CN108362629A - Escherichia coli O 157:The rapid detection method and kit of the single viable bacterias of H7 - Google Patents
Escherichia coli O 157:The rapid detection method and kit of the single viable bacterias of H7 Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1434—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its optical arrangement
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N2015/1486—Counting the particles
Abstract
A kind of Escherichia coli O 157:The single viable bacteria rapid sensitive quantitative detecting methods of H7 and detection kit.Using increasing the pre-treating methods such as bacterium, enrichment and purifying by Escherichia coli O 157:H7 carries out enriching and purifying, then specific immunofluorescence antibody is used to mark Escherichia coli O 157:H7, and be aided with film permselectivity fluorescent dye and distinguish dead bacterium and viable bacteria to mark, then flow cytometer showed technology detection is carried out, by counting different fluorescence signals, directly obtain Escherichia coli O 157:The concentration of H7 viable bacterias.The present invention detection method have the advantages that accurate quantitative analysis, high specificity, high sensitivity, detection time it is short, it is easy to operate, life or death bacterium can be distinguished.
Description
Technical field：
The invention belongs to Microbiological detection of foods fields, are related to Escherichia coli O 157:The Quantitative detection of H7, especially
The rapid detection method and kit of single viable bacteria.
Background technology：
Escherichia coli O 157:H7(Escherichia coli O157:H7 it is) a kind of important food-borne pathogens, belongs to
It is most representative serotype in enterohemorrhagic escherichia coli in enterobacteriaceae Escherichia.Its infective dose is very
It is low, minimum 10 viable bacterias may Infective, mainly cause the hemorrhagic diarrhea and enteritis of people, and concurrent haemolytic uraemic is comprehensive
Close disease, thrombotic thrombocytopenic purpura etc., it is serious can causing death.Therefore it fast and accurately can quantitatively detect to eat
Escherichia coli O 157 in product and water:H7 viable bacterias, food-safe and guarantee people's health have very important significance.
Culture-based method detects Escherichia coli O 157:H7 time and effort consumings need 18 hours or more time that could complete to detect.
Existing Escherichia coli O 157:The Fast Detection Technique of H7 has the following disadvantages：
1, dead bacterium and viable bacteria be cannot distinguish between
It is well known that only viable bacteria just have it is pathogenic, so if do not know viable bacteria amount, even quantitative detection, also very
The gradient of infection difficult to estimate for counting the bacterium.
But current domestic and international mainstream technology, including immunological method, molecular biology method, Luminex and streaming meter
Number method etc..Not only detection time is still longer, but also is difficult to differentiate between life or death bacterium.
Because immunological method is the principle to be reacted to each other by antigen-antibody, it can only detect total bacterial antigens egg
In vain；And molecular biology method is can only to detect total bacterial nucleic acid by gene magnification principle；Luminex is by anti-
Body capture bacterial antigens are detected by antibody labelled antigen again, can only be detected and be obtained bacterial surface antigen concentration；Previously build
Vertical streaming counting method can only be marked by antibody capture bacterium, then by bacterial nucleic acid dyestuff, can only detect to obtain total
Bacterial concentration.Above method cannot distinguish between dead bacterium and viable bacteria.
2, detection sensitivity is less than the minimum infective dose (10CFU) of the bacterium, can not detect single viable bacteria
Escherichia coli O 157:The minimum infective dose of H7 is 10CFU, the detection of existing Luminex and streaming counting method
Limit is all 103CFU/mL or more cannot be satisfied Escherichia coli O 157:The basic need of H7 detections.
For example, Chinese patent CN201611201541.6, Escherichia coli O 157 is quickly detected with flow cytometry:H7.Behaviour
It is by Escherichia coli O 157 as step:H7(E.coli O157:H7 after) being dyed with SybrGreen I stostes, then it is thin with B
Born of the same parents capture various concentration Escherichia coli O 157:H7 establishes regression equation by the corresponding fluorescence intensity of flow cytomery,
Then it detects according to operating procedure after obtaining the fluorescence intensity of the Escherichia coli to be measured of B cell capture, is calculated by journey every time
To Escherichia coli O 157:H7 concentration.
But the Escherichia coli O 157 that this method detects:H7 is the total concentration of dead bacterium and viable bacteria.In addition, the patent
Detection limit is 4.9 × 104CFU/mL.If only single bacterium colony, can not detect.
