CN109762871A - A kind of mixture by single sulfonic acid tetrazolium and PMS derivative is used for the purposes and its detection method of microorganism detection - Google Patents
A kind of mixture by single sulfonic acid tetrazolium and PMS derivative is used for the purposes and its detection method of microorganism detection Download PDFInfo
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Abstract
The invention belongs to technical field of biomedical detection, more particularly to single sulfonic acid tetrazolium is used for the purposes and method of microorganism detection；It is nontoxic using the mixture reagent of novel single sulfonic acid tetrazolium and PMS derivative, can be incubated for altogether with microorganism, have the characteristics of color change real-time monitoring bacterium, provide a kind of detection method of quick bacteria property；Detection reagent selects the mono- sulfonic acid tetrazolium detection architecture of EZMTT, and good linear dose-relationship is presented on bacterial number living；Test agent passes through single sulfonic acid tetrazolium reactant in the absorbance value of 450nm, obtains EC50Curve carries out bacterial action evaluation.
Description
Technical field
The invention belongs to technical field of biomedical detection, more particularly to by single sulfonic acid tetrazolium and PMS derivative
Mixture is used for the purposes and method of microorganism detection.
Background technique
Mycobacterium tuberculosis (M.tuberculosis), is commonly called as tubercle bacillus, is to cause pathogen lungy.It can invade
Each organ of whole body, but be most common with pulmonary tuberculosis.Tuberculosis is still important infectious disease so far.Estimate 1/3 sense in world population
Contaminate mycobacterium tuberculosis.According to WHO, every year there are about 8,000,000 new cases generation, at least 3,000,000 people die of the disease.China is built
The death rate reaches 200-300 people/100,000 before state, occupies first of various disease death reasons, and living standards of the people improve after the founding of the state, health
Condition improvement, has especially carried out mass prevention and mass treatment, the universal bcg vaccination of children, and morbidity and mortality lungy greatly drop
It is low.It should be noted that some areas are because of the reasons such as AIDS, drug abuse, the application of immunosuppressor, excessive drinking and poverty, hair in the world
Sick rate is again on the rise.There are Bacillus tuberculosis and bacillus tuberculosis bovis to human disease, anonymous mycobacteria can also
Cause similar tuberculosis sample lesion, but rare.
The elongated slightly curved song of tubercle bacillus, the garden Duan Jidun, size about 1~4 × 0.4um have pod in single or branched arrangement
Film, atrichia, without brood cell.In outmoded lesion and culture, form is not often true to type, and can be in granular form, spherical, the stub of string
Shape, long filament shape etc..Generally common Qi-Ni Shi Na Shi (Ziehl-Neelsen) the acid fast staining dyeing of tubercle bacillus, tuberculosis bar
Bacterium dyes red, other nonacidfast bacteriums and cytoplasm matter etc. are blue.The acid-resisting of tubercle bacillus depends on institute in cell wall
Residue containing mycolic acid is related with the integrality of cell wall lamina propria.The detection method of tubercle bacillus is few, traditional direct detection
Method has plate smear, Grain-negative, fluorescence colour, Ziehi-Neelsen stain, micro- sem observation etc., indirectly there is PCR detection method,
Fluorescent immune method etc., operating procedure is various, and detection of complex, the time is long, at high cost etc..
Therefore, clinical diagnosis takes the appropriate sample of lesions position mainly according to the type of tubercle bacillus affection.Such as pneumonia knot
Core, which is taken, coughs up phlegm and (preferably takes cough up phlegm for the first time in the morning, picking band blood or phlegm)；Kidney or tuberculosis of bladder with sterile urethral catheterization or take
Mud-stream urine；Intestinal tuberculosis takes stool sample, and tubercular meningitis carries out spinal column puncture and takes cerebrospinal fluid；Pyothorax, pleurisy, abdomen
Film inflammation or tuberculosis of bone marrow etc., which then puncture, takes pus.
(1) direct smear dyes
Coughing up phlegm can direct smear.It is dyed with luxuriant~Na Shi method, if microscopy finds acid fast bacteria, it may be possible to tubercle bacillus,
But it should usually report: " finding acid fast bacteria ", because non-pathogenic resistance acid-fast bacilli may be contaminated in sample, only according to form
Dyeing not can determine that it is tubercle bacillus, need to further be separately cultured identification.If tubercle bacillus amount is few in sample, miscellaneous bacteria and impurity are more
When, direct smear is not easy to detect (generally require every milliliter of sputum contain 100,000, tubercle bacillus or more could detect), should be concentrated
After collecting bacterium, then smear staining microscopy, to improve detection positive rate.
