CN105759031B - A kind of quick determination method for campylobacter jejuni - Google Patents
A kind of quick determination method for campylobacter jejuni Download PDFInfo
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- CN105759031B CN105759031B CN201610156407.2A CN201610156407A CN105759031B CN 105759031 B CN105759031 B CN 105759031B CN 201610156407 A CN201610156407 A CN 201610156407A CN 105759031 B CN105759031 B CN 105759031B
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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
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- 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
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- G01N33/531—Production of immunochemical test materials
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Abstract
The present invention provides a kind of quick determination method for campylobacter jejuni, this method is first combined campylobacter jejuni 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 judgement sample jejuni.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 marker 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 at the same time, 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 campylobacter jejuni.
Background technology
Campylobacter jejuni (Campylobacter), belongs to spiral Cordycepps, Gram-negative, thalline hettocyrtosis like comma shape,
1.5~5 μm long, 0.2~0.8 μm wide, thalline one or both ends amphitrichous, movement is vivaciously.The strain is in 1973 from diarrhoeal diseases
Separated first in human faecal mass, be one of Main Pathogenic Bacteria for causing mankind's diarrhea.Campylobacter jejuni has endotoxin to attack small intestine
Cause chordapsus with colorectal mucosa, can also cause outbreak of epidemic or the mass food poisoning of diarrhea.Incubation period is generally 3~5
My god, the pathogenic position to people is jejunum, ileum and colon.Cardinal symptom is diarrhea and abdominal pain, is generated heat sometimes, occasionally has vomiting and takes off
Water.Bacterium can be entered blood flow by intestinal mucosa and cause septicemia and other organs to infect sometimes, such as meningitis, arthritis, renal plevis kidney
Inflammation etc..This bacterium of infection of pregnant women can cause to miscarry, premature labor, and neonate can be made to be contaminted.In this case, establish sensitive, fast
The detection method of speed is the important technology premise of effective prevention campylobacter jejuni.
It is mainly in the prior art biochemical identification method for campylobacter jejuni detection method, but detection time is long, reagent
Of high cost, especially campylobacter jejuni micro- aerobic characteristic, is separately cultured it increasingly difficult, it is necessary to the micro- aerobic bar of extra maintenance
The facility and reagent of part, in recent years, with the development of molecular biology, DNA sequencing technology obtains in the detection of campylobacter jejuni
To being widely applied, such method sensitiveness is higher, specific relatively strong, but defect also more prominent on the one hand its cost compared with
Height, operating procedure is relatively cumbersome, time-consuming longer in addition, therefore the commercial application of such method is restricted all the time.In recent years,
Enzyme linked immunosorbent assay (ELISA) receives more and more attention in the detection of campylobacter jejuni, and such method is because having
Quickly, it is sensitive, special, accurate, can quantify, is easy to operate, without valuable instrument and equipment, it is and of less demanding to sample purity, because
This detection especially suitable for batch samples.However, the ELISA method for detecting campylobacter jejuni in the prior art is generally based on
Antibody or antigen the catalysis hydrogen peroxide generation hydroxy radical of horseradish peroxidase-labeled, and then aoxidize colourless chemical colour reaction bottom
Thing tetramethyl biphenyl diamines (TMB) forms blue product, then using terminate liquid (2M H2SO4) terminate reaction form yellow solution
Absorbance is recorded at 450nm.Such method is because its colored intensity is relatively low, therefore detection sensitivity is relatively low, when to be measured
Easily there is false negative result when object content is relatively low in sample, so as to can not meet the requirement of practical application.
In recent years, some new signal transduction mechanisms have been reported for substituting the signal transduction mechanism use of traditional ELISA
In the sensitivity for improving ELISA, as radiommunoassay substrate, chemical luminous substrate, fluorogenic substrate and resonance colloidal gold 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, select which kind of label enzyme and its coupling method with antibody, bottom of developing the color
The selection of thing and specific luminescent method；It in effect aspect, should ensure the sensitivity of chromogenic reaction, and should meet colored intensity
With the linear relationship of object content.Therefore, it is especially clever to lift its detection for the ELISA detection method of campylobacter jejuni
Method for the purpose of quick property is improved, and has prominent technical difficulty.
The content of the invention
A kind of it is contemplated that technological deficiency for the prior art, there is provided quick detection side for campylobacter jejuni
Method, it is relatively low to solve the campylobacter jejuni ELISA detection method precision of the prior art.
