CN105785020B - A kind of quick determination method for bacillus cereus - Google Patents
A kind of quick determination method for bacillus cereus Download PDFInfo
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Abstract
The invention provides a kind of quick determination method for bacillus cereus, this method is first combined bacillus cereus with coated polyclonal antibody, then biotinylated monoclonal antibody is connected, reconnect the catalase C100 of marked by streptavidin, decomposing hydrogen dioxide solution is catalyzed by catalase, the fluorescent quenching of the cadmium telluride quantum dot to mercaptopropionic acid modification is reduced, according to the height of fluorescence intensity come the concentration of bacillus cereus in judgement sample.This method is based on double antibodies sandwich Enzyme-multiplied immune technique, and employ biotin avidin system be used for react amplification.What is more important, due to present invention employs new antibody labeling enzyme (catalase C100) and more sensitive fluorogenic substrate (cadmium telluride quantum dot), and it have matched effective reaction condition, so that detection sensitivity is significantly improved, cost, lifting detection efficiency are reduced simultaneously, therefore there is good promotion prospect.
Description
Technical field
The present invention relates to technical field of microbial detection, further to the Antigen Detection Techniques based on ELISA, specifically relates to
A kind of and quick determination method for bacillus cereus.
Background technology
Bacillus cereus (Bacillus cereus), category bacillus, Gram-positive, no pod membrane are facultative good
Oxygen, 20~45 DEG C of growth temperature range.It is widely present in soil, water, air and animal intestinal tract.Bacillus cereus can draw
Play food poisoning, symptom includes Nausea and vomiting, stomachache etc., and direct morbid substance is its metabolite enterotoxin, waxy brood cell's bar
Enterotoxin caused by bacterium has two kinds：One of which is heat resistant type enterotoxin, and 30min still has pathogenicity under 100 DEG C of environment, then
In addition Bacillus cereus itself gemma has prominent environmental resistance, therefore the strain serious threat human health.At this
In the case of kind, the important technology premise that sensitive, quick detection method is effective preventing and treating bacillus cereus is established.
It is mainly in the prior art biochemical identification method (such as GB/T4789.14- for bacillus cereus detection method
2014), but detection time length, reagent cost are high, and the detection method being separately cultured has certain contingency, it is therefore desirable to
Experiment sample is increased to ensure that testing result is correct.In recent years, with the development of molecular biology, DNA sequencing technology is waxy
It is widely used in the detection of bacillus, such method sensitiveness is higher, specific relatively strong, but defect is also more prominent
It is higher to go out on the one hand its cost, operating procedure is relatively cumbersome, time-consuming longer in addition, therefore the commercial application of such method is all the time
It is restricted.In recent years, enzyme linked immunosorbent assay (ELISA) receives increasing weight in the detection of bacillus cereus
Depending on, such method because with it is quick, sensitive, special, accurate, can quantify, it is easy to operate, without valuable instrument and equipment, and to sample
Product purity requirement is not high, is therefore particularly suitable for the detection of batch samples.However, bacillus cereus is detected in the prior art
ELISA method generally the antibody based on horseradish peroxidase-labeled or antigen catalysis hydrogen peroxide generation hydroxy radical, and then
Aoxidize colourless chemical colour reaction substrate tetramethyl biphenyl diamines (TMB) and form blue product, then using terminate liquid (2M H2SO4)
Terminating reaction forms yellow solution and absorbance is recorded at 450nm.Such method detects spirit because its colored intensity is relatively low
Sensitivity is relatively low, false negative result easily occurs when object content is relatively low in testing sample, so as to meet actual answer
It is required that.
In recent years, some new signal transduction mechanisms have been reported for substituting traditional ELISA signal transduction mechanism use
In the sensitivity for improving ELISA, as radiommunoassay substrate, chemical luminous substrate, fluorogenic substrate and resonance collaurum are molten
Liquid etc..However, the structure of luminescence system needs to take into full account the molecule biological property of detected object, such as in method layer
Face, the coating of antibody and its binding ability with antigen, the coupling method from which kind of label enzyme and its with antibody, bottom of developing the color
The selection of thing and specific luminescent method；In effect aspect, the sensitivity of chromogenic reaction should be ensured, colored intensity should be met again
With the linear relationship of object content.Therefore, for the ELISA detection method of bacillus cereus, especially to lift its detection
Method for the purpose of sensitivity is improved, and has prominent technical difficulty.
The content of the invention
A kind of it is contemplated that technological deficiency for prior art, there is provided quick detection side for bacillus cereus
Method, it is relatively low to solve the bacillus cereus ELISA detection method precision of prior art.
Another technical problem to be solved by the present invention is that prior art is used for the ELISA side of bacillus cereus antigen detection
In method, the enzyme sensitivity of labelled antibody is relatively low.
The invention solves another technical problem be prior art be used for bacillus cereus antigen detection ELISA side
In method, Color Appearance System sensitivity is relatively low.
The invention solves another technical problem be when using catalase C100 as antibody labeling enzyme to waxy gemma bar
When bacterium antigen performs ELISA detections, concrete technology method is simultaneously indefinite.
The invention solves another technical problem be when using catalase C100 as antibody labeling enzyme, using hydrogen peroxide and
When the cadmium telluride quantum dot of mercaptopropionic acid modification performs ELISA detections as substrate to bacillus cereus antigen, testing result
It is bad with the linear relationship of antigen concentration.