The sterile cerebrospinal fluid directly taken, urethral catheterization or midstream urine can be directly with centrifugation collection bacterium.It coughs up phlegm and stool sample
Concentration collection bacterium is more because containing miscellaneous function bacterium, need to use 4%NaOH or 3%HCL or 6%H2SO4Processing, then, with centrifugal by tuberculosis
Bacillus concentration is gathered in tube bottom, then taking precipitate smear is made acid-fast stain inspection, is separately cultured or animal experiment.
(2) it is separately cultured
Growth of bacillus tubercle is slow, and culture period is long, when the sediment that collection bacterium is concentrated with acid-base neutralization, is inoculated in solid culture
On base, to seal with wax, mouth prevents drying.37 DEG C culture 4~6 weeks after inspection result.According to slow growth, bacterium colony drying, graininess,
For cheese color as cauliflower-shaped, thallus dyeing acid-resisting is strong, and majority is tubercle bacillus.If bacterium colony, thallus dyeing are not all true to type, then may
For anonymous mycobacteria, test should be further differentiated.
(3) animal experiment
Common cavy or suslik identify solidifying isolated culture and toxicity test like tubercle bacillus.Concentration collection bacterium of learning from else's experience is handled
1.0 milliliters of sample to be injected in cavy groin subcutaneous, through 3~4 weeks breeding observings, such as there is regional glandular enlargement, it is thin
Or tuberculin test is positive, it can timely dissect: if after observation 6~8 weeks, having not yet to see hair patient, also want dissect.It should be infused when dissect
Whether there is or not tuberculosis for the internal organs such as meaning observation lymph node, liver, spleen, lung.
Containing NAD (P) H, therefore common tetrazolium detects living cells, but traditional MTT, CCK8, WST8, MTS method pair
Cell is toxic can only to detect a few houres.There is plurality of articles report for CCK8 and WST8, CCK8 is detected with tumour cell, hair
After now educating 12 hours altogether, cell inactivates substantially.
Summary of the invention
The present invention, which has studied, a kind of to be established a kind of detection that sensitivity is quick and easy using EZMTT reagent and grows bacterium slowly
The diagnostic method of (such as tubercle bacillus).NAD(P)+/ NAD (P) H is a kind of important cofactors, is present in all bacteriums,
It participates in a variety of life processes, including energetic supersession, mitochondrial function, calcium homeostasis, oxidative stress, gene tune as reaction medium
Section, immune function, aging and cell death etc..NAD(P)+/ NAD (P) H is often detected with tetrazolium, but traditional MTT,
CCK8, WST8, MTS method are toxic to cell, can only detect a few houres, and cause cell death.Therefore, a kind of based on thin
Born of the same parents are horizontal, it is sensitive it is nontoxic, can the detection method of prolonged incubation carry out microbioassay, keep in scientific research, environment, medical diagnosis
Deng application in have the function of key.
Experimental principle: single sulfonic acid tetrazolium is tried as a kind of novel single sulfonic acid tetrazotized zole compound in electronics coupled
In the presence of agent 1-methoxy PMS, it can be restored by Intramitochondrial dehydrogenase and generate the orange-yellow of high water soluble
Formazan product (formazan) measures 450nm wavelength OD value by microplate reader.Living cells quantity is more, then absorbance is higher.
Single corresponding formazan product height of sulfonic acid tetrazolium is water-soluble, easy to operate, the non-toxic, sensitivity to microorganism
There is inoxidizability drug screening not generate false positive, therefore quickly detect in clinical bacteria for height, unknown bacterial multiplication measurement,
The applications such as bacterial drug resistance measurement above have broad prospects.
It is nontoxic using novel single sulfonic acid tetrazolium reagent, can be incubated for altogether with microorganism, have the characteristics of color change real-time
Bacterium is monitored, a kind of detection method of quick bacteria property is provided.Detection reagent selects the mono- sulfonic acid tetrazolium detection of EZMTT
Good linear dose-relationship is presented on bacterial number living in system；Test agent passes through single sulfonic acid tetrazolium reactant and exists
The absorbance value of 450nm, obtains EC50Curve carries out bacterial action evaluation.
Detection method of the invention is a kind of super-sensitive detection method based on bacterium.It is provided in the method for the present invention
The design of mixture of single sulfonic acid tetrazolium and phenazinium methyl sulfate (PMS) derivative, can be with slip-knot to microorganism nontoxicity
Core bacillus is incubated for several weeks jointly, does not influence microbial activity, easy to operate, and quickly, single sulfonic acid tetrazolium reagent is mild for detection
And the cell after detecting can be further used for subsequent experimental through rinsing；Single sulfonic acid tetrazolium reagent is not easy anti-with reducing substances
It answers, interferes small；The reagent is applied widely, including the detection after environmental microorganism enrichment, all kinds of detections of Clinical microorganism.
