CN110283878A - ATP bioluminescence lgCA-lgIAThe method for marking bent method detection anti-bacteria stainless steel bacteria resistance energy - Google Patents

ATP bioluminescence lgCA-lgIAThe method for marking bent method detection anti-bacteria stainless steel bacteria resistance energy Download PDF

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CN110283878A
CN110283878A CN201910200149.7A CN201910200149A CN110283878A CN 110283878 A CN110283878 A CN 110283878A CN 201910200149 A CN201910200149 A CN 201910200149A CN 110283878 A CN110283878 A CN 110283878A
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sample
atp
group
bacteria
value
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李文杰
曹彦忠
崔宗岩
李学民
杨志伟
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INSPECTION AND QUARANTINE TECHNOLOGY CENTER OF QINHUANGDAO ENTRY-EXIT INSPECTION AND QUARANTINE BUREAU
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INSPECTION AND QUARANTINE TECHNOLOGY CENTER OF QINHUANGDAO ENTRY-EXIT INSPECTION AND QUARANTINE BUREAU
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2304/00—Chemical means of detecting microorganisms
    • C12Q2304/60—Chemiluminescent detection using ATP-luciferin-luciferase system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/245—Escherichia (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/25—Shigella (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/255—Salmonella (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/305—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F)
    • G01N2333/31—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F) from Staphylococcus (G)

Abstract

The present invention relates to a kind of method of anti-bacteria stainless steel antibacterium performance detection, detecting step includes: sample preparation and pretreatment；Lectotype selection and reagent, culture medium are prepared；Culture presevation, activation and bacteria suspension preparation；ATP log concentration value lgCARelative intensity of fluorescence logarithm lgIAMark Qu Jianli and inoculation bacterium solution viable bacteria ATP concentration CACalibration；Sample inoculation, culture and elution recycling；Recovered liquid IAMeasurement and its viable bacteria ATP concentration CAAnd TAIt calculates；Antibiotic rate R or antibacterial activity value A is calculated；Evaluation of result；Its special feature is that: accurate quantitative test can be carried out to the stainless steel bacteria resistance with R or A characterization using ATP fluophotometer.Present invention provide that control sample and antimicrobial sample after elution recycling contact 0h and culture for 24 hours, measure recovered liquid IAAnd with lgIACharacterization and calculating R or A；Evaluation of result foundation is provided.The ATP bioluminescence lgC for the anti-bacteria stainless steel antibacterium performance detection that the present invention researches and developsA‑lgIACalibration curve method will be promoted with the advanced detection technique support product quality of science.

Description

ATP bioluminescence lgCA-lgIAMark bent method detection anti-bacteria stainless steel bacteria resistance energy Method
Technical field
The present invention relates to the antibacterium performance test methods of anti-bacteria stainless steel, specifically a kind of application ATP fluophotometer The ATP biology that can be carried out accurate quantitative detection to the anti-bacteria stainless steel bacteria resistance with antibiotic rate R or antibacterial activity value A characterization is glimmering Light lgCA-lgIACalibration curve method belongs to stainless steel antibacterial functions detection technique field.
Background technique
Stainless steel is closely bound up with human lives, is widely used in all various aspects such as industry and the people's livelihood；In recent years, by science and technology Progressive and consumption demand traction, as new structure/function integration material coating type anti-bacteria stainless steel, surface modified version Anti-bacteria stainless steel, composite bactericidal stainless steel, alloy-type anti-bacteria stainless steel come into being.It is predicted according to authoritative institution, 70% or more The stainless steel market in the fields such as Medical Devices, kitchen bathroom, public utility can be substituted by anti-bacteria stainless steel；Along with antimicrobial technology In the application of stainless steel industry and universal, relevant standardisation requires gradually to bring into schedule.
Currently, anti-biotic material performance detection technical system is mainly for bacterium and fungi, various countries' bacteria resistance energy both at home and abroad Test method principle is mostly based on the colony counting method being separately cultured to bacterium, first is that the pad pasting training that Japanese Industrial Standards propose Support and impregnate quantitative test method；Two are derived from the antibacterial around-France of U.S. textile industry；Third is that international oscillation flask method；Four It is derived from the dropping method of U.S. textile enterprise；Fifth is that Japanese enterprises are directed to the coverslip method of photocatalyst-type anti-biotic product research and development.It cuts To currently, China has issued and implemented three anti-bacteria stainless steel standards: YB/T 4171-2008 " copper-bearing antibacterial stainless steel ", GB/T 24170.1-2009 " stainless steel with antibacterial surface part 1: electrochemical process ", SN/T 2399-2010 " antibacterial metallic materials evaluation Method ".But there are following common problems in technology contents and practical application for existing detection method: first is that involved experimental strain mistake It is few, it is difficult to meet the research and development of new product and Quality Control demand that enterprise grows with each passing hour；Second is that experimentation is cumbersome, relevant operation is by laboratory technician Professional experiences influence, and test error is big, lack comparativity；Third is that test period between 48 hours~72 hours, time and warp It helps at high cost.In recent years, increasingly mature in the development of international Bacteria Detection technical field ATP fluorescence analysis, with traditional Plating Correlation compared to ATP testing result is 98%, and accuracy is high and can realize quick detection；Developed country uses the method In HACCP.Domestic ATP correlative study is started late, and is drawn by application demand, and existing ATP fluorescence detector has become China and defends Raw department specified health supervision and the relevant special inspecting equipment of food safety.Current foreign countries' anti-biotic material performance detection technology It studies to quantification, rapid and summary trend development, focuses on testing result accuracy and comparativity；ATP fluorescence is used for reference Analysis principle formulates ISO 20743:2007-2013 " Textiles-Determination of antibacterial Activity of textile products (the antibacterium performance measurement of textile-antibiotic finish textile) " and ISO 13629-1:2012《Textiles-Determination of antifungal activity of textile Products.Part1Luminescence (measurement of textile-antibiotic finish textile fungicidal properties) ", it is specified that with sample The fluorimetry of the anti-thin/mould performance of ATP changes of contents characterization after inoculation, but it is only applicable to water imbibition and control sample Carefully/mould increasing value > 0 textile material or poromerics, in key technologies sides such as viable bacteria way of recycling, test conditions for validity Face is not suitable for having the materials such as stainless steel, the glass of unwetted property hard surface and control sample bacterium increasing value < 0；Simultaneously not Uncertainty of measurement assessment is provided.In addition, the standard method dependent antimicrobial performance characterization parameter is more single, it is living to only relate to antibacterial Property 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 anti-bacteria stainless steel Performance Testing Technology.The art of this patent highway route design connects Rail is international, and detection method belongs to the whole world and initiates, 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 anti-bacteria stainless steel bacteria resistance can be carried out the ATP bioluminescence lgC of detectionA-lgIACalibration curve method can solve Certainly anti-bacteria stainless steel or even other field anti-biotic material and product bacteria resistance can accurate quantitative test problems.
In order to solve the above technical problems, the technical solution adopted by the present invention is that:
A kind of detection method of anti-bacteria stainless steel bacteria resistance energy, comprising: (1) sample preparation and pretreatment；(2) equipment is selected Type and reagent, culture medium are prepared；(3) culture presevation, activation and bacteria suspension preparation；(4) ATP log concentration value lgCAIt is relatively glimmering Luminous intensity logarithm lgIAStandard curve is established and inoculation bacterium solution viable bacteria ATP concentration CACalibration；(5) sample inoculation, culture and elution Recycling；(6) reclaim liquid phase is to fluorescence intensity level IAMeasurement；(7) recovered liquid viable bacteria ATP concentration CAAnd TACalculate and antibiotic rate R and Antibacterial activity value A is calculated；(8) evaluation of result；It is characterized in that, using ATP fluophotometer to antibiotic rate R or antibacterial activity The anti-bacteria stainless steel bacteria resistance of value A characterization can be carried out the ATP bioluminescence lgC of accurate quantitative testA-lgIACalibration curve method, It is specific:
In reclaim liquid phase to fluorescence intensity level IAIn measurement:
Quantity, size and the pre-treatment requirement for specifying control sample and antimicrobial sample, testing standard bacterial strain is passed on, After activation, takes continuous switching 2 times Fresh bacterial cultures to prepare bacteria suspension and primary dcreening operation is carried out to its bacterium number.It is trained with test strain Nutrient 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 × 10-9mol/L、 7.0×10-8mol/L、7.0×10-7Mol/L is (if instrument upper limit of detection is unable to reach 10-7Mol/L magnitude or high standard Curve it is linear poor, its ATP concentration series can be changed to 7.0 × 10-10mol/L、7.0×10-9mol/L、7.0×10- 8) and 4.2 × 10 mol/L-11mol/L、4.2×10-10mol/L、4.2×10-9Mol/L, and measure its relative intensity of fluorescence value IA；Draw the lgC of two respective concentrationsA-lgIAStandard curve is derived from fitting equation Y=aA0X+bA0(high concentration), Y =aAX+bA(low concentration) and linearly dependent coefficient RA0 2(high concentration), RA 2(low concentration).It is 6.0 × 10 to bacteria total amount8CFU/ After the bacteria suspension of mL carries out 10 times of gradient dilutions, its relative intensity of fluorescence value I is measuredA, according to high standard fitting equation Y =aA0X+bA0Calculate corresponding viable bacteria ATP concentration CA；It is adjusted to obtain CARange is 5.0 × 10-8Mol/L~9.0 × 10- 8The inoculation bacterium solution of mol/L.Then, 0.3mL bacterium solution is added dropwise respectively to each group control sample and antimicrobial sample surface to be measured, immediately Elution recycling is carried out to 6 groups of 0h contact samples with 4.7mL eluent；Using the relatively glimmering of ATP fluorescent spectrophotometer measuring recovered liquid Light intensity value IAC0ij、IAT0ij, according to low concentration calibration curve equation formula Y=aAX+bACalculate the ATP concentration C of its viable bacteriaAOijWith TAOij.Meanwhile contacting sample for 24 hours for 6 groups and being sealed in sterilized petri dishes, (37 ± 1) DEG C, (90 ± 2) %RH culture ± 2h for 24 hours Afterwards；Remained on surface bacterium is recycled using mode identical with 0h contact sample and measures the relative intensity of fluorescence value of its recovered liquid IACtij、IATtij, calculate corresponding viable bacteria ATP concentration CAtijAnd TAtij；
In antibiotic rate R and antibacterial activity value A is calculated:
According to ATP low concentration standard curve lgCA-lgIALinear equation Y=aAX+bA, after being contacted with 0h and being cultivated for 24 hours Every control sample and antimicrobial sample reclaim liquid phase are to fluorescent strength determining valueWithBased on number According to；In the case where testing condition for validity, its bacterium increasing value F after cultivating for 24 hours is calculatedij、GijAnd antibiotic rate RijAnd antibacterial activity Value Aij；To the R of every group of sampleijAnd AijArithmetic mean of instantaneous value is taken to obtain corresponding RiAnd Ai；The antibacterial of every batch of anti-bacteria stainless steel sample Rate R and antibacterial activity value A is its 3 groups of sample RiAnd AiArithmetic mean of instantaneous value；And the clear related data revision of the convention and uncertainty of measurement It is required that；
In evaluation of result:
With reference to health industry common practice and the requirement of dependent antimicrobial product standard, determine that antibacterium grading performance determines mark It is quasi-；As the antibiotic rate R of certain group (part) anti-bacteria stainless steel samplei(Rij) or antibacterial activity value Ai(Aij) and other two groups (four) samples When the bacteria resistance of product can be compared to a levels be at least differed, one group of (part) sample is extracted again and repeats to test；Calculate its warp ATP bioluminescence lgCA─lgIAThe antibiotic rate or antibacterial activity value that calibration curve method measures.If two groups of front and back (part) antibacterial is not Steel sample antibacterium performance level of becoming rusty is identical, then abandons it；Take other two groups (four) remaining sample antibiotic rate Ri(Rij) or antibacterial Activity value Ai(Aij) arithmetic mean of instantaneous value as batch (group) anti-bacteria stainless steel sample bacteria resistance can evaluation result.
