CN107421939A - A kind of reagent and its application process of Quantitative detection Susceptibility to antibiotics - Google Patents
A kind of reagent and its application process of Quantitative detection Susceptibility to antibiotics Download PDFInfo
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- CN107421939A CN107421939A CN201710875113.XA CN201710875113A CN107421939A CN 107421939 A CN107421939 A CN 107421939A CN 201710875113 A CN201710875113 A CN 201710875113A CN 107421939 A CN107421939 A CN 107421939A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
- G01N21/763—Bioluminescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56938—Staphylococcus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/9446—Antibacterials
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- 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)
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- 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
A kind of kit of Quantitative detection Susceptibility to antibiotics, having can direct detection bacterium ATP reagent；Once add the latter step of the reagent and realize the cracking of bacterium and ATP release and detection.The kit of the present invention can obtain drug sensitive experiment result in 2 hours.It is low to hardware requirement, any micro-pore plate type luminometer can be used, and automatic sample handling system need not be used.The inventive method is easy to operate, step sample-adding operation, it is not necessary to be injected separately into lysate and ATP detection reagents.MIC value can be determined, and whether reports resistance simultaneously.
Description
Technical field：
Reagent and its application process the present invention relates to a kind of quick detection pathogenic microorganism to Susceptibility to antibiotics.
Background technology：
Conventional sensitivity testing to antibacterials (the Antimicrobial that Clinical microorganism laboratory uses at present
Susceptibility Tests, AST) method mainly have disk diffusion method, dilution method, E-test methods etc..These methods by
Result judge is carried out in observing by the naked eye microorganism growing state, it is often necessary to which cultivating 16 more than hour could report susceptibility
As a result.
Chinese patent《Quick medicine-sensitive detection kit》(ZL201120469811) drug sensitive test is carried out using dilution method, led to
Cross color developing detection method and shorten the experiment used time, but because the sensitivity of color developing detection method is relatively low, need 9-11 hours to obtain
To drug sensitivity tests.
Although self-reacting device drastically increases the speed of drug sensitive test, still need to obtain susceptibility knot at least 4 hours
Fruit.And these instrument costs are higher, basic hospital and relevant clinical section office use and are limited.
Such as the Biomerieux self-reacting devices of VITEK 2, need obtain drug sensitivity tests at the soonest within 4 hours.It is other to face
The self-reacting device of bed detection also includes：BD Phoenix-100 automatic bacterials identification/susceptibility system, Beckman-
Coulter MicroScan microbial identifications and Analysis of Drug Susceptibility system and Thermo Scientific Sensititre
ARIS 2X ID/AST systems etc..These instruments generally using the dynamic variation of turbidimetry observation bacterial number, are limited to compare
The muting sensitivity of turbid method, the bacterial concentration needed for drug sensitive test is larger, and needs to cultivate more than 4h.
Wu Xiao etc. establishes a kind of side of quick detection bacteria antibiotic susceptibility using atriphos-biloluminescence method
Method, based on micro broth dilution method, the bacterium after culture a period of time is detected using ATP biloluminescence methods, passes through calculating
Whether luminance compared with blank judges the resistance of bacterium.Because ATP bioluminescent detections sensitivity is high, can contract
Short culture used time, the sentence read result in 4h, and (ATP biloluminescence method detection bacterium susceptibility technologies are built without high concentration bacterium solution
Vertical and application,《Zhejiang clinical medicine》The 10th phase of volume 17 in October, 2015：1680).
But the above method still has a disadvantage that：
First, the hardware configuration of detector is had higher requirements：
The micro-pore plate type luminometer of the less outfit of basic medical unit must be used, and need optional equipment automatic
Sampling system.
Second, operation is cumbersome, therefore the technology is more difficult in basic hospital use：
1) need successively to be injected separately into two kinds of reagents by automatic sample handling system
Because this method needs to be separately added into two kinds of reagents：Bacterial lysate and ATP detection reagents, are lighted by microwell plate
The automatic sample handling system of detector successively injects lysate and ATP detection reagents；Need at least equipped with two auto injection systems
System.