Another technical problem to be solved by the present invention is that the prior art is used for the ELISA method of campylobacter jejuni antigen detection
In, the enzyme sensitivity of labelled antibody is relatively low.
The invention solves another technical problem be the prior art be used for campylobacter jejuni antigen detection ELISA method
In, Color Appearance System sensitivity is relatively low.
The invention solves another technical problem be when using catalase C100 as antibody marker enzyme to campylobacter jejuni
When antigen performs ELISA detections, concrete technology method is simultaneously indefinite.
The invention solves another technical problem be when using catalase C100 as antibody marker enzyme, using hydrogen peroxide and
Mercaptopropionic acid modification cadmium telluride quantum dot as substrate to campylobacter jejuni antigen perform ELISA detection when, testing result and
The linear relationship of antigen concentration is bad.
The invention solves another technical problem be when using catalase C100 as antibody marker enzyme, using hydrogen peroxide and
Mercaptopropionic acid modification cadmium telluride quantum dot as substrate to campylobacter jejuni antigen perform ELISA detect when, during wash
It is ineffective.
To realize above technical purpose, the present invention uses following technical scheme：
A kind of quick determination method for campylobacter jejuni, this method belong to double antibodies sandwich enzyme-linked immunization, this method
It is the detection for campylobacter jejuni antigen, 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 reaction process, hydrogen peroxide decomposes under catalase effect, quenches so as to reduce fluorescence
The effect of going out, realizes and shines.
Preferably, this method comprises the following steps：
1) it is coated with Antibodies of Campylobacter Jejuni；
2) take step 1) be coated with after antibody, mix with sample to be tested after in 35~39 DEG C of light protected environments react 40~
80min, washing；
3) biotinylated campylobacter jejuni monoclonal antibody mixing is then added, is reacted in 35~39 DEG C of light protected environments
40~80min, washing；
4) then add marked by streptavidin catalase C100 mixing, in 35~39 DEG C of light protected environments react 40~
80min, washing；
5) hydrogen peroxide solution mixing of the concentration for 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 sample to be tested jejuni content, using concentration known in practical operation
And multigroup campylobacter jejuni titer of distribution gradient draws fluorescence intensity-bacteria concentration standard curve by above method, then
The jejunum campylobacter bacterial content of sample to be tested is calculated from standard curve using the fluorescence intensity of sample to be tested.Concrete operation method can
With any selection of general technology general knowledge according to the art.Multigroup campylobacter jejuni titer of above-mentioned gradient distribution, can
To select 10 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 campylobacter jejuni monoclonal antibody of change and the antigen antibody complex of solid phase；Step 4) utilizes biotin-chain
Mould Avidin 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 be reused prior to more than equilibrium at room temperature 30min before use；In step 2)
The addition of sample to be tested is preferably 80~120 μ L/ holes, more optimizedly 100 μ L/ holes；Biotinylated jejunum is curved in step 3)
The addition of aspergillus monoclonal antibody is preferably 80~120 μ L/ holes, more optimizedly 100 μ L/ holes；Streptavidin in step 4)
The addition of the catalase C100 of mark is preferably 80~120 μ L/ holes, more optimizedly 100 μ L/ holes；Hydrogen peroxide solution in step 5)
Addition be preferably 80~120 μ L/ holes, more optimizedly 100 μ L/ holes；The cadmium telluride quantum that mercaptopropionic acid is modified in step 6)
The addition of point 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
Campylobacter jejuni 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, more preferably be 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 campylobacter jejuni monoclonal antibody is to be prepared by the following method
's：Prepare containing 1~3mg/mL campylobacter jejunis monoclonal antibody, the PBS solution of 0.07~0.08mg/mL biotins, in keeping away
30~60min is reacted under optical condition, the concentration of the PBS solution is 0.005~0.02mol/L, and then dialysis removes biotin,
Obtain the biotinylated campylobacter jejuni monoclonal antibody.It can further perform following preferred：The biotin 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 purification, 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 purification 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 microplate reader 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 solution, 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
Campylobacter jejuni monoclonal antibody, refers to biotin being covalently attached to answering of being obtained after campylobacter jejuni monoclonal antibody molecule
Compound.The catalase C100 of the marked by streptavidin, refers to carry out Streptavidin and catalase C100 obtained by covalent bond
Compound.