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, it is dense to have reached viable bacteria ATP The modernization of degree measurement and stainless steel antibacterium performance detection；Can effectively reduce human factor in experimentation influences, and avoids The generally existing large error of traditional plate culture；It can ensure the quantification of testing result, while increase substantially detection The accuracy of data；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 lgCA- relative intensity of fluorescence logarithm lgIAStandard curve；Construct anti-bacteria stainless steel bacteria resistance energy ATP bioluminescence The mathematical model of real-time quantitative analysis method.Meanwhile country variant consumer perceptions habit is taken into account, take antibiotic rate R and antibacterial Activity value A improves the science and versatility of detection method as correlated performance evaluation index.
(3) innovative: compared with the conventional method that existing operation is numerous, error is big, the period is long, 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 stainless The precision of steel antibacterium performance test results 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 lgCA、lgIAStandard curve quantitative analysis method based on linear relationship, innovation is simultaneously Enrich bacteria suspension viable bacteria concentration characterization and measuring method, specify control sample, standard liquid concentration, determination step, calculation formula, The technology contents such as uncertainty；The pioneering recovered liquid viable bacteria relative intensity of fluorescence value I directly measured with instrumentAIt evaluates as a result Form calculates and determines antibiotic rate R or antibacterial activity value A, and by group and a between-group variation coefficient CV to investigate measurement not true Fixed degree, technically has centainly perspective.
(5) operability: ATP fluophotometer is cheap, it is easy to operate, be widely used, this patent establish with sample Method is simple for the stainless steel bacteria resistance energy ATP fluorometric investigation that the variation of inoculation viable bacteria ATP concentration is attached most importance to before and after product culture, Description of Related Art is clear and specific, should be readily appreciated that and grasps；Have stronger operability in implementation process, is suitable for not With professional standards Experiment on Microbiology personnel, it 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 stainless steel bacteria resistance energy detection technique realization generalization, simultaneously Reference can be provided to the product scopes bacteria resistance energy detection technique research such as ceramics, glass.
Further, preferred embodiment of the invention is:
The sample preparation and pretreatment carries out in the steps below:
(1) control sample: in addition to not having antibacterial coating, control sample classification, metallic matrix, technique and appearance, ruler Very little, quantity etc. is identical with anti-bacteria stainless steel sample to be measured；Each strain test uses 6 groups of samples；Wherein 0h contact and for 24 hours Culture experiment respectively uses 3 groups, every group of 5 samples；
(2) antimicrobial sample: after the processing of the electrochemical methods such as electroplated, chemical plating, antibacterial coating is formed not on surface Become rusty Steel material and product；Sample cover surface is uniform and has certain adhesive strength, no any damage for reaching substrate metal； Having a size of (50 ± 2) mm × (50 ± 2) mm, thickness is not more than 5mm.Antimicrobial sample quantity is identical as control sample, and every group to be measured Sample selects one group of control sample as object of reference and effectively identifies；
(3) sample pre-treatments: impregnating 5min in 75% ethanol solution for control sample and antimicrobial sample before experiment, and With the abundant cleaning sample of sterile water to remove ethyl alcohol on superclean bench；Sample surface to be measured is put into upwards after natural drying It is 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 meter, matched reagent box, Special test tube etc. is spent, wherein ATP fluophotometer wavelength range 300nm~650nm, ATP Concentration Testing range 10-13Mol/L~10-7mol/L；(20~50) DEG C ± 1 DEG C, (50~95) %RH The constant temperature and humidity incubator of ± 2%RH；The pressure steam sterilizer of (121 ± 2) DEG C, (103 ± 5) kPa；- 20 DEG C~-70 DEG C Low temperature refrigerator；0 DEG C~8 DEG C of refrigerating box；The electronic balance of sensibility reciprocal 0.001g；The ultrasonic wave of frequency range (30~50) kHz is clear Wash device；The vortex oscillator of the range of speeds (500~3000) r/min；The pH meter of precision ± 0.1 (25 DEG C)；Electric furnace；
(3) material utensil: the sterile measuring pipette of 1mL, 10mL；0.05mL, 0.1mL, 0.2mL, 1mL, 5mL, 10mL (meter Error is measured less than single track changeable fluid liquid-transfering gun and sterile liquid transfer gun head 1%)；The sterile conical flask of capacity 250mL, 500mL； The sterile petri dish of diameter 90mm；Maxwell bacterium standard opacity tube and mating test tube；Sterile test tube；Diameter connects no more than 4mm's Kind ring；L stick；Alcolhol burner；Sterilize tweezers；Medical adhesive tape；Cotton ball soaked in alcohol (75%)；The ruler of scale division value 1mm；Thermometer；The stopwatch of precision 0.01s；A6 white copy paper；0.7mm core black gel ink pen；
(4) reagent: 75% ethanol solution；(121 DEG C of high pressure sterilization 15min, 5 DEG C~10 DEG C are deposited 85% physiological saline Put 30d)；
(5) medium/liquid (commercially available medium/liquid can be used): through 121 DEG C of high pressure sterilizations after matched medium/liquid packing 15min, 2 DEG C~8 DEG C storage 30d；
Nutrient agar (NA): 5g beef extract, 10g peptone, 5g sodium chloride, 15g agar are dissolved by heating in 1000mL water In, adjust pH to 7.0~7.2；
Nutrient broth (NB): 3g beef extract, 10g peptone, 5g sodium chloride are dissolved by heating in 1000mL water, pH is adjusted To 7.0~7.2；
Salmonella preservation is with brain heart oxoid meat soup (BHI): by 10g peptone, 5g sodium chloride, 2.5g phosphoric acid hydrogen two Sodium, 2g glucose dissolve by heating in 500mL cattle heart leachate, adjust pH to 7.4 ± 0.2；
The culture solution of escherichia coli and Shigella/staphylococcus aureus and salmonella: by 1:500/1:100 Nutrient broth (NB) mixed with 85% sterile saline solution, adjust pH to 7.0~7.2；
(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 standard reagent stoste: by 60.5mg trinosin (C10H14O13P3Na2·3H2O) it is dissolved in 100mL In water, being configured to concentration is 1 × 10-3The solution of mol/L, -20 DEG C of freezings are sealed 6 months；
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 nutrient broth can be down to 10 in 15min by liquid-13Mol/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；
ATP fluorescent reagent: by 16mg luciferase (EC:1.13.12.7), the D- fluorescein of 12.6mg, 56mg cow's serum Albumen is dissolved in the ATP fluorescent reagent buffer solution of 30mL, and 15min is stored at room temperature after mixing, is used in 3h；
Culture presevation, activation and the bacteria suspension preparation, carries out in the steps below:
(1) test strain: escherichia coli ATCC 8739；Staphylococcus aureus ATCC 6538；Salmonella ATCC 14028；Shigella CMCC 51105 (or provided by national corresponding culture presevation administrative center and can be traced to the source other Strain)；
(2) culture presevation: opening freeze-drying lactobacillus pipe with sterile working, and adequate nutrition meat soup (sramana is added with capillary syring Salmonella BHI broth), pressure-vaccum makes strain melt dispersion for several times.Then, a little test strain suspension is instilled and 5mL is housed In the test tube of~10mL nutrient broth, (37 ± 1) DEG C culture for 24 hours~48h；As slant preservation bacterium, (5 ± 1) DEG C storage (does not surpass Spend 1 month), inoculation times were no more than for 14 generations；
(3) actication of culture: slant preservation bacterium is transferred nutrient agar slant medium, and (37 ± 1) DEG C culture 18h~for 24 hours； Switching 1 time daily, continuous switching are no more than 15d.Test is trained using the 3rd generation~the 14th generation and in the interior Fresh bacterial transferred for 24 hours Support object；
(4) prepared by bacteria suspension: scraping a small amount of Fresh bacterial from strain activation and culture base with oese, test strain is added Bacteria suspension is made in culture solution；1000r/min shakes test tube 1min or with 30cm amplitude of oscillation shaking test tube 80 times, it is ensured that bacterial suspension is equal It is even.Appropriate bacteria suspension is moved in sterile test tube matched with Maxwell standard opacity tube, it is white in A6 with 0.7mm core black gel ink pen The parallel lines that 3 length are 10cm are drawn on color copy paper；Estimate test tube and standard opacity tube (nominal concentration 6.0 × 108CFU/ ML culture solution is added dropwise until the two turbidity is identical in differences in turbidity)；
The ATP log concentration value lgCARelative intensity of fluorescence logarithm lgIAStandard curve is established and inoculation bacterium solution is living Bacterium ATP concentration CACalibration carries out in the steps below:
(1) standard serial solution is determining and prepared by ATP bioluminescence test specimens: using test strain culture 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 × 10-9mol/L、7.0×10-8mol/L、 7.0×10-7Mol/L is (if instrument upper limit of detection is unable to reach 10-7Mol/L magnitude or high standard curve it is linear compared with Its ATP concentration series can be changed to 7.0 × 10 by difference-10mol/L、7.0×10-9mol/L、7.0×10-8) and 4.2 × 10 mol/L-11mol/L、4.2×10-10mol/L、4.2×10-9Mol/L is simultaneously mixed.Then, with sterilizing liquid-transfering gun by the ATP standard of 0.1mL Serial solution is moved to respectively in three sterile test tubes, and phosphate buffer of the 0.9mL containing 0.037% sucrose is successively added dropwise and mixes It is even；The mixed solution of 0.1mL various concentration is moved to respectively in three instrument sterile test tubes again, as ATP bioluminescence Test Duplicate Samples；
(2) relative intensity of fluorescence value IAMeasurement: according to relative intensity of fluorescence value I in this patentAMeasuring method, with test organisms After kind culture solution is as blank sample liquid validation instrument and reagent set background；According to the sequence of concentration from low to high, to various concentration The ATP that 0.1mL is added dropwise in three Duplicate Samples of ATP standard solution respectively extracts reagent, and the ATP fluorescence examination of 0.1mL is instilled after mixing Agent；It mixes again, with its relative intensity of fluorescence value of ATP fluorescent spectrophotometer measuring and records immediately and (ensure each link operating time Unanimously, cross contamination is avoided).Each Duplicate Samples minute is no more than 15s, with each three Duplicate Samples of concentration ATP standard solution The arithmetic mean of instantaneous value of relative intensity of fluorescence value is its IAMeasured value；
(3) ATP log concentration value lgCARelative intensity of fluorescence logarithm lgIAStandard curve is established: with ATP standard series The relative intensity of fluorescence logarithm lgI of solutionAAs abscissa, with its corresponding ATP log concentration value lgCAFor ordinate work Figure；Calibration curve is carried out to mathematical relationship between the two, draws the lgC of two respective concentrationsA-lgIAStandard curve；And it applies Least square fitting method is derived from the linear equation Y=a of curveA0X+bA0(high concentration), Y=aAX+bA(low concentration) and phase Close coefficients RA0 2(high concentration), RA 2(low concentration).Work as RA0 2And RA 2>=0.98, it when confidence level >=0.95, is done according to this patent Measurement it is effective；
