, 2 DEG C~8 DEG C storage 30d；

One dextrose culture-medium of potato (mycotic spore actication of culture and its total plate count plate count use): 300g is new Fresh potato removes the peel stripping and slicing, boils 20min~30min in 1000mL water；Filter to take juice, into filtrate be added 20g glucose, 1000mL is settled to after 0.1g chloramphenicol, 20g agar.

2.6ATP fluorescence reaction reagent (or with commercial reagent): in addition to phosphate buffer solution, matched ATP fluorescence reaction examination - 20 DEG C~-70 DEG C of agent preservations, use in 6 months；

ATP fluorescent reagent: 0.7mg luciferase, the D- fluorescein of 12.6mg, 56mg bovine serum albumin are dissolved in 30mL ATP fluorescent reagent buffer solution, be stored at room temperature 15min after mixing, used in 3h.

(3) culture presevation, activation and spore suspension preparation

3.1 test strains: aspergillus niger ATCC 16404；Chaetomium globosum ATCC 6205；Penicillium chrysogenum ATCC 9179 (or by Other strains that national Culture Collection is provided and can be traced to the source).

3.2 culture presevation: mould test strain is inoculated in simultaneously by potato-dextrose culture-medium inclined-plane with sterile working Indicate the date, 28 DEG C~30 DEG C culture to inclined-planes cover with mycotic spore (7d~14d)；3 DEG C~10 DEG C preservation 4 months.

3.3 actication of culture: with oese scrape preservation bacterium spore, be inoculated with potato-dextrose culture-medium inclined-plane, 28 DEG C ~30 DEG C of culture 7d~14d are until generate a large amount of spores.Mold species test tube plug must not be extracted before spore suspension by preparing, and every Only for spore liquid of preparation after test tube opening, suspension is prepared using the mycotic spore newly cultivated every time.

The preparation of 3.4 spore suspensions: the sterile water of 10mL is added into strain test tube, gently scrapes media surface with oese The spore stoste injection of wash-off is equipped with the sterile taper of jumping a queue of 15 beades and 45mL Cha Shi culture solution by fresh mycotic spore In bottle, 3000r/min shakes test tube 2min, breaks up spore ball, mixes spore liquid.Then, sterile absorbent cotton or eight layers of yarn will be covered with The glass funnel of cloth is placed on conical flask, and filtering spore suspension removes mycelia and culture medium fragment；By filtrate move to sterilizing from In heart pipe, 8000r/min separating treatment at least 10min, removes supernatant at room temperature；It is heavy that spore is cleaned with 50mL Cha Shi culture solution again Starch is simultaneously centrifuged, after repeated washing 3 times, with the spore sediment after the dilution centrifugation of Cha Shi culture solution.Every kind of test mould is pressed Spore suspension is prepared according to the above method, the spore liquid of each test strain is mixed in equal volume；0 DEG C~7 DEG C storages, 4d is interior to be used.

(4) OD value-viable bacteria content logarithm lgC_BStandard curve is established and inoculating spores liquid C_BCalibration

The measurement of 4.1 bacterium solution OD values: primary dcreening operation is carried out using spore total amount of the blood counting chamber to mixing spore suspension, uses Cha Shi Culture solution is 6.0 × 10 to concentration⁸The mixing spore liquid of CFU/mL carries out 1:10,1:15,1:20 and is serially diluted, and with culture solution As blank control sample liquid, zeroing correction is carried out to microplate reader.Then, the spore liquid of the above-mentioned different dilutions of 100 μ L is distinguished 96 hole flat-bottomed plates are injected, each dilution does 3 multiple holes；It is added dropwise the MTT solution of 20 μ L to every hole, (28 ± 1) DEG C, (95 ± 2) after %RH cultivates 30min；Maximum absorption band is scanned in 560nm~610nm wave-length coverage, determines maximum absorption wavelength； The viable bacteria OD value of each hole mixed solution is measured, the viable bacteria OD value of different dilution spore suspensions is its 3 multiple holes OD measured values Arithmetic mean of instantaneous value.Meanwhile the method referring to as defined in national standard GB 4789.15-2016, it is appropriate to carry out to above-mentioned mixing spore suspension After dilution, in (28 ± 1) DEG C culture 3d, and bacterium colony counting is carried out to plate, demarcate its viable bacteria content C_B。

4.2OD value-viable bacteria content logarithm lgC_BStandard curve is established and inoculating spores liquid C_BCalibration: with different dilutions The viable bacteria OD value of spore suspension is as ordinate, with its viable bacteria content logarithm lgC_BFor abscissa, OD value-lgC is drawn_BStandard Curve；The linear equation Y=a of curve is derived from using least square fitting method₀X+b₀And coefficient R₀ ².Work as R₀ ²≥ 0.98, when confidence level >=0.95, the spore liquid viable bacteria content C that is measured according to this patent method_BEffectively.

(5) ATP log concentration value lgC_ARelative intensity of fluorescence logarithm lgI_AStandard curve is established and inoculation bacterium solution viable bacteria ATP concentration C_ACalibration

The determination of 5.1ATP standard serial solution and its test specimens preparation: ATP fluorescent reagent buffer solution is by 1.0 × 10^- ³The ATP standard stock solution of mol/L is diluted to high and low concentration standard serial solution: 7.0 × 10^-8mol/L、7.0×10^-7mol/L、 7.0×10^-6Mol/L and 2.1 × 10^-9mol/L、2.1×10^-8mol/L、2.1×10^-7mol/L.It then, will with sterilizing liquid-transfering gun The above-mentioned ATP standard solution of 0.1mL is moved to respectively in three sterile test tubes, and 0.05mL sterile water and 0.35mL physiology salt is successively added dropwise Aqueous solution simultaneously mixes, as level-one ATP standard solution test specimens；0.1mL level-one ATP standard solution test specimens are moved to respectively again It is each that 0.4mL physiological saline is added dropwise and mixes in three sterile test tubes, as second level ATP standard solution test specimens；And it is moved with sterilizing Liquid rifle moves to the second level ATP standard solution test specimens of 0.1mL various concentration in three instrument sterile test tubes respectively, as ATP standard solution tests Duplicate Samples.

5.2 blank background value calibrations: the aqueous solution, 0.35mL with sterilizing liquid-transfering gun by 0.1mL containing 0.05% wetting agent are raw The ATP lysate of reason salt water and 0.05mL are separately added into same branch sterile test tube, and 3000r/min shakes test tube 30s；It stands Level-one blank sample is used as after 10min~20min；0.1mL level-one blank sample is moved in an other sterile test tube again, is added dropwise 0.4mL physiological saline simultaneously mixes, as second level blank sample.Then, 0.1mL second level blank sample is moved to respectively with sterilizing liquid-transfering gun In three instrument sterile test tubes, as skip test Duplicate Samples；The ATP that 0.1mL is successively added dropwise into three Duplicate Samples is mentioned Reagent is taken, instills the ATP fluorescent reagent of 0.1mL after mixing again.3000r/min shakes test tube 5s, uses ATP fluophotometer immediately Measure its relative intensity of fluorescence value I_AAnd it records and (ensures that each link operating time is consistent, avoid cross contamination).Each Duplicate Samples Minute is no more than 15s, using the arithmetic mean of instantaneous value of three skip test Duplicate Samples relative intensity of fluorescence values as instrument and examination Agent group background values (or calibrating background according to instrument operation instruction).