For another example, Chinese patent CN201110360550.0 discloses a kind of side of liquid-phase chip detection Escherichia coli O 157
Method
Do not use flow cytometer showed that can not distinguish dead bacterium and viable bacteria using liquid-phase chip；And obtained testing result is not
It is absolute quantification method, is relative quantification.
In addition, the detection limit of CN201110360550.0 methods is 103CFU/mL.And the minimum infective dose of the bacterium is
10CFU.So the method is difficult practical application.
Therefore, quickly detection, high sensitivity (detection limit reaches single bacterium), and viable count can be detected, it is Escherichia coli
O157:The actual needs and technological difficulties of H7 detections.
Invention content：
The first object of the present invention is to provide a kind of Escherichia coli O 157:The quantitative side of detection of the rapid sensitive of the single viable bacterias of H7
Method, that is, this method allows for distinguishing life or death bacterium, and detection limit can reach the sensitivity level for detecting single viable bacteria.
The second object of the present invention is to provide the detection kit for reaching above-mentioned purpose.
There are three key problem in technology points for this method tool：When how enriching and purifying Escherichia coli O 157:H7, second is that how special
Ground identifies Escherichia coli O 157:H7, third, how to accurately distinguish Escherichia coli O 157:Life or death bacterium in H7.
In view of the above-mentioned problems, present inventor has performed following work：
Using enrichment liquid by Escherichia coli O 157:H7 is enriched with.Using enrichment liquid by original Escherichia coli O 157:H7 bacterium
Liquid is diluted to a series of bacterium solution of various concentrations.Concentration is about 1CFU/mL, 10CFU/mL, 100CFU/mL, 1000CFU/mL,
10000CFU/mL, and plate count is carried out to it.Then 37 DEG C are carried out to the bacterium solution of above-mentioned various concentration to cultivate, and every
0.5h carries out a plate count.Draw Escherichia coli O 157:The response curve of H7 viable bacteria concentrations and incubation time, obtains the side of recurrence
Journey is as follows：
Y=X/ (2^ (1.9919t-2.3391) * 10)
In formula,
Y is Escherichia coli O 157 in sample to be tested:The original concentration of H7, CFU/mL；
X is the Escherichia coli O 157 that flow type analyzer detects:H7 concentration, CFU/mL；
T is Zengjing Granule time, h.
Using coupling has the Escherichia coli O 157 of Green fluorescent dye:The antibody of H7, to Escherichia coli O 157:H7 is carried out
Specificity fluorescent marks.By comparing Escherichia coli O 157 monoclonal antibody (MAB) and polyclonal antibody (PAB) in this method
In quality, filtered out the antibody of best results, and best reaction condition is obtained by optimization.
The red fluorescence dyestuff with film permselectivity is used, to dead Escherichia coli O 157:H7 carries out fluorescence
Label.And the accuracy of its life or death bacterium dyeing is verified.And optimize Escherichia coli O 157:H7 double fluorescence labeling items
Part.
Accordingly, the present invention establishes for the first time a kind of quantitatively detecting Escherichia coli using flow cytometer showed technology rapid sensitive
O157:The method of H7 viable bacterias, it is characterized in that：Before being detected, pre-treatment first is carried out to measuring samples；The pre-treatment is
Increasing bacterium, enrichment and purification process are carried out, Escherichia coli O 157 is made:H7 enriching and purifyings.
The increasing bacterium, method are that enrichment liquid is added in sample to be tested, then 37 DEG C of culture 5h or more filter, collect filtrate.
Can use those skilled in the art commonly various culture solutions as enrichment liquid.
Preferably enrichment liquid formula is：3~30g/L of peptone, 0.5~5g/L of powdered beef, 0.5~5g/L of sodium chloride, Portugal
Grape sugar 0.5~5g/L, pH 7.2~7.6.
The enrichment, method are to centrifuge above-mentioned filtrate, discard supernatant liquid, retain the precipitation of bottom
The purifying, method are PBS to be added in above-mentioned precipitation precipitation, the sample bacterium solution purified is resuspended.
The detection is to use double immunofluorescense Escherichia coli O 157:H7, then carry out flow cytometer showed technology detection；Pass through
Different fluorescence signals are counted, Escherichia coli O 157 is directly obtained:The concentration of H7 viable bacterias.
The double immunofluorescense is to carry out specific immunofluorescence antibody label, and be aided with film permselectivity fluorescence dye
Material label distinguishes dead bacterium and viable bacteria.