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-campylobacter jejuni monoclonal antibody, anti-campylobacter jejuni polyclonal antibody, Streptavidin, catalase C100 can be in
Market is bought.
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 sample to be tested can be vegetables, when sample to be tested is vegetables, first carry out following operation again into
Row detection：(1) vegetables bought are weighed 1mg to be put in sterile test tube, sterilize 1h in super-clean bench, then smashs to pieces；(2) add
The bacterium solution of 9mL PBS (pH 7.4,0.01mol/L) and 1mL, acutely shakes 30 minutes；(3) to take supernatant to add immunomagnetic beads dense
Contracting enrichment is spare for 1mL.
The present invention is detected using enzyme-linked immunologic adsorption test method.For detecting the novel fluorescence of campylobacter jejuni
The principle of ELISA detection method is：Campylobacter jejuni is combined with coated polyclonal antibody, then plus biotinylated list
Clonal antibody, along with SA@CAT (the catalase C100 of marked by streptavidin, similarly hereinafter), 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 jejunum in judgement sample
The concentration of Campylobacter spp.If the jejunum campylobacter bacteria concentration in sample is high, fluorescence intensity is high；Conversely, then fluorescence intensity is low.That is fluorescence
The height of intensity and the concentration of the bacterium in sample are positively correlated.This method can be directly used for the campylobacter jejuni in detection romaine lettuce.
The detection method of the present invention is easy to operate, and detection is sensitive, accurate, quick, suitable for the detection of batch samples.
Had the advantages that using technical solution of the present invention：
1st, the method for the present invention uses the horseradish peroxidase in more sensitive new catalase substitution conventional ELISA method
Enzyme, can greatly save cost.
2nd, the method for the present invention 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 label 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 will not be described in detail in following embodiments to belonging to known structure or function.
Approximating language used in following embodiments can be used for quantitative expression, show do not changing the feelings of basic function
Quantity is allowed to have certain variation under condition.Therefore, to be not limited to this with the modified numerical value of the language such as " about ", " left and right " institute accurate
Numerical value is in itself.In certain embodiments, the scope for allowing its modified numerical value in positive and negative 10 (10%) " about " is represented
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 related with the precision of measuring instrument.
In addition to being defined, technical and scientific term used has and fields technology people of the present invention in following embodiments
The identical meanings that member is commonly understood by.
Test reagent consumptive material used, is routine biochemistry reagent unless otherwise specified in following embodiments；The experiment
Method, is conventional method unless otherwise specified；Quantitative test in following embodiments, is respectively provided with three repeated experiments, as a result
It is averaged；% in following embodiments, is mass percentage unless otherwise instructed.
In following embodiments, the campylobacter jejuni monoclonal antibody and polyclonal antibody of Anti-Biotin are bought in Wuxi
De Baier Bioisystech Co., Ltd；The biotin (Cat.No.B5161), Streptavidin (Cat.No.S4762), touch
Enzyme (cat.No.C100) and substrate solution A hydrogen peroxide (35%, cat.No.349887) are bought public in U.S. Sigma-Aldrich
Department.The catalase C100 refers to catalase (cat.No.C100).
(jejunum of the novel fluorescence ELISA detection method of present invention detection campylobacter jejuni in romaine lettuce is detected of embodiment 1
Bend the application in bacterial content)
When novel fluorescence ELISA detection method of the present invention is used to detect the jejunum campylobacter bacterial content in romaine lettuce, by following
Step is implemented：Sample pre-treatments, be 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 spare.