(4) it is inoculated with the viable bacteria ATP concentration C of bacterium solutionACalibration: according to relative intensity of fluorescence value I in this patentAMeasuring method is right Bacteria total amount is 6.0 × 10 after the turbidimetry primary dcreening operation of Maxwell8The relative fluorescence of 10 times of gradient dilution liquid of bacteria suspension of CFU/mL is strong Angle value IAIt is measured；According to high standard fitting equation Y=aA0X+bA0It calculates and demarcates its viable bacteria ATP concentration CA.Through The adjustment of test strain culture solution, obtains CARange is 5.0 × 10-8Mol/L~9.0 × 10-8The inoculation bacterium solution of mol/L, measurement is simultaneously Record relative intensity of fluorescence value I of the 0.3mL inoculation bacterium solution after the dilution of 4.7mL eluent in 1minA0；
Sample inoculation, culture and the elution recycling, carries out in the steps below:
(1) sample inoculation culture: 0.3mL inoculation bacterium solution is added dropwise respectively in each group control sample and is resisted with sterilizing liquid-transfering gun (with curve lgC on bacterium sample surface to be measuredA-lgIACalibration is derived from same branch test strain stoste test tube with bacterium solution, and 2 DEG C ± 0.2 DEG C save, use in 2h), bacterium solution is smeared uniformly with L stick (attachment inoculation bacterium solution but do not hang drop), makes its covering sample whole table Face.Ware lid is covered, is sealed the plate for contacting sample for 24 hours equipped with 6 groups with medical adhesive tape；(37 ± 1) DEG C, (90 ± 2) %RH training Support ± 2h for 24 hours；
(2) elution recycling: (0.2% sterile surfaces activity can be added using 4.7mL test strain culture solution as eluent Agent), after 6 groups of 0h contact sample inoculation bacteriums, sufficiently eluted using solid or liquid swab method immediately；6 groups cultivate for 24 hours Sample uses type of elution identical with 0h contact sample to recycle bacterium.Solid swab method is to be soaked with saturation eluent with head Aseptic cotton carrier is uniformly embrocated 10 times in each sample surfaces back and forth anyhow respectively, and rotates with it cotton swab；Cut off experimenter's hand Cotton swab is placed in the sterile test tube equipped with remaining eluent, as recovered liquid (if recovered liquid after mixing by the swab stick of contact portion Less than 5mL, eluent is added to 5mL).Liquid swab method draws 4.7mL eluent with sterilizing liquid-transfering gun, in plate repeatedly It rinses each sample surfaces at least 4 times, sufficiently elute and moves into washing lotion in sterile test tube, as recovered liquid (if returning after mixing Liquid is received less than 5mL, adds eluent to 5mL)；
The reclaim liquid phase is to fluorescence intensity level IAMeasurement carries out in the steps below:
(1) instrument and reagent set relative intensity of fluorescence background value calibration: use sterilizing liquid-transfering gun by 0.1mL test strain culture The ATP lysate of liquid, 0.8mL phosphate buffer solution and 0.1mL containing 0.037% sucrose is separately added into same branch sterile test tube In and mix well, then the above-mentioned mixed solution of 0.1mL is successively moved in three instrument sterile test tubes, stand 5min~ 30min, as skip test Duplicate Samples.The ATP that 0.1mL is added dropwise respectively into three Duplicate Samples extracts reagent, instills after mixing The ATP fluorescent reagent of 0.1mL；It mixes again, uses its relative intensity of fluorescence value of ATP fluorescent spectrophotometer measuring I immediatelyAAnd it records (ensuring that each link operating time is consistent, avoid cross contamination).Each Duplicate Samples minute is no more than 15s, with three blank The arithmetic mean of instantaneous value of Duplicate Samples relative intensity of fluorescence value is tested (or to use according to pertinent instruments as instrument and reagent set background values Illustrate to calibrate background values)；
(2) reclaim liquid phase is to fluorescence intensity level IAMeasurement: it if blank reagent group background level meets instrument requirement, uses The ATP lysate for phosphate buffer solution and 0.1mL of the liquid-transfering gun by 0.1mL recovered liquid, 0.8mL containing 0.037% sucrose that sterilize Be separately added into same branch sterile test tube, after mixing well by the above-mentioned mixed solution of 0.1mL successively move to three instruments without In bacterium test tube, 5min~30min is stood, as ATP fluorometric investigation Duplicate Samples.It is added dropwise 0.1mL's respectively into three Duplicate Samples ATP extracts reagent, and the ATP fluorescent reagent of 0.1mL is instilled after mixing；It mixes again, uses its phase of ATP fluorescent spectrophotometer measuring immediately To fluorescence intensity level IAAnd it records and (ensures that each link operating time is consistent, avoid cross contamination).Each Duplicate Samples minute No more than 15s, using the arithmetic mean of instantaneous value of three ATP fluorometric investigation Duplicate Samples relative intensity of fluorescence values as the I of recovered liquidAMeasurement Value (or instrument matched reagent box is used, it is required according to operation instructions, directly upper machine measures the opposite of three Duplicate Samples of recovered liquid Fluorescence intensity level IA)；
The recovered liquid viable bacteria ATP concentration CAAnd TAIt calculates and antibiotic rate R and antibacterial activity value A is calculated, by following steps It is rapid to carry out:
(1) the viable bacteria ATP concentration C of recovered liquidAAnd TAIt calculates
According to ATP low concentration standard curve lgCA-lgIALinear equation Y=aAX+bA, calculate control sample and antibacterial After sample is contacted through 0h and is cultivated for 24 hours, the viable bacteria ATP concentration C of recovered liquidAAnd TA；Relevant calculation (uses instrument matched reagent box When, the viable bacteria ATP concentration C of 1mL recovered liquid is calculated according to its practical sample volumeAAnd TA) see formula (1)~(12):
In formula:
CA0And CAtThe average ATP concentration that -3 groups of 0h are contacted and control sample recycles viable bacteria after cultivating for 24 hours, unit are to rub You are every liter (mol/L)；
CA0iAnd CAtiThe average ATP concentration that-every group 0h is contacted and control sample recycles viable bacteria after cultivating for 24 hours, unit are Mole every liter (mol/L)；Sample group i=1,2,3；
CA0ijAnd CAtijThe ATP concentration that-every 0h is contacted and control sample recycles viable bacteria after cultivating for 24 hours, 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 cultivating for 24 hours 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；
aA- low concentration standard curve lgCA-lgIASlope；bA- low concentration standard curve lgCA-lgIAIn vertical axis intercept；
TA0And TAtThe average ATP concentration that -3 groups of 0h are contacted and antimicrobial sample recycles viable bacteria after cultivating for 24 hours, unit are to rub You are every liter (mol/L)；
TA0iAnd TAtiThe average ATP concentration that-every group 0h is contacted and antimicrobial sample recycles viable bacteria after cultivating for 24 hours, unit are Mole every liter (mol/L)；Sample group i=1,2,3；
TA0ijAnd TAtijThe ATP concentration that-every 0h is contacted and antimicrobial sample recycles viable bacteria after cultivating for 24 hours, 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 cultivating for 24 hours 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 4.7mL eluent of every 0h contact control sample Interior relative intensity of fluorescence measured value is close, i.e.,Every after cultivating for 24 hours control sample recovered liquid relative fluorescence it is strong Spend the logarithm of measured valueThat is CAtij≥0.1×CA0ij.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) bacterium increasing value Fij、GijIt calculates
Every control sample and antimicrobial sample after cultivating for 24 hours, bacterium increasing value Fij、GijRespectively according to formula (13), (14) it calculates:
In formula:
FijAnd GijThe bacterium increasing value of-every control sample and antimicrobial sample after cultivating for 24 hours, sample group i=1, 2,3；Every group of sample number into spectrum j=1,2,3,4,5；
CA0ijAnd CAtijThe ATP concentration that-every 0h is contacted and control sample recycles viable bacteria after cultivating for 24 hours, 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 cultivating for 24 hours 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；
TA0ijAnd TAtijThe ATP concentration that-every 0h is contacted and antimicrobial sample recycles viable bacteria after cultivating for 24 hours, 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 cultivating for 24 hours 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；
aA- low concentration standard curve lgCA-lgIASlope；
(4) antibiotic rate R is calculated
It tests under condition for validity, the antibiotic rate R of every anti-bacteria stainless steel sampleij, every group and every batch of sample antibiotic rate Ri It is calculated respectively according to formula (15), (16), (17) with R:
In formula:
RijThe antibiotic rate of-every anti-bacteria stainless steel sample, %；Sample group i=1,2,3；Every group of sample number into spectrum j=1, 2,3,4,5；
RiThe antibiotic rate of-every group anti-bacteria stainless steel sample, %；Sample group i=1,2,3；
R-every batch of anti-bacteria stainless steel sample antibiotic rate, %；
CAtijAnd TAtij- every antimicrobial sample and control sample recycle the average ATP concentration of viable bacteria after cultivating for 24 hours, 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 cultivating for 24 hours, 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；
aA- low concentration standard curve lgCA-lgIASlope；
(5) antibacterial activity value A is calculated
It tests under condition for validity, the antibacterial activity value A of every anti-bacteria stainless steel sampleij, every group and every batch of sample antibacterial Activity value AiIt is calculated respectively according to formula (18), (19), (20) with A:
In formula:
AijThe antibacterial activity value of-every anti-bacteria stainless steel sample；Sample group i=1,2,3；Every group of sample number into spectrum j=1, 2,3,4,5；
AiThe antibacterial activity value of-every group anti-bacteria stainless steel sample, sample group i=1,2,3；
A-every batch of anti-bacteria stainless steel sample antibacterial activity value；
FijAnd GijThe bacterium increasing value of-every control sample and antimicrobial sample after cultivating for 24 hours, 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 cultivating for 24 hours 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；
With- every 0h contact and after cultivating for 24 hours 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；
aA- low concentration standard curve lgCA-lgIASlope；
(6) data revision of the convention requirement: to the viable bacteria ATP of ATP standard serial solution concentration and inoculation bacterium solution, sample recovered liquid Concentration CAWhen being demarcated, with reference to the data revision of the convention regulation in GB 4789.2-2016 in relation to clump count；Work as CALess than 100mol/ When L, " rounding up " round numbers；Work as CAWhen not less than 100mol/L, preceding 2 bit digital is taken after the 3rd bit digital " rounding up ", after Face replaces digit with 0；It can also be indicated with 10 exponential form, " rounding up " uses two effective digitals afterwards.Control sample and After antimicrobial sample is contacted through 0h and cultivated for 24 hours, reclaim liquid phase is to fluorescent strength determining value round numbers, antibiotic rate Rij、Ri, R calculate As a result three effective digitals are taken；Bacterium increasing value Fij、GijWith antibacterial activity value Aij、Ai, A calculated result take two effective digitals；
(7) uncertainty of measurement: this patent method, which mainly passes through, to be calculated control sample and antimicrobial sample and contacts through 0h and for 24 hours 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 that ATP fluorimetric assay for biological materials method, which is applied to stainless steel bacteria resistance, to be surveyed The reproducibility of examination；In regulation group and between-group variation coefficient CV≤10%.