The Immunofluorescent Antibody is that Green fluorescent dye of the fluorescence emission spectrum in 501nm~540nm passes through chemical base
The crosslinked Escherichia coli O 157 polyclonal antibody of group or monoclonal antibody can identify specific marker large intestine bar with specific marker
Bacterium O157:H7；
Film permselectivity fluorescent dye is red fluorescence dyestuff of the fluorescence emission spectrum in 601nm~640nm, Ke Yibiao
Remember dead Escherichia coli O 157:H7.
It is described to distinguish dead bacterium and viable bacteria, method are as follows：
Using the antibody of above-mentioned Green fluorescent dye coupling to Escherichia coli O 157:H7 carries out specific marker, then uses
Dead bacterium is marked above-mentioned film permselectivity red fluorescence dyestuff, finally by the fluorescence channel of flow type analyzer
It is detected.
There is the Escherichia coli O 157 monoclonal of fluorescein isothiocynate anti-for example, coupling can be added in the bacterium solution of purifying
Body (FITC-MAB) and propidium iodide (PI) are incubated after mixing.
If only detecting green fluorescence, it is determined as that the bacterium detected is Escherichia coli O 157:H7 viable bacterias；
If being detected simultaneously by red and green two kinds of fluorescence, it is determined as that the bacterium detected is Escherichia coli O 157:
The dead bacterium of H7；
If green fluorescence is not detected, it is judged to that Escherichia coli O 157 is not present:H7.
So passing through the above method, you can complete Escherichia coli O 157:The qualitative and quantitative detection of H7 life or death bacterium.
The flow type analyzer detection：Detection parameters setting is as follows：Exciting light is respectively 450~500nm, and analyze speed is
1~15 μ L/min, detection time are 15~300s, and by double fluorescence channels, analysis calculates Escherichia coli O 157 viable bacteria concentration X.
The analysis calculates, and by this field conventional method, usually obtains Escherichia coli O 157 by flow type analyzer first:
H7 viable bacterias number and analysis sample volume, to which the Escherichia coli O 157 that flow type analyzer detects be calculated:H7 concentration, so
Escherichia coli O 157 in sample to be tested is calculated further according to following equation afterwards:The original concentration of H7 viable bacterias.
Escherichia coli O 157 in sample to be tested is calculated as follows:The original concentration of H7 viable bacterias
Y=X/ (2^ (1.9919t-2.3391) * 10)
In formula,
Y is Escherichia coli O 157 in sample to be tested:The original concentration of H7, CFU/mL；
X is the Escherichia coli O 157 that flow type analyzer detects:H7 concentration, CFU/mL；
T is Zengjing Granule time, h
It is primary actually detected concrete operations of the invention below.
1, the increasing bacterium enrichment of sample to be tested：
225mL enrichment liquids are added in 25mL or 25g samples to be tested, 5h or more are cultivated for 37 DEG C in isothermal vibration incubator.
2, the filtering of sample to be tested：
Sample to be tested is filtered using the filter membrane in 2.7 μm~15 μm of apertures, retains filtrate.
3, in sample bacterium purification enrichment；
12000 × g of about 250mL filtrates is centrifuged into 5min.Supernatant liquid is discarded, the precipitation of bottom is retained.It is added 2.5mL's
Precipitation is resuspended in PBS, is transferred in new centrifuge tube, the sample bacterium solution purified.
4, FITC-MAB (ultimate density is 1 μ g/mL) and PI (ultimate density is 1 μ g/mL), mixing is added in the bacterium solution purified
After be protected from light be incubated 15min.
5, flow type analyzer detects：The setting of flow type analyzer detection parameters is as follows：Exciting light is 488nm, and analyze speed is
10.5 μ L/min, detection time 30s, by the bis- fluorescence channels of 488-Grn-488-Red, analysis calculates Escherichia coli O 157 and lives
Bacteria concentration.
6, interpretation of result by step 5 detection gained viable bacteria concentration X values and step 1 in Zengjing Granule time t values, substitution side
In journey Y=X/ (2^ (1.9919t-2.3391) * 10), the Y of gained is Escherichia coli O 157 in sample to be tested:H7's is original dense
Degree.