2) using flash type luminescence reagent, it is necessary to detect luminous value at once after ATP detection reagents are added
The lighting time interval of flash type luminescence reagent is shorter, and luminous intensity is very fast with time attenuation ratio, it is necessary to is adding
" luminous value is detected at once ", otherwise influence testing result after ATP detection reagents.
Third, can not meet the needs of MIC (minimum inhibitory concentration) measure：
The concentration gradient of the antibacterials set in this method is few, can not determine MIC (minimum inhibitory concentration).Therefore the party
Method is qualitative report sensitiveness, is not provided with definite MIC value.And quantitative result MIC value is for instructing clinician to use
Medicine is then even more important.
Therefore, it is necessary to develop a kind of relatively low to equipment requirement, and the quick of MIC (minimum inhibitory concentration) value can be determined
Drug susceptibility test method and kit, be easy to the detection in time of situation of all-level hospitals and relevant clinical section office and monitoring susceptibility
As a result, simple, quick solution is provided for antibacterial therapy.
The content of the invention
The purpose of the present invention is to develop a kind of kit of quick detection Susceptibility to antibiotics, can not only be shorter
Drug sensitivity tests are provided in time, and definite MIC (minimum inhibitory concentration) value is provided, and it is low to hardware requirement.
The invention provides a kind of kit of new quick detection Susceptibility to antibiotics, the kit is with micro meat soup
Based on dilution method, include antibacterials plate, culture medium and bacterial ATP detection reagent., will be to be measured thin during using the kit
The culture medium that bacterium is provided with kit is adjusted to suitable concentration and is added in antibacterials plate, after cultivating 2 hours, by bacterial ATP
Detection reagent is added in antibacterials plate, and then antibacterials plate is put into multi-functional plate reader and detects luminous intensity, root
MIC value is determined according to relative luminous intensity, and reports sensitive, intermediary or resistance.
The invention provides a kind of kit of quick detection Susceptibility to antibiotics, it is characterized in that,：
1) having can direct detection bacterium ATP reagent：
The reagent is the detection reagent containing bacterial ATP releasing agent and bioluminescence material, can realize bacterium with a step
Cracking and ATP release and detection.
The bioluminescence material is wide variety of glow-type bioluminescence material；
Although the main component and luciferase and D- luciferins of wide variety of glow-type bioluminescence material, the ratio of the two is not
Together, the mass ratio of both materials is 1:4 to 1:6, preferably 1:4.7.
Preferable bacterial ATP detection reagent contains bacterial ATP releasing agent, buffer solution, luciferase, D- luciferins and stably
Agent etc..
Wherein, the bacterial ATP releasing agent is preferably surfactant, is pricked selected from cetyl trimethylammonium bromide, benzene
One or more during oronain, Chlorhexidine and Qula are logical.
Preferable bacterial ATP detection reagent is containing chlorhexidine acetate (0.15%-1.20%), triton x-100
(1.5%-2.5%), luciferase (0.02%-0.05%) and D- luciferins (0.1%-0.2%) aqueous solution.The percentage
Than being mass ratio.
Bacterial ATP detection reagent used consists of the following composition in an example of the present invention：N-2- hydroxyethyl piperazines-
N'-2- ethyl sulfonic acids (150-250mM), cetyl trimethylammonium bromide (0.06%-0.10%), chlorhexidine acetate (0.15%-
1.2%), Qula leads to (1.5%-2.5%), magnesium chloride (0.3%-0.5%), ethylenediamine tetra-acetic acid (0.7%-1.0%), fluorination
Sodium (0.05%-0.1%), sodium pyrophosphate (0.01%-0.02%), luciferase (0.02%-0.05%), D- luciferins
(0.1%-0.2%) and water.