5 control samples, 5 antimicrobial samples and 5 control samples after cultivating for 24 hours 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 are trained for 24 hours After supporting, the relative intensity of fluorescence measured value of each ATP test Duplicate Samples):
15 control samples, 15 antimicrobial samples and 15 control samples, 15 after cultivating for 24 hours 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 for 24 hours After culture, the relative intensity of fluorescence measured value of each ATP test Duplicate Samples):
5 control samples, 5 antimicrobial samples and 5 control samples after cultivating for 24 hours of every group of 0h contact, 5 it is anti- Bacterium sample, standard deviation of the reclaim liquid phase to fluorescent strength determining valueAnd Respectively 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 are trained for 24 hours After supporting, the relative intensity of fluorescence measured value of each ATP test Duplicate Samples):
15 control samples, 15 antimicrobial samples and 15 control samples, 15 after cultivating for 24 hours that 3 groups of 0h are contacted Part antimicrobial sample, standard deviation of the reclaim liquid phase to fluorescent strength determining valueAndRespectively according to public affairs 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 through 0h contact and for 24 hours After culture, the relative intensity of fluorescence measured value of each ATP test Duplicate Samples):
The evaluation of result of the anti-bacteria stainless steel sample bacteria resistance energy, 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:
If using antibiotic rate R as correlated performance evaluation index: working as Rij/Ri(staphylococcus aureus when/R < 90% With salmonella Rij/Ri/ R < 80%), sample is without antibacterial actions；As 90%≤Rij/Ri(golden yellow grape when/R < 99% 80%≤R of coccus and salmonellaij/Ri/ R < 90%), sample has antibacterial actions；As 99%≤Rij/Ri/ R < 99.9% When (90%≤R of staphylococcus aureus and salmonellaij/Ri/ R < 99%), sample antibacterial actions are stronger；Work as Rij/Ri/R (staphylococcus aureus and salmonella R when >=99.9%ij/Ri/ R >=99%), sample antibacterial actions are extremely strong；
If using antibacterial activity value A as correlated performance evaluation index: working as Aij/Ai(Staphylococcus aureus when/A < 1.0 Bacterium and salmonella Aij/Ai/ A < 0.5), sample is without antibacterial actions；As 1.0≤Aij/Ai(Staphylococcus aureus when/A < 2.0 0.5≤A of bacterium and salmonellaij/Ai/ A < 1.0), sample has antibacterial actions；As 2.0≤Aij/AiIt is (golden yellow when/A < 3.0 1.0≤A of color staphylococcus and salmonellaij/Ai/ A < 2.0), sample antibacterial actions are stronger；Work as Aij/Ai(gold when/A >=3.0 Staphylococcus aureus and salmonella Aij/Ai/ A >=2.0), sample antibacterial actions are extremely strong；
(2) as the antibiotic rate R of certain group (part) anti-bacteria stainless steel samplei(Rij) or antibacterial activity value Ai(Aij) and other two groups When the bacteria resistance of (four) sample can be compared to a levels be at least differed, one group of (part) sample is extracted again and repeats to test； It is calculated through ATP bioluminescence lgCA─lgIAThe antibiotic rate or antibacterial activity value that calibration curve method measures.If two groups of front and back is anti- Bacterium stainless steel sample antibacterium performance level is identical, then abandons it；Take other two groups (four) remaining sample antibiotic rate Ri(Rij) or Antibacterial activity value Ai(Aij) arithmetic mean of instantaneous value as batch (group) anti-bacteria stainless steel sample bacteria resistance can evaluation result；
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 is illustrated so that the bacteria resistance of alloy-type anti-bacteria stainless steel sink sample can detect as an example.
Specific detection method carries out in the steps below:
(1) sample preparation and pretreatment
1.1 control samples: in addition to not having antibacterial coating, control sample classification, metallic matrix, technique and appearance, ruler Very little, quantity etc. is identical with anti-bacteria stainless steel sample to be measured；Each strain test uses 6 groups of samples；Wherein 0h contact and for 24 hours Culture experiment respectively uses 3 groups, every group of 5 samples.
1.2 antimicrobial samples: after the processing of the electrochemical methods such as electroplated, chemical plating, antibacterial coating is formed not on surface Become rusty Steel material and product；Sample cover surface is uniform and has certain adhesive strength, no any damage for reaching substrate metal； Having a size of (50 ± 2) mm × (50 ± 2) mm, thickness is not more than 5mm.Antimicrobial sample quantity is identical as control sample, and every group to be measured Sample selects one group of control sample as object of reference and effectively identifies.
1.3 sample pre-treatments: impregnating 5min in 75% ethanol solution for control sample and antimicrobial sample before experiment, and With the abundant cleaning sample of sterile water to remove ethyl alcohol on superclean bench；Sample surface to be measured is put into upwards after natural drying It is 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 meter, matched reagent box, Special test tube etc. is spent, wherein ATP fluophotometer wavelength range 300nm~650nm, ATP Concentration Testing range 10-13Mol/L~10-7mol/L；(20~50) DEG C ± 1 DEG C, (50~95) %RH The constant temperature and humidity incubator of ± 2%RH；The pressure steam sterilizer of (121 ± 2) DEG C, (103 ± 5) kPa；- 20 DEG C~-70 DEG C Low temperature refrigerator；0 DEG C~8 DEG C of refrigerating box；The electronic balance of sensibility reciprocal 0.001g；The ultrasonic wave of frequency range (30~50) kHz is clear Wash device；The vortex oscillator of the range of speeds (500~3000) r/min；The pH meter of precision ± 0.1 (25 DEG C)；Electric furnace.
2.3 material utensils: the sterile measuring pipette of 1mL, 10mL；0.05mL, 0.1mL, 0.2mL, 1mL, 5mL, 10mL (meter Error is measured less than single track changeable fluid liquid-transfering gun and sterile liquid transfer gun head 1%)；The sterile conical flask of capacity 250mL, 500mL； The sterile petri dish of diameter 90mm；Maxwell bacterium standard opacity tube and mating test tube；Sterile test tube；Diameter connects no more than 4mm's Kind ring；L stick；Alcolhol burner；Sterilize tweezers；Medical adhesive tape；Cotton ball soaked in alcohol (75%)；The ruler of scale division value 1mm；Thermometer；The stopwatch of precision 0.01s；A6 white copy paper；0.7mm core black gel ink pen.
2.4 reagents: 75% ethanol solution；(121 DEG C of high pressure sterilization 15min, 5 DEG C~10 DEG C are deposited 85% physiological saline Put 30d).
2.5 medium/liquids (can use commercially available medium/liquid): through 121 DEG C of high pressure sterilizations after matched medium/liquid packing 15min, 2 DEG C~8 DEG C storage 30d；
Nutrient agar (NA): 5g beef extract, 10g peptone, 5g sodium chloride, 15g agar are dissolved by heating in 1000mL water In, adjust pH to 7.0~7.2；
Nutrient broth (NB): 3g beef extract, 10g peptone, 5g sodium chloride are dissolved by heating in 1000mL water, pH is adjusted To 7.0~7.2；
Salmonella preservation is with brain heart oxoid meat soup (BHI): by 10g peptone, 5g sodium chloride, 2.5g phosphoric acid hydrogen two Sodium, 2g glucose dissolve by heating in 500mL cattle heart leachate, adjust pH to 7.4 ± 0.2；
The culture solution of escherichia coli and Shigella/staphylococcus aureus and salmonella: by 1:500/1:100 Nutrient broth (NB) mixed with 85% sterile saline solution, adjust pH to 7.0~7.2.
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；
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 standard reagent stoste: by 60.5mg trinosin (C10H14O13P3Na2·3H2O) it is dissolved in 100mL In water, being configured to concentration is 1 × 10-3The solution of mol/L, -20 DEG C of freezings are sealed 6 months；
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 nutrient broth can be down to 10 in 15min by liquid-13Mol/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；
ATP fluorescent reagent: by 16mg luciferase (EC:1.13.12.7), the D- fluorescein of 12.6mg, 56mg cow's serum Albumen is dissolved in the ATP fluorescent reagent buffer solution of 30mL, and 15min is stored at room temperature after mixing, is used in 3h.
(3) culture presevation, activation and bacteria suspension preparation
3.1 test strains: escherichia coli ATCC 8739；Staphylococcus aureus ATCC 6538；Salmonella ATCC 14028；Shigella CMCC 51105 (or provided by national corresponding culture presevation administrative center and can be traced to the source other Strain).
3.2 culture presevation: opening freeze-drying lactobacillus pipe with sterile working, and adequate nutrition meat soup (sramana is added with capillary syring Salmonella BHI broth), pressure-vaccum makes strain melt dispersion for several times.Then, a little test strain suspension is instilled and 5mL is housed In the test tube of~10mL nutrient broth, (37 ± 1) DEG C culture for 24 hours~48h；As slant preservation bacterium, (5 ± 1) DEG C storage (does not surpass Spend 1 month), inoculation times were no more than for 14 generations.
3.3 actication of culture: slant preservation bacterium is transferred nutrient agar slant medium, and (37 ± 1) DEG C culture 18h~for 24 hours； Switching 1 time daily, continuous switching are no more than 15d.Test is trained using the 3rd generation~the 14th generation and in the interior Fresh bacterial transferred for 24 hours Support object.
The preparation of 3.4 bacteria suspensions: a small amount of Fresh bacterial is scraped from strain activation and culture base with oese, test strain is added Bacteria suspension is made in culture solution；1000r/min shakes test tube 1min or with 30cm amplitude of oscillation shaking test tube 80 times, it is ensured that bacterial suspension is equal It is even.Appropriate bacteria suspension is moved in sterile test tube matched with Maxwell standard opacity tube, it is white in A6 with 0.7mm core black gel ink pen The parallel lines that 3 length are 10cm are drawn on color copy paper；Estimate test tube and standard opacity tube (nominal concentration 6.0 × 108CFU/ ML culture solution is added dropwise until the two turbidity is identical in differences in turbidity).
(4) ATP log concentration value lgCARelative intensity of fluorescence logarithm lgIAStandard curve is established and inoculation bacterium solution viable bacteria ATP concentration CACalibration
4.1 standard serial solutions are determining and prepared by ATP bioluminescence test specimens: using test strain culture 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 × 10-9mol/L、7.0×10-8mol/L、 7.0×10-7Mol/L is (if instrument upper limit of detection is unable to reach 10-7Mol/L magnitude or high standard curve it is linear compared with Its ATP concentration series can be changed to 7.0 × 10 by difference-10mol/L、7.0×10-9mol/L、7.0×10-8) and 4.2 × 10 mol/L-11mol/L、4.2×10-10mol/L、4.2×10-9Mol/L is simultaneously mixed.Then, with sterilizing liquid-transfering gun by the ATP standard of 0.1mL Serial solution is moved to respectively in three sterile test tubes, and phosphate buffer of the 0.9mL containing 0.037% sucrose is successively added dropwise and mixes It is even；The mixed solution of 0.1mL various concentration is moved to respectively in three instrument sterile test tubes again, as ATP bioluminescence Test Duplicate Samples.
4.2 relative intensity of fluorescence value IAMeasurement: according to relative intensity of fluorescence value I in this patentAMeasuring method, with test organisms After kind culture solution is as blank sample liquid validation instrument and reagent set background；According to the sequence of concentration from low to high, to various concentration The ATP that 0.1mL is added dropwise in three Duplicate Samples of ATP standard solution respectively extracts reagent, and the ATP fluorescence examination of 0.1mL is instilled after mixing Agent；It mixes again, with its relative intensity of fluorescence value of ATP fluorescent spectrophotometer measuring and records immediately and (ensure each link operating time Unanimously, cross contamination is avoided).Each Duplicate Samples minute is no more than 15s, with each three Duplicate Samples of concentration ATP standard solution The arithmetic mean of instantaneous value of relative intensity of fluorescence value is its IAMeasured value.