The present inventor demonstrates the effect of this detection method：
The Escherichia coli O 157 that 10 times are serially diluted simultaneously using this method and colony counting method:H7 bacterium solutions are examined
It surveys, and the two result is compared.As a result, it has been found that：This method has good linear pass with plate count method acquired results
System illustrates that this method has good accuracy, and the Monitoring lower-cut of method is 1CFU/mL.
According to the detection method that aforementioned present invention is established, the present invention also provides a kind of Escherichia coli O 157s:H7 viable bacterias are fast
Fast accurate quantitative analysis detection kit, the kit include following component：
The mixed liquor of Immunofluorescent Antibody and film permselectivity fluorescent dye is stored in and is protected from light EP pipes；
Escherichia coli O 157:H7 reference cultures (strain number CICC 21530), lyophilized powder are stored in brown cillin bottle；
E. coli k12 reference culture (strain number ATCC 10798), lyophilized powder are stored in brown cillin bottle；
The filter membrane in 2.7 μm~15 μm apertures；
Enrichment liquid (3~30g/L of peptone, 0.5~5g/L of powdered beef, 0.5~5g/L of sodium chloride, 0.5~5g/ of glucose
L, pH 7.2~7.6), it is stored in and is protected from light plastic bottle.
Escherichia coli O 157 is carried out with the kit of the present invention:The operating method of H7 detections, it is characterized in that：
1, the increasing bacterium enrichment of sample to be tested：
225mL enrichment liquids are added in 25mL or 25g samples to be tested, 5h or more are cultivated for 37 DEG C in isothermal vibration incubator.
2, the filtering of sample to be tested：
Sample to be tested is filtered using the filter membrane in 2.7 μm~15 μm of apertures, retains filtrate.
3, in sample bacterium purification enrichment；
12000 × g of about 250mL filtrates is centrifuged into 5min.Supernatant liquid is discarded, the precipitation of bottom is retained.It is added 2.5mL's
Precipitation is resuspended in PBS, is transferred in new centrifuge tube, the sample bacterium solution purified.
4, FITC-MAB (ultimate density is 1 μ g/mL) and PI (ultimate density is 1 μ g/mL), mixing is added in the bacterium solution purified
After be protected from light be incubated 15min.
5, flow type analyzer detects：The setting of flow type analyzer detection parameters is as follows：Exciting light is 488nm, and analyze speed is
10.5 μ L/min, detection time 30s, by the bis- fluorescence channels of 488-Grn-488-Red, analysis calculates Escherichia coli O 157 and lives
Bacteria concentration.
6, interpretation of result by step 5 detection gained viable bacteria concentration X values and step 1 in Zengjing Granule time t values, substitution side
In journey Y=X/ (2^ (1.9919t-2.3391) * 10), the Y of gained is Escherichia coli O 157 in sample to be tested:H7's is original dense
Degree.
Being verified by experiments this method not only can Escherichia coli O 157 in qualitative detection liquid sample:H7 whether there is,
It can also quantify and detect Escherichia coli O 157:The Exact concentrations of H7 life or death bacterium (see embodiment 3).
The present invention have the advantages that following innovation and：
1, detection sensitivity is high, can detect single viable bacteria
Increase the pre-treating methods such as bacterium, enrichment and purifying, Escherichia coli O 157 by increase:The detection of H7 is limited down to 1CFU.
This method is absolute quantification method.It is single bacterium, the Escherichia coli after fluorescent staining that it, which detects object,
O157:H7 directly counts large intestine bar living by the detection zone of flow type analyzer by counting the bacterium of different fluorescence one by one
Bacterium O157:H7 number.
2 while detecting Escherichia coli O 157:H7 whether there is and the Exact concentrations of its life or death bacterium
By using FITC-MAB and PI to Escherichia coli O 157 to be checked:H7 carries out double fluorescence labeling, can examine simultaneously
Survey Escherichia coli O 157:H7 whether there is and the Exact concentrations of its life or death bacterium.
Directly against Escherichia coli O 157:H7 is dyed, only special by the Immunofluorescent Antibody with green fluorescence
Property label Escherichia coli O 157:H7, then by the red dead bacterium of film permselectivity dye marker (because only that dead bacterium can just send out
Raw cell wall rupture, causes red fluorescence dyestuff to enter cell poststaining).If bacterium simultaneously by green and red-label,
It is then dead Escherichia coli O 157:H7；If a bacterium is only by Green Marker, for Escherichia coli O 157 living:H7.
3, simple and convenient, time-consuming short, most of samples can complete detection in 6h.