The bacterial ATP detection reagent of the present invention can realize bacterial lysate and ATP detection reagents in the prior art with a step
The function of two kinds of reagents.During detection, it is not necessary to first inject lysate, reinject ATP detection reagents, can not only reduce operation step
Suddenly, it is not required that configuration automatic sample handling system.
2) there is the antibacterials plate of series concentration gradient
There are the antibacterials of series concentration gradient, the series concentration gradient is every kind of in the antibacterials plate of the present invention
Medicine has the concentration of ten or more.
For different detection medicines specific concentration value according to be actually needed setting.Such as with reference to CLSI file configurations,
Concentration range includes the explanation break listed by CLSI files, and includes the value of Quality Control bacterium, can meet the needs of MIC measure.
There is bigger difference in the specific concentration gradient of antibacterials, such as piperazine draws west because of the property of antibacterials
The concentration of woods is 12800,6400,3200,1600,800,400,200,100,50 and 25 μ g/mL, and the concentration of Ciprofloxacin is
200th, 100,50,25,12.5,6.25,3.12,1.56,0.78 and 0.39 μ g/mL.
3) any instrument with luminous detection function can be used to be detected
It can be carried out detecting using any instrument with luminous detection function, obtain required result.And it is not required to
Want optional equipment automatic sampling apparatus.
Such as, the micro-pore plate type luminometer that can not only use prior art to use, can also use basic medical unit
There is the multi-functional plate reader (see the embodiment of the present invention) of outfit, which kind of instrument all not need optional equipment to enter automatically using
Sample system.
Because the bacterial ATP detection reagent of the present invention is wide variety of glow-type luminescence reagent, luminous intensity is relatively steady after adding reagent
Determine and can continue for some time, it is not necessary to detected immediately after adding reagent, the detection reagent is added to antibacterials when in use
After plate, then antibacterials plate is put into instrument and detected, it is possible to do not need automatic sample handling system.
Multi-functional plate reader is the instrument that there are outfit in most basic medical unit and relevant clinical section office.And micro-pore plate type
Luminometer is customizations instrument, said mechanism Disposing rate than relatively low.
And the detection that documents method does not have required by prompting can realize its method using multi-functional plate reader is smart
Degree.
A variety of antibacterials can be set according to being actually needed in the antibacterials plate that the kit of the present invention provides；Antibacterial
The species of medicine changes according to the species of bacterium to be measured, and every kind of antibacterials include multiple concentration, the forms of antibacterials according to
Depending on the concrete condition of different pharmaceutical.In the case of conditions permit, preferable drug form is the drying for anchoring at orifice plate bottom
Powdery or graininess, it is easy to the keeping of kit to transport.
The culture medium that the kit of the present invention provides is the standard with reference to CLSI (U.S. clinical and laboratory standards institute),
Depending on the change of the species of bacterium to be measured, Mueller-Hinton meat soups (CAMHB), the RPMI- of regulation cation can be selected
Culture medium needed for 1640 broth bouillons or other drug sensitive tests.
The application process of kit of the present invention is：Tested bacteria is added to after cultivating 2h in antibacterials plate, by institute
State bacterial ATP detection reagent to be added in antibacterials plate, then whole antibacterials plate is put into multi-functional plate reader and examined
Luminous intensity is surveyed, calculates relative luminous intensity of the antibacterials hole with respect to the positive hole without antibacterials.
The above-mentioned tested bacteria being added in antibacterials plate is that the tested bacteria of required concentration is adjusted to culture medium, described
Required concentration, is decided according to the actual requirements, such as with reference to CLSI standards determine suitable concentration (concentration of non-severe bacteria is about 5 ×
105CFU/mL)。
The computational methods of relative luminous intensity are as follows：
Relative luminous intensity=(antibacterials hole luminous intensity/positive hole luminous intensity) × 100%
The MIC value (minimum inhibitory concentration) of the medicine is obtained from the minimum drug concentration of relative luminous intensity≤80%, together
When with reference to CLSI standards resistance whether conclusion obtained.The break concentration provided according to CLSI, when MIC≤sensitive break, is treated
It is sensitive to the antibacterials to survey bacterium；When MIC >=resistance break, bacterium to be measured is to antibiotic resistance；When MIC is between two breaks
Between when, bacterium to be measured is intermediary to the susceptibility of the antibacterials.