5.3ATP standard solution relative intensity of fluorescence value I_AMeasurement: from low to high according to concentration, to various concentration ATP The ATP that 0.1mL is added dropwise in three test Duplicate Samples of standard solution respectively extracts reagent, and the ATP for instilling 0.1mL after mixing again is glimmering Light reagent；3000r/min shakes test tube 5s, uses its relative intensity of fluorescence value of ATP fluorescent spectrophotometer measuring I immediately_AAnd it records (really It is consistent to protect each link operating time, avoids cross contamination).Each Duplicate Samples minute is no more than 15s, with each concentration ATP mark The arithmetic mean of instantaneous value of three Duplicate Samples relative intensity of fluorescence values of quasi- solution is its I_AMeasured value.

5.4ATP log concentration value lgC_ARelative intensity of fluorescence logarithm lgI_AStandard curve is established: with various concentration ATP The relative intensity of fluorescence logarithm lgI of standard solution_AAs abscissa, with its ATP log concentration value lgC_AFor ordinate mapping； Calibration curve is carried out to mathematical relationship between the two, draws the lgC of two respective concentrations_A-lgI_AStandard curve；And application is most Small two multiply the linear equation Y=a that fitting process is derived from curve_A0X+b_A0(high concentration), Y=a_AX+b_AIt is (low concentration) and related Coefficients R_A0 ²(high concentration), R_A ²(low concentration).Work as R_A0 ²And R_A ²>=0.98, it when confidence level >=0.95, is done according to this patent Measurement is effective.

5.5 inoculation bacterium solution viable bacteria ATP concentration Cs_ACalibration: with Cha Shi culture solution to viable bacteria content C_BIt is 1.0 × 10⁸CFU/mL ~5.0 × 10⁸The mould mixing spore liquid of CFU/mL carries out continuous 10 times of gradient dilutions, measures its relative intensity of fluorescence value I_A；Root According to high standard fitting equation Y=a_A0X+b_A0It calculates and demarcates corresponding viable bacteria ATP concentration C_A, through Cha Shi culture solution tune It is whole, obtain C_ARange is 5.0 × 10^-7Mol/L~9.0 × 10^-7The inoculation bacterium solution of mol/L measures and records 0.3mL inoculation bacterium solution Relative intensity of fluorescence value I after the dilution of 4.6mL eluent in 1min_A0。

(6) sample inoculation, culture and elution recycling

6.1 inoculated and cultureds: antibacterial rubber sample carries out antibacterial tests after completing air conditioning in 1h, with sterilizing liquid-transfering gun 0.3mL inoculation bacterium solution is added dropwise respectively (with lgC to each group control sample and antimicrobial sample surface to be measured_A-lgI_ACalibration curve spore Sub- liquid is derived from same branch mixing spore liquid test tube, and 0 DEG C of ± 1 DEG C of preservation, 2h is interior to be used), (adhered to inoculation bacterium solution with L stick but do not hung Drop) bacterium solution is smeared uniformly, so that it is covered sample whole surface.Ware lid is covered, sample will be contacted equipped with 6 groups of 48h with medical adhesive tape The plate of product seals；(28 ± 2) DEG C, (95 ± 2) %RH cultivate 48h ± 2h.

6.2 elution recycling: after 6 groups of 0h contact sample inoculation moulds, 4.6mL eluent is drawn with sterilizing liquid-transfering gun immediately (i.e. Cha Shi culture solution), each sample inoculation surface of repeated flushing at least 4 times in plate, sufficiently elution are simultaneously anti-with liquid transfer gun head Mould washing lotion in multiple pressure-vaccum plate, until lawn again moves into washing lotion in sterile test tube after being broken up；3000r/min shakes test tube 2min, as the recovered liquid of sample to be tested (if recovered liquid adds eluent to 4.9mL) less than 4.9mL after mixing.Each group Sample after 48h is cultivated uses type of elution identical with 0h contact sample to recycle mould.

(7) reclaim liquid phase is to fluorescence intensity level I_AMeasurement

7.1 instruments and reagent set relative intensity of fluorescence background value calibration: according to lgC_A-lgI_ABlank when standard curve is established Background calibration methods, with the wetting agent solution of Cha Shi culture solution substitution 0.05%, determining instrument and reagent set background values.

7.2 reclaim liquid phases are to fluorescence intensity level I_AMeasurement: if background level meets instrument requirement, with sterilizing liquid-transfering gun The ATP lysate of 4.9mL recovered liquid and 0.1mL is separately added into same branch sterile test tube, 3000r/min shakes test tube 30s； After being stored at room temperature 20min, then the ATP extraction reagent of 5.0mL is added dropwise and mixes；It is stored at room temperature 10min.It will with sterilizing liquid-transfering gun The above-mentioned mixed solution of 0.1mL is moved to respectively in three instrument sterile test tubes, tests Duplicate Samples as ATP bioluminescence.To The ATP fluorescent reagent of 0.1mL is respectively added dropwise in three Duplicate Samples, 3000r/min shakes test tube 5s, uses ATP fluophotometer immediately Measure its relative intensity of fluorescence value I_AAnd it records and (ensures that each link operating time is consistent, avoid cross contamination).Each Duplicate Samples Minute is no more than 15s, using the arithmetic mean of instantaneous value of three ATP bioluminescences test Duplicate Samples relative intensity of fluorescence value as to The I of sample recovered liquid_AMeasured value.

(8) recovered liquid viable bacteria ATP concentration C_AAnd T_AIt calculates and antibiotic rate R and antibacterial activity value A is calculated

8.1 recovered liquid viable bacteria ATP concentration Cs_AAnd T_AIt calculates

In formula:

With- every 0h contact and after 48h is cultivated antimicrobial sample recovered liquid relative intensity of fluorescence measured value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5.

8.2 test conditions for validity

If recovered liquid and 0.3mL inoculation the bacterium solution 1min after the dilution of 4.6mL eluent of every 0h contact control sample Interior relative intensity of fluorescence measured value is close, i.e.,Every after 48h is cultivated control sample recovered liquid relative fluorescence it is strong Spend the logarithm of measured valueThat is C_Atij≥0.1×C_A0ij.Then when 3 groups of 0h contact control sample reclaim liquid phase To fluorescent strength determining valueGroup in and when between-group variation coefficient CV≤10% (involved calculation formula is shown in this patent correlation Uncertainty of measurement requirement), the measurement carried out according to this patent method is effective.

8.3 mould increasing value F_ij、G_ijIt calculates

In formula:

a_A- low concentration standard curve lgC_A-lgI_ASlope.

8.4 antibiotic rate R are calculated

In formula:

R-every batch of antibacterial rubber sample antibiotic rate, %；

a_A- low concentration standard curve lgC_A-lgI_ASlope.

8.5 antibacterial activity value A are calculated

In formula:

A-every batch of antibacterial rubber sample antibacterial activity value；

a_A- low concentration standard curve lgC_A-lgI_ASlope.

8.6 data revisions of the convention requirement: calibration mycotic spore liquid viable bacteria content C_BAnd ATP concentration of standard solution and inoculation bacterium Liquid, sample recovered liquid viable bacteria ATP concentration C_AWhen, it is provided with reference to the data revision of the convention in GB 4789.2-2016 in relation to clump count, when C_BLess than 100CFU/mL or C_AWhen less than 100mol/L, " rounding up " round numbers；Work as C_BNot less than 100CFU/mL or C_AIt is not small When 100mol/L, take preceding 2 bit digital after the 3rd bit digital " rounding up ", behind with 0 replace digit；Can also with 10 index Form indicates that " rounding up " uses two effective digitals afterwards.Control sample and antimicrobial sample through 0h contact and 48h culture after, The relative intensity of fluorescence measured value round numbers of recovered liquid, antibiotic rate R_ij、R_i, R calculated result take three effective digitals；Mould increases Value F_ij、G_ijWith antibacterial activity value A_ij、A_i, A calculated result take two effective digitals.