4, it is accompanied with Escherichia coli O 157 in kit:H7 reference cultures and e. coli k12 reference culture as a contrast,
It can ensure the exactness and accuracy of testing result to the full extent.
Description of the drawings
Fig. 1 be in embodiment 1 monoclonal antibody and polyclonal antibody respectively with various concentration Escherichia coli O 157:H7 is incubated
Flow type analyzer testing result afterwards；
Fig. 2 is the Escherichia coli O 157 that flow type analyzer detects different life or death bacteria concentration ratios in embodiment 3:H7 bacterium solutions
Flow type analyzer testing result；
Fig. 3 is the linear relationship of the testing result of this method and classic flat-plate method of counting in embodiment 4.
Specific implementation mode
With reference to specific example, the present invention is further explained.It should be understood that these embodiments be merely to illustrate the present invention and
It is not used in and limits the scope of the invention.In addition to being defined, technical and scientific term used in following embodiment has and this hair
The identical meanings that bright one of ordinary skill in the art are commonly understood by.
Experiment reagent consumptive material used in following embodiment is unless otherwise specified conventional biochemical reagent.Following implementation
Test method without specific conditions in example, usually according to normal condition in condition, or the suggestion according to manufacturer
Condition.Bacterial strain involved in embodiment belongs to the prior art, and those skilled in the art can be easy to from open commercial channel
It obtains.
Approximating language used in following embodiment can be used for quantifying bidding documents, show in the feelings for not changing basic function
Quantity is allowed to have certain variation under condition.Therefore, it is not limited to this accurately with the modified numerical value of the language such as " about ", " left and right " institute
Numerical value itself.In some cases, approximating language may be related with the precision of measuring instrument.
The comparison of embodiment 1 Escherichia coli O 157 monoclonal antibody and polyclonal antibody
One, materials and methods
1. Escherichia coli O 157:H7 reference cultures, number CICC21530 are purchased from Chinese industrial Microbiological Culture Collection pipe
Reason center.
2. by cultured Escherichia coli O 157:12000 × g of H7 bacterium solutions centrifuges 5min, abandons supernatant, and bacterium mud uses PBS weights
It is outstanding.With PBS to the Escherichia coli O 157 of resuspension:H7 carries out 10 times and is serially diluted.
3. taking two group of 10 Escherichia coli O 157 being serially diluted again:H7 bacterium solutions, concentration are about 103~108CFU/mL.One
The Escherichia coli O 157 monoclonal antibody (FITC-MAB) of final concentration of 1 μ g/mL is added in group bacterium solution；It is added in another group dense eventually
Degree is the Escherichia coli O 157 polyclonal antibody (FITC-PAB) of 1 μ g/mL.It is protected from light and is incubated 15min.
4. using flow type analyzer, the sample of above-mentioned dyeing is detected.Parameter setting is as follows：Exciting light is 488nm,
Analyze speed is 10.5 μ L/min, detection time 30s.
Two, experimental result
10 times of Escherichia coli O 157s being serially diluted:After H7 bacterium solutions are incubated with FITC-PAB, examined with flow type analyzer
It surveys, as a result as shown in Figure 1A.It can be seen that with the reduction of bacteria concentration, green fluorescence wave crest is gradually moved to right until bacterial concentration is down to about
106CFU/mL。
10 times of Escherichia coli O 157s being serially diluted:After H7 bacterium solutions are incubated with FITC-MAB, examined with flow type analyzer
It surveys, as a result such as Figure 1B.It can be seen that FITC-MAB can be to the Escherichia coli O 157 of high concentration:H7 carries out fluorescent marker, and does not have
The decaying of fluorescence intensity.
The comparison of the above results shows monoclonal antibody and the reaction efficiency higher of Escherichia coli O 157.Therefore the present invention
Escherichia coli O 157 monoclonal antibody (FITC-MAB) is selected.
The verification of 2 Escherichia coli O 157 monoclonal antibody specificity of embodiment
One, materials and methods
1. Escherichia coli O 157:H7 reference cultures, number CICC 21530；Escherichia coli reference culture, number ATCC
25922；Escherichia coli reference culture, number ATCC 13706；Escherichia coli reference culture, number CMCC 44102；Large intestine bar
Bacterium K12 reference cultures, number ATCC 10798；Staphylococcus aureus reference culture, number ATCC 6538P；Enterobacter cloacae
Reference culture, number ATCC 13047；Clostridium perfringen reference culture, number ATCC 13048；Citrobacter freundii standard
Bacterial strain, number ATCC 43864.The above bacterial strain is purchased from Chinese industrial Microbiological Culture Collection administrative center.