The invention solves another technical problem be when using catalase C100 as antibody labeling enzyme, using hydrogen peroxide and
Mercaptopropionic acid modification cadmium telluride quantum dot as substrate to bacillus cereus antigen perform ELISA detect when, during wash
Wash ineffective.
To realize above technical purpose, the present invention uses following technical scheme：
A kind of quick determination method for bacillus cereus, this method belong to double antibodies sandwich ELISA, the party
Method is the detection for bacillus cereus antigen, and the enzyme that labelled antibody is used in this method is catalase C100.
Preferably, substrate includes the cadmium telluride quantum dot that hydrogen peroxide and mercaptopropionic acid are modified in this method.Wherein dioxygen
Water is as cadmium telluride quantum dot fluorescence quenching, and in course of reaction, hydrogen peroxide decomposes under catalase effect, is quenched so as to reduce fluorescence
The effect of going out, realize luminous.
Preferably, this method comprises the following steps：
1) it is coated with bacillus cereus antibody；
2) take step 1) be coated with after antibody, mix with testing sample after in 35~39 DEG C of light protected environments react 40~
80min, washing；
3) biotinylated bacillus cereus monoclonal antibody mixing is then added, it is anti-in 35~39 DEG C of light protected environments
40~80min is answered, is washed；
4) then add marked by streptavidin catalase C100 mixing, in 35~39 DEG C of light protected environments react 40~
80min, washing；
5) the hydrogen peroxide solution mixing that concentration is 8~12 μm of ol/L is then added, is reacted in 35~39 DEG C of light protected environments
20~40min；
6) then add mercaptopropionic acid modification cadmium telluride quantum dot mixing, in 35~39 DEG C of light protected environments react 10~
20min；
7) detecting step 6) product fluorescence intensity.
The fluorescence intensity is used to react bacillus cereus content in testing sample, using known dense in practical operation
Spend and multigroup bacillus cereus titer of distribution gradient passes through above method and draws fluorescence intensity-bacteria concentration standard song
Line, the fluorescence intensity of testing sample is recycled to calculate the bacillus cereus content of testing sample from standard curve.Specific behaviour
Making method can be according to any selection of general technology general knowledge of the art.Multigroup bacillus cereus of above-mentioned gradient distribution
Titer, 10 can be selected respectively1CFU/mL、102CFU/mL、103CFU/mL、104CFU/mL、105CFU/mL、106CFU/mL。
In the optimal technical scheme, step 2) is used for the antigen antibody complex for obtaining solid phase；Step 3) realizes biotin
Connection between the bacillus cereus monoclonal antibody of change and the antigen antibody complex of solid phase；Step 4) using biotin-
Streptavidin system realizes the connection with catalase C100；Step 5) enzymatic decomposing hydrogen dioxide solution；Step 6) realizes chromogenic reaction.
In the optimal technical scheme：Each reagent can first can reuse before prior to more than equilibrium at room temperature 30min；Step 2)
The addition of middle testing sample is preferably 80~120 μ L/ holes, more optimizedly 100 μ L/ holes；It is biotinylated waxy in step 3)
The addition of bacillus monoclonal antibody is preferably 80~120 μ L/ holes, more optimizedly 100 μ L/ holes；Strepto- parent in step 4)
Addition with the catalase C100 of element mark is preferably 80~120 μ L/ holes, more optimizedly 100 μ L/ holes；Hydrogen peroxide in step 5)
The addition of solution is preferably 80~120 μ L/ holes, more optimizedly 100 μ L/ holes；The cadmium telluride that mercaptopropionic acid is modified in step 6)
The addition of quantum dot is preferably 30~70 μ L/ holes, more optimizedly 50 μ L/ holes.
Preferably, step 1) specifically includes following operation：
A coating buffer, dilution) are used as using the carbonate buffer solution of 0.04~0.06mol/L, pH9.4~9.8 on ELISA Plate
Bacillus cereus polyclonal antibody is to 9~11 μ g/mL；
B cleaning solution washing ELISA Plate is utilized after) removing the liquid on ELISA Plate, adds bovine serum albumin(BSA) confining liquid,
1~3h is closed in 35~39 DEG C, then discards confining liquid.
It can further perform following preferred：The concentration of the bovine serum albumin(BSA) is 0.3~0.7%, more optimizedly
0.5%；The addition of confining liquid is 320~360 μ L/ holes, and more excellent is 340 μ L/ holes；Discard the ELISA Plate after confining liquid in
Dry at room temperature, and after 2~6 DEG C of preservations.
Preferably, step A) in the addition of coating buffer be 80~120 μ g/ holes, 8~12h is stood after dilution and is performed again
Step B).
Preferably, step 3) the biotinylated bacillus cereus monoclonal antibody is to be prepared by the following method
's：Prepare containing 1~3mg/mL bacillus cereus monoclonal antibody, the PBS solution of 0.07~0.08mg/mL biotins, in
30~60min is reacted under the conditions of lucifuge, the concentration of the PBS solution is 0.005~0.02mol/L, and then dialysis removes biology
Element, that is, obtain the biotinylated bacillus cereus monoclonal antibody.It can further perform following preferred：The biology
Element is active esterification biotin；The time of the dialysis is 66~78h, more optimizedly 72h.