Including following content: a kind of that the mixture of single sulfonic acid tetrazolium and PMS derivative is used for microorganism detection
Purposes, by the design of mixture of single sulfonic acid tetrazolium and phenazinium methyl sulfate (PMS) derivative for growing microorganism slowly
Prolonged incubation, tracking growth, the purposes for growing doubling time detection, cellular drug resistance, Enrichment of bacteria indicator.
A kind of method for mixing analyte detection microorganism of list sulfonic acid tetrazolium and PMS derivative, the form formula
Concentration containing single sulfonic acid tetrazolium is 0.1~10mM, and the concentration of phenazinium methyl sulfate (PMS) derivative is 1~250 μM,
The dosage form is non-toxic and does not influence microorganism growth, can be 1 hour~30 days with the long-term co-incubation of bacterium, the finger as bacterium living
Show agent；
Single sulfonic acid tetrazolium chemical formula are as follows:
；Described phenazinium methyl sulfate (PMS) derivative includes:
Straight chain, branch, the cyclic substituents of R=1-18 carbon atom, it may include other N, O, S, P, Si5 and halogen etc. are former
Son.Such as Methyl-PMS, ethyl-PMS, hexyl-PMS, Cyclohexyl-PMS, C18-PMS etc.,
Preferably, the mixture by single sulfonic acid tetrazolium and PMS derivative is used for the purposes of microorganism detection,
The microorganism includes: pathogenic tubercle bacillus, and corynebacterium diphtheriae, Mycobacterium leprae produce water bacterium door Aquificae, thermobacillus door
Therm, thermally desulfurizing bacillus door Thermodesulfobacteria, abnormal cocci-Thermus door Deinococcus-Tmus produce
Golden bacterium door Chrysiogenetes, green curved bacterium door Chloroflexi, hot germ door Thermomicrobia, nitrification spirillum door
Nitrospirae, deferrization bacillus door Deferribacteres, Cyanophyta Cyanobacteria, green bacterium door Chlorobi, deformation
Bacterium door Proteobacteria, Firmicutes Firmicutes, actinomyces door Actinobacteria float mould door
Planctomycetes), Chlamydia door Chlamydiae, conveyor screw door Spirochaetes, cellulomonas door
Fibrobacteres, acidfast bacilli door Acidobacteria, Bacteroidetes Bacteroidetes, Flavobacterium door Flteria, sheath
Rouge bacillus door Sphingobacteria, Fusobacterium door Fusobacria, wart germ door Verrucomicrob, net group bacterium door
Dictyoglomi, bud monad door Gemm, staphylococcus aureus (the golden aerobic coccus bacterium of Portugal's Grain-positive), epidermis grape ball
Bacterium, α type Streptococcus hemolyticus, β type Streptococcus hemolyticus, non-Streptococcus hemolyticus, pneumococcus, enterococcus, the aerobic ball of Gram-negative
Bacterium for example meningococcus, gonococcus, mora catarrh bacterium, Gram-negative aerobasilus such as acinetobacter (acinetobacter anitratus,
Acinetobacter lwoffii), pseudomonas (Pseudomonas aeruginosa and other pseudomonads), Bacillus foecalis alkaligenes, Brucella, pertussis
Bacillus, Legionnella；Buttiauxella such as enterobacteriaceae lactobacteriaceae (Escherichia coli, citrobacter category, detection of Salmonella
Category, Shigella, Klebsiella, Serratia, Proteus, Pu Lufeideng Pseudomonas, Morganella), yersinia pestis with
And Bacillus influenzae；Vibrionaceae such as comma bacillus, ElTor vibrios, vibrio parahaemolytious, Aeromonas hydrophila；Grain-positive anaerobism ball
Bacterium such as peptococcus, peptostreptococcus；Grain-negative anaerobic cocci such as Fei Shi coccus, such as fragile class bar of gram-negative facultatively anacrobic rod
Bacterium, fusiform bacilarmature；Form bacterium such as bacillus anthracis, wax-shaped bacillus, clostridium tetani, Bacillus perfringens, the meat poisoning bar of brood cell
Bacterium, clostridium difficile；The gram negative bacilli such as listerisa monocytogenes in mjme, erysipelothrix ruhsiopathiae of brood cell are not formed.
Preferably, the method for the mixing analyte detection microorganism by single sulfonic acid tetrazolium and PMS derivative, including
Following steps:
Step 1: by microbionation in the saturating of the various types of cells culture medium for filling the single sulfonic acid tetrazolium detection reagent of addition
Isotropic disk, inoculating cell 1 to 105A every ml；
It is bred 1 hour or more Step 2: being put into incubator, every 1 hour-a few weeks, measures the absorbance of 450nm, marked
Directrix curve observes color change and dose response.