8.7 uncertainties of measurement: this patent method, which mainly passes through, calculates control sample and antimicrobial sample through 0h contact and 48h After culture, reclaim liquid phase is in the group of fluorescent strength determining value and between-group variation coefficient CV=σ ÷ μ × 100% (μ, σ and C V calculated result remains into 2 significant digits), judge for ATP fluorimetric assay for biological materials method to be applied to the test of rubber fungicidal properties Reproducibility；In regulation group and between-group variation coefficient CV≤10%.

5 control samples, 5 antimicrobial samples and 5 control samples after 48h is cultivated of every group of 0h contact, 5 it is anti- Bacterium sample, arithmetic mean of instantaneous value of the reclaim liquid phase to fluorescent strength determining valueAnd Respectively according to formula (21), (22) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k= 1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are contacted through 0h and 48h is trained After supporting, the relative intensity of fluorescence measured value of each Duplicate Samples):

5 control samples, 5 antimicrobial samples and 5 control samples after 48h is cultivated of every group of 0h contact, 5 it is anti- Bacterium sample, the standard deviation of the relative intensity of fluorescence measured value of recovered liquidAndRespectively according to public affairs Formula (25) and (26) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k =1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are through 0h contact and 48h After culture, the relative intensity of fluorescence measured value of each Duplicate Samples):

(9) evaluation of result

9.1, with reference to health industry common practice and the requirement of dependent antimicrobial product standard, take following classification criterion:

Such as using antibacterial activity value A as correlated performance evaluation index: working as A_ij/A_iWhen/A < 0.5, sample is without mould proof work With；As 0.5≤A_ij/A_iWhen/A < 1.0, sample has mildew-proof function；As 1.0≤A_ij/A_iWhen/A < 2.0, sample mildew-proof function It is moderate；As 2.0≤A_ij/A_iWhen/A < 3.0, sample mildew-proof function is stronger；Work as A_ij/A_iWhen/A >=3.0, sample mildew-proof function pole By force.

9.2 work as the antibiotic rate R of certain group (part) antibacterial rubber sample_i(R_ij) or antibacterial activity value A_i(A_ij) and other two groups When the fungicidal properties of (four) sample is compared to a levels are at least differed, one group of (part) sample is extracted again and repeats to test；Meter It is calculated through ATP bioluminescence lgC_A─lgI_AThe antibiotic rate or antibacterial activity value that calibration curve method measures.If two groups of front and back antibacterial Rubber sample fungicidal properties level is identical, then abandons it；Take other two groups (four) remaining sample antibiotic rate R_i(R_ij) or antibacterial work Property value A_i(A_ij) evaluation result of the arithmetic mean of instantaneous value as batch (group) the antibacterial rubber sample fungicidal properties.

Detection qu alification, instrument and equipment used in the present embodiment and test equipment, medium/liquid, chemical reagent, reference culture:

(1) bio-safety qualification

In the two stage biological safety experiment room that institute registration number is CNAS BL0059, by having secondary advanced techniques academic title Microorganism detection professional complete related experiment.

(2) instrument and equipment and test equipment

2.1 two stage biological safety cabinets: 1300 series of secondary B2 type Biohazard Safety Equipment of Thermo Scientific, workbench Surface area 0.55m², capacity 1130m³/ h, filter efficiency 99.99% (0.3 μm).

2.2 superclean benches: American blend Thermo Scientific^TM Heraguard^TM, model ECO ultra-clean work Make platform, inside width × depth × height=920mm × 585mm × 645mm；Air velocity is 0.15m/s~0.25m/s and 0.36m/s ~0.45m/s.

2.3ATP bioluminescence rapid detection system: the portable system SURE ATP fluorescence of Hygiena company, the U.S. Detector, box containing matched reagent, plastics Special test tube etc.；ATP content detection lower limit 4 × 10^-18Mol/ml, the inspection of microorganism total amount Rising limit 1.0CFU/ml, RLU range of readings 0~9999, detection time 10s, 1000 times/s of sampling rate, measurement error ± 5%.

2.4 microplate reader: the full-automatic microplate reader of American blend Thermo Scientific, model Multiskan FC, wave Long range (340~850) nm ± 1nm, range of readings (0.0~6.0) Abs ± 0.001Abs；The corresponding measurement accuracy of 405nm is ± 1% (0.0Abs~3.0Abs) or ± 2% (3.0Abs~4.0Abs).

2.5 Photobiology microscopes: have the adjustable eyepiece of 10 times of wide visual fields and 4 times, 10 times, 20 times, 40 times and 100 times The tri- mesh research grade biomicroscope of Olympus BX43 of flat-field achromatic objective lens.

2.6 constant temperature and humidity incubators: Guan Sen biotechnology (Shanghai) Co., Ltd. model WS-380H, volume 380L, temperature control The constant temperature and humidity incubator of ± 0.8 DEG C of range (0~50) DEG C, wet range (30~95) %RH ± 2%RH of control.

2.7 constant water bath box: the electric heating constant temperature sink of upper sea base Wei test apparatus equipment Co., Ltd model DK-S420, Volume 15L, ± 0.5 DEG C of temperature control range (5~99.9) DEG C, timing range 1min~999min.

2.8 constant temperature oscillators: the permanent model WS-380H in Shanghai one, ± 0.1 DEG C of temperature control range (4~65) DEG C, frequency of oscillation (40~300) r/min, timing range (1~99) h.

2.9 refrigerated centrifuges: the vertical refrigerated centrifuge of Beijing Xin Nuolihua Instrument Ltd. model DL7M-12L turns Fast 8000r/min ± 20r/min, 12300 × g of relative centrifugal force；Capacity 14400ml, timing range 1s~99h59min99s, ± 1 DEG C of temperature control range (- 20~40) DEG C, centrifuge tube specification Ф 74mm × 168mm.

2.10 pressure steam sterilizers: Japanese Sanyo company model MLS-3780 autoclave, volume 75L, sterilizing temperature ± 2 DEG C, maximum pressure 0.235MPa of degree (105~135) DEG C, timer (1~250) min.

2.11 low temperature refrigerators: the superfreeze storage of Zhong Kemeiling low temperature Science and Technology Co., Ltd. model DW-HL398 Case, dischargeable capacity 398L, -10 DEG C~-86 DEG C of storage temperature.

2.12 refrigerators: the cryogenic box ultralow temperature ice of the glad experimental instruments and equipment limited model HXL-25-250AD of Dongguan City sky Case, ± 0.1 DEG C of refrigerating box (2~10) DEG C, ± 0.1 DEG C of household freezer (- 30~20) DEG C.

2.13 electronic balances: the electronic balance of Japanese Shimadzu model AUX220, range 220g, precision ± 0.1mg.

2.14 ultrasonic cleaners: the supersonic wave cleaning machine of Shanghai Yi Jing ultrasonic instrument Co., Ltd model YQ-120C, Capacity 3.2L, supersonic frequency 40KHz/28KHz/25KHz, timing range 1min~30min/99min.

2.15 vortex oscillators: the vortex oscillator of American blend Coleparmer Votex-Genie2, the range of speeds (500~3000) r/min ± 3r/min, timing range 1s~60s.