Preferably, the catalase C100 of the step 4) marked by streptavidin is prepared by the following method：Prepare
PBS solution containing 0.5~2mg/mL Streptavidins, the concentration of the PBS solution is 0.005~0.02mol/L, is then added
Enter SM (PEG)24React 30~60min, then gel column purify, collect filtrate add thereto catalase C100 to final concentration 1~
3mg/mL, that is, obtain the catalase C100 of the marked by streptavidin.In the optimal technical scheme：Gel column purifying link is used
In the unnecessary crosslinking agent of removal；Wherein protein content can first be detected and mix the solution containing albumen by collecting obtained filtrate
Catalase C100 is added after conjunction.
Preferably, the cadmium telluride quantum dot of step 6) the mercaptopropionic acid modification is prepared by the following method：Match somebody with somebody
It is precursor to make the solution received containing 8~12mmol/L cadmium nitrates, 20~28mmol/L mercaptopropionic acids, 3~7mmol/L hydrogen tellurides
Solution, the pH of the precursor solution is 11~11.5, by the precursor solution heating water bath to 93~97 DEG C, that is, obtains the mercapto
The cadmium telluride quantum dot of base propionic acid modification.
Preferably, the detection of fluorescence intensity is realized using ELIASA in step 7), and excitation wavelength 310nm, hair
The a length of 590nm of ejected wave.
Preferable on the basis of any of the above technical scheme, the washing is rinsed or soaked using cleaning solution, described to wash
It is the PBST solution containing 0.3~0.7% (v/v) Tween-20 to wash liquid, the concentration of the PBST solution for 0.005~
0.02mol/L, the pH of the PBST solution is 7.0~7.5.
In above technical scheme, catalase (Catalase) is also known as catalase, the catalase used in the present invention
C100 refers exclusively to catalase produced by Sigma-Aldrich, model cat.No.C100.It is described biotinylated
Bacillus cereus monoclonal antibody, refer to obtain after biotin is covalently attached into bacillus cereus monoclonal antibody molecule
Compound.The catalase C100 of the marked by streptavidin, refer to Streptavidin and catalase C100 carrying out covalent bond
The compound of gained.
In above technical scheme, ELISA Plate can select 96 hole black fluorescent ELISA Plates.96 hole black fluorescent ELISA Plates, it is raw
Thing element, anti-bacillus cereus monoclonal antibody, anti-bacillus cereus polyclonal antibody, Streptavidin, catalase C100 are equal
It can be bought in market.
Preferably, the washing is that the cleaning solution in 320~360 μ L/ holes is added into enzyme mark hole, washing 3~4 times, often
10~30s of minor tick.
Preferably, the testing sample can be vegetables, when testing sample is vegetables, first carries out following operation and enter again
Row detection：(1) vegetables bought are weighed into 1mg to be put in sterile test tube, sterilize 1h in super-clean bench, then smashs to pieces；(2) add
9mL PBS (pH 7.4,0.01mol/L) and 1mL bacterium solution, acutely shake 30 minutes；(3) to take supernatant to add immunomagnetic beads dense
Contracting enrichment is standby for 1mL.
The present invention is detected using enzyme-linked immunologic adsorption test method.For detecting the novel fluorescence of bacillus cereus
The principle of ELISA detection method is：Bacillus cereus is combined with coated polyclonal antibody, then plus biotinylated
Monoclonal antibody, along with SA CAT (the catalase C100 of marked by streptavidin, similarly hereinafter), dioxygen moisture is catalyzed by catalase
Solution, the fluorescent quenching of the cadmium telluride quantum dot to mercaptopropionic acid modification is reduced, according to the height of fluorescence intensity come in judgement sample
The concentration of bacillus cereus.If the bacillus cereus concentration in sample is high, fluorescence intensity is high；Conversely, then fluorescence intensity
It is low.That is the concentration of the height of fluorescence intensity and the bacterium in sample is into positive correlation.This method can be directly used for detecting the wax in romaine lettuce
Sample bacillus.The detection method of the present invention is easy to operate, and detection is sensitive, accurate, quick, suitable for the inspection of batch samples
Survey.
Had the advantages that using technical solution of the present invention：
1st, the inventive method uses the horseradish peroxidase in more sensitive new catalase substitution conventional ELISA method
Enzyme, cost can be greatlyd save.
2nd, the inventive method uses more sensitive new fluorogenic substrate (cadmium telluride quantum dot that mercaptopropionic acid is modified)
Substitute the chemical colour reaction substrate in conventional ELISA method, its detection sensitivity can be greatly improved, relative to traditional ELISA extremely
The 2-3 orders of magnitude are improved less.
Brief description of the drawings
Fig. 1 is detection method and the conventional detection method using horseradish peroxidase as antibody labeling thing enzyme
Principle comparison diagram.
Fig. 2 is fluorescence intensity in the embodiment of the present invention 1-bacteria concentration canonical plotting.
Fig. 3 is fluorescence intensity in the embodiment of the present invention 2-bacteria concentration canonical plotting.
Embodiment
The embodiment of the present invention will be described in detail below.In order to avoid excessive unnecessary details,
It is will not be described in detail in following examples to belonging to known structure or function.
Approximating language used in following examples can be used for quantitative expression, show do not changing the feelings of basic function
Under condition quantity can be allowed to have certain variation.Therefore, it is accurate that the numerical value corrected with the language such as " about ", " left and right " is not limited to this
Numerical value is in itself.In certain embodiments, the numerical value for " about " representing to allow its amendment is in the scope of positive and negative 10 (10%)
Interior change, such as, " about 100 " represent can be any numerical value between 90 to 110.In addition, " the about first numerical value arrives
In the statement of second value ", the first and second numerical value two values are at about corrected.In some cases, approximating language
May be relevant with the precision of measuring instrument.