Preferably, the method for the mixing analyte detection microorganism by single sulfonic acid tetrazolium and PMS derivative, by this
The mixture of single sulfonic acid tetrazolium and PMS derivative is used to detect the doubling time of tubercle bacillus, includes the following steps:
The transparent of the nutrient chemical that single sulfonic acid tetrazolium detection reagent is added is filled Step 1: tested bacteria is inoculated in
Plate, inoculated bacteria, 2 times of dilutions are put into incubator and cultivate 1 hour；
Step 2: detecting within every 6~24 hours the absorbance of 450nm for weeks on end, the time that observation signal multiplication needs is just
It is the doubling time of tubercle bacillus.
Preferably, the method for the mixing analyte detection microorganism by single sulfonic acid tetrazolium and PMS derivative, by this
The mixture of single sulfonic acid tetrazolium and PMS derivative is used to detect the Resistance detection of tubercle bacillus, includes the following steps:
Drug such as antibiotic, list containing culture medium sulfonic acid tetrazolium inspection is added Step 1: tested bacteria is inoculated in and is filled
The transparent panel of the specific bacteria culture medium of test agent, inoculated bacteria are put into incubator and cultivate 1 hour；
Step 2: the absorbance of every a few week detection 450nm of 6~24 hours continuitys, observation signal variation judge that cell is raw
Long inhibition or cell death.
Preferably, the method for the mixing analyte detection microorganism by single sulfonic acid tetrazolium and PMS derivative, by this
The mixture of single sulfonic acid tetrazolium and PMS derivative is used to detect the quick detection of tubercle bacillus, includes the following steps:
Step 1: biological sample 0.1~10ml of sputum after enzymolysis processing, is diluted 1~5 times with aqua sterilisa, centrifugal enrichment
Tubercle bacillus；
Step 2: high activity culture medium, antibiotic and single sulfonic acid tetrazolium detection reagent is added in the tubercle bacillus of enrichment,
Breed 1 hour~15 days, the absorbances of a few week detection 450nm of every 6 hours continuitys, at the same observation signal multiplication need when
Between；
Step 3: quantify tubercle bacillus quantity using standard curve, and by the signal multiplication time, difference tubercle bacillus and
Other bacteriums.
Preferably, the method for the mixing analyte detection microorganism by single sulfonic acid tetrazolium and PMS derivative, by this
The mixture of single sulfonic acid tetrazolium and PMS derivative diagnoses tubercle bacillus, the screening of enrichment and inhibitor.
Specifically, microorganism detection method is grown to slow based on EZMTT, corresponding culture is used according to different microorganisms
Base.It is the experimental method of different researchs below:
1. tubercle bacillus is in fluid nutrient medium, after 37 DEG C of stationary culture saturations, it is inoculated in and fills addition four nitrogen of single sulfonic acid
96 orifice plates (2 times of dilutions) of azoles salt detection reagent culture medium, 37 DEG C of cultivations measure the absorbance of 450nm daily, carry out t1/2 times
Increase time detection.
Tubercle bacillus culture medium: including but is not limited to Russell medium, and coulee occasion time egg medium revives and leads to culture medium,
Middle brook 7H9 (7H10,7H11) culture medium, Bactec 460TM TB culture medium etc..
2. acquiring 5~1000ml of sample in varying environment, single sulfonic acid tetrazole containing nutrient chemical is added in high speed centrifugation enrichment
Salt detection reagent is bred 1 hour or more, measures the absorbance and time effect of 450nm, carries out Bacteria Detection characterization and evaluation living.
3. acquiring sputum, enzymatic hydrolysis is added single sulfonic acid tetrazolium detection reagent containing nutrient chemical, each antibiotics and breeds,
The absorbance of 450nm is measured, the dosage effect and time effect evaluation of bacterial action detection are carried out.Quickly detection microorganism and micro-
The drug resistance of biology.
4. Escherichia coli are incubated in bacterial liquid culture medium, 37 DEG C, 220~250rpm shaking table culture are inoculated in and fill
96 orifice plates (2 times of dilutions) of culture medium, are added single the cultivation of sulfonic acid tetrazolium detection reagent 1 hour or more, continuously survey within every 1 hour
Determine the absorbance of 450nm, carries out the detection of t1/2 doubling time.
Bacterial liquid culture medium can with LB culture medium be (1.0% tryptone, 0.5% yeast extract, 1.0%NaCl,
pH 7.4)
5. streptomycete, in fermentation medium, 28 DEG C, 220~250rpm shaking table culture are inoculated in and fill the 96 of culture medium
Orifice plate is added single the cultivation of sulfonic acid tetrazolium detection reagent 1 hour or more, the absorbance of every 1 hour METHOD FOR CONTINUOUS DETERMINATION 450nm, into
The row t1/2 doubling time is detected.