4.3ATP log concentration value lgCARelative intensity of fluorescence logarithm lgIAStandard curve is established: with ATP standard series The relative intensity of fluorescence logarithm lgI of solutionAAs abscissa, with its corresponding ATP log concentration value lgCAFor ordinate work Figure；Calibration curve is carried out to mathematical relationship between the two, draws the lgC of two respective concentrationsA-lgIAStandard curve；And it applies Least square fitting method is derived from the linear equation Y=a of curveA0X+bA0(high concentration), Y=aAX+bA(low concentration) and phase Close coefficients RA0 2(high concentration), RA 2(low concentration).Work as RA0 2And RA 2>=0.98, it when confidence level >=0.95, is done according to this patent Measurement it is effective.
The viable bacteria ATP concentration C of 4.4 inoculation bacterium solutionsACalibration: according to relative intensity of fluorescence value I in this patentAMeasuring method is right Bacteria total amount is 6.0 × 10 after the turbidimetry primary dcreening operation of Maxwell8The relative fluorescence of 10 times of gradient dilution liquid of bacteria suspension of CFU/mL is strong Angle value IAIt is measured；According to high standard fitting equation Y=aA0X+bA0It calculates and demarcates its viable bacteria ATP concentration CA.Through The adjustment of test strain culture solution, obtains CARange is 5.0 × 10-8Mol/L~9.0 × 10-8The inoculation bacterium solution of mol/L, measurement is simultaneously Record relative intensity of fluorescence value I of the 0.3mL inoculation bacterium solution after the dilution of 4.7mL eluent in 1minA0。
(5) sample inoculation, culture and elution recycling
5.1 sample inoculation cultures: 0.3mL inoculation bacterium solution is added dropwise respectively in each group control sample and is resisted with sterilizing liquid-transfering gun (with curve lgC on bacterium sample surface to be measuredA-lgIACalibration is derived from same branch test strain stoste test tube with bacterium solution, and 2 DEG C ± 0.2 DEG C save, use in 2h), bacterium solution is smeared uniformly with L stick (attachment inoculation bacterium solution but do not hang drop), makes its covering sample whole table Face.Ware lid is covered, is sealed the plate for contacting sample for 24 hours equipped with 6 groups with medical adhesive tape；(37 ± 1) DEG C, (90 ± 2) %RH training Support ± 2h for 24 hours.
5.2 elution recycling: (0.2% sterile surfaces activity can be added using 4.7mL test strain culture solution as eluent Agent), after 6 groups of 0h contact sample inoculation bacteriums, sufficiently eluted using solid or liquid swab method immediately；6 groups cultivate for 24 hours Sample uses type of elution identical with 0h contact sample to recycle bacterium.Solid swab method is to be soaked with saturation eluent with head Aseptic cotton carrier is uniformly embrocated 10 times in each sample surfaces back and forth anyhow respectively, and rotates with it cotton swab；Cut off experimenter's hand Cotton swab is placed in the sterile test tube equipped with remaining eluent, as recovered liquid (if recovered liquid after mixing by the swab stick of contact portion Less than 5mL, eluent is added to 5mL).Liquid swab method draws 4.7mL eluent with sterilizing liquid-transfering gun, in plate repeatedly It rinses each sample surfaces at least 4 times, sufficiently elute and moves into washing lotion in sterile test tube, as recovered liquid (if returning after mixing Liquid is received less than 5mL, adds eluent to 5mL).
(6) reclaim liquid phase is to fluorescence intensity level IAMeasurement
6.1 instruments and reagent set relative intensity of fluorescence background value calibration: use sterilizing liquid-transfering gun by 0.1mL test strain culture The ATP lysate of liquid, 0.8mL phosphate buffer solution and 0.1mL containing 0.037% sucrose is separately added into same branch sterile test tube In and mix well, then the above-mentioned mixed solution of 0.1mL is successively moved in three instrument sterile test tubes, stand 5min~ 30min, as skip test Duplicate Samples.The ATP that 0.1mL is added dropwise respectively into three Duplicate Samples extracts reagent, instills after mixing The ATP fluorescent reagent of 0.1mL；It mixes again, uses its relative intensity of fluorescence value of ATP fluorescent spectrophotometer measuring I immediatelyAAnd it records (ensuring that each link operating time is consistent, avoid cross contamination).Each Duplicate Samples minute is no more than 15s, with three blank The arithmetic mean of instantaneous value of Duplicate Samples relative intensity of fluorescence value is tested (or to use according to pertinent instruments as instrument and reagent set background values Illustrate to calibrate background values).
6.2 reclaim liquid phases are to fluorescence intensity level IAMeasurement: it if blank reagent group background level meets instrument requirement, uses The ATP lysate for phosphate buffer solution and 0.1mL of the liquid-transfering gun by 0.1mL recovered liquid, 0.8mL containing 0.037% sucrose that sterilize Be separately added into same branch sterile test tube, after mixing well by the above-mentioned mixed solution of 0.1mL successively move to three instruments without In bacterium test tube, 5min~30min is stood, as ATP fluorometric investigation Duplicate Samples.It is added dropwise 0.1mL's respectively into three Duplicate Samples ATP extracts reagent, and the ATP fluorescent reagent of 0.1mL is instilled after mixing；It mixes again, uses its phase of ATP fluorescent spectrophotometer measuring immediately To fluorescence intensity level IAAnd it records and (ensures that each link operating time is consistent, avoid cross contamination).Each Duplicate Samples minute No more than 15s, using the arithmetic mean of instantaneous value of three ATP fluorometric investigation Duplicate Samples relative intensity of fluorescence values as the I of recovered liquidAMeasurement Value (or instrument matched reagent box is used, it is required according to operation instructions, directly upper machine measures the opposite of three Duplicate Samples of recovered liquid Fluorescence intensity level IA)。
(7) recovered liquid viable bacteria ATP concentration CAAnd TAIt calculates and antibiotic rate R and antibacterial activity value A is calculated
The viable bacteria ATP concentration C of 7.1 recovered liquidsAAnd TAIt calculates
According to ATP low concentration standard curve lgCA-lgIALinear equation Y=aAX+bA, calculate control sample and antibacterial After sample is contacted through 0h and is cultivated for 24 hours, the viable bacteria ATP concentration C of recovered liquidAAnd TA；Relevant calculation (uses instrument matched reagent box When, the viable bacteria ATP concentration C of 1mL recovered liquid is calculated according to its practical sample volumeAAnd TA) see formula (1)~(12):
In formula:
CA0And CAtThe average ATP concentration that -3 groups of 0h are contacted and control sample recycles viable bacteria after cultivating for 24 hours, unit are to rub You are every liter (mol/L)；
CA0iAnd CAtiThe average ATP concentration that-every group 0h is contacted and control sample recycles viable bacteria after cultivating for 24 hours, unit are Mole every liter (mol/L)；Sample group i=1,2,3；
CA0ijAnd CAtijThe ATP concentration that-every 0h is contacted and control sample recycles viable bacteria after cultivating for 24 hours, 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 cultivating for 24 hours 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；
aA- low concentration standard curve lgCA-lgIASlope；bA- low concentration standard curve lgCA-lgIAIn vertical axis intercept；
TA0And TAtThe average ATP concentration that -3 groups of 0h are contacted and antimicrobial sample recycles viable bacteria after cultivating for 24 hours, unit are to rub You are every liter (mol/L)；
TA0iAnd TAtiThe average ATP concentration that-every group 0h is contacted and antimicrobial sample recycles viable bacteria after cultivating for 24 hours, unit are Mole every liter (mol/L)；Sample group i=1,2,3；
TA0ijAnd TAtijThe ATP concentration that-every 0h is contacted and antimicrobial sample recycles viable bacteria after cultivating for 24 hours, 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 cultivating for 24 hours 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.
7.2 test conditions for validity
If recovered liquid and 0.3mL inoculation the bacterium solution 1min after the dilution of 4.7mL eluent of every 0h contact control sample Interior relative intensity of fluorescence measured value is close, i.e.,Every after cultivating for 24 hours control sample recovered liquid relative fluorescence it is strong Spend the logarithm of measured valueThat is CAtij≥0.1×CA0ij.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.
7.3 bacterium increasing value Fij、GijIt calculates
Every control sample and antimicrobial sample after cultivating for 24 hours, bacterium increasing value Fij、GijRespectively according to formula (13), (14) it calculates:
In formula:
FijAnd GijThe bacterium increasing value of-every control sample and antimicrobial sample after cultivating for 24 hours, sample group i=1, 2,3；Every group of sample number into spectrum j=1,2,3,4,5；
CA0ijAnd CAtijThe ATP concentration that-every 0h is contacted and control sample recycles viable bacteria after cultivating for 24 hours, 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 cultivating for 24 hours 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；
TA0ijAnd TAtijThe ATP concentration that-every 0h is contacted and antimicrobial sample recycles viable bacteria after cultivating for 24 hours, 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 cultivating for 24 hours 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；
aA- low concentration standard curve lgCA-lgIASlope.
7.4 antibiotic rate R are calculated
It tests under condition for validity, the antibiotic rate R of every anti-bacteria stainless steel sampleij, every group and every batch of sample antibiotic rate Ri It is calculated respectively according to formula (15), (16), (17) with R:
In formula:
RijThe antibiotic rate of-every anti-bacteria stainless steel sample, %；Sample group i=1,2,3；Every group of sample number into spectrum j=1, 2,3,4,5；
RiThe antibiotic rate of-every group anti-bacteria stainless steel sample, %；Sample group i=1,2,3；
R-every batch of anti-bacteria stainless steel sample antibiotic rate, %；
CAtijAnd TAtij- every antimicrobial sample and control sample recycle the average ATP concentration of viable bacteria after cultivating for 24 hours, 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 cultivating for 24 hours, 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；
aA- low concentration standard curve lgCA-lgIASlope.
7.5 antibacterial activity value A are calculated
It tests under condition for validity, the antibacterial activity value A of every anti-bacteria stainless steel sampleij, every group and every batch of sample antibacterial Activity value AiIt is calculated respectively according to formula (18), (19), (20) with A:
In formula:
AijThe antibacterial activity value of-every anti-bacteria stainless steel sample；Sample group i=1,2,3；Every group of sample number into spectrum j=1, 2,3,4,5；
AiThe antibacterial activity value of-every group anti-bacteria stainless steel sample, sample group i=1,2,3；
A-every batch of anti-bacteria stainless steel sample antibacterial activity value；
FijAnd GijThe bacterium increasing value of-every control sample and antimicrobial sample after cultivating for 24 hours, 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 cultivating for 24 hours 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；
With- every 0h contact and after cultivating for 24 hours 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；
aA- low concentration standard curve lgCA-lgIASlope.
7.6 data revisions of the convention requirement: the concentration of ATP standard serial solution and the viable bacteria ATP of inoculation bacterium solution, sample recovered liquid Concentration CAWhen being demarcated, with reference to the data revision of the convention regulation in GB 4789.2-2016 in relation to clump count；Work as CALess than 100mol/ When L, " rounding up " round numbers；Work as CAWhen not less than 100mol/L, preceding 2 bit digital is taken after the 3rd bit digital " rounding up ", after Face replaces digit with 0；It can also be indicated with 10 exponential form, " rounding up " uses two effective digitals afterwards.Control sample and After antimicrobial sample is contacted through 0h and cultivated for 24 hours, reclaim liquid phase is to fluorescent strength determining value round numbers, antibiotic rate Rij、Ri, R calculate As a result three effective digitals are taken；Bacterium increasing value Fij、GijWith antibacterial activity value Aij、Ai, A calculated result take two effective digitals.
7.7 uncertainties of measurement: this patent method, which mainly passes through, to be calculated control sample and antimicrobial sample and contacts through 0h and for 24 hours 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 that ATP fluorimetric assay for biological materials method, which is applied to stainless steel bacteria resistance, to be surveyed The reproducibility of examination；In regulation group and between-group variation coefficient CV≤10%.