(2) it is detected above-mentioned sample jejuni content with detection method
Take the ELISA Plate for being coated with anti-jejunum campylobacter bacterial content polyclonal antibody, add 100 μ L/ holes of standard items/sample to pair
In the micropore answered；Take the ELISA Plate for being coated with anti-campylobacter jejuni polyclonal antibody, this 100 μ L/ hole of sample-adding to corresponding micropore
In, gently vibration mixes, and reacts 60min with being placed after cover board membrane cover plate in 37 DEG C of light protected environments；Cover board film carefully is opened, by hole
Interior liquid drying, with 340 μ L/ holes of wash operating solution, fully washing 4 times, per minor tick 10s, is patted dry (after patting dry not with blotting paper
The bubble being eliminated can be poked with original pipette tips), add 100 μ L/ of biotinylation campylobacter jejuni monoclonal antibody
Hole, gently vibration mix, and react 60min with being placed after cover board membrane cover plate in 37 DEG C of light protected environments；Cover board film carefully is opened, by hole
Interior liquid drying, with 340 μ L/ holes of wash operating solution, fully washing 3 times, per minor tick 10s, is patted dry (after patting dry not with blotting paper
The bubble being eliminated can be poked with original pipette tips), SA CAT are added, gently vibration mixes, with being put after cover board membrane cover plate
Put in 37 DEG C of light protected environments and react 60min；Cover board film carefully is opened, liquid in hole is dried, with 340 μ L/ holes of wash operating solution,
Fully washing 3 times, per minor tick 10s, pats dry that (bubble not being eliminated after patting dry can be stabbed with original pipette tips with blotting paper
It is broken).Substrate solution A hydrogen peroxide (10 μm of ol/L) dilution is added, 100 μ L/ holes, gently vibration mixes, with cover board membrane cover plate postposition
30min is reacted in 37 DEG C of light protected environments；Add the 50 μ L/ of cadmium telluride quantum dot substrate solution of fluorogenic substrate liquid B mercaptopropionic acids modification
Hole, gently vibration mix, and with 15min is reacted in cover board membrane cover plate 37 DEG C of light protected environments of postposition, set the fluorescence microplate reader (U.S.
Thermo Varioskan Flash all-wave lengths multi-function microplate reader) in excitation wavelength it is 310nm, launch wavelength is at 590nm
Detection, measures per hole fluorescent value (data are please run through in 5min)；With the concentration pair of the fluorescent value of standard items test and standard items
Numeric renderings standard curve, reference standard curve calculate the content of sample jejuni.
The preparation of the coated 96 hole black fluorescent ELISA Plate of anti-campylobacter jejuni polyclonal antibody is to use 0.05mol/L
Carbonate (CBS) buffer solution of pH 9.6 (buys campylobacter jejuni polyclonal antibody in the Sino-German Bai Er in Wuxi as coating buffer
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, use is dilute
340 μ L/ holes of concentrated cleaning solution after releasing, board-washing time, 30s/ times；Then adding 0.5% bovine serum albumin(BSA), (BSA, buys in U.S.
Sigma-Aldrich companies of state, cat.No.A4737) closing, 340 μ L/ holes, 37 DEG C of placement 2h, discard confining liquid, after patting dry
(25 DEG C) dry between ELISA Plate places constant temperature；It will be preserved after sampling observation is qualified at 4 DEG C of ELISA Plate vacuum sealing postposition.
The campylobacter jejuni debita spissitudo gradient is respectively 100CFU/mL、101CFU/mL、102CFU/mL、
103CFU/mL、104CFU/mL、105CFU/mL。
The biotinylated campylobacter jejuni monoclonal antibody obtains in the following manner：1mg campylobacter jejunis is single
Clonal 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), makes antibody
Final concentration of 2mg/mL, room temperature lucifuge reaction 45min.Reaction product is dialysed 72h in the PBS solution of 0.01mol/L, removes trip
From biotin；After dialysis, sample is freeze-dried to obtain biotinylated campylobacter jejuni monoclonal antibody, dispensed ,-
20 DEG C of preservations.
The SA@CAT are obtained in the following manner：SA PBS (pH 8.6,0.01mol/L) are dissolved as 1mg/mL, then
Add 10 μ L SM (PEG)24(Thermo：22104,82.8mg/mL) 45min is reacted, after reaction with gel column (Thermo：
43230) isolate and purify, unnecessary crosslinking agent of going out；The liquid of collection is measured 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 campylobacter jejuni monoclonal antibody working solution：Using active esterification biotin and jejunum
Campylobacter spp monoclonal antibody is coupled, and 1 is diluted to PBS (0.01M, pH7.4):230.
The preparation of the SA@CAT working solutions：Be coupled 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, the PBST of 0.01mol/L, pH
It is worth between scope 7.0-7.5.
(3) analysis result
With the bacterium solution 10 of 6 campylobacter jejuni 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 fluorescence intensity at 590nm.
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.
Percentage fluorescence rate is quenched as ordinate, it is bent that standard is drawn with campylobacter jejuni bacteria concentration (CFU/mL) abscissa
Line, obtains linear equation.Standard curve is y=-2.822Log (x)+78.031, R2=0.9808, see attached 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 jejunum in sample to be multiplied by its corresponding extension rate
The actual concentrations of Campylobacter spp.