Streptomycete culture medium: 3.0% soluble starch, 1.0% sucrose, 0.5% glucose, 0.8% maltose, 1.2%
Soybean cake powder, 0.4% peptone, 1.2% fish meal, 0.6% yeast powder, 0.2% (NH4)2S04, 0.35%MgSO4, 0.02%
KH2P04, 0.4%CaC03, 0.075%NaN03, pH 7.0
Experimental principle
In the presence of electronics coupled reagent phenazine Methylsulfate (PMS) derivative, single sulfonic acid tetrazolium inspection
Test agent can be restored by dehydrogenase in NAD (P) H system or cell mitochondrial, generate the orange-yellow formazan of high water soluble
Product (formazan), there is UV absorption at 450nm.
Detection method of the invention is a kind of super-sensitive detection method based on cell.It is used in the method for the present invention
The formula of single sulfonic acid tetrazolium and phenazinium methyl sulfate (PMS) derivative is suitable for and slow life bacterial growth nontoxicity
The common incubation up to several weeks of long bacterium, for the vigor of detection bacterium, growth cycle and Resistance detection.
Currently, the method for the present invention the reproducibility of (i.e. single sulfonic acid tetrazolium detection reagent measuring method) be using the z factor come
It is assessed, as positive control value and not, celliferous culture medium is used as yin to nontoxic tubercle bacillus (20000, every hole cell)
Property control value.As shown in the following figure, negative control group shows that background peaks are 0.127 ± 0.014Abs, and positive controls numerical value
For 1.021 ± 0.104Abs, therefore, the z factor values of single sulfonic acid tetrazolium detection reagent are 0.7；Show measuring method of the present invention
It is repeated more preferable；Specific effect is shown in Fig. 3.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention without any creative labor, may be used also for those of ordinary skill in the art
To obtain other drawings based on these drawings.
Fig. 1 is to optimize EZMTT detection kit, the effect figure of signal increment variation for tubercle bacillus in embodiment 1
Fig. 2-1,2-2,2-3,2-4 are in embodiment 2 with comparing nontoxic type EZMTT, CCK8, MTS detection reagent and biography
The effect figure of OD60 bacterial concentration detection method of uniting detection tubercle bacillus signal increment variation；
Wherein tulase extension rate is followed successively by 21-211；
EZMTT, cells grown signal increase increase with time, and the cell of extremely low concentration can also be detected；
Cck8, cells grown signal is seldom increase with time, and the cell of slightly lower concentration cannot be detected；
MTS educates 10 days altogether under similarity condition with cell, no signal variation；
OD600 also cannot get result.
Fig. 3 is that tubercle bacillus detects the value-added time effect figure of signal in embodiment 3.
Fig. 4 is embodiment 4, and the weight of single sulfonic acid tetrazolium detection reagent detection tubercle bacillus vigor is assessed using the z factor
Existing property effect diagram.
Fig. 5 is the comparison of tubercle bacillus fluorescence method and EZMTT method in embodiment 5, and clinical bacteria is in combination with its feature times
Increase time progress quick diagnosis.
Fig. 6 is that different compounds are shown by growth inhibition or cytotoxicity progress cell viability evaluation effect in embodiment 6
It is intended to.
Fig. 7 is the linear dose response detection figure of Bacillus coli cells quantity in embodiment 7.
Fig. 8 is the value-added time effect figure of E. coli detection signal in embodiment 8；Since the resolution ratio of color is lower,
It is described herein, is corresponding in turn to 0-6h from the bottom to top in 7 lines.
Fig. 9 is Drug Resistance of E. coli lab diagram in embodiment 9.
Figure 10 is the linear dose response detection figure of 10 Streptomyces cell quantity of embodiment.
Specific embodiment
The present invention will be further described in detail below with reference to the embodiments, following embodiment be explanation of the invention and
The invention is not limited to following embodiments.
Embodiment 1
The optimization of tubercle bacillus EZMTT detection reagent, includes the following steps
Tubercle bacillus is inoculated in fluid nutrient medium (Middle brook 7H9), is directly inoculated in 96 holes of the culture medium filled
Plate mixes different single sulfonic acid tetrazole and PMS derivative, and single sulfonic acid tetrazole takes 1mM and PMS derivative to take 100 μM,
And after being cultivated 5 days with sealed membrane sealing.According to Reagent Protocol, absorbance is measured in 450nm and 600nm, carries out cell growth letter
Number detection.
Embodiment 2
The methods comparison of tubercle bacillus cell viability detection, includes the following steps
Tubercle bacillus (1000 every hole) is inoculated in fluid nutrient medium (Middle brook 7H9), is directly inoculated in the training filled
Support base 96 orifice plates, add different tetrazole detection reagents: CCK8, MTS, EZMTT, and with sealed membrane sealing culture 15 days after.