5 control samples, 5 antimicrobial samples and 5 control samples after cultivating for 24 hours 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 are trained for 24 hours 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 cultivating for 24 hours 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 for 24 hours After culture, the relative intensity of fluorescence measured value of each Duplicate Samples):
5 control samples, 5 antimicrobial samples and 5 control samples after cultivating for 24 hours of every group of 0h contact, 5 it is anti- Bacterium sample, standard deviation of the reclaim liquid phase to fluorescent strength determining valueAnd Respectively 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 are trained for 24 hours 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 cultivating for 24 hours that 3 groups of 0h are contacted Part antimicrobial sample, standard deviation of the reclaim liquid phase to fluorescent strength determining valueAndRespectively according to public affairs 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 through 0h contact and for 24 hours After culture, the relative intensity of fluorescence measured value of each Duplicate Samples):
(8) evaluation of result
8.1, with reference to health industry common practice and the requirement of dependent antimicrobial product standard, take following classification criterion:
If using antibiotic rate R as correlated performance evaluation index: working as Rij/Ri(staphylococcus aureus when/R < 90% With salmonella Rij/Ri/ R < 80%), sample is without antibacterial actions；As 90%≤Rij/Ri(golden yellow grape when/R < 99% 80%≤R of coccus and salmonellaij/Ri/ R < 90%), sample has antibacterial actions；As 99%≤Rij/Ri/ R < 99.9% When (90%≤R of staphylococcus aureus and salmonellaij/Ri/ R < 99%), sample antibacterial actions are stronger；Work as Rij/Ri/R (staphylococcus aureus and salmonella R when >=99.9%ij/Ri/ R >=99%), sample antibacterial actions are extremely strong；
If using antibacterial activity value A as correlated performance evaluation index: working as Aij/Ai(Staphylococcus aureus when/A < 1.0 Bacterium and salmonella Aij/Ai/ A < 0.5), sample is without antibacterial actions；As 1.0≤Aij/Ai(Staphylococcus aureus when/A < 2.0 0.5≤A of bacterium and salmonellaij/Ai/ A < 1.0), sample has antibacterial actions；As 2.0≤Aij/AiIt is (golden yellow when/A < 3.0 1.0≤A of color staphylococcus and salmonellaij/Ai/ A < 2.0), sample antibacterial actions are stronger；Work as Aij/Ai(gold when/A >=3.0 Staphylococcus aureus and salmonella Aij/Ai/ A >=2.0), sample antibacterial actions are extremely strong.
8.2 work as the antibiotic rate R of certain group (part) anti-bacteria stainless steel samplei(Rij) or antibacterial activity value Ai(Aij) and other two groups When the bacteria resistance of (four) sample can be compared to a levels be at least differed, one group of (part) sample is extracted again and repeats to test； It is calculated through ATP bioluminescence lgCA─lgIAThe antibiotic rate or antibacterial activity value that calibration curve method measures.If two groups of front and back is anti- Bacterium stainless steel sample antibacterium performance level is identical, then abandons it；Take other two groups (four) remaining sample antibiotic rate Ri(Rij) or Antibacterial activity value Ai(Aij) arithmetic mean of instantaneous value as batch (group) anti-bacteria stainless steel sample bacteria resistance can evaluation result.
Laboratory biosafety qualification, instrument and equipment, medium/liquid, chemical reagent, reference culture used in the present embodiment:
(1) Laboratory biosafety 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
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 ScientificTM HeraguardTM, 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 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.
2.5 pressure steam sterilizers: the autoclave of Japanese Sanyo company model MLS-3780, volume 75L, sterilizing ± 2 DEG C of temperature (105~135) DEG C, maximum pressure 0.235MPa, timing range (1~250) min.
2.6 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.
2.7 refrigerating boxes: the Medical refrigerator of Zhong Kemeiling low temperature Science and Technology Co., Ltd. model YC-968L effectively holds Long-pending 968L, ± 0.1 DEG C of storage temperature (2~8) DEG C.
2.8 electronic balances: the electronic balance of Japanese Shimadzu model AUX220, range 220g, precision ± 0.1mg.
2.9 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.
2.10 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.
2.11pH 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.
2.12 electric furnaces: the 1KW closed temp.-adjustable electric furnace of Changzhou Deco Instrument Ltd. model DLD-1KW.
(3) medium/liquid
The medium/liquid of Beijing overpass technical concern Co., Ltd production.
(4) chemical reagent
Trinosin standard items and prepare ATP fluorescent reagent buffer solution, ATP lysate, ATP extracting solution and A series of biochemical reagents needed for ATP fluorescent reagent are purchased from U.S.'s Amresco product of Shanghai Jin Pan Biotechnology Co., Ltd agency Board.
(5) reference culture
Escherichia coli ATCC 8739 and staphylococcus aureus ATCC 6538 is purchased from Chinese industrial microorganism fungus kind Preservation administrative center.
The detection data and result of the present embodiment calculate:
Using ATP fluophotometer through lgCA-lgIACalibration curve method can be carried out the bacteria resistance of antibacterial stainless steel sample Detection, coherent detection data and Calculation of Measuring Uncertainty result are shown in Table the (inspection of Shigella and salmonella of 1~table 4 respectively It is close with escherichia coli and the testing result of staphylococcus aureus respectively to survey result).
1 relative intensity of fluorescence measured value of table and antibiotic rate R, antibacterial activity value A calculated result (escherichia coli)
2 relative intensity of fluorescence value Calculation of Measuring Uncertainty result (escherichia coli) of table
3 relative intensity of fluorescence measured value of table and antibiotic rate R, antibacterial activity value A calculated result (staphylococcus aureus)
4 relative intensity of fluorescence Calculation of Measuring Uncertainty result (staphylococcus aureus) 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 (9)

1. a kind of detection method of anti-bacteria stainless steel bacteria resistance energy, comprising: (1) sample preparation and pretreatment；(2) lectotype selection And reagent, culture medium are prepared；(3) culture presevation, activation and bacteria suspension preparation；(4) ATP log concentration value lgCARelative fluorescence Intensity logarithm lgIAStandard curve is established and inoculation bacterium solution viable bacteria ATP concentration CACalibration；(5) it sample inoculation, culture and elutes back It receives；(6) reclaim liquid phase is to fluorescence intensity level IAMeasurement；(7) recovered liquid viable bacteria ATP concentration CAAnd TAIt calculates and antibiotic rate R and anti- Bacterium activity value A is calculated；(8) evaluation of result；It is characterized in that, using ATP fluophotometer to antibiotic rate R or antibacterial activity value The anti-bacteria stainless steel bacteria resistance of A characterization can be carried out the ATP bioluminescence lgC of accurate quantitative testA-lgIACalibration curve method, tool Body:
In reclaim liquid phase to fluorescence intensity level IAIn measurement:
Quantity, size and the pre-treatment requirement for specifying control sample and antimicrobial sample, testing standard bacterial strain is passed on, is activated Afterwards, it takes continuous switching 2 times Fresh bacterial cultures to prepare bacteria suspension and primary dcreening operation is carried out to its bacterium number, with test strain culture 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 × 10-9mol/L、7.0× 10-8mol/L、7.0×10-7Mol/L is (if instrument upper limit of detection is unable to reach 10-7Mol/L magnitude or high standard curve It is linear poor, its ATP concentration series can be changed to 7.0 × 10-10mol/L、7.0×10-9mol/L、7.0×10-8mol/L) With 4.2 × 10-11mol/L、4.2×10-10mol/L、4.2×10-9Mol/L, and measure its relative intensity of fluorescence value IA；Draw two The lgC of respective concentrationA-lgIAStandard curve is derived from fitting equation Y=aA0X+bA0(high concentration), Y=aAX+bAIt is (low Concentration) and linearly dependent coefficient RA0 2(high concentration), RA 2(low concentration) is 6.0 × 10 to bacteria total amount8The bacteria suspension of CFU/mL After carrying out 10 times of gradient dilutions, its relative intensity of fluorescence value I is measuredA, according to high standard fitting equation Y=aA0X+bA0It pushes away Calculate corresponding viable bacteria ATP concentration CA；It is adjusted to obtain CARange is 5.0 × 10-8Mol/L~9.0 × 10-8The inoculation bacterium of mol/L Then 0.3mL bacterium solution is added dropwise to each group control sample and antimicrobial sample surface to be measured in liquid respectively, use 4.7mL eluent pair immediately 6 groups of 0h contact samples carry out elution recycling；Using the relative intensity of fluorescence value I of ATP fluorescent spectrophotometer measuring recovered liquidAC0ij、 IAT0ij, according to low concentration calibration curve equation formula Y=aAX+bACalculate the ATP concentration C of its viable bacteriaAOijAnd TAOij, meanwhile, by 6 groups Contact sample is sealed in sterilized petri dishes for 24 hours, and (37 ± 1) DEG C, (90 ± 2) %RH culture are for 24 hours after ± 2h；Sample is contacted using with 0h The identical mode of product recycles remained on surface bacterium and measures the relative intensity of fluorescence value I of its recovered liquidACtij、IATtij, calculate corresponding Viable bacteria ATP concentration CAtijAnd TAtij；
In antibiotic rate R and antibacterial activity value A is calculated:
According to ATP low concentration standard curve lgCA-lgIALinear equation Y=aAX+bA, with 0h contact and for 24 hours cultivate after every it is right Product and antimicrobial sample reclaim liquid phase are to fluorescent strength determining value in the same old wayWithAs basic data；It is trying It tests under condition for validity, calculates its bacterium increasing value F after cultivating for 24 hoursij、GijAnd antibiotic rate RijWith antibacterial activity value Aij；It is right The R of every group of sampleijAnd AijArithmetic mean of instantaneous value is taken to obtain corresponding RiAnd Ai；The antibiotic rate R of every batch of anti-bacteria stainless steel sample and anti- Bacterium activity value A is its 3 groups of sample RiAnd AiArithmetic mean of instantaneous value；And the clear related data revision of the convention and uncertainty of measurement require；
In evaluation of result:
With reference to health industry common practice and the requirement of dependent antimicrobial product standard, antibacterium grading performance criterion is determined；When The antibiotic rate R of certain group (part) anti-bacteria stainless steel samplei(Rij) or antibacterial activity value Ai(Aij) with other two groups (four) samples When bacteria resistance can be compared to a levels be at least differed, one group of (part) sample is extracted again and repeats to test；It is raw through ATP to calculate it Object fluorescence lgCA─lgIAThe antibiotic rate or antibacterial activity value that calibration curve method measures, if two groups of front and back (part) anti-bacteria stainless steel Sample antibacterium performance level is identical, then abandons it；Take other two groups (four) remaining sample antibiotic rate Ri(Rij) or antibacterial activity Value Ai(Aij) arithmetic mean of instantaneous value as batch (group) anti-bacteria stainless steel sample bacteria resistance can evaluation result.
2. the detection method of anti-bacteria stainless steel bacteria resistance energy according to claim 1, which is characterized in that the sample Preparation and pre-treatment carry out in the steps below:
(1) control sample: in addition to not having antibacterial coating, control sample classification, metallic matrix, technique and appearance, size, number Amount etc. is identical with anti-bacteria stainless steel sample to be measured；Each strain test uses 6 groups of samples；Wherein 0h is contacted and is cultivated for 24 hours Test each with 3 groups, every group of 5 samples；
(2) antimicrobial sample: after the processing of the electrochemical methods such as electroplated, chemical plating, the stainless steel of antibacterial coating is formed on surface Material and product；Sample cover surface is uniform and has certain adhesive strength, no any damage for reaching substrate metal；Size For (50 ± 2) mm × (50 ± 2) mm, thickness is not more than 5mm, and antimicrobial sample quantity is identical as control sample, every group of sample to be tested It selects one group of control sample as object of reference and effectively identifies；
(3) sample pre-treatments: control sample and antimicrobial sample are impregnated into 5min in 75% ethanol solution before experiment, and super With the abundant cleaning sample of sterile water to remove ethyl alcohol on net workbench；Sample surface to be measured is put into upwards after natural drying sterile It is spare in plate.