In addition to being defined, technology used and scientific terminology have and art technology people of the present invention in following examples
The identical meanings that member is commonly understood by.
Test reagent consumptive material used, is routine biochemistry reagent unless otherwise specified in following examples；The experiment
Method, it is conventional method unless otherwise specified；Quantitative test in following examples, it is respectively provided with and repeats to test three times, as a result
Average；% in following examples, it is weight/mass percentage composition unless otherwise instructed.
In following examples, the bacillus cereus monoclonal antibody and polyclonal antibody of Anti-Biotin are bought in Wuxi
Sino-German Bai Er Bioisystech Co., Ltd；Described biotin (Cat.No.B5161), Streptavidin (Cat.No.S4762),
Catalase (cat.No.C100) and substrate solution A hydrogen peroxide (35%, cat.No.349887) are bought in U.S. Sigma-Aldrich
Company.Described catalase C100 refers to catalase (cat.No.C100).
(wax of the novel fluorescence ELISA detection method of present invention detection bacillus cereus in romaine lettuce is detected of embodiment 1
Application in sample bacillus content)
When novel fluorescence ELISA detection method of the present invention is used to detect bacillus cereus content in romaine lettuce, by with
Lower step is implemented：Sample pre-treatments, detected with detection method, analysis result.
(1) sample pre-treatments
The romaine lettuce sample 1g handled well is taken, is added in the sterile PBS of 9ml, adds 1ml bacterium solutions, acutely concussion 30 minutes；
It is 1mL liquid to take supernatant to add immunomagnetic beads to carry out enrichment concentration, is taken out standby.
(2) detect bacillus cereus content in above-mentioned sample with detection method
The ELISA Plate for being coated with anti-bacillus cereus content polyclonal antibody is taken, adds the μ L/ holes of standard items/sample 100 to arrive
In corresponding micropore；The ELISA Plate for being coated with anti-bacillus cereus polyclonal antibody is taken, this 100 μ L/ hole of sample-adding are corresponding to
In micropore, gently vibration mixes, and reacts 60min with being placed after cover plate membrane cover plate in 37 DEG C of light protected environments；Cover plate film carefully is opened,
Liquid in hole is dried, with the μ L/ holes of wash operating solution 340, fully washing 4 times, per minor tick 10s, is patted dry and (patted dry with blotting paper
The bubble not being eliminated afterwards can be poked with original pipette tips), add biotinylation bacillus cereus monoclonal antibody
100 μ L/ holes, gently vibration mix, and react 60min with being placed after cover plate membrane cover plate in 37 DEG C of light protected environments；Carefully open cover plate
Film, liquid in hole is dried, with the μ L/ holes of wash operating solution 340, fully washing 3 times, per minor tick 10s, patted dry with blotting paper
(bubble not being eliminated after patting dry can be poked with original pipette tips), SA CAT are added, gently vibration mixes, with cover plate film
Placed after cover plate in 37 DEG C of light protected environments and react 60min；Cover plate film carefully is opened, liquid in hole is dried, uses wash operating solution
340 μ L/ holes, fully washing 3 times, per minor tick 10s, pat dry that (bubble that is not eliminated after patting dry is available to be not used with blotting paper
The pipette tips crossed are poked).Substrate solution A hydrogen peroxide (10 μm of ol/L) dilution, 100 μ L/ holes are added, gently vibration mixes, and uses cover plate
30min is reacted in the rearmounted 37 DEG C of light protected environments of membrane cover plate；Add the cadmium telluride quantum dot bottom of fluorogenic substrate liquid B mercaptopropionic acids modification
The μ L/ holes of thing liquid 50, gently vibration mix, and with 15min is reacted in the rearmounted 37 DEG C of light protected environments of cover plate membrane cover plate, set luciferase mark
Instrument (U.S. Thermo Varioskan Flash all-wave lengths multi-function microplate reader) is 310nm in excitation wavelength, and launch wavelength is
Detect, determined per hole fluorescent value (data are please run through in 5min) at 590nm；With the fluorescent value and standard items of standard items test
Log concentration value draws standard curve, and reference standard curve calculates the content of bacillus cereus in sample.
The preparation of the coated 96 hole black fluorescent ELISA Plate of anti-bacillus cereus polyclonal antibody is to use
0.05mol/L pH 9.6 carbonate (CBS) buffer solution as coating buffer, by bacillus cereus polyclonal antibody (buy in
Wuxi Zhongde Bore Bioisystech Co., Ltd) 10 μ g/mL are interpreted into, 100 μ L/ holes, 4 DEG C stand overnight, and take out ELISA Plate and get rid of
Liquid in plate, with the μ L/ holes of concentrated cleaning solution 340 after dilution, board-washing is secondary, 30s/ times；Then 0.5% bovine serum albumin(BSA) is added
(BSA, buying in Sigma-Aldrich, cat.No.A4737) is closed, 340 μ L/ holes, 37 DEG C of placement 2h, discards envelope
Liquid is closed, (25 DEG C) dry between the ELISA Plate after patting dry places constant temperature；Inspect by random samples it is qualified after will be protected at rearmounted 4 DEG C of ELISA Plate vacuum sealing
Deposit.