Embodiment 2 (using horseradish peroxidase as antibody marker enzyme, is used as the campylobacter jejuni of chromogenic substrate using TMB
ELISA detection method)
When traditional ELISA detection method is used to detect the jejunum campylobacter bacterial content in romaine lettuce and beef items, by following
Step is implemented：Sample pre-treatments, be 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 spare.
(2) it is detected above-mentioned sample jejuni content with tradition ELISA detection method
Take the ELISA Plate for being coated with anti-jejunum campylobacter bacterial content polyclonal antibody, add 100 μ L/ holes of standard items/sample to pair
In the micropore answered；Take the ELISA Plate for being coated with anti-campylobacter jejuni polyclonal antibody, this 100 μ L/ hole of sample-adding to corresponding micropore
In, gently vibration mixes, and reacts 60min with being placed after cover board membrane cover plate in 37 DEG C of light protected environments；Cover board film carefully is opened, by hole
Interior liquid drying, with 340 μ L/ holes of wash operating solution, fully washing 4 times, per minor tick 10s, is patted dry (after patting dry not with blotting paper
The bubble being eliminated can be poked with original pipette tips), add 100 μ L/ of biotinylation campylobacter jejuni monoclonal antibody
Hole, gently vibration mix, and react 60min with being placed after cover board membrane cover plate in 37 DEG C of light protected environments；Cover board film carefully is opened, by hole
Interior liquid drying, with 340 μ L/ holes of wash operating solution, fully washing 3 times, per minor tick 10s, is patted dry (after patting dry not with blotting paper
The bubble being eliminated can be poked with original pipette tips), SA HRP are added, gently vibration mixes, with being put after cover board membrane cover plate
Put in 37 DEG C of light protected environments and react 60min；Cover board film carefully is opened, liquid in hole is dried, with 340 μ L/ holes of wash operating solution,
Fully washing 3 times, per minor tick 10s, pats dry that (bubble not being eliminated after patting dry can be stabbed with original pipette tips with blotting paper
It is broken)；TMB nitrite ions, 100 μ L/ holes are added, gently vibration is mixed, reacted with cover board membrane cover plate 37 DEG C of light protected environments of postposition
15min；50 μ L/ holes of terminate liquid are added, gently vibration mixes, and setting microplate reader detects at 450nm or at dual wavelength 450nm,
Measure is per hole absorbance (data are please run through in 5min)；The absorbance size of sample to be tested and standard items is contrasted, it is quantitative
Analyze the residual quantity of the campylobacter jejuni in sample to be tested.
(3) analysis result
With the bacterium solution 10 of 6 campylobacter jejuni 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 mark
The photon absorbing intensity average value (diplopore) of quasi- product or sample subtracts the photon absorbing intensity value of first standard (0 standard), then except in standard
The photon absorbing intensity average value (diplopore) of product or sample, i.e. percentage absorptance (%)=(B-B0)/B × 100%, wherein B are standard
The photon absorbing intensity average value (diplopore) of product or sample, B0For the photon absorbing intensity value of first standard (0 standard).
Using standard items percentage absorptance as ordinate, standard song is drawn for abscissa with jejunum campylobacter bacteria concentration (CFU/mL)
Line, obtains linear equation.Standard curve is y=9.9009Log (x) -115.74, R2=0.9738, see attached drawing 3.This method
Lowest detection line is defined as absorptance of the percentage absorptance more than 0CFU/mL and adds three standard deviations.It is bent by the standard
Line computation obtains lowest detection and is limited to 5*105CFU/mL.When carrying out actual sample detection, by the percentage fluorescence rate of sample ((B-
B0)/B × 100%) value substitute into standard curve in, from standard curve read corresponding to sample concentration, it is corresponding dilute to be multiplied by its
Release the actual concentrations that multiple is sample jejuni.
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 to also demonstrate that the new E LISA methods of the present invention are applicable to detect with high sensitivity
The material that conventional ELISA method can detect.
Embodiment 3
A kind of quick determination method for campylobacter jejuni, this method belong to double antibodies sandwich enzyme-linked immunization, this method
It is the detection for campylobacter jejuni antigen, the enzyme that labelled antibody is used in this method is catalase C100.