It is to 30 kinds different that ETEC and staphylococcus aureus have detected using the kit of the present invention respectively
The sensitiveness of antibacterials；10 plants of enterobacterias are have detected respectively to ampicillin and cephazoline, 10 plants of staphylococcuses pair simultaneously
The sensitiveness of erythromycin and penicillin.Detection above can report MIC value in 2h, and be reported with CLSI standard method
Value compared to all CLSI standards allow error range in.
The kit of the present invention can not only be quantified and detected, can also qualitative detection.
In practical application, if only needing that antibiotic resistance whether conclusion rapidly learnt, without obtaining MIC
It is more easy with the kit operation of the present invention in the case of value：Only use negative hole, positive hole and the antibacterial of antibacterials plate
Medicine break concentration hole (including sensitive break and resistance break), it is consistent when remaining operation is with above-mentioned quantitatively detection.
Concrete operation method is：Tested bacteria is added to after cultivating 2h in antibacterials plate, the bacterial ATP is detected
Reagent is added in antibacterials plate, is then put into antibacterials plate and the luminous strong of corresponding aperture is detected in multi-functional plate reader
Degree, calculate relative luminous intensity of the antibacterials hole with respect to the positive hole without antibacterials.Antibacterials break concentration hole reference
CLSI standards determine that different microorganisms and antibacterials have bigger difference.Such as detection enterobacteriaceae is to ammonia benzyl
Negative hole, positive hole, 400 μ g/mL (sensitive break) and 1600 μ g/mL (resistance break) hole are chosen during the sensitiveness in XiLin；
Negative hole, positive hole, 100 μ g/mL (sensitive break) and 400 μ g/ are chosen when detecting enterobacteriaceae to the sensitiveness of cephazoline
ML (resistance break) hole.
The computational methods of relative luminous intensity are as follows：
Relative luminous intensity=(antibacterials hole luminous intensity/positive hole luminous intensity) × 100%
When sensitive break and the relative luminous intensity of resistance break concentration all≤80%, then tested microorganism is to the antimicrobial
Thing is sensitive；As the relative luminous intensity ＞ 80% of sensitive break concentration, and relative luminous intensity≤80% of resistance break concentration,
Then tested microorganism is to the antibacterials sensitivity intermediary；When sensitive break and the relative luminous intensity of resistance break concentration all
＞ 80%, then tested microorganism is to the antibiotic resistance.
It is an advantage of the invention that：
1st, drug sensitive experiment result is more rapidly obtained
Compared with documents, saving of time half.Drug sensitive experiment result can be obtained within 2 hours.
2nd, hardware requirement is low, can meet basic hospital and the needs of relevant clinical section office
Micro-pore plate type luminometer or the conventional multi-functional plate reader with luminous detection function can be used to carry out
Monitoring, it is not necessary to use automatic sample handling system.
3rd, it is easy to operate
One step sample-adding operation, it is not necessary to be injected separately into lysate and ATP detection reagents.
4th, MIC value can be determined, and whether reports resistance simultaneously
It is not only qualitative determination drug sensitivity tests, but obtains MIC value, clinician can be helped to select in good time properly
Medicine, avoid producing or aggravating the resistance of bacterium early.
Brief description of the drawings：
The luminous value of the ETEC detection of Fig. 1 varying numbers；
The luminous value of the staphylococcus aureus detection of Fig. 2 varying numbers.
Embodiment
Following instance is merely to illustrate and explain the present invention, and can not limit the present invention.Those skilled in the art are according to the present invention
Principle and method be possible to reach the effect of the present invention with other different medicines, bacterium and ATP detection reagents.