Described bacillus cereus debita spissitudo gradient is respectively 100CFU/mL、101CFU/mL、102CFU/mL、
103CFU/mL、104CFU/mL、105CFU/mL。
The biotinylated bacillus cereus monoclonal antibody obtains in the following manner：By the waxy gemma bars of 1mg
Bacterium monoclonal antibody is diluted with PBS (pH 8.6,0.01mol/L), and 50 μ L of addition, which are vivaciously esterified biotin (0.76mg/mL), to be made to resist
The final concentration of 2mg/mL of body, room temperature lucifuge reaction 45min.Reaction product is dialysed 72h in 0.01mol/L PBS solution, is gone
Except free biotin；After dialysis terminates, sample is freeze-dried to obtain biotinylated bacillus cereus monoclonal antibody,
Packing, -20 DEG C of preservations.
The SA@CAT are obtained in the following manner：SA is dissolved as 1mg/mL with PBS (pH 8.6,0.01mol/L), then
Add 10 μ L SM (PEG)24(Thermo：22104,82.8mg/mL) 45min is reacted, reaction uses gel column (Thermo after terminating：
43230) isolate and purify, unnecessary crosslinking agent of going out；The liquid of collection is determined with Nanodrop, will have being mixed with solution for albumen
Close, be then added in 5mg catalase solution (final concentration 2mg/mL).The sample of acquisition is freeze-dried, packing, -20 DEG C of preservations.
The preparation of the biotinylation bacillus cereus monoclonal antibody working solution：Using active esterification biotin and wax
Sample bacillus monoclonal antibody is coupled what is obtained, and 1 is diluted to PBS (0.01M, pH7.4):230.
The preparation of the SA@CAT working solutions：Be coupled what is obtained using Streptavidin and catalase, with PBS (0.01M,
PH7.4) it is diluted to 1:300.
The preparation of the substrate solution A hydrogen peroxide：10 μm of ol/L are diluted to PBS (0.01mol/L, pH7.4).The sulfydryl
The preparation of the cadmium telluride quantum dot fluorogenic substrate liquid of propionic acid modification：Method, 1 is diluted to PBS (0.01mol/L, pH7.4):
400。
The concentrated cleaning solution is 10 times of concentrated cleaning solutions, and it includes 0.5% Tween-20,0.01mol/L PBST, pH
It is worth between scope 7.0-7.5.
(3) analysis result
With the bacterium solution 10 of 6 bacillus cereus various concentrations of above-mentioned preparation1CFU/mL、102CFU/mL、103CFU/
mL、104CFU/mL、105CFU/mL、106CFU/mL.It is 310nm in excitation wavelength, launch wavelength is that detection fluorescence is strong at 590nm
Degree.
It is quenched the calculating of percentage fluorescence rate, percentage fluorescence rate equal to first standard (0 mark is quenched in standard items or sample
It is accurate) fluorescence intensity level subtract standard items or sample fluorescence intensity level average value (diplopore), then except in first standard (0
Standard), i.e. percentage fluorescence rate (%)=(F0-F)/F0× 100%, wherein F0For the fluorescence intensity of first standard (0 standard)
Value, F are the average value (diplopore) of the fluorescence intensity level of standard items or sample.
So that percentage fluorescence rate is quenched as ordinate, it is bent that standard is drawn with bacillus cereus bacteria concentration (CFU/mL) abscissa
Line, obtain linear equation.Standard curve is y=-2.822Log (x)+78.031, R2=0.9808, see accompanying drawing 2.This method
The fluorescent value that minimum detection limit is defined as 0CFU/mL adds three standard deviations.Lowest detection is calculated to obtain by the standard curve
Line is 5*101CFU/mL.When carrying out actual sample detection, by the fluorescent value ((F of sample0-F)/F0× 100%) value substitutes into mark
In directrix curve, the concentration of sample corresponding to reading from standard curve, it is waxy in sample to be multiplied by its corresponding extension rate
The actual concentrations of bacillus.
Embodiment 2 (using horseradish peroxidase as antibody labeling enzyme, is used as the bacillus cereus of chromogenic substrate using TMB
ELISA detection method)
When traditional ELISA detection method is used to detect bacillus cereus content in romaine lettuce and beef items, by with
Lower step is implemented：Sample pre-treatments, detected with traditional ELISA detection method, analysis result.
(1) sample pre-treatments
The romaine lettuce sample 1g handled well is taken, is added in the sterile PBS of 9ml, adds 1ml bacterium solutions, acutely concussion 30 minutes；
It is 1mL liquid to take supernatant to add immunomagnetic beads to carry out enrichment concentration, is taken out standby.