2. pair above-mentioned all bacterial strains carry out rejuvenation and amplification, and are diluted to suitable concentration (about 106CFU/mL)。
3. FITC-MAB (ultimate density is 1 μ g/mL) is separately added into above-mentioned bacterium solution, it is protected from light and is incubated 15min.Then with stream
Formula analyzer is detected.
Two, experimental result
Escherichia coli O 157 monoclonal antibody is reacted with the bacterium solution of different strains, then flow type analyzer carries out again
Its specificity is detected, the results are shown in Table 1, only Escherichia coli O 157:H7 detects green fluorescence, shows Escherichia coli O 157
Monoclonal antibody has good specificity.Also indicate that this method has the advantages that high specificity simultaneously.
The specificity experiments result of 1 Escherichia coli O 157 monoclonal antibody of table
The verification of 3 Escherichia coli O 157 life or death bacterium detection accuracy of embodiment
One, materials and methods
1. by cultured Escherichia coli O 157:12000 × g of H7 bacterium solutions centrifuges 5min, abandons supernatant, and bacterium mud uses PBS weights
It is outstanding, obtain viable bacteria bacterium solution.1mL viable bacteria bacterium solutions are drawn, isopropanol (ultimate density 70%) is added and handles 30min.Centrifugation is abandoned later
Supernatant, bacterium mud are resuspended using PBS, obtain dead bacterium bacterium solution.
2. by Escherichia coli O 157:H7 life or death bacterium bacterium solutions are with different volume ratio mixing (0/10,1/9,5/5,9/1,10/
0).Then flow cytomery sample is used.
Two, experimental result
By various concentration than Escherichia coli O 157:H7 life or death bacterium mix, and are detected with flow type analyzer, gained
The results are shown in Figure 2.The result shows that the Escherichia coli O 157 of flow cytometer detection:The ratio value of H7 life or death bacterium is tapped with actual value ten
Closely, illustrate that this method can detect the Escherichia coli O 157 in liquid with accurate quantitative analysis:H7 life or death bacterium.
4 present invention Escherichia coli O 157 in detecting pure water of embodiment:The application of H7 contents
One, materials and methods
1. by cultured Escherichia coli O 157:12000 × g of H7 bacterium solutions centrifuges 5min, abandons supernatant, and bacterium mud uses PBS weights
It is outstanding.
2. with sterile purified water to Escherichia coli O 157:H7 bacterium solutions carry out 10 times and are serially diluted, and the present invention is respectively adopted
Detection method and national standard in escherichia coli colony counting method quantitative detection is carried out to sample, analyze the linear of this method with
Sensitivity.Two, experimental result
By Escherichia coli O 157:H7 bacterium solutions are diluted to concentration 10 with sterile purified water0~104The sample of CFU/mL, will
225mL enrichment liquids are added in 25mL pure water, and 5h are cultivated for 37 DEG C in isothermal vibration incubator.Culture terminates, will about 250mL pure water
12000 × g of sample centrifuges 5min.Supernatant liquid is discarded, the precipitation of bottom is retained.Precipitation, transfer is resuspended in the PBS that 2.5mL is added
Into new centrifuge tube, the sample bacterium solution that is purified.
Through increasing bacterium, enrichment and sample is detected using the method for the present invention and colony counting method simultaneously after purification.As a result it shows
Show (table 2), pure water sample is after increasing bacterium 5h and enriching and purifying, Escherichia coli O 157:The concentration of H7 is 2.57 × 103~1.12
×108In CFU/mL measurement ranges, the method for the present invention and colony counting method testing result are almost the same.Large intestine after increasing bacterium enrichment
Bacillus O157:The concentration of H7 is more than 108When CFU/mL, due to the limitation of flow type analyzer itself, exceed its upper limit of detection.When big
Enterobacteria O157:The concentration of H7 is less than 103When CFU/mL, flow cytometer showed result and colony counting method deviation are larger.By bacteria concentration
103~108The testing result of CFU/mL, is depicted as standard curve, as shown in figure 3, between flow cytometer detection method and colony counting method
Linear good (R2=0.9972) it, by Zengjing Granule time 5h in the viable bacteria concentration X values and step 1 obtained by flow cytometer showed, substitutes into
In equation Y=X/ (2^ (1.9919t-2.3391) * 10), the Y of gained is Escherichia coli O 157 in sample to be tested:H7's is original dense
Degree.The detection range of this method is 1~5.69 × 104CFU/mL, sensitivity 1CFU/mL.