3. the detection method of anti-bacteria stainless steel bacteria resistance energy according to claim 1, which is characterized in that the equipment Type selecting 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, matched reagent box, Special test tube etc., wherein ATP fluophotometer wavelength range 300nm~650nm, ATP Concentration Testing range 10-13Mol/L~10-7mol/L；(20~50) DEG C ± 1 DEG C, (50~95) %RH The constant temperature and humidity incubator of ± 2%RH；The pressure steam sterilizer of (121 ± 2) DEG C, (103 ± 5) kPa；- 20 DEG C~-70 DEG C Low temperature refrigerator；0 DEG C~8 DEG C of refrigerating box；The electronic balance of sensibility reciprocal 0.001g；The ultrasonic wave of frequency range (30~50) kHz is clear Wash device；The vortex oscillator of the range of speeds (500~3000) r/min；The pH meter of precision ± 0.1 (25 DEG C)；Electric furnace；
(3) material utensil: the sterile measuring pipette of 1mL, 10mL；(metering misses by 0.05mL, 0.1mL, 0.2mL, 1mL, 5mL, 10mL Difference is less than single track changeable fluid liquid-transfering gun and sterile liquid transfer gun head 1%)；The sterile conical flask of capacity 250mL, 500mL；Diameter The sterile petri dish of 90mm；Maxwell bacterium standard opacity tube and mating test tube；Sterile test tube；Diameter is not more than the oese of 4mm； L stick；Alcolhol burner；Sterilize tweezers；Medical adhesive tape；Cotton ball soaked in alcohol (75%)；The ruler of scale division value 1mm； Thermometer；The stopwatch of precision 0.01s；A6 white copy paper；0.7mm core black gel ink pen；
(4) reagent: 75% ethanol solution；85% physiological saline (121 DEG C of high pressure sterilization 15min, 5 DEG C~10 DEG C storages 30d)；
(5) medium/liquid (commercially available medium/liquid can be used): through 121 DEG C of high pressure sterilization 15min after the packing of matched medium/liquid, 2 DEG C~8 DEG C of storage 30d；
Nutrient agar (NA): 5g beef extract, 10g peptone, 5g sodium chloride, 15g agar are dissolved by heating in 1000mL water, adjusted Save pH to 7.0~7.2；
Nutrient broth (NB): by 3g beef extract, 10g peptone, 5g sodium chloride dissolve by heating in 1000mL water, adjust pH to 7.0~7.2；
Salmonella preservation is with brain heart oxoid meat soup (BHI): by 10g peptone, 5g sodium chloride, 2.5g disodium hydrogen phosphate, 2g Glucose dissolves by heating in 500mL cattle heart leachate, adjusts pH to 7.4 ± 0.2；
The culture solution of escherichia coli and Shigella/staphylococcus aureus and salmonella: by the battalion of 1:500/1:100 It supports meat soup (NB) to mix with 85% sterile saline solution, adjusts pH to 7.0~7.2；
(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 standard reagent stoste: by 60.5mg trinosin (C10H14O13P3Na2·3H2O it) is dissolved in 100mL water, Being configured to concentration is 1 × 10-3The solution of mol/L, -20 DEG C of freezings are sealed 6 months；
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 nutrient broth can be down to 10 in 15min-13Mol/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, pH to 12.0 ± 0.5 is adjusted；
ATP fluorescent reagent: by 16mg luciferase (EC:1.13.12.7), the D- fluorescein of 12.6mg, 56mg bovine serum albumin It is dissolved in the ATP fluorescent reagent buffer solution of 30mL, 15min is stored at room temperature after mixing, is used in 3h.
4. the detection method of anti-bacteria stainless steel bacteria resistance energy according to claim 1, which is characterized in that the strain Preservation, activation and bacteria suspension preparation, carry out in the steps below:
(1) test strain: escherichia coli ATCC 8739；Staphylococcus aureus ATCC 6538；Salmonella ATCC 14028；Shigella CMCC 51105 (or other strains that is provided and can be traced to the source by national corresponding culture presevation administrative center)；
(2) culture presevation: opening freeze-drying lactobacillus pipe with sterile working, and adequate nutrition meat soup (salmonella is added with capillary syring With BHI broth), pressure-vaccum make for several times strain melt dispersion, then, by a little test strain suspension instill equipped with 5mL~ In the test tube of 10mL nutrient broth, (37 ± 1) DEG C culture for 24 hours~48h；As slant preservation bacterium, (5 ± 1) DEG C storage (is no more than 1 month), inoculation times were no more than for 14 generations；
(3) actication of culture: slant preservation bacterium is transferred nutrient agar slant medium, and (37 ± 1) DEG C culture 18h~for 24 hours；Daily Switching 1 time, continuously switching is no more than 15d, and test is using the 3rd generation~the 14th generation and in the interior Fresh bacterial cultures transferred for 24 hours；
(4) prepared by bacteria suspension: scraping a small amount of Fresh bacterial from strain activation and culture base with oese, test strain culture is added Bacteria suspension is made in liquid；1000r/min shakes test tube 1min or with 30cm amplitude of oscillation shaking test tube 80 times, it is ensured that and bacterial suspension is uniform, Appropriate bacteria suspension is moved in sterile test tube matched with Maxwell standard opacity tube, with 0.7mm core black gel ink pen in A6 white The parallel lines that 3 length are 10cm are drawn on copy paper；Estimate test tube and standard opacity tube (nominal concentration 6.0 × 108CFU/mL) Differences in turbidity, be added dropwise culture solution until the two turbidity it is identical until.
5. the detection method of anti-bacteria stainless steel bacteria resistance energy according to claim 1, which is characterized in that the ATP Log concentration value lgCARelative intensity of fluorescence logarithm lgIAStandard curve is established and inoculation bacterium solution viable bacteria ATP concentration CACalibration, It carries out in the steps below:
(1) standard serial solution is determining and prepared by ATP bioluminescence test specimens: using test strain culture solution by 1.0 × 10-3mol/ The ATP standard stock solution of L is diluted to high and low concentration standard serial solution: 7.0 × 10-9mol/L、7.0×10-8mol/L、7.0× 10-7Mol/L is (if instrument upper limit of detection is unable to reach 10-7Mol/L magnitude or high standard curve it is linear poor, can will Its ATP concentration series is changed to 7.0 × 10-10mol/L、7.0×10-9mol/L、7.0×10-8) and 4.2 × 10 mol/L-11mol/ L、4.2×10-10mol/L、4.2×10-9Mol/L is simultaneously mixed, then, with sterilizing liquid-transfering gun that the ATP standard series of 0.1mL is molten Liquid is moved to respectively in three sterile test tubes, and phosphate buffer of the 0.9mL containing 0.037% sucrose and mixing is successively added dropwise；Again will The mixed solution of 0.1mL various concentration is moved to respectively in three instrument sterile test tubes, parallel as the test of ATP bioluminescence Sample；
(2) relative intensity of fluorescence value IAMeasurement: according to relative intensity of fluorescence value I in this patentAMeasuring method is trained with test strain After nutrient solution is as blank sample liquid validation instrument and reagent set background；According to the sequence of concentration from low to high, marked to various concentration ATP The ATP that 0.1mL is added dropwise in three Duplicate Samples of quasi- solution respectively extracts reagent, and the ATP fluorescent reagent of 0.1mL is instilled after mixing；Again Secondary mixing, immediately with its relative intensity of fluorescence value of ATP fluorescent spectrophotometer measuring and record (ensure that each link operating time is consistent, Avoid cross contamination), each Duplicate Samples minute is no more than 15s, relatively glimmering with each three Duplicate Samples of concentration ATP standard solution The arithmetic mean of instantaneous value of light intensity value is its IAMeasured value；
(3) ATP log concentration value lgCARelative intensity of fluorescence logarithm lgIAStandard curve is established: with ATP standard serial solution Relative intensity of fluorescence logarithm lgIAAs abscissa, with its corresponding ATP log concentration value lgCAFor ordinate mapping；It is right Mathematical relationship between the two carries out calibration curve, draws the lgC of two respective concentrationsA-lgIAStandard curve；And application is minimum Two multiply the linear equation Y=a that fitting process is derived from curveA0X+bA0(high concentration), Y=aAX+bA(low concentration) and phase relation Number RA0 2(high concentration), RA 2(low concentration), works as RA0 2And RA 2>=0.98, when confidence level >=0.95, the survey done according to this patent It is fixed effective；
(4) it is inoculated with the viable bacteria ATP concentration C of bacterium solutionACalibration: according to relative intensity of fluorescence value I in this patentAMeasuring method, to through wheat Bacteria total amount is 6.0 × 10 after family name's turbidimetry primary dcreening operation8The relative intensity of fluorescence value I of 10 times of gradient dilution liquid of bacteria suspension of CFU/mLA It is measured；According to high standard fitting equation Y=aA0X+bA0It calculates and demarcates its viable bacteria ATP concentration CA, through test organisms Kind culture solution adjustment, obtains CARange is 5.0 × 10-8Mol/L~9.0 × 10-8The inoculation bacterium solution of mol/L, measures and records 0.3mL is inoculated with relative intensity of fluorescence value I of the bacterium solution after the dilution of 4.7mL eluent in 1minA0。
6. the detection method of anti-bacteria stainless steel bacteria resistance energy according to claim 1, which is characterized in that the sample Inoculation, culture and elution recycling, carry out in the steps below:
(1) sample inoculation culture: 0.3mL inoculation bacterium solution is added dropwise respectively in each group control sample and antibacterial sample with sterilizing liquid-transfering gun (with curve lgC on product surface to be measuredA-lgIACalibration is derived from same branch test strain stoste test tube, 2 DEG C of ± 0.2 DEG C of guarantors with bacterium solution Deposit, used in 2h), bacterium solution is smeared uniformly with L stick (attachment is inoculated with bacterium solution but does not hang drop), it is made to cover sample whole surface, Ware lid is covered, is sealed the plate for contacting sample for 24 hours equipped with 6 groups with medical adhesive tape；(37 ± 1) DEG C, (90 ± 2) %RH culture 24h±2h；
(2) elution recycling: using 4.7mL test strain culture solution as eluent (0.2% sterile surfaces activating agent can be added), 6 After group 0h contact sample inoculation bacterium, sufficiently eluted using solid or liquid swab method immediately；6 groups of samples cultivated for 24 hours Bacterium is recycled using type of elution identical with 0h contact sample, solid swab method is to be soaked with the sterile of saturation eluent with head Cotton swab is uniformly embrocated 10 times in each sample surfaces back and forth anyhow respectively, and rotates with it cotton swab；Cut off the contact of experimenter's hand Cotton swab is placed in the sterile test tube equipped with remaining eluent, as recovered liquid (if recovered liquid is insufficient after mixing by partial swab stick 5mL, addition eluent to 5mL), liquid swab method draw 4.7mL eluent, the repeated flushing in plate with sterilizing liquid-transfering gun It each sample surfaces at least 4 times, sufficiently elutes and moves into washing lotion in sterile test tube, as recovered liquid (if recovered liquid after mixing Less than 5mL, eluent is added to 5mL).