(2) detect bacillus cereus content in above-mentioned sample with traditional ELISA detection method
The ELISA Plate for being coated with anti-bacillus cereus content polyclonal antibody is taken, adds the μ L/ holes of standard items/sample 100 to arrive
In corresponding micropore；The ELISA Plate for being coated with anti-bacillus cereus polyclonal antibody is taken, this 100 μ L/ hole of sample-adding are corresponding to
In micropore, gently vibration mixes, and reacts 60min with being placed after cover plate membrane cover plate in 37 DEG C of light protected environments；Cover plate film carefully is opened,
Liquid in hole is dried, with the μ L/ holes of wash operating solution 340, fully washing 4 times, per minor tick 10s, is patted dry and (patted dry with blotting paper
The bubble not being eliminated afterwards can be poked with original pipette tips), add biotinylation bacillus cereus monoclonal antibody
100 μ L/ holes, gently vibration mix, and react 60min with being placed after cover plate membrane cover plate in 37 DEG C of light protected environments；Carefully open cover plate
Film, liquid in hole is dried, with the μ L/ holes of wash operating solution 340, fully washing 3 times, per minor tick 10s, patted dry with blotting paper
(bubble not being eliminated after patting dry can be poked with original pipette tips), SA HRP are added, gently vibration mixes, with cover plate film
Placed after cover plate in 37 DEG C of light protected environments and react 60min；Cover plate film carefully is opened, liquid in hole is dried, uses wash operating solution
340 μ L/ holes, fully washing 3 times, per minor tick 10s, pat dry that (bubble that is not eliminated after patting dry is available to be not used with blotting paper
The pipette tips crossed are poked)；TMB nitrite ions are added, 100 μ L/ holes, gently vibration mixes, with the rearmounted 37 DEG C of lucifuge rings of cover plate membrane cover plate
15min is reacted in border；Terminate liquid 50 μ L/ holes are added, gently vibration mixes, and setting ELIASA is at 450nm or dual wavelength 450nm
Place's detection, is determined per hole absorbance (data are please run through in 5min)；The absorbance for contrasting testing sample and standard items is big
It is small, the residual quantity of the bacillus cereus in quantitative analysis testing sample.
(3) analysis result
With the bacterium solution 10 of 6 bacillus cereus various concentrations of above-mentioned preparation1CFU/mL、102CFU/mL、103CFU/
mL、104CFU/mL、105CFU/mL、106CFU/mL.The percent absorption of the calculating of percentage absorptance, standard items or sample is equal to
The photon absorbing intensity average value (diplopore) of standard items or sample subtracts the photon absorbing intensity value of first standard (0 standard), then except in mark
The photon absorbing intensity average value (diplopore) of quasi- product or sample, i.e. percentage absorptance (%)=(B-B0)/B × 100%, wherein B are mark
The photon absorbing intensity average value (diplopore) of quasi- product or sample, B0For the photon absorbing intensity value of first standard (0 standard).
It is that abscissa draws standard with bacillus cereus concentration (CFU/mL) using standard items percentage absorptance as ordinate
Curve, obtain linear equation.Standard curve is y=9.9009Log (x) -115.74, R2=0.9738, see accompanying drawing 3.This method
Lowest detection line be defined as percentage absorptance and add three standard deviations more than 0CFU/mL absorptance.Pass through the standard
Curve calculate lowest detection is limited to 5*105CFU/mL.When carrying out actual sample detection, by the percentage fluorescence rate of sample ((B-
B0)/B × 100%) value substituted into standard curve, read from standard curve corresponding to sample concentration, be multiplied by dilute corresponding to it
Release the actual concentrations that multiple is bacillus cereus in sample.
By the contrast of embodiment 1 and embodiment 2 it can be found that the new E LISA methods sensitivity of the present invention is up to classical
10000 times of ELISA method, while it is any with high-sensitivity detection to also demonstrate that new E LISA methods of the invention are applicable to
The material that conventional ELISA method can detect.
Embodiment 3
A kind of quick determination method for bacillus cereus, this method belong to double antibodies sandwich ELISA, the party
Method is the detection for bacillus cereus antigen, and the enzyme that labelled antibody is used in this method is catalase C100.
On the basis of above technical scheme, meet following condition：
Substrate includes the cadmium telluride quantum dot that hydrogen peroxide and mercaptopropionic acid are modified in this method.
Specific detection method comprises the following steps：
1) it is coated with bacillus cereus antibody；
2) antibody after step 1) coating is taken, mixes after reacting 40min in 35 DEG C of light protected environments, washes with testing sample
Wash；
3) biotinylated bacillus cereus monoclonal antibody mixing is then added, is reacted in 35 DEG C of light protected environments
40min, washing；
4) the catalase C100 mixing of marked by streptavidin is then added, 40min is reacted in 35 DEG C of light protected environments, washes
Wash；
5) the hydrogen peroxide solution mixing that concentration is 8 μm of ol/L is then added, reacts 20min in 35 DEG C of light protected environments；
6) the cadmium telluride quantum dot mixing of mercaptopropionic acid modification is then added, reacts 10min in 35 DEG C of light protected environments；
7) detecting step 6) product fluorescence intensity.
Wherein step 1) specifically includes following operation：
A) using 0.04mol/L, pH9.4 carbonate buffer solution as coating buffer on ELISA Plate, waxy gemma bar is diluted
For bacterium polyclonal antibody to 9 μ g/mL, the addition of coating buffer is 80 μ g/ holes, and 8h is stood after dilution；
B cleaning solution washing ELISA Plate is utilized after) removing the liquid on ELISA Plate, adds bovine serum albumin(BSA) confining liquid,
1h is closed in 35 DEG C, then discards confining liquid.
Step 3) the biotinylated bacillus cereus monoclonal antibody is prepared by the following method：Preparation contains
There are 1mg/mL bacillus cereus monoclonal antibody, the PBS solution of 0.07mg/mL biotins, reacted under the conditions of lucifuge
30min, the concentration of the PBS solution is 0.005mol/L, and then dialysis removes biotin, that is, obtains the biotinylated wax
Sample bacillus monoclonal antibody.