Embodiment 1 prepares bacterial ATP detection reagent
Bacterial ATP detection reagent is prepared according to following steps：
(1) 100mL 150-250mM N-2- hydroxyethyl piperazine-N'-2- second sulphurs are prepared using freshly prepared high purity water
Sour (HEPES) solution；Addition 0.15-1.20g chlorhexidine acetates, 1.5-2.5mL triton x-100s (Triton X-100),
0.3-0.5g Magnesium dichloride hexahydrates, the ethylenediamine hydrate Sequestrene AAs (EDTA) of 0.7-1.0g bis-；Use 1M sodium hydroxide
After regulation pH value of solution is 7.5, the membrane filtration using 0.22 μm is degerming；Add 20-50mg luciferases (Sigma) and 100-
200mg D- luciferins (Sigma), dissolving, -20 DEG C of preservations.
Or (2) prepare 100mL 150-250mM N-2- hydroxyethyl piperazine-N'-2- second using freshly prepared high purity water
Sulfonic acid (HEPES) solution；Add 0.06-0.10g cetyl trimethylammonium bromides (CTAB), 0.15-1.20g acetic acid chlorine oneself
It is fixed, 1.5-2.5mL triton x-100s (Triton X-100), 0.3-0.5g Magnesium dichloride hexahydrates, the hydration second two of 0.7-1.0g bis-
Amine Sequestrene AA (EDTA), 0.05-0.10g sodium fluorides and the dissolving of 0.01-0.02g sodium pyrophosphates；Use 1M hydroxide
After sodium regulation pH value of solution is 7.5, the membrane filtration using 0.22 μm is degerming；Add 20-50mg luciferases (Sigma) and 100-
200mg D- luciferins (Sigma), dissolving, -20 DEG C of preservations.
Again or (3) prepare 100mL 200mM N-2- hydroxyethyl piperazine-N'-2- second sulphurs using freshly prepared high purity water
Sour (HEPES) solution；0.08g cetyl trimethylammonium bromides (CTAB), 0.16g chlorhexidine acetates are added, 1mL Qulas lead to X-
100 (Triton X-100), 0.4g Magnesium dichloride hexahydrates, the ethylenediamine hydrate Sequestrene AAs (EDTA) of 0.86g bis-, 0.08g
Sodium fluoride and the dissolving of 0.01g sodium pyrophosphates；After sodium hydrate regulator solution pH using 1M is 7.5,0.22 μm of filter membrane is used
Filtration sterilization；Add 35.8mg luciferases (Sigma) and 168mg D- luciferins (Sigma), dissolving, -20 DEG C of preservations.
The bacterial ATP detection reagent detection bacterium of embodiment 2
In order to investigate the performance of bacterial ATP detection reagent, recovery ETEC (ATCC 25922) and golden yellow Portugal
For grape coccus (ATCC 29213) on TSA plating mediums, 37 DEG C are cultivated 18-24h.It is with CAMHB meat soups that two kinds of bacterium difference are dilute
A series of concentration are interpreted into, the dilution bacterium solution of certain volume is pipetted into blank porous plate, adds in the embodiment 1 of certain volume and match somebody with somebody
The bacterial ATP detection reagent of system, it is put into multi-functional plate reader (PerkinElmer), detects the luminous value in each hole, each concentration
Repeat three holes.
Tables 1 and 2 respectively be detect varying number ETEC and staphylococcus aureus luminous value, Fig. 1
And shown in Fig. 2 be detection luminous intensity and bacterial number between relation.From the figure, it can be seen that within a large range
Luminous intensity and bacterial population are into positive correlation.In view of background and its standard deviation, luminescence method detection ETEC and gold
The minimum detection limit of staphylococcus aureus can reach 25CFU and 20CFU respectively.The result shows the bacterial ATP detection prepared
Reagent can apply to the detection of bacterium, and have very high detection sensitivity, can detect the bacterium of very low quantity.