According to Reagent Protocol, absorbance is measured in the optimal wavelength of corresponding reagent, carries out the detection of cell growth signals.MTS cannot be examined
Suspension cell is surveyed, it is small that CCK8 cannot track signal intensity, and MTS cannot track that signal intensity is small, and the EZMTT of optimization has very strong letter
Number, and signal is grown always.
Embodiment 3
The detection of tubercle bacillus cell doubling time, includes the following steps
Tubercle bacillus is inoculated in fluid nutrient medium (Middle brook 7H9), and 37 DEG C are cultivated 72 hours, due to cell mass
It is poly- to use OD600Detection, therefore after breaing up, it is directly inoculated in and fills the culture medium that single sulfonic acid tetrazolium detection reagent is added
96 orifice plates, successively twice of dilution, and sealed with sealed membrane.The absorbance of measurement 450nm daily, carries out cell growth time effect
Detection.It is shown under anaerobic in Fig. 2 series, the doubling time of tubercle bacillus is about 24 hours.
Embodiment 4
Tubercle bacillus (1000000 cells) is inoculated with or is not inoculated in 96 orifice plates for filling 7H9 fluid nutrient medium.Then again
Single sulfonic acid tetrazolium detection reagent is added to cultivate 5 days, measures the absorbance of 450nm.Fig. 3 shows EZMTT method good reliability (z
Factor=7).
Embodiment 5
Biological sample (100ul) inoculation and (Middle brook 7H9) fluid nutrient medium (band anaerobic containing tubercle bacillus
The liquid 7ml of fluorescence indicator and EZMTT detection agent), it is put into incubator, detection anaerobic fluorescence indicator is shown and EZMTT becomes
Color, closing are cultivated, and measure the absorbance of 450nm daily, carry out the detection of cell growth time effect.Fig. 5 shows that EZMTT can join
It closes and uses, EZMTT Coloring Time was than anaerobic fluorescence indicator report sun time advance 2 days.Therefore it can be used for the quick of tubercle bacillus
Diagnosis.
Embodiment 6
Biological sample (100ul) inoculation and (Middle brook 7H9) fluid nutrient medium (liquid containing tubercle bacillus
7ml), it is not added or adds compound (0-20uM)) it is put into incubator incubation 4 days, add EZMTT to measure the absorbance of 450nm daily, into
The detection of row cell growth time effect.Fig. 6 shows EZMTT discoloration display antibody-resistant bacterium, and the doubling time shows tubercle bacillus
Long growth cycle is extraordinary fast diagnosis method.
Embodiment 7
Bacillus coli cells vigor testing methods, include the following steps
Escherichia coli are inoculated in LB solid medium, 37 DEG C of climatic chamber overnight incubations；1:10~1:20 ratio spreads cultivation
In LB liquid medium, 37 DEG C, 220rpm, cultivate 16~22 hours in shaking table；To after the time, OD is measured600It is 3~5, inoculation
In 96 orifice plates for filling culture medium, successively twice of dilution.It adds single the cultivation of sulfonic acid tetrazolium detection reagent 1 hour or more,
Measure the absorbance of 450nm；Single sulfonic acid tetrazolium is not added in another group of diluting cells, directly uses OD600It is living to carry out cell for tracking
The comparison of power evaluation method.Fig. 7 shows that EZMTT method and OD600 method have relatively good correlation.
Embodiment 8
The all samples of embodiment 8, the absorbance for measuring 450nm per hour carry out the detection of cell growth time effect.
Fig. 8, display EZMTT method have preferably linear and smaller error amount, accurately show that the E.co l i doubling time is 0.5 hour.
Embodiment 9
The Escherichia coli (the not drug resistant 1000-2000 cell of drug resistance) for crossing liquid culture, which are inoculated in, fills LB Liquid Culture
96 orifice plates of base.Add antibiotic (Kan；0-50ug/ml), it then adds single sulfonic acid tetrazolium detection reagent and cultivates 1
Hour or more, measure the absorbance of 450nm.Fig. 9 shows that EZMTT method can the sensitive drug resistance for quickly and easily detecting microorganism
Property.
Embodiment 10
Streptomyces cell vigor testing methods, include the following steps
Streptomycete is inoculated in streptomycete culture medium, 28 DEG C, 250rpm, cultivates 18~22 hours in shaking table；To after the time, survey
Obtain OD600It is 3~5, is inoculated in 96 orifice plates for filling culture medium, successively twice of dilution.Add single sulfonic acid tetrazolium detection examination
Agent is cultivated 0~1 hour, and the absorbance of 450nm is measured, and is carried out cell viability and is evaluated Figure 10.
In addition, it should be noted that, the specific embodiments described in this specification, product reagent named title etc. can
With difference.The equivalent or simple change that all principles described according to the invention patent design are done, is included in the invention patent
In protection scope.Those skilled in the art can do various repair to described specific embodiment
Change or supplement or be substituted in a similar manner, without departing from structure of the invention or surmounts the claims and defined
Range, be within the scope of protection of the invention.