2.16pH meter: the accurate pH meter of Shanghai precision instrumentation Co., Ltd model MP512-03, range ability (- 2.000~19.999) pH ± 0.002pH, ± 0.4 DEG C of temperature range (- 10~110) DEG C.

2.17 electric furnaces: the 1KW closed temp.-adjustable electric furnace of Changzhou Deco Instrument Ltd. model DLD-1KW.

2.18 cutting machines: the full-automatic slicing machine of Yancheng City Kun Hao machinery plant model LH-500D, cutoff length (0.1~ 9999.9) mm ± 0.05mm, cut width (1~500) mm ± 0.05mm；Upper speed 3m/s, lower speed 4m/s.

2.19 flexible sanding belts: laboratory home-built equipment, polish line speeds 20m/s ± 5m/s；Grinding wheel model C -60-P- 4-V, diameter 150mm, linear velocity range 10m/s~12m/s, abrasive material are the black silicon carbide of granularity 60.

2.20 blood counting chambers: the blood counting chamber that refinement specification in Shanghai is 25 × 16.

(3) medium/liquid

The medium/liquid of Beijing overpass technical concern Co., Ltd production.

(4) chemical reagent

Tetramethyl azo azoles salt, trinosin standard items and preparation ATP fluorescent reagent buffer solution, ATP cracking A series of biochemical reagents needed for liquid, ATP extracting solution and ATP fluorescent reagent are purchased from Shanghai Jin Pan Biotechnology Co., Ltd agency's U.S.'s Amresco brand.

(5) reference culture

Aspergillus niger ATCC 16404, Chaetomium globosum ATCC 6205, penicillium chrysogenum ATCC 9179 are purchased from the common micro- life of China Object culture presevation administrative center.

The detection data and result of the present embodiment calculate:

Using ATP fluophotometer through lgC_A-lgI_ACalibration curve method examines the fungicidal properties of antibacterial rubber sample It surveys, coherent detection data and Calculation of Measuring Uncertainty the results are shown in Table 1~table 2.

1 relative intensity of fluorescence measured value of table and antibiotic rate R, antibacterial activity value A calculated result

2 relative intensity of fluorescence Calculation of Measuring Uncertainty result of table

The above description is only an embodiment of the present invention, is not intended to limit the scope of the invention, all to utilize this hair Equivalent transformation made by bright description, is applied directly or indirectly in other relevant technical fields, and is included in this hair In bright scope of patent protection.

Claims

1. a kind of detection method of antibacterial rubber fungicidal properties, comprising: (1) sample preparation and pretreatment；(2) lectotype selection and examination Agent, culture medium are prepared；(3) culture presevation, activation and spore suspension preparation；(4) OD value-viable bacteria content logarithm lgC_BStandard is bent Line is established；(5) ATP log concentration value lgC_ARelative intensity of fluorescence logarithm lgI_AStandard curve is established and inoculation bacterium solution viable bacteria ATP concentration C_ACalibration；(6) sample inoculation, culture and elution recycling；(7) reclaim liquid phase is to fluorescence intensity level I_AMeasurement；(8) it recycles Liquid viable bacteria ATP concentration C_AAnd T_AIt calculates and antibiotic rate R and antibacterial activity value A is calculated；(9) evaluation of result；It is characterized in that, using ATP fluophotometer carries out accurate quantitative test to the antibacterial rubber fungicidal properties with antibiotic rate R or antibacterial activity value A characterization ATP bioluminescence lgC_A-lgI_ACalibration curve method, specific:

In reclaim liquid phase to fluorescence intensity level I_AIn measurement:

Preparation, size, quantity and the pre-treatment requirement for specifying control sample and antimicrobial sample, by the standard bacteria of mould test strain After strain is passed on, activated, fresh mycotic spore culture is taken to prepare spore suspension；OD value-is established using MTT colorimetric analysis Viable bacteria content logarithm lgC_BStandard curve is derived from the linear equation Y=a of curve₀X+b₀And coefficient R₀ ²；And it is right The spore suspension of different dilutions carries out viable bacteria content C_BCalibration, then, with ATP fluorescent reagent buffer solution by 1.0 × 10^- ³The ATP standard stock solution of mol/L is diluted to high and low concentration standard serial solution: 7.0 × 10^-8mol/L、7.0×10^-7mol/L、 7.0×10^-6Mol/L and 2.1 × 10^-9mol/L、2.1×10^-8mol/L、2.1×10^-7Mol/L measures its relative intensity of fluorescence Value I_A；Draw the lgC of two respective concentrations_A-lgI_AStandard curve is derived from fitting equation Y=a_A0X+b_A0(high concentration), Y=a_AX+b_A(low concentration) and linearly dependent coefficient R_A0 ²(high concentration), R_A ²(low concentration), meanwhile, with Cha Shi culture solution to viable bacteria Content C_BIt is 1.0 × 10⁸CFU/mL~5.0 × 10⁸The mould mixing spore liquid of CFU/mL carries out continuous 10 times of gradient dilutions, surveys Fixed its relative intensity of fluorescence value I_A, and according to high standard fitting equation Y=a_A0X+b_A0Calculate corresponding viable bacteria ATP concentration C_A；It is adjusted to obtain C_ARange is 5.0 × 10^-7Mol/L~9.0 × 10^-7Then the inoculation bacterium solution of mol/L is waited for each group sample It surveys surface and 0.3mL inoculation bacterium solution is added dropwise respectively, elution recycling is carried out to 6 groups of 0h contact samples with 4.6mL eluent immediately, is measured The relative intensity of fluorescence value I of recovered liquid_AC0ij、I_AT0ij, according to low concentration calibration curve equation formula Y=a_AX+b_ACalculate its viable bacteria ATP concentration C_AOijAnd T_AOij, meanwhile, the control sample and antimicrobial sample that 6 groups are sealed in sterilized petri dishes (28 ± 2) DEG C, After cultivating 48h ± 2h under conditions of (95 ± 2) %RH；Recycling remained on surface bacterium is eluted using mode identical with 0h contact sample And measure the relative intensity of fluorescence value I of recovered liquid_ACtij、I_ATtij, calculate corresponding viable bacteria ATP concentration C_AtijAnd T_Atij；

In antibiotic rate R and antibacterial activity value A is calculated:

According to ATP low concentration standard curve lgC_A-lgI_ALinear equation Y=a_AX+b_A, contacted with 0h and after 48h is cultivated it is every The relative intensity of fluorescence measured value of part control sample and antimicrobial sample recovered liquidWith Based on number According to；In the case where testing condition for validity, its mould increasing value F after 48h is cultivated is calculated_ij、G_ijAnd antibiotic rate R_ijAnd antibacterial activity Value A_ij；To the R of every group of sample_ijAnd A_ijArithmetic mean of instantaneous value is taken to obtain corresponding R_iAnd A_i；The antibiotic rate R of every batch of antibacterial rubber sample It is its three groups of sample R with antibacterial activity value A_iAnd A_iArithmetic mean of instantaneous value；And the clear related data revision of the convention and uncertainty of measurement are wanted It asks；

In evaluation of result:

With reference to health industry common practice and the requirement of dependent antimicrobial product standard, determine that fungicidal properties is classified criterion；When certain The antibiotic rate R of group (part) antibacterial rubber sample_i(R_ij) or antibacterial activity value A_i(A_ij) mould proof with other two groups (four) samples When performance is compared to a levels are at least differed, one group of (part) sample is extracted again and repeats to test；It is calculated through ATP bioluminescence lgC_A─lgI_AThe antibiotic rate or antibacterial activity value that calibration curve method measures, if two groups of front and back (part) antibacterial rubber sample is mould proof Performance level is identical, then abandons it；Take other two groups (four) remaining sample antibiotic rate R_i(R_ij) or antibacterial activity value A_i(A_ij) Evaluation result of the arithmetic mean of instantaneous value as batch (group) the antibacterial rubber sample fungicidal properties.