Table 1 detects the luminous value of varying number ETEC
Table 2 detects the luminous value of varying number staphylococcus aureus
| Bacterial population (CFU) | Luminous intensity (RLU) |
| 0 | 95.3±20.8 |
| 20 | 464.7±56.1 |
| 200 | 1472.3±167.7 |
| 2000 | 13032.0±1926.1 |
| 20000 | 122109.3±2550.2 |
| 200000 | 1552810.0±78042.5 |
Embodiment 3 detects bacterial number after antibiotic effect
In order to detect the change that antibiotic acts on bacterial number after 2h, recovery ETEC (ATCC 25922) and gold
For staphylococcus aureus (ATCC 29213) on TSA plating mediums, 37 DEG C are cultivated 18-24h.Made in 96 orifice plates with No. 1 row
For negative control, No. 6 row are used as positive control；A rows are cephazolines, B and E rows are Piperacillins, C and G rows are Netilmicins,
D rows are lavo-ofloxacins, and F rows are minocyclines.50 μ L CAMHB meat soups are added in 1 to No. 6 Lie Ge holes of orifice plate, will be anti-
Raw element concentration (the μ g/mL of cephazoline 4, the μ g/mL of Piperacillin 16, the μ g/ of Netilmicin 2 needed for using CAMHB broth dilutions
ML, the μ g/mL of lavo-ofloxacin 0.125, the μ g/mL of minocycline 1), add the antibiotic dilution of the 50 above-mentioned concentration of μ L in No. 2 row
Into corresponding row, antibiotic to No. 3-5 row, No. 5 50 unnecessary μ L solution of row are diluted using the method for doubling dilution step by step and gone
Fall.The bacteria suspension that 0.5 maxwell unit turbidity is directly made in several bacterium colonies in meat soup is selected from the agar plate of culture, is gone forward side by side
One step uses 150 times of broth dilution；A-D rows add ETEC dilution, and E-G rows add staphylococcus aureus dilution
Liquid, 50 μ L meat soups are added in No. 1 row, the dilution bacterium solution for being separately added into 50 μ L is arranged at No. 2-6.After 37 DEG C of culture 2h, add in each hole
Enter 100 μ L bacterial ATP detection reagent, be put into multi-functional plate reader, record the luminous value in each hole.3 pieces of experiment repetition is porous
Plate, data are handled.
Table 3 is that various concentrations antibiotic acts on the relative luminous intensity detected after 2h, due to luminous intensity and bacterial number
Between positive correlation, the change of relative luminous intensity reflects the change of bacterial number.Antibiotic concentration increases, relative luminous
Intensity decreases, then it is considered that the reduction of the quantity of bacterium, this result show, the change of the bacterial number after antibiotic effect 2h
Change can be detected by luminescence method, while show to be expected to report in 2h using the bacterial ATP detection reagent of the present invention
Accuse drug sensitivity tests.
Table 3 detects the relative luminous intensity of bacterium after the effect of various concentrations antibiotic
The preparation of the Susceptibility to antibiotics quick detection kit of embodiment 4
Comprising as follows：
1) antibacterials plate：
Preparation method：In 384 orifice plates, No. 1 is classified as negative control, and No. 24 are classified as positive control, and No. 2-11 arranges and No. 14-23
Row are antibiotic row.Cephazoline is made into the dense of 3200,1600,800,400,200,100,50,25,12.5,6.25 μ g/mL
Degree, the 2-11 holes of A rows are added separately to, per the μ L of hole 0.5.OXA is made into 1600,800,400,200,100,50,
25th, 12.5,6.25,3.125 μ g/mL concentration, the 14-23 holes of A rows are added separately to, per the μ L of hole 0.5.Similarly will be other
The antibacterials of concentration are added in corresponding hole, last negative pressure drying.
2) 10mL bacterial ATPs detection reagent one bottle (composition is for example foregoing).
3) 20mL one bottle of CAMHB meat soups (or other meat soups, different culture media is equipped with according to the difference of detection bacterium).