The catalase C100 of the step 4) marked by streptavidin is prepared by the following method：Preparation contains 0.5mg/
The PBS solution of mL Streptavidins, the concentration of the PBS solution is 0.005mol/L, then adds SM (PEG)24Reaction
30min, then gel column purifying, collect filtrate and add catalase C100 to final concentration 1mg/mL thereto, that is, obtain the strepto-
The catalase C100 of Avidin mark.
The cadmium telluride quantum dot of step 6) the mercaptopropionic acid modification is prepared by the following method：Preparation contains
The solution that 8mmol/L cadmium nitrates, 20mmol/L mercaptopropionic acids, 3mmol/L hydrogen tellurides are received is precursor solution, the precursor solution
PH be 11, by the precursor solution heating water bath to 93 DEG C, that is, obtain the cadmium telluride quantum dot of mercaptopropionic acid modification.
The detection of fluorescence intensity is realized using ELIASA in step 7), excitation wavelength 310nm, and launch wavelength is
590nm。
The washing is rinsed or soaked using cleaning solution, and the cleaning solution contains 0.3% (v/v) Tween-20
PBST solution, the concentration of the PBST solution is 0.005mol/L, and the pH of the PBST solution is 7.0.
Embodiment 4
A kind of quick determination method for bacillus cereus, this method belong to double antibodies sandwich ELISA, the party
Method is the detection for bacillus cereus antigen, and the enzyme that labelled antibody is used in this method is catalase C100.
On the basis of above technical scheme, meet following condition：
Substrate includes the cadmium telluride quantum dot that hydrogen peroxide and mercaptopropionic acid are modified in this method.
Specific detection method comprises the following steps：
1) it is coated with bacillus cereus antibody；
2) antibody after step 1) coating is taken, mixes after reacting 80min in 39 DEG C of light protected environments, washes with testing sample
Wash；
3) biotinylated bacillus cereus monoclonal antibody mixing is then added, is reacted in 39 DEG C of light protected environments
80min, washing；
4) the catalase C100 mixing of marked by streptavidin is then added, 80min is reacted in 39 DEG C of light protected environments, washes
Wash；
5) the hydrogen peroxide solution mixing that concentration is 12 μm of ol/L is then added, reacts 40min in 39 DEG C of light protected environments；
6) the cadmium telluride quantum dot mixing of mercaptopropionic acid modification is then added, reacts 20min in 39 DEG C of light protected environments；
7) detecting step 6) product fluorescence intensity.
Wherein step 1) specifically includes following operation：
A) using 0.06mol/L, pH9.8 carbonate buffer solution as coating buffer on ELISA Plate, waxy gemma bar is diluted
For bacterium polyclonal antibody to 11 μ g/mL, the addition of coating buffer is 120 μ g/ holes, and 12h is stood after dilution；
B cleaning solution washing ELISA Plate is utilized after) removing the liquid on ELISA Plate, adds bovine serum albumin(BSA) confining liquid,
3h is closed in 39 DEG C, then discards confining liquid.
Step 3) the biotinylated bacillus cereus monoclonal antibody is prepared by the following method：Preparation contains
There are 3mg/mL bacillus cereus monoclonal antibody, the PBS solution of 0.08mg/mL biotins, reacted under the conditions of lucifuge
60min, the concentration of the PBS solution is 0.02mol/L, and then dialysis removes biotin, that is, obtains the biotinylated wax
Sample bacillus monoclonal antibody.
The catalase C100 of the step 4) marked by streptavidin is prepared by the following method：Preparation contains 2mg/mL
The PBS solution of Streptavidin, the concentration of the PBS solution is 0.02mol/L, then adds SM (PEG)2460min is reacted, and
Gel column purifies afterwards, collects filtrate and adds catalase C100 to final concentration 3mg/mL thereto, that is, obtains the Streptavidin mark
The catalase C100 of note.
The cadmium telluride quantum dot of step 6) the mercaptopropionic acid modification is prepared by the following method：Preparation contains
The solution that 12mmol/L cadmium nitrates, 28mmol/L mercaptopropionic acids, 7mmol/L hydrogen tellurides are received is precursor solution, and the precursor is molten
The pH of liquid is 11.5, by the precursor solution heating water bath to 97 DEG C, that is, obtains the cadmium telluride quantum of the mercaptopropionic acid modification
Point.
The detection of fluorescence intensity is realized using ELIASA in step 7), excitation wavelength 310nm, and launch wavelength is
590nm。
The washing is rinsed or soaked using cleaning solution, and the cleaning solution contains 0.7% (v/v) Tween-20
PBST solution, the concentration of the PBST solution is 0.02mol/L, and the pH of the PBST solution is 7.5.
Embodiment 5
A kind of quick determination method for bacillus cereus, this method belong to double antibodies sandwich ELISA, the party
Method is the detection for bacillus cereus antigen, and the enzyme that labelled antibody is used in this method is catalase C100.
On the basis of above technical scheme, meet following condition：
Substrate includes the cadmium telluride quantum dot that hydrogen peroxide and mercaptopropionic acid are modified in this method.