2. the detection method of antibacterial rubber fungicidal properties according to claim 1, which is characterized in that the sample preparation And pre-treatment, it carries out in the steps below:

(1) control sample: being injection moulded by hygienic level high-density polyethylene, quantity, size and thickness and antibacterial rubber to be measured Sample is identical；

(2) antimicrobial sample: by antibacterial rubber material to be measured or product (for cured sizing material) be placed in (23 ± 2) DEG C, (50 ± 5) the no less than air conditioning of 16h is carried out in the environment of %RH, it is ensured that room air free flow simultaneously touches its each surface, ruler Very little is (50 ± 2) mm × (50 ± 2) mm, with a thickness of 1.0mm ± 0.1mm, 2.0mm ± 0.2mm or 4.0mm ± 0.2mm；Work as sample It when thickness is greater than 5mm, need to be cut with leather slicer or similar functions cutting machine, and with flexible sanding belt to sample injustice Whole place polishes, and each strain test uses 6 groups of samples；Wherein 0h contact and 48h culture experiment respectively use 3 groups, every group of 5 samples Product select 1 control sample as object of reference respectively and effectively identify；

(3) sample pre-treatments: control sample and antimicrobial sample are impregnated into 1min in 70% ethanol solution before experiment, and super With the abundant cleaning sample of sterile water, to remove ethyl alcohol, (person that is unsuitable for ethanol disinfection can directly be rushed with sterile water on net workbench It washes)；Sample surface to be measured is put into upwards after natural drying spare in sterilized petri dishes.

3. the detection method of antibacterial rubber fungicidal properties according to claim 1, which is characterized in that the lectotype selection And reagent, culture medium are prepared, and are carried out in the steps below:

(1) General Requirement: test analytical reagents and meet tertiary effluent as defined in GB/T 6682-2008 (distilled water or go from Sub- water), laboratory has the safe qualification of two stage biological, and personnel have regular Microbiological Lab's working experience；

(2) instrument and equipment: the superclean bench of two stage biological safety cabinet or lustration class not less than 100；Fluorophotometric containing ATP The ATP bioluminescence rapid detection system of meter, Special test tube etc., ATP fluophotometer wavelength range 300nm~650nm, ATP Concentration Testing range 10^-11Mol/L~10^-6mol/L；Wave-length coverage 400nm~760nm, range of readings 0.0Abs~4.0Abs's Microplate reader；Amplification factor 40 ×~400 × Photobiology microscope；(25~30) DEG C ± 1 DEG C, (20~95) %RH ± 2% The constant temperature and humidity incubator of RH；46 DEG C ± 1 DEG C of constant water bath box；Revolving speed >=8000r/min centrifuge and mating centrifuge tube； The vortex oscillator of the range of speeds (500~3000) r/min；The pressure steam sterilizer of (121 ± 2) DEG C, (103 ± 5) kPa；- 20 DEG C~-80 DEG C of low temperature refrigerator；0 DEG C~10 DEG C of refrigerating box；The electronic balance of sensibility reciprocal 0.001g；Frequency range (30~50) The ultrasonic cleaner of kHz；The pH meter of precision ± 0.1 (25 DEG C)；Electric furnace；Have counteredge, changeable blade or rotation to cut out The cutting machine of knife and feed roller and cleavable different-thickness film；Surface abrasive is scratched for electronic/machinery of aluminium oxide or silicon carbide Property sanding belt；

(3) material utensil: blood counting chamber and dedicated coverslip；The sterile measuring pipette of 1mL, 10mL；0.05mL,0.1mL, The single track changeable fluid liquid-transfering gun and sterile liquid transfer gun head of 0.2mL, 1mL, 5mL, 10mL (measurement error is less than 1%)；Aperture is not Syringe-driven filter greater than 0.45 μm；The sterile conical flask and bottle stopper of capacity 100mL, 250mL, 500mL；The flat culture in 96 holes Plate；The sterile petri dish of diameter 90mm；Glass funnel；Sterile cock test tube；Diameter is not more than the oese of 4mm；L stick；Diameter The bead of 5mm；Alcolhol burner；Sterilize tweezers；Medical adhesive tape；Absorbent cotton and gauze for biochemistry detection；Aseptic filter paper；Thermometer；The stopwatch of precision 0.01s；

(4) reagent: 70% ethanol solution；MTT (tetramethyl azo azoles salt) solution (0.45 μm of filter membrane of 5mg/mL, pH value 7.4 Filtration sterilization, 4 DEG C~6 DEG C are kept in dark place 15d)；121 DEG C of high pressure sterilization 30min, 5 DEG C~10 DEG C storages after following reagent packing The physiological saline of 30d:85%；N monomethyl ethanesulfonic acid, Tween 80, two pungent sulfonation sodium succinates are chosen any one kind of them, and are configured to 0.05% Wetting agent solution；

(5) medium/liquid (commercially available medium/liquid can be used): 121 DEG C of high pressure sterilization 30min after the packing of matched medium/liquid, 2 DEG C ~8 DEG C of storage 30d；

Cha Shi culture solution (preparation of mycotic spore liquid, bacteria suspension dilution and sample elution use): by 2g sodium nitrate, 1g phosphoric acid hydrogen two Potassium, 0.5g potassium chloride, 0.5g magnesium sulfate, 0.01g ferrous sulfate, 30g sucrose, which are dissolved by heating, contains 0.05% wetting agent in 1000mL Aqueous solution in, adjust pH to 6.0~6.5 (25 DEG C)；

One dextrose culture-medium of potato (mycotic spore actication of culture and its total plate count plate count use): by the fresh horse of 300g Bell potato removes the peel stripping and slicing, boils 20min~30min in 1000mL water；Juice is filtered to take, 20g glucose, 0.1g are added into filtrate 1000mL is settled to after chloramphenicol, 20g agar；

(6) ATP fluorescence reaction reagent (or with commercial reagent): in addition to phosphate buffer solution, matched ATP fluorescence reaction reagent- 20 DEG C~-70 DEG C preservations, use in 6 months；

Dilution buffer: 0.005mol/L and the disodium phosphate soln for containing 0.037% sucrose adjust pH to 7.2 ± 0.2； 121 DEG C of high pressure sterilizations 15min, 2 DEG C~8 DEG C storage 30d；

ATP fluorescent reagent buffer solution: by 1117mg trishydroxymethylaminomethane, 183mg disodium ethylene diamine tetraacetate, 808mg Magnesium acetate, 6.7mg dithiothreitol, the beta-cyclodextrin of 25000mg and 925mg glucose dissolve by heating in 250mL water, adjust Save pH to 7.5 ± 0.2；It is used in 8h；

ATP lysate: by 4.6 international units/ml apyrase (EC:3.6.1.5) and 46 international units/ml Adenosine phosphate deaminase (EC:3.5.4.6 or EC:3.5.4.17), 37mg sucrose, that 20mg bovine serum albumin is dissolved in 10mL is dense Degree is in the 2-morpholine ethane sulfonic acid buffer solution of 0.05mol/L, and adjusting use in pH to 6.0 ± 0.5,8h, (1mL lysate exists The ATP concentration in Sharpe culture solution can be down to 10 in 15min^-11Mol/L or less)；

ATP extracting solution: 45mg trishydroxymethylaminomethane is dissolved by heating in 9.8ml water, the benzene for being 10% with 0.2ml concentration After pricking the mixing of oronain solution, adjust pH to 12.0 ± 0.5 (fungal cell ATP extraction efficiency is not less than 80%)；

ATP fluorescent reagent: 0.7mg luciferase, the D- fluorescein of 12.6mg, 56mg bovine serum albumin are dissolved in 30mL's ATP fluorescent reagent buffer solution is stored at room temperature 15min, uses in 3h after mixing.