The kit of embodiment 5 is applied to the MIC value for detecting a variety of antibacterials
The MIC value for detecting a variety of antibacterials to investigate kit to be applied to, recovery ETEC (CMCC
44103) and staphylococcus aureus (ATCC 25923) is on TSA plating mediums, and 37 DEG C are cultivated 18-24h.Use antimicrobial
Thing sensitiveness quick detection kit is used for quickly detecting MIC value：Several bacterium colonies are selected from the agar plate of culture directly to exist
The bacteria suspension of 0.5 maxwell unit turbidity is made in CAMHB meat soups, and further uses 300 times of broth dilution；In antibacterials plate
In, feminine gender row add 25 μ L meat soups, and 25 μ L dilution bacterium solution is separately added into antibacterials row and positive row；37 DEG C of culture 2h
Afterwards, 25 μ L bacterial ATP detection reagent is added in each hole, is put into multi-functional plate reader, records the luminous value in each hole；To data
Handled, be defined as detecting MIC value with the Cmin of relative luminous intensity≤80%.Micro broth dilution method is used simultaneously
Tested, determine standard MIC value.
Shown in table 4 is comparison of the kit detection Multiple Classes of Antibiotics to bacterium MIC value and the MIC of standard method detection.
As a result show, kit detection method is identical with the MIC value majority of standard law, although occurring the poor multiple proportions gradient of result once in a while
Situation, but the methodology allowable error of standard method is one multiple proportions gradient of deviation, it can be said that the knot of kit detection method
Fruit and dilution method are consistent.The kit of the present invention is the MIC value that can apply to detect common Multiple Classes of Antibiotics.
The MIC results (μ g/mL) of the kit detection method of table 4 and standard law measure
The kit of embodiment 6 is applied to the MIC value of detection multi-strain bacteria
In order to investigate the situation that kit is applied to detection multi-strain bacteria, recovering experiment bacterial strain is on TSA plating mediums, and 37
DEG C culture 18-24h.MIC value is used for quickly detecting using Susceptibility to antibiotics quick detection kit：Put down from the agar of culture
The bacteria suspension that 0.5 maxwell unit turbidity is directly made in several bacterium colonies in CAMHB meat soups is selected in ware, and further uses meat soup
300 times of dilution；In antibacterials plate, feminine gender row add 25 μ L meat soups, and 25 μ L are separately added into antibacterials row and positive row
Dilution bacterium solution；After 37 DEG C of culture 2h, 25 μ L bacterial ATP detection reagent is added in each hole, is put into multi-functional plate reader, is remembered
Record the luminous value in each hole；Data are handled, MIC value is detected by determination of the Cmin of relative luminous intensity≤80%.
Tested simultaneously using micro broth dilution method, determine standard MIC value.
Table 5 and table 6 are using kit detection enterobacteria and staphylococcic MIC value and the ratio with standard method respectively
Compared with.Testing result shows that kit detection method is identical with the MIC value majority of standard law, although occurring poor one of result times once in a while
Methodology allowable error than the situation of gradient, but standard method is one multiple proportions gradient of deviation, it can be said that kit is examined
The result of survey method and dilution method are consistent.The kit of the present invention goes for the measure of the MIC value of multi-strain bacteria.
The MIC results (μ g/mL) of the kit detection method of table 5 and standard law measure
The MIC results (μ g/mL) of the kit detection method of table 6 and standard law measure
Claims (10)
1. a kind of kit of Quantitative detection Susceptibility to antibiotics, it is characterized in that, having can directly detection bacterium ATP
Reagent；It is described directly to detect, it is once to add the latter step of the reagent to realize the cracking of bacterium and ATP release and detection.
2. the kit described in claim 1, it is characterized in that, also contain the antibacterials plate with series concentration gradient, it is described
Series concentration gradient is the concentration that every kind of medicine has ten or more, and the medicine is bonded to orifice plate bottom.
3. the kit described in claim 1, it is characterized in that, it any instrument with luminous detection function can be used to be examined
Survey, and automatic sampling apparatus need not be equipped with.