2 this method of table and Escherichia coli O 157 in colony counting method detection pure water:The result of H7
5 present invention Escherichia coli O 157 in detecting milk of embodiment:The application of H7 contents
One, materials and methods
1. by cultured Escherichia coli O 157:12000 × g of H7 bacterium solutions centrifuges 5min, abandons supernatant, and bacterium mud uses PBS weights
It is outstanding.Its concentration is detected with flow type analyzer, result is 1.11 × 109CFU/ml.With sterilizing PBS solution to Escherichia coli O 157:
H7 bacterium solutions carry out 10 times and are serially diluted.
3. by Escherichia coli O 157:In H7 artificial infections to milk, it is prepared into the milk sample of artificial contamination, it is a concentration of
100~104CFU/mL。
4. sample is carried out increasing bacterium, enrichment and purifying.
5. rapidly purifying the fat and protein body in kit removal milk sample using microorganism in milk.To purifying
Sample afterwards is handled as follows：It takes the milk samples of 0.5mL to be after purification placed in 2mL centrifuge tubes, then takes microorganism in milk
The sample treatment liquid 1mL for rapidly purifying kit is placed in sample, overturns mixing 20 times, 12000 × g repeatedly, centrifuges 5min.It abandons
Upper-layer fat and middle level clear liquid are gone, the precipitation of bottom is retained.Precipitation is resuspended in the PBS solution that 500 μ L are added, and is transferred to new centrifugation
Guan Zhong, adds the PBS solution of 1mL, and after mixing, 12000 × g centrifuges 5min.
6. Escherichia coli O 157 monoclonal antibody (ultimate density is 1 μ g/mL) is added for the bacterium solution of purifying and propidium iodide is (most
Final concentration of 1 μ g/ml), it is protected from light and is incubated 15min.
7. flow type analyzer detects：The Escherichia coli O 157 having to fluorescent marker:H7 carries out flow cytometer detection.
Two, experimental result
The Escherichia coli O 157 of various concentration gradient is inoculated in milk:25mL artificial contamination's milk samples are added H7
225mL enrichment liquids, 37 DEG C of culture 5h in isothermal vibration incubator.Culture terminates, will the 12000 × g centrifugations of about 250mL milk samples
5min.Supernatant liquid is discarded, the precipitation of bottom is retained.Precipitation is resuspended in the PBS that 2.5mL is added, and is transferred in new centrifuge tube, obtains
To the sample bacterium solution of purifying.Then fat and egg in microorganism fast purifying kit removal milk sample in milk are used again
White particle.
And its concentration is detected using the detection method in the present invention.The results are shown in Table 3,
Through increasing bacterium, enrichment and sample is detected using the method for the present invention and colony counting method simultaneously after purification.As a result it shows
Show (table 4), milk sample is after increasing bacterium 5h and enriching and purifying, Escherichia coli O 157:The concentration of H7 is 103~108Between CFU/mL
When, detection method accuracy of the invention is good.It will be in the viable bacteria concentration X values and step 1 obtained by flow cytometer showed when Zengjing Granule
Between 5h, substitute into equation Y=X/ (2^ (1.9919t-2.3391) * 10), the Y of gained is Escherichia coli O 157 in sample to be tested:
The original concentration of H7.The detection range of this method is 1~5.64 × 104CFU/mL, sensitivity 1CFU/mL.
3 this method of table detects Escherichia coli O 157 in milk sample:The accuracy of H7
6 Escherichia coli O 157 of embodiment:H7 viable bacterias quick and precisely immue quantitative detection reagent box
Including following component：
The mixed liquor of Immunofluorescent Antibody and film permselectivity fluorescent dye is stored in and is protected from light EP pipes；
Escherichia coli O 157:H7 reference cultures (strain number CICC 21530), lyophilized powder are stored in brown cillin bottle；
E. coli k12 reference culture (strain number ATCC 10798), lyophilized powder are stored in brown cillin bottle；
The filter membrane in 2.7 μm~15 μm apertures；
Enrichment liquid is stored in and is protected from light plastic bottle.