4. the detection method of antibacterial rubber fungicidal properties according to claim 1, which is characterized in that the strain is protected Hiding, activation and spore suspension preparation, carry out in the steps below:

(1) test strain: aspergillus niger ATCC 16404；Chaetomium globosum ATCC 6205；Penicillium chrysogenum ATCC 9179 (or by country Other strains that Culture Collection is provided and can be traced to the source)；

(2) culture presevation: mould test strain is inoculated in by potato-dextrose culture-medium inclined-plane with sterile working and is indicated Date, 28 DEG C~30 DEG C culture to inclined-planes cover with mycotic spore (7d~14d)；3 DEG C~10 DEG C preservation 4 months；

(3) actication of culture: with oese scrape preservation bacterium spore, be inoculated with potato-dextrose culture-medium inclined-plane, 28 DEG C~30 DEG C culture 7d~14d must not extract mold species test tube plug, every test tube before preparing spore suspension until generate a large amount of spores Only for spore liquid of preparation after opening, suspension is prepared using the mycotic spore newly cultivated every time；

(4) prepared by spore suspension: the sterile water of 10mL being added into strain test tube, the fresh of media surface is gently scraped with oese The spore stoste injection of wash-off is equipped with the sterile conical flask of jumping a queue of 15 beades and 45mL Cha Shi culture solution by mycotic spore In, 3000r/min shakes test tube 2min, breaks up spore ball, then sterile absorbent cotton or eight layers of gauze will be covered with by mixing spore liquid Glass funnel be placed on conical flask, filtering spore suspension removes mycelia and culture medium fragment；Filtrate is moved into sterilizing centrifugation Guan Zhong, 8000r/min separating treatment at least 10min, removes supernatant at room temperature；Spore precipitating is cleaned with 50mL Cha Shi culture solution again Object is simultaneously centrifuged, after repeated washing 3 times, with Cha Shi culture solution dilution centrifugation after spore sediment, every kind of test mould according to The above method prepares spore suspension, and the spore liquid of each test strain is mixed in equal volume；0 DEG C~7 DEG C storages, 4d is interior to be used.

5. the detection method of antibacterial rubber fungicidal properties according to claim 1, which is characterized in that the OD value-work Bacterial content logarithm lgC_BStandard curve is established and inoculating spores liquid C_BCalibration carries out in the steps below:

(1) bacterium solution OD value measures: carrying out primary dcreening operation using spore total amount of the blood counting chamber to mixing spore suspension, is cultivated with Cha Shi Liquid is 6.0 × 10 to concentration⁸The mixing spore liquid of CFU/mL carries out 1:10,1:15,1:20 and is serially diluted, and using culture solution as Blank control sample liquid carries out zeroing correction to microplate reader, and then, the spore liquid of the above-mentioned different dilutions of 100 μ L is injected separately into 96 hole flat-bottomed plates, each dilution do 3 multiple holes；It is added dropwise the MTT solution of 20 μ L to every hole, (28 ± 1) DEG C, (95 ± 2) after %RH cultivates 30min；Maximum absorption band is scanned in 560nm~610nm wave-length coverage, determines maximum absorption wavelength；It surveys The viable bacteria OD value of fixed each hole mixed solution, the viable bacteria OD value of different dilution spore suspensions are the calculation of its 3 multiple holes OD measured values Art average value, meanwhile, the method referring to as defined in national standard GB 4789.15-2016 carries out above-mentioned mixing spore suspension appropriate dilute After releasing, in (28 ± 1) DEG C culture 3d, and bacterium colony counting is carried out to plate, demarcate its viable bacteria content C_B；

(2) OD value-viable bacteria content logarithm lgC_BStandard curve is established and inoculating spores liquid C_BCalibration: with different dilution spores The viable bacteria OD value of suspension is as ordinate, with its viable bacteria content logarithm lgC_BFor abscissa, OD value-lgC is drawn_BStandard curve； The linear equation Y=a of curve is derived from using least square fitting method₀X+b₀And coefficient R₀ ², work as R₀ ²>=0.98, it sets Letter is horizontal >=0.95 when, the spore liquid viable bacteria content C that is measured according to this patent method_BEffectively.

6. the detection method of antibacterial rubber fungicidal properties according to claim 1, which is characterized in that the ATP concentration Logarithm lgC_ARelative intensity of fluorescence logarithm lgI_AStandard curve is established and inoculation bacterium solution viable bacteria ATP concentration C_ACalibration, is pressed State step progress:

(1) determination of ATP standard serial solution and its test specimens preparation: ATP fluorescent reagent buffer solution is by 1.0 × 10^-3mol/L ATP standard stock solution be diluted to high and low concentration standard serial solution: 7.0 × 10^-8mol/L、7.0×10^-7mol/L、7.0×10^-6Mol/L and 2.1 × 10^-9mol/L、2.1×10^-8mol/L、2.1×10^-7Mol/L then will be on 0.1mL with sterilizing liquid-transfering gun It states ATP standard solution to move to respectively in three sterile test tubes, 0.05mL sterile water and 0.35mL normal saline solution is successively added dropwise And mix, as level-one ATP standard solution test specimens；0.1mL level-one ATP standard solution test specimens are moved into three nothings respectively again It is each that 0.4mL physiological saline is added dropwise and mixes in bacterium test tube, as second level ATP standard solution test specimens；And it will with sterilizing liquid-transfering gun The second level ATP standard solution test specimens of 0.1mL various concentration are moved to respectively in three instrument sterile test tubes, are marked as ATP Quasi- solution testing Duplicate Samples；

(2) blank background value calibration: aqueous solution, 0.35mL physiology salt with sterilizing liquid-transfering gun by 0.1mL containing 0.05% wetting agent The ATP lysate of water and 0.05mL are separately added into same branch sterile test tube, and 3000r/min shakes test tube 30s；Stand 10min Level-one blank sample is used as after~20min；0.1mL level-one blank sample is moved in an other sterile test tube again, it is raw that 0.4mL is added dropwise Reason salt water simultaneously mixes, and as second level blank sample, then, 0.1mL second level blank sample is moved to three Zhi Yi respectively with sterilizing liquid-transfering gun In device special sterile test tube, as skip test Duplicate Samples；The ATP that 0.1mL is successively added dropwise into three Duplicate Samples extracts reagent, Instill the ATP fluorescent reagent of 0.1mL after mixing again, 3000r/min shakes test tube 5s, immediately with ATP fluorescent spectrophotometer measuring its Relative intensity of fluorescence value I_AAnd record and (ensure that each link operating time is consistent, avoid cross contamination), when each Duplicate Samples measure Between be no more than 15s, using the arithmetic mean of instantaneous value of three skip test Duplicate Samples relative intensity of fluorescence values as instrument and reagent set sheet Floors (or calibrating background according to instrument operation instruction)；