7. the detection method of anti-bacteria stainless steel bacteria resistance energy according to claim 1, which is characterized in that the recycling Liquid phase is to fluorescence intensity level IAMeasurement carries out in the steps below:
(1) instrument and reagent set relative intensity of fluorescence background value calibration: with sterilizing liquid-transfering gun by 0.1mL test strain culture solution, The ATP lysate of phosphate buffer solution and 0.1mL of the 0.8mL containing 0.037% sucrose is separately added into same branch sterile test tube And mix well, then the above-mentioned mixed solution of 0.1mL is successively moved in three instrument sterile test tubes, standing 5min~ 30min, as skip test Duplicate Samples, the ATP that 0.1mL is added dropwise respectively into three Duplicate Samples extracts reagent, instills after mixing The ATP fluorescent reagent of 0.1mL；It mixes again, uses its relative intensity of fluorescence value of ATP fluorescent spectrophotometer measuring I immediatelyAAnd it records (ensuring that each link operating time is consistent, avoid cross contamination), each Duplicate Samples minute is no more than 15s, with three blank The arithmetic mean of instantaneous value of Duplicate Samples relative intensity of fluorescence value is tested (or to use according to pertinent instruments as instrument and reagent set background values Illustrate to calibrate background values)；
(2) reclaim liquid phase is to fluorescence intensity level IAMeasurement: if blank reagent group background level meets instrument requirement, with sterilizing The ATP lysate of phosphate buffer solution and 0.1mL of the liquid-transfering gun by 0.1mL recovered liquid, 0.8mL containing 0.037% sucrose is distinguished It is added in same branch sterile test tube, the above-mentioned mixed solution of 0.1mL is successively moved into three sterile examinations of instrument after mixing well Guan Zhong stands 5min~30min, and as ATP fluorometric investigation Duplicate Samples, the ATP that 0.1mL is added dropwise respectively into three Duplicate Samples is mentioned Reagent is taken, the ATP fluorescent reagent of 0.1mL is instilled after mixing；It mixes again, with ATP fluorescent spectrophotometer measuring, it is relatively glimmering immediately Light intensity value IAAnd record and (ensure that each link operating time is consistent, avoid cross contamination), each Duplicate Samples minute does not surpass 15s is crossed, using the arithmetic mean of instantaneous value of three ATP fluorometric investigation Duplicate Samples relative intensity of fluorescence values as the I of recovered liquidAMeasured value (or instrument matched reagent box is used, it is required according to operation instructions, directly upper machine measures the relatively glimmering of three Duplicate Samples of recovered liquid Light intensity value IA)。
8. the detection method of anti-bacteria stainless steel bacteria resistance energy according to claim 1, which is characterized in that the recycling Liquid viable bacteria ATP concentration CAAnd TAIt calculates and antibiotic rate R and antibacterial activity value A is calculated, carry out in the steps below:
(1) the viable bacteria ATP concentration C of recovered liquidAAnd TAIt calculates
According to ATP low concentration standard curve lgCA-lgIALinear equation Y=aAX+bA, calculate control sample and antimicrobial sample After contacting through 0h and cultivating for 24 hours, the viable bacteria ATP concentration C of recovered liquidAAnd TA；Relevant calculation (when using instrument matched reagent box, root The viable bacteria ATP concentration C of 1mL recovered liquid is calculated according to its practical sample volumeAAnd TA) see formula (1)~(12):
In formula:
CA0And CAt- 3 groups of 0h contact and after cultivating for 24 hours control sample recycling viable bacteria average ATP concentration, unit is mole every It rises (mol/L)；
CA0iAnd CAtiThe average ATP concentration that-every group 0h is contacted and control sample recycles viable bacteria after cultivating for 24 hours, unit are mole Every liter (mol/L)；Sample group i=1,2,3；
CA0ijAnd CAtij- every 0h contact and after cultivating for 24 hours 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 cultivating for 24 hours 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；
aA- low concentration standard curve lgCA-lgIASlope；bA- low concentration standard curve lgCA-lgIAIn vertical axis intercept；
TA0And TAt- 3 groups of 0h contact and after cultivating for 24 hours antimicrobial sample recycling viable bacteria average ATP concentration, unit is mole every It rises (mol/L)；
TA0iAnd TAtiThe average ATP concentration that-every group 0h is contacted and antimicrobial sample recycles viable bacteria after cultivating for 24 hours, unit are mole Every liter (mol/L)；Sample group i=1,2,3；
TA0ijAnd TAtij- every 0h contact and after cultivating for 24 hours 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 cultivating for 24 hours 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 4.7mL eluent in 1min Relative intensity of fluorescence measured value is close, i.e.,Every control sample recovered liquid relative intensity of fluorescence survey after cultivating for 24 hours The logarithm of definite valueThat is CAtij≥0.1×CA0ij, 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) bacterium increasing value Fij、GijIt calculates
Every control sample and antimicrobial sample after cultivating for 24 hours, bacterium increasing value Fij、GijIt is counted respectively according to formula (13), (14) It calculates:
In formula:
FijAnd GijThe bacterium increasing value of-every control sample and antimicrobial sample after cultivating for 24 hours, sample group i=1,2,3；Often Group sample number into spectrum j=1,2,3,4,5；
CA0ijAnd CAtij- every 0h contact and after cultivating for 24 hours 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 cultivating for 24 hours 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；
TA0ijAnd TAtij- every 0h contact and after cultivating for 24 hours 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 cultivating for 24 hours 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；
aA- low concentration standard curve lgCA-lgIASlope；
(4) antibiotic rate R is calculated
It tests under condition for validity, the antibiotic rate R of every anti-bacteria stainless steel sampleij, every group and every batch of sample antibiotic rate RiWith R points It is not calculated according to formula (15), (16), (17):
In formula:
RijThe antibiotic rate of-every anti-bacteria stainless steel sample, %；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3, 4,5；
RiThe antibiotic rate of-every group anti-bacteria stainless steel sample, %；Sample group i=1,2,3；
R-every batch of anti-bacteria stainless steel sample antibiotic rate, %；
CAtijAnd TAtij- every antimicrobial sample and control sample recycle the average ATP concentration of viable bacteria after cultivating for 24 hours, 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 after cultivating for 24 hours, 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；
aA- low concentration standard curve lgCA-lgIASlope；
(5) antibacterial activity value A is calculated
It tests under condition for validity, the antibacterial activity value A of every anti-bacteria stainless steel sampleij, every group and every batch of sample antibacterial activity Value AiIt is calculated respectively according to formula (18), (19), (20) with A:
In formula:
AijThe antibacterial activity value of-every anti-bacteria stainless steel sample；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3, 4,5；
AiThe antibacterial activity value of-every group anti-bacteria stainless steel sample, sample group i=1,2,3；
A-every batch of anti-bacteria stainless steel sample antibacterial activity value；
FijAnd GijThe bacterium increasing value of-every control sample and antimicrobial sample after cultivating for 24 hours, sample group i=1,2,3；Often Group sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and after cultivating for 24 hours 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；
With- every 0h contact and after cultivating for 24 hours 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；
aA- low concentration standard curve lgCA-lgIASlope；
(6) data revision of the convention requirement: the concentration and inoculation bacterium solution, the viable bacteria ATP of sample recovered liquid to ATP standard serial solution are dense Spend CAWhen being demarcated, with reference to the data revision of the convention regulation in GB 4789.2-2016 in relation to clump count；Work as CALess than 100mol/L When, " rounding up " round numbers；Work as CAWhen not less than 100mol/L, preceding 2 bit digital is taken after the 3rd bit digital " rounding up ", after Face replaces digit with 0；Can also be indicated with 10 exponential form, " rounding up " afterwards use two effective digitals, control sample and After antimicrobial sample is contacted through 0h and cultivated for 24 hours, reclaim liquid phase is to fluorescent strength determining value round numbers, antibiotic rate Rij、Ri, R calculate As a result three effective digitals are taken；Bacterium increasing value Fij、GijWith antibacterial activity value Aij、Ai, A calculated result take two effective digitals；
(7) uncertainty of measurement: this patent method mainly passes through calculating control sample and antimicrobial sample and contacts through 0h and cultivate for 24 hours 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 ATP fluorimetric assay for biological materials method being applied to the performance test of stainless steel antibacterium Reproducibility；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 cultivating for 24 hours 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 formulaWithAfter respectively every group of control sample and antimicrobial sample are contacted through 0h and cultivated for 24 hours, respectively The relative intensity of fluorescence measured value of Duplicate Samples):
15 control samples, 15 antimicrobial samples and 15 control samples after cultivating for 24 hours 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 are trained for 24 hours 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 cultivating for 24 hours that every group of 0h is contacted Product, standard deviation of the reclaim liquid phase to fluorescent strength determining valueAnd Respectively according to formula (25) and (26) (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 formulaWithAfter respectively every group of control sample and antimicrobial sample are contacted through 0h and cultivated for 24 hours, respectively The relative intensity of fluorescence measured value of Duplicate Samples):
15 control samples, 15 antimicrobial samples and 15 control samples after cultivating for 24 hours of 3 groups of 0h contact, 15 it is anti- Bacterium sample, standard deviation of the reclaim liquid phase to fluorescent strength determining valueAndRespectively 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 are trained for 24 hours After supporting, the relative intensity of fluorescence measured value of each Duplicate Samples):
9. the detection method of anti-bacteria stainless steel bacteria resistance energy according to claim 1, which is characterized in that the result Evaluation 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:
If using antibiotic rate R as correlated performance evaluation index: working as Rij/Ri(staphylococcus aureus and sand when/R < 90% Door Salmonella Rij/Ri/ R < 80%), sample is without antibacterial actions；As 90%≤Rij/Ri(staphylococcus aureus when/R < 99% With 80%≤R of salmonellaij/Ri/ R < 90%), sample has antibacterial actions；As 99%≤Rij/RiWhen/R < 99.9% (90%≤R of staphylococcus aureus and salmonellaij/Ri/ R < 99%), sample antibacterial actions are stronger；Work as Rij/Ri/R≥ (staphylococcus aureus and salmonella R when 99.9%ij/Ri/ R >=99%), sample antibacterial actions are extremely strong；
If using antibacterial activity value A as correlated performance evaluation index: working as Aij/AiWhen/A < 1.0 (staphylococcus aureus and Salmonella Aij/Ai/ A < 0.5), sample is without antibacterial actions；As 1.0≤Aij/AiWhen/A < 2.0 (staphylococcus aureus and 0.5≤A of salmonellaij/Ai/ A < 1.0), sample has antibacterial actions；As 2.0≤Aij/Ai(golden yellow Portugal when/A < 3.0 1.0≤A of grape coccus and salmonellaij/Ai/ A < 2.0), sample antibacterial actions are stronger；Work as Aij/AiIt is (golden yellow when/A >=3.0 Staphylococcus and salmonella Aij/Ai/ A >=2.0), sample antibacterial actions are extremely strong；
(2) as the antibiotic rate R of certain group (part) anti-bacteria stainless steel samplei(Rij) or antibacterial activity value Ai(Aij) and other two group (four Part) bacteria resistance of sample when can be compared to a levels are at least differed, extracts one group of (part) sample again and repeats to test；It calculates It is through ATP bioluminescence lgCA─lgIAThe antibiotic rate or antibacterial activity value that calibration curve method measures, if two groups of front and back antibacterial is not Steel sample antibacterium performance level of becoming rusty is identical, then abandons it；Take other two groups (four) remaining sample antibiotic rate Ri(Rij) or antibacterial Activity value Ai(Aij) arithmetic mean of instantaneous value as batch (group) anti-bacteria stainless steel sample bacteria resistance can evaluation result.
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