Claims (8)
1. the purposes that a kind of mixture by single sulfonic acid tetrazolium and PMS derivative is used for microorganism detection, it is characterised in that:
By the microorganism that the design of mixture of single sulfonic acid tetrazolium and phenazinium methyl sulfate (PMS) derivative is slow for the speed of growth
Prolonged incubation, tracking growth, growth doubling time detection, cellular drug resistance, Enrichment of bacteria indicator purposes.
2. a kind of method of the mixing analyte detection microorganism of list sulfonic acid tetrazolium and PMS derivative, it is characterised in that: described
Concentration of the form formula containing single sulfonic acid tetrazolium is 0.1~10mM, and the concentration of phenazinium methyl sulfate (PMS) derivative is 1
~250 μM, the dosage form is non-toxic and does not influence microorganism growth, can be 1 hour~30 days with the long-term co-incubation of bacterium, as work
The indicator of bacterium；
Single sulfonic acid tetrazolium chemical formula are as follows:
Described phenazinium methyl sulfate (PMS) derivative includes:
Straight chain, branch, the cyclic substituents of R=1-18 carbon atom, it may include the atoms such as other N, O, S, P, Si5 and halogen.
3. the mixture according to claim 1 by single sulfonic acid tetrazolium and PMS derivative is used for microorganism detection
Purposes, it is characterised in that: the microorganism includes: pathogenic tubercle bacillus, and corynebacterium diphtheriae, Mycobacterium leprae produce water bacterium door
Aquificae, thermobacillus door Therm, thermally desulfurizing bacillus door Thermodesulfobacteria, abnormal cocci-Thermus door
Deinococcus-Tmus, pan bacterium door Chrysiogenetes, green curved bacterium door Chloroflexi, hot germ door
Thermomicrobia, nitrification spirillum door Nitrospirae, deferrization bacillus door Deferribacteres, Cyanophyta
Cyanobacteria, green bacterium door Chlorobi, Proteobacteria Proteobacteria, Firmicutes Firmicutes, actinomyces
Door Actinobacteria floats mould door Planctomycetes), Chlamydia door Chlamydiae, conveyor screw door
Spirochaetes, cellulomonas door Fibrobacteres, acidfast bacilli door Acidobacteria, Bacteroidetes
Bacteroidetes, Flavobacterium door Flteria, sphingolipid bacillus Sphingobacteria, Fusobacterium door Fusobacria, wart
Germ door Verrucomicrob, net group bacterium door Dictyoglomi, bud monad door Gemm, staphylococcus aureus (golden Portugal's leather
Blue positive aerobic coccus bacterium), staphylococcus epidermis, α type Streptococcus hemolyticus, β type Streptococcus hemolyticus, non-Streptococcus hemolyticus, pneumonia
Coccus, enterococcus, the aerobic coccus of Gram-negative such as meningococcus, gonococcus, mora catarrh bacterium, the aerobic bar of Gram-negative
Bacterium such as acinetobacter (acinetobacter anitratus, Acinetobacter lwoffii), pseudomonas (Pseudomonas aeruginosa and other false unit cells
Bacterium), Bacillus foecalis alkaligenes, Brucella, Bordetella pertussis, Legionnella；Buttiauxella such as enterobacteriaceae lactobacteriaceae
(Escherichia coli, citrobacter category, Salmonella, Shigella, Klebsiella, Serratia, Proteus, Pu Lu
Phenanthrene steps on Pseudomonas, Morganella), yersinia pestis and Bacillus influenzae；Vibrionaceae such as comma bacillus, ElTor vibrios, secondary haemolysis arc
Bacterium, Aeromonas hydrophila；Grain-positive anaerobic cocci such as peptococcus, peptostreptococcus；Grain-negative anaerobic cocci such as Fei Shi
Coccus, gram-negative facultatively anacrobic rod such as bacteroides fragilis, fusiform bacilarmature；Formed brood cell bacterium for example bacillus anthracis, wax-shaped bacillus,
Clostridium tetani, Bacillus perfringens, clostridium botulinum, clostridium difficile；The gram negative bacilli such as monocyte of brood cell is not formed
Increasing property Listeria, erysipelothrix ruhsiopathiae.
4. the method for the mixing analyte detection microorganism according to claim 2 by single sulfonic acid tetrazolium and PMS derivative,
It is characterized by comprising following steps:
Step 1: by microbionation in the transparent of the various types of cells culture medium for filling the single sulfonic acid tetrazolium detection reagent of addition
Plate, inoculating cell 1 to 105A every ml；
It is bred 1 hour or more Step 2: being put into incubator, every 1 hour-a few weeks, measures the absorbance of 450nm, obtain standard song
Line observes color change and dose response.