(3) ATP standard solution relative intensity of fluorescence value I_AMeasurement: from low to high according to concentration, to various concentration ATP standard The ATP that 0.1mL is added dropwise in three test Duplicate Samples of solution respectively extracts reagent, instills the ATP fluorescence examination of 0.1mL after mixing again Agent；3000r/min shakes test tube 5s, uses its relative intensity of fluorescence value of ATP fluorescent spectrophotometer measuring I immediately_AAnd it records and (ensures each The link operating time is consistent, avoids cross contamination), each Duplicate Samples minute is no more than 15s, molten with each concentration ATP standard The arithmetic mean of instantaneous value of three Duplicate Samples relative intensity of fluorescence values of liquid is its I_AMeasured value；

(4) ATP log concentration value lgC_ARelative intensity of fluorescence logarithm lgI_AStandard curve is established: with various concentration ATP standard The relative intensity of fluorescence logarithm lgI of solution_AAs abscissa, with its ATP log concentration value lgC_AFor ordinate mapping；To two Mathematical relationship between person carries out calibration curve, draws the lgC of two respective concentrations_A-lgI_AStandard curve；And application minimum two Multiply the linear equation Y=a that fitting process is derived from curve_A0X+b_A0(high concentration), Y=a_AX+b_A(low concentration) and related coefficient R_A0 ²(high concentration), R_A ²(low concentration), works as R_A0 ²And R_A ²>=0.98, when confidence level >=0.95, the measurement done according to this patent Effectively；

(5) it is inoculated with bacterium solution viable bacteria ATP concentration C_ACalibration: with Cha Shi culture solution to viable bacteria content C_BIt is 1.0 × 10⁸CFU/mL~5.0 ×10⁸The mould mixing spore liquid of CFU/mL carries out continuous 10 times of gradient dilutions, measures its relative intensity of fluorescence value I_A；According to height Concentration standard curve equation Y=a_A0X+b_A0It calculates and demarcates corresponding viable bacteria ATP concentration C_A, adjust, obtain through Cha Shi culture solution To C_ARange is 5.0 × 10^-7Mol/L~9.0 × 10^-7The inoculation bacterium solution of mol/L measures and records 0.3mL inoculation bacterium solution warp Relative intensity of fluorescence value I after the dilution of 4.6mL eluent in 1min_A0。

7. the detection method of antibacterial rubber fungicidal properties according to claim 1, which is characterized in that the sample connects Kind, culture and elution recycling, carry out in the steps below:

(1) inoculated and cultured: antibacterial rubber sample carries out antibacterial tests after completing air conditioning in 1h, with sterilizing liquid-transfering gun to each 0.3mL inoculation bacterium solution is added dropwise (with lgC in group control sample and antimicrobial sample surface to be measured respectively_A-lgI_ACalibration curve spore liquid It is derived from same branch mixing spore liquid test tube, 0 DEG C of ± 1 DEG C of preservation, 2h is interior to be used), it will with L stick (adhere to inoculation bacterium solution but do not hang drop) Bacterium solution is smeared uniformly, so that it is covered sample whole surface, is covered ware lid, will contact the flat of sample equipped with 6 groups of 48h with medical adhesive tape Ware sealing；(28 ± 2) DEG C, (95 ± 2) %RH cultivate 48h ± 2h；

(2) elution recycling: after 6 groups of 0h contact sample inoculation moulds, 4.6mL eluent is drawn with sterilizing liquid-transfering gun immediately and (is examined Family name's culture solution), each sample inoculation surface of repeated flushing at least 4 times in plate are sufficiently eluted and are blown repeatedly with liquid transfer gun head Mould washing lotion in plate is inhaled, until lawn again moves into washing lotion in sterile test tube after being broken up；3000r/min shakes test tube 2min, as the recovered liquid of sample to be tested (if recovered liquid adds eluent to 4.9mL), each group less than 4.9mL after mixing Sample after 48h is cultivated uses type of elution identical with 0h contact sample to recycle mould.

8. the detection method of antibacterial rubber fungicidal properties according to claim 1, which is characterized in that the reclaim liquid phase To fluorescence intensity level I_AMeasurement carries out in the steps below:

(1) instrument and reagent set relative intensity of fluorescence background value calibration: according to lgC_A-lgI_ABlank background when standard curve is established Calibration method, with the wetting agent solution of Cha Shi culture solution substitution 0.05%, determining instrument and reagent set background values；

(2) reclaim liquid phase is to fluorescence intensity level I_AMeasurement:, will with sterilizing liquid-transfering gun if background level meets instrument requirement The ATP lysate of 4.9mL recovered liquid and 0.1mL are separately added into same branch sterile test tube, and 3000r/min shakes test tube 30s；Room After temperature stands 20min, then the ATP extraction reagent of 5.0mL is added dropwise and mixes；It is stored at room temperature 10min, it will with sterilizing liquid-transfering gun The above-mentioned mixed solution of 0.1mL is moved to respectively in three instrument sterile test tubes, tests Duplicate Samples as ATP bioluminescence, to The ATP fluorescent reagent of 0.1mL is respectively added dropwise in three Duplicate Samples, 3000r/min shakes test tube 5s, uses ATP fluophotometer immediately Measure its relative intensity of fluorescence value I_AAnd record and (ensure that each link operating time is consistent, avoid cross contamination), each Duplicate Samples Minute is no more than 15s, using the arithmetic mean of instantaneous value of three ATP bioluminescences test Duplicate Samples relative intensity of fluorescence value as to The I of sample recovered liquid_AMeasured value.

9. the detection method of antibacterial rubber fungicidal properties according to claim 1, which is characterized in that the recovered liquid is living Bacterium ATP concentration C_AAnd T_AIt calculates and antibiotic rate R and antibacterial activity value A is calculated, carry out in the steps below:

(1) recovered liquid viable bacteria ATP concentration C_AAnd T_AIt calculates

According to ATP low concentration standard curve lgC_A-lgI_ALinear equation Y=a_AX+b_A, calculate each group sample through 0h contact and The viable bacteria ATP concentration C of recovered liquid after 48h culture_AAnd T_A, relevant calculation is shown in formula (1)~(12):

In formula:

C_A0And C_At- 3 groups of 0h contact and after 48h is cultivated control sample recycling viable bacteria average ATP concentration, unit is mole every It rises (mol/L)；

C_A0ijAnd C_Atij- every 0h contact and after 48h is cultivated control sample recycling viable bacteria ATP concentration, unit is mole every It rises (mol/L)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

With- every 0h contact and after 48h is cultivated control sample recovered liquid relative intensity of fluorescence measured value, RLU； Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

T_A0And T_At- 3 groups of 0h contact and after 48h is cultivated antimicrobial sample recycling viable bacteria average ATP concentration, unit is mole every It rises (mol/L)；

T_A0ijAnd T_Atij- every 0h contact and after 48h is cultivated antimicrobial sample recycling viable bacteria ATP concentration, unit is mole every It rises (mol/L)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

With- every 0h contact and after 48h is cultivated antimicrobial sample recovered liquid relative intensity of fluorescence measured value, RLU； Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

(2) condition for validity is tested

If the recovered liquid and 0.3mL inoculation bacterium solution of every 0h contact control sample are after the dilution of 4.6mL eluent in 1min Relative intensity of fluorescence measured value is close, i.e.,Every control sample recovered liquid relative intensity of fluorescence survey after 48h is cultivated The logarithm of definite valueThat is C_Atij≥0.1×C_A0ij, then when 3 groups of 0h contact control sample reclaim liquid phases are to glimmering Luminous intensity measured valueGroup in and when between-group variation coefficient CV≤10% (involved calculation formula is shown in this patent measurement of correlation Uncertainty requirement), the measurement carried out according to this patent method is effective；