4. the kit described in claim 1, the reagent is containing bacterial ATP releasing agent and wide variety of glow-type bioluminescence material
Detection reagent.
5. the kit described in claim 4, the bacterial ATP releasing agent is surfactant, selected from cetyl trimethyl
One or more during ammonium bromide, benzalkonium chloride, Chlorhexidine and Qula are logical；
The wide variety of glow-type bioluminescence material is the material containing luciferase, D- luciferins, is characterized in：Luciferase and D- fluorescents
The mass ratio of element is 1:4~6.
6. the kit described in claim 5, the mass ratio of luciferase and D- luciferins is 1:4.7.
7. the application method of the kit described in claim 1, it is characterized in that：Tested bacteria is added in antibacterials plate and trained
After supporting, described in addition can direct detection bacterium ATP detection reagent, whole antibacterials plate is put into detector and detects each hole
Luminous intensity, then calculate the relative luminous intensity of antibacterials holes with respect to the positive hole without antibacterials；
It is characterized in that：
1) step sample-adding operation, it is not necessary to first inject lysate；
2) do not require to detect immediately after adding reagent；
3) auto injection equipment is not needed；
4) result is obtained after cultivating 2 hours.
8. the application method described in claim 7, the detector is any instrument with luminous detection function.
9. the application method described in claim 7, used anti-when only needing qualitative acquisition antibiotic resistance whether conclusion
Bacterium medicine plate need not have series concentration gradient, only antibacterials break concentration；Contrast negative hole, positive hole and antimicrobial
The luminous intensity in thing break concentration hole, remaining operation are consistent with the method for claim 7；The antibacterials break is quick
Feel two concentration of break and resistance break.
10. a kind of method of Quantitative detection Susceptibility to antibiotics, it is characterized in that, using can directly detection bacterium ATP
Reagent；It is described directly to detect, it is once to add the latter step of the reagent to realize the cracking of bacterium and ATP release and detection.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
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| CN201710875113.XA CN107421939A (en) | 2017-09-25 | 2017-09-25 | A kind of reagent and its application process of Quantitative detection Susceptibility to antibiotics |
| PCT/CN2018/102508 WO2019056926A1 (en) | 2017-09-25 | 2018-08-27 | Reagent for rapidly and quantitatively detecting antimicrobial susceptibility and application method thereof |
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| CN201710875113.XA CN107421939A (en) | 2017-09-25 | 2017-09-25 | A kind of reagent and its application process of Quantitative detection Susceptibility to antibiotics |
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| WO (1) | WO2019056926A1 (en) |
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| WO2019056926A1 (en) * | 2017-09-25 | 2019-03-28 | 江苏中新医药有限公司 | Reagent for rapidly and quantitatively detecting antimicrobial susceptibility and application method thereof |
| CN110699421A (en) * | 2019-11-12 | 2020-01-17 | 郑州安图生物工程股份有限公司 | Method for rapidly detecting drug-resistant phenotype of strain |
| CN110904185A (en) * | 2019-12-25 | 2020-03-24 | 武汉市农业科学院 | Quick detection kit for drug sensitivity of duck pathogenic bacteria based on iodine nitro tetrazole color development |
| CN110964781A (en) * | 2019-12-25 | 2020-04-07 | 武汉市农业科学院 | ATP bioluminescence-based quick detection kit for drug sensitivity of duck pathogenic bacteria |
| CN111705107A (en) * | 2020-07-13 | 2020-09-25 | 浙江工业大学 | Antibiotic susceptibility and compound antibacterial activity detection method based on EZMTT reagent |
| CN113845798A (en) * | 2021-09-15 | 2021-12-28 | 丹娜(天津)生物科技股份有限公司 | Antibacterial drug ink and method for preparing ester drug sensitive test strip by using same |
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| CN110658234B (en) * | 2019-09-30 | 2021-02-26 | 浙江大学 | Disposable quick-dismantling type pollutant impedance detection device |
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| WO2019056926A1 (en) | 2019-03-28 |
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