Claims (10)
1. a kind of quickly detecting Escherichia coli O 157 using flow cytometer showed technology:The method of the single viable bacterias of H7, it is characterized in that：Into
Before row detection, pre-treatment first is carried out to measuring samples；The pre-treatment is to carry out increasing bacterium, enrichment and purification process.
2. method described in claim 1, the increasing bacterium are to cultivate sample to be tested addition enrichment liquid, then filter, receive
Collect filtrate；The enrichment is to centrifuge above-mentioned filtrate, discards supernatant liquid, retains the precipitation of bottom；The purifying, being will be upper
It states precipitation and PBS resuspension precipitations, the sample bacterium solution purified is added.
3. method described in claim 1, the detection are to use double fluorescence labeling Escherichia coli O 157:H7, then divided with streaming
Analyzer carries out flow cytometer showed technology detection；By counting different fluorescence signals, Escherichia coli O 157 is obtained:The detection of H7 viable bacterias is dense
Degree.
4. the method described in claim 3, Escherichia coli O 157 in sample to be tested:The original concentration of H7 viable bacterias is counted as follows
It calculates：
Y=X/ (2^ (1.9919t-2.3391) * 10)
In formula,
Y is Escherichia coli O 157 in sample to be tested:The original concentration of H7, CFU/mL；
X is the Escherichia coli O 157 that flow type analyzer detects:H7 concentration, CFU/mL；
T is Zengjing Granule time, h.
5. the technical indicator of the method described in claim 3, the flow type analyzer is：Fluorescence sensitivity<10MESF, particle ruler
It is very little<40nm, fluorescence resolution ratio RSD<2%, sample analysis rate>10000events/min.
6. the method described in claim 3, the flow type analyzer detection parameters are as follows：Analyze speed is 1~15 μ L/min, inspection
The survey time is 15~300s.
7. the method described in claim 3, the double fluorescence labeling are to carry out specific immunofluorescence antibody label and film selection
Permeability fluorochrome label distinguishes dead bacterium and viable bacteria.
8. the method described in claim 3, the Immunofluorescent Antibody is green of the fluorescence emission spectrum in 501nm~540nm
Fluorescent dye passes through the crosslinked Escherichia coli O 157 polyclonal antibody of chemical group or monoclonal antibody；The film permselectivity
Fluorescent dye is red fluorescence dyestuff of the fluorescence emission spectrum in 601nm~640nm.
9. a kind of Escherichia coli O 157:H7 viable bacterias quick and precisely immue quantitative detection reagent box, it is characterized in that containing enrichment liquid, immune glimmering
Photoactivated antibody and film permselectivity fluorescent dye.
10. the kit described in claim 9, also contains following component：
Escherichia coli O 157:H7 reference cultures；
E. coli k12 reference culture；
The filter membrane in 2.7 μm~15 μm apertures；
The Immunofluorescent Antibody and film permselectivity fluorescent dye are the mixed liquors of the two；
The enrichment liquid formula is：3~30g/L of peptone, 0.5~5g/L of powdered beef, 0.5~5g/L of sodium chloride, glucose 0.5
~5g/L；PH 7.2~7.6.
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| CN113122469A (en) * | 2021-02-26 | 2021-07-16 | 中国计量科学研究院 | Bacterial standard substance for calibration of living cell online detector and preparation method thereof |
| CN113687072A (en) * | 2021-09-10 | 2021-11-23 | 佛山墨赛生物技术有限公司 | Test strip, kit and detection method for quantitative detection of viable bacteria of Escherichia coli O157H 7 |
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| CN110607343A (en) * | 2019-09-02 | 2019-12-24 | 重庆三峡学院 | Method for detecting live bacteria by double-dyeing method |
| CN112980916A (en) * | 2019-12-16 | 2021-06-18 | 中国计量科学研究院 | Rapid quantitative detection method and kit for trace staphylococcus aureus viable bacteria in dairy products |
| CN113122469A (en) * | 2021-02-26 | 2021-07-16 | 中国计量科学研究院 | Bacterial standard substance for calibration of living cell online detector and preparation method thereof |
| CN113687072A (en) * | 2021-09-10 | 2021-11-23 | 佛山墨赛生物技术有限公司 | Test strip, kit and detection method for quantitative detection of viable bacteria of Escherichia coli O157H 7 |
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