(3) mould increasing value F_ij、G_ijIt calculates

Every control sample and antimicrobial sample are after 48h is cultivated, mould increasing value F_ij、G_ijIt is counted respectively according to formula (13), (14) It calculates:

In formula:

F_ijAnd G_ijThe mould increasing value of-every control sample and antimicrobial sample after 48h is cultivated, sample group i=1,2,3；Often Group sample number into spectrum j=1,2,3,4,5；

C_A0ijAnd C_AtijThe average ATP concentration that-every 0h is contacted and control sample recycles viable bacteria after 48h is cultivated, unit are to rub You are every liter (mol/L)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

With- every 0h contact and after 48h is cultivated control sample recovered liquid relative intensity of fluorescence measured value, RLU； Group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

T_A0ijAnd T_AtijThe average ATP concentration values that-every 0h is contacted and antimicrobial sample recycles viable bacteria after 48h is cultivated, unit It is mole every liter (mol/L)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

a_A- low concentration standard curve lgC_A-lgI_ASlope；

(4) antibiotic rate R is calculated

It tests under condition for validity, the antibiotic rate R of every antibacterial rubber sample_ij, every group and every batch of sample antibiotic rate R_iDistinguish with R It is calculated according to formula (15), (16), (17):

In formula:

R_ijThe antibiotic rate of-every antibacterial rubber sample, %；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4, 5；

R-every batch of antibacterial rubber sample antibiotic rate, %；

C_AtijAnd T_Atij- every antimicrobial sample and control sample recycle the average ATP concentration of viable bacteria after 48h is cultivated, and unit is Mole every liter (mol/L)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

With- every antimicrobial sample and control sample are after 48h is cultivated, the relative intensity of fluorescence measured value of recovered liquid, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

a_A- low concentration standard curve lgC_A-lgI_ASlope；

(5) antibacterial activity value A is calculated

It tests under condition for validity, the antibacterial activity value A of every antibacterial rubber sample_ij, every group and every batch of sample antibacterial activity value A_iIt is calculated respectively according to formula (18), (19), (20) with A:

In formula:

A_ijThe antibacterial activity value of-every antibacterial rubber sample；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4, 5；

A-every batch of antibacterial rubber sample antibacterial activity value；

a_A- low concentration standard curve lgC_A-lgI_ASlope；

(6) data revision of the convention requirement: calibration mycotic spore liquid viable bacteria content C_BAnd ATP concentration of standard solution and inoculation bacterium solution, sample Recovered liquid viable bacteria ATP concentration C_AWhen, with reference to the data revision of the convention regulation in GB 4789.2-2016 in relation to clump count, work as C_BIt is less than 100CFU/mL or C_AWhen less than 100mol/L, " rounding up " round numbers；Work as C_BNot less than 100CFU/mL or C_AIt is not less than When 100mol/L, take preceding 2 bit digital after the 3rd bit digital " rounding up ", behind with 0 replace digit；Can also with 10 index shape Formula indicates that " rounding up " is returned afterwards using two effective digitals, control sample and antimicrobial sample after 0h is contacted and 48h is cultivated Receive the relative intensity of fluorescence measured value round numbers of liquid, antibiotic rate R_ij、R_i, R calculated result take three effective digitals；Mould increasing value F_ij、G_ijWith antibacterial activity value A_ij、A_i, A calculated result take two effective digitals；

(7) uncertainty of measurement: this patent method, which mainly passes through, calculates control sample and antimicrobial sample through 0h contact and 48h culture Afterwards, reclaim liquid phase in the group of fluorescent strength determining value and between-group variation coefficient CV=σ ÷ μ × 100% (μ, σ and CV count Calculate result and remain into 2 significant digits), judge the reproduction that ATP fluorimetric assay for biological materials method is applied to the test of rubber fungicidal properties Property；In regulation group and between-group variation coefficient CV≤10%；

5 control samples, 5 antimicrobial samples and 5 control samples, the 5 antibacterial samples after 48h is cultivated that every group of 0h is contacted Product, arithmetic mean of instantaneous value of the reclaim liquid phase to fluorescent strength determining valueAnd Respectively according to formula (21), (22) (sample group i=1,2,3 is calculated；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k=1,2,3； In formulaWithRespectively every group of control sample and antimicrobial sample are after 0h is contacted and 48h is cultivated, respectively The relative intensity of fluorescence measured value of Duplicate Samples):

15 control samples, 15 antimicrobial samples and 15 control samples after 48h is cultivated of 3 groups of 0h contact, 15 it is anti- Bacterium sample, arithmetic mean of instantaneous value of the reclaim liquid phase to fluorescent strength determining valueAndRespectively according to formula (23), (24) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k= 1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are contacted through 0h and 48h is trained After supporting, the relative intensity of fluorescence measured value of each Duplicate Samples):

5 control samples, 5 antimicrobial samples and 5 control samples, the 5 antibacterial samples after 48h is cultivated that every group of 0h is contacted Product, the standard deviation of the relative intensity of fluorescence measured value of recovered liquidAndRespectively according to formula (25) and (26) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k= 1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are contacted through 0h and 48h is trained After supporting, the relative intensity of fluorescence measured value of each Duplicate Samples):

15 control samples, 15 antimicrobial samples and 15 control samples after 48h is cultivated of 3 groups of 0h contact, 15 it is anti- Bacterium sample, the standard deviation of the relative intensity of fluorescence measured value of recovered liquidAndRespectively according to formula (27) and (28) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k= 1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are contacted through 0h and 48h is trained After supporting, the relative intensity of fluorescence measured value of each Duplicate Samples):

10. the detection method of antibacterial rubber fungicidal properties according to claim 1, which is characterized in that the result is commented Valence carries out in the steps below:

Such as using antibiotic rate R as correlated performance evaluation index: working as R_ij/R_iWhen/R < 80%, sample is without mildew-proof function；When 80% ≤R_ij/R_iWhen/R < 90%, sample has mildew-proof function；As 90%≤R_ij/R_iWhen/R < 99%, sample mildew-proof function is moderate； As 99%≤R_ij/R_iWhen/R < 99.9%, sample mildew-proof function is stronger；Work as R_ij/R_iWhen/R >=99.9%, sample mildew-proof function pole By force；

Such as using antibacterial activity value A as correlated performance evaluation index: working as A_ij/A_iWhen/A < 0.5, sample is without mildew-proof function；When 0.5≤A_ij/A_iWhen/A < 1.0, sample has mildew-proof function；As 1.0≤A_ij/A_iWhen/A < 2.0, sample mildew-proof function is moderate； As 2.0≤A_ij/A_iWhen/A < 3.0, sample mildew-proof function is stronger；Work as A_ij/A_iWhen/A >=3.0, sample mildew-proof function is extremely strong；

(2) as the antibiotic rate R of certain group (part) antibacterial rubber sample_i(R_ij) or antibacterial activity value A_i(A_ij) and other two groups (four) When the fungicidal properties of sample is compared to a levels are at least differed, one group of (part) sample is extracted again and repeats to test；Calculate its warp ATP bioluminescence lgC_A─lgI_AThe antibiotic rate or antibacterial activity value that calibration curve method measures, if two groups of front and back antibacterial rubber sample Product fungicidal properties level is identical, then abandons it；Take other two groups (four) remaining sample antibiotic rate R_i(R_ij) or antibacterial activity value A_i (A_ij) evaluation result of the arithmetic mean of instantaneous value as batch (group) the antibacterial rubber sample fungicidal properties.