Specific detection method comprises the following steps：
1) it is coated with bacillus cereus antibody；
2) antibody after step 1) coating is taken, mixes after reacting 60min in 37 DEG C of light protected environments, washes with testing sample
Wash；
3) biotinylated bacillus cereus monoclonal antibody mixing is then added, is reacted in 37 DEG C of light protected environments
60min, washing；
4) the catalase C100 mixing of marked by streptavidin is then added, 60min is reacted in 37 DEG C of light protected environments, washes
Wash；
5) the hydrogen peroxide solution mixing that concentration is 10 μm of ol/L is then added, reacts 30min in 37 DEG C of light protected environments；
6) the cadmium telluride quantum dot mixing of mercaptopropionic acid modification is then added, reacts 15min in 37 DEG C of light protected environments；
7) detecting step 6) product fluorescence intensity.
Embodiment 6
A kind of quick determination method for bacillus cereus, this method belong to double antibodies sandwich ELISA, the party
Method is the detection for bacillus cereus antigen, and the enzyme that labelled antibody is used in this method is catalase C100, this method midsole
Thing includes the cadmium telluride quantum dot that hydrogen peroxide and mercaptopropionic acid are modified.
Embodiment 7
A kind of quick determination method for bacillus cereus, this method belong to double antibodies sandwich ELISA, the party
Method is the detection for bacillus cereus antigen, and the enzyme that labelled antibody is used in this method is catalase C100.
Embodiments of the invention are described in detail above, but the content is only presently preferred embodiments of the present invention,
It is not intended to limit the invention.All all any modification, equivalent and improvement done in the application range of the present invention etc., all should
Within protection scope of the present invention.
Claims (8)
1. a kind of quick determination method for bacillus cereus, this method belongs to double antibodies sandwich ELISA, this method
It is the detection for bacillus cereus antigen, the enzyme that labelled antibody is used in this method is catalase C100, substrate in this method
The cadmium telluride quantum dot modified including hydrogen peroxide and mercaptopropionic acid.
2. the quick determination method according to claim 1 for bacillus cereus, it is characterised in that including following step
Suddenly：
1) it is coated with bacillus cereus antibody；
2) antibody after step 1) coating is taken, is mixed with testing sample after 40~80min of reaction in 35~39 DEG C of light protected environments,
Washing；
3) biotinylated bacillus cereus monoclonal antibody mixing is then added, reacts 40 in 35~39 DEG C of light protected environments
~80min, washing；
4) the catalase C100 mixing of marked by streptavidin is then added, 40~80min is reacted in 35~39 DEG C of light protected environments,
Washing；
5) the hydrogen peroxide solution mixing that concentration is 8~12 μm of ol/L is then added, react 20 in 35~39 DEG C of light protected environments~
40min；
6) then add mercaptopropionic acid modification cadmium telluride quantum dot mixing, in 35~39 DEG C of light protected environments react 10~
20min；
7) detecting step 6) product fluorescence intensity.
3. the quick determination method according to claim 2 for bacillus cereus, it is characterised in that step 1) is specific
Including following operation：
A it is) waxy as coating buffer, dilution using the carbonate buffer solution of 0.04~0.06mol/L, pH9.4~9.8 on ELISA Plate
Bacillus polyclonal antibody is to 9~11 μ g/mL；
B cleaning solution washing ELISA Plate is utilized after) removing the liquid on ELISA Plate, bovine serum albumin(BSA) confining liquid is added, in 35
~39 DEG C of 1~3h of closing, then discard confining liquid.
4. the quick determination method according to claim 3 for bacillus cereus, it is characterised in that step A) in wrap
It is 80~120 μ L/ holes by the addition of liquid, 8~12h is stood after dilution and performs step B again).
5. the quick determination method according to claim 2 for bacillus cereus, it is characterised in that step 3) is described
Biotinylated bacillus cereus monoclonal antibody is prepared by the following method：Preparation contains the waxy buds of 1~3mg/mL
Spore bacillus monoclonal antibody, the PBS solution of 0.07~0.08mg/mL biotins, 30~60min, institute are reacted under the conditions of lucifuge
The concentration for stating PBS solution is 0.005~0.02mol/L, and then dialysis removes biotin, that is, obtains described biotinylated waxy
Bacillus monoclonal antibody.
6. the quick determination method according to claim 2 for bacillus cereus, it is characterised in that step 4) is described
The catalase C100 of marked by streptavidin is prepared by the following method：Preparation contains 0.5~2mg/mL Streptavidins
PBS solution, the concentration of the PBS solution is 0.005~0.02mol/L, then adds SM (PEG)2430~60min is reacted, and
Gel column purifies afterwards, collects filtrate and adds catalase C100 to 1~3mg/mL of final concentration thereto, that is, obtains the Streptavidin
The catalase C100 of mark.
7. the quick determination method according to claim 2 for bacillus cereus, it is characterised in that step 6) is described
The cadmium telluride quantum dot of mercaptopropionic acid modification is prepared by the following method：Prepare containing 8~12mmol/L cadmium nitrates, 20~
28mmol/L mercaptopropionic acids, the solution of 3~7mmol/L sodium hydrogen tellurides are precursor solution, the pH of the precursor solution for 11~
11.5, by the precursor solution heating water bath to 93~97 DEG C, that is, obtain the cadmium telluride quantum dot of the mercaptopropionic acid modification.
8. the quick determination method according to claim 2 for bacillus cereus, it is characterised in that glimmering in step 7)
The detection of luminous intensity utilizes ELIASA realization, excitation wavelength 310nm, launch wavelength 590nm.
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