5. the method for the mixing analyte detection microorganism according to claim 2 by single sulfonic acid tetrazolium and PMS derivative,
The mixture of the list sulfonic acid tetrazolium and PMS derivative is used to detect the doubling time of tubercle bacillus, it is characterised in that packet
Include following steps:
Step 1: tested bacteria to be inoculated in the transparent panel for filling the nutrient chemical that single sulfonic acid tetrazolium detection reagent is added, connect
Kind bacterium, 2 times of dilutions are put into incubator and cultivate 1 hour；
Step 2: detecting within every 6~24 hours the absorbance of 450nm for weeks on end, the time that observation signal multiplication needs is exactly to tie
The doubling time of core bacillus.
6. the method for the mixing analyte detection microorganism according to claim 2 by single sulfonic acid tetrazolium and PMS derivative,
The mixture of the list sulfonic acid tetrazolium and PMS derivative is used to detect the Resistance detection of tubercle bacillus, it is characterised in that
Include the following steps:
Drug such as antibiotic, list containing culture medium sulfonic acid tetrazolium detection examination is added Step 1: tested bacteria is inoculated in and is filled
The transparent panel of the specific bacteria culture medium of agent, inoculated bacteria are put into incubator and cultivate 1 hour；
Step 2: the absorbance of every a few week detection 450nm of 6~24 hours continuitys, observation signal variation judge cell growth suppression
System or cell death.
7. the method for the mixing analyte detection microorganism according to claim 2 by single sulfonic acid tetrazolium and PMS derivative,
The mixture of the list sulfonic acid tetrazolium and PMS derivative is used for the quick detection of tubercle bacillus in clinical sample, feature
It is to include the following steps:
Step 1: biological sample 0.1~10ml of sputum after enzymolysis processing, is diluted 1~5 times with aqua sterilisa, centrifugal enrichment tuberculosis
Bacillus；
Step 2: high activity culture medium, antibiotic and single sulfonic acid tetrazolium detection reagent is added in the tubercle bacillus of enrichment, breed
1 hour~15 days, the absorbance of every a few week detection 450nm of 24 hours continuitys, while observing the time required for cell doubles；
Step 3: quantifying tubercle bacillus quantity using standard curve, and by 450nm signal, determines the doubling time, distinguish tuberculosis
Bacillus and other bacteriums.
8. the method for the mixing analyte detection microorganism according to claim 2 by single sulfonic acid tetrazolium and PMS derivative,
The mixture of the list sulfonic acid tetrazolium and PMS derivative diagnoses tubercle bacillus or other clinical bacterias, is enriched with
With the screening of inhibitor.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111304278A (en) * | 2020-02-24 | 2020-06-19 | 杭州伽玛生物科技有限公司 | Drug sensitive kit |
| CN111321195A (en) * | 2020-03-14 | 2020-06-23 | 浙江工业大学 | EZMTT-based method for continuously determining cancer cell proliferation inhibition effect |
| CN111500670A (en) * | 2020-04-20 | 2020-08-07 | 杭州伽玛生物科技有限公司 | High-throughput drug sensitivity detection kit and use method and application thereof |
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| CN101551382A (en) * | 2009-05-12 | 2009-10-07 | 镇江旋光生化有限公司 | Cell viability detection kit and preparation method and application thereof |
| CN103808678A (en) * | 2014-02-25 | 2014-05-21 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Method and kit for measuring cell activity and application of kit |
| CN105418526A (en) * | 2015-11-09 | 2016-03-23 | 杭州伽玛生物科技有限公司 | Compound with mono-sulfophenyl tetrazole structure and application of compound with mono-sulfophenyl tetrazole structure |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101551382A (en) * | 2009-05-12 | 2009-10-07 | 镇江旋光生化有限公司 | Cell viability detection kit and preparation method and application thereof |
| CN103808678A (en) * | 2014-02-25 | 2014-05-21 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Method and kit for measuring cell activity and application of kit |
| CN105418526A (en) * | 2015-11-09 | 2016-03-23 | 杭州伽玛生物科技有限公司 | Compound with mono-sulfophenyl tetrazole structure and application of compound with mono-sulfophenyl tetrazole structure |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111304278A (en) * | 2020-02-24 | 2020-06-19 | 杭州伽玛生物科技有限公司 | Drug sensitive kit |
| CN111321195A (en) * | 2020-03-14 | 2020-06-23 | 浙江工业大学 | EZMTT-based method for continuously determining cancer cell proliferation inhibition effect |
| CN111500670A (en) * | 2020-04-20 | 2020-08-07 | 杭州伽玛生物科技有限公司 | High-throughput drug sensitivity detection kit and use method and application thereof |
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