On the basis of above technical scheme, meet the following conditions：
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 Antibodies of Campylobacter Jejuni；
2) antibody after step 1) coating is taken, mixes after reacting 40min in 35 DEG C of light protected environments, washes with sample to be tested
Wash；
3) biotinylated campylobacter jejuni 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) hydrogen peroxide solution mixing of the concentration for 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 the carbonate buffer solution of 0.04mol/L, pH9.4 as coating buffer on ELISA Plate, campylobacter jejuni is diluted
For 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 campylobacter jejuni monoclonal antibody is prepared by the following method：Preparation contains
1mg/mL campylobacter jejunis monoclonal antibody, the PBS solution of 0.07mg/mL biotins, react 30min under the conditions of lucifuge, institute
The concentration for stating PBS solution is 0.005mol/L, and then dialysis removes biotin, that is, obtains the biotinylated campylobacter jejuni
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 purification, collects filtrate and adds 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 microplate reader 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 campylobacter jejuni, this method belong to double antibodies sandwich enzyme-linked immunization, this method
It is the detection for campylobacter jejuni antigen, the enzyme that labelled antibody is used in this method is catalase C100.
On the basis of above technical scheme, meet the following conditions：
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 Antibodies of Campylobacter Jejuni；
2) antibody after step 1) coating is taken, mixes after reacting 80min in 39 DEG C of light protected environments, washes with sample to be tested
Wash；
3) biotinylated campylobacter jejuni 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) hydrogen peroxide solution mixing of the concentration for 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 the carbonate buffer solution of 0.06mol/L, pH9.8 as coating buffer on ELISA Plate, campylobacter jejuni is diluted
For 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 campylobacter jejuni monoclonal antibody is prepared by the following method：Preparation contains
3mg/mL campylobacter jejunis monoclonal antibody, the PBS solution of 0.08mg/mL biotins, react 60min under the conditions of lucifuge, institute
The concentration for stating PBS solution is 0.02mol/L, and then dialysis removes biotin, that is, obtains the biotinylated campylobacter jejuni
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 purification afterwards, collects filtrate and adds catalase C100 to final concentration 3mg/mL thereto, that is, obtain 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 microplate reader 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 campylobacter jejuni, this method belong to double antibodies sandwich enzyme-linked immunization, this method
It is the detection for campylobacter jejuni antigen, the enzyme that labelled antibody is used in this method is catalase C100.
On the basis of above technical scheme, meet the following conditions：
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 Antibodies of Campylobacter Jejuni；
2) antibody after step 1) coating is taken, mixes after reacting 60min in 37 DEG C of light protected environments, washes with sample to be tested
Wash；
3) biotinylated campylobacter jejuni 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) hydrogen peroxide solution mixing of the concentration for 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 campylobacter jejuni, this method belong to double antibodies sandwich enzyme-linked immunization, this method
It is the detection for campylobacter jejuni antigen, the enzyme that labelled antibody is used in this method is catalase C100, substrate bag in this method
Include hydrogen peroxide and the cadmium telluride quantum dot of mercaptopropionic acid modification.
Embodiment 7
A kind of quick determination method for campylobacter jejuni, this method belong to double antibodies sandwich enzyme-linked immunization, this method
It is the detection for campylobacter jejuni antigen, the enzyme that labelled antibody is used in this method is catalase C100.
The embodiment of the present invention is 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., should all
Within protection scope of the present invention.
Claims (9)
1. a kind of quick determination method of non-disease diagnostic purpose for campylobacter jejuni, it is enzyme-linked that this method belongs to double antibodies sandwich
Immunization, this method are the detections for campylobacter jejuni antigen, and the enzyme that labelled antibody is used in this method is catalase C100, should
Substrate includes the cadmium telluride quantum dot that hydrogen peroxide and mercaptopropionic acid are modified in method.
2. the quick determination method of the non-disease diagnostic purpose according to claim 1 for campylobacter jejuni, its feature
It is to comprise the following steps：
1) it is coated with Antibodies of Campylobacter Jejuni；
2) antibody after step 1) coating is taken, is mixed with sample to be tested after 40~80min of reaction in 35~39 DEG C of light protected environments,
Washing；
3) mixing of biotinylated campylobacter jejuni monoclonal antibody is then added, react 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) hydrogen peroxide solution mixing of the concentration for 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 of the non-disease diagnostic purpose according to claim 2 for campylobacter jejuni, its feature
It is that step 1) specifically includes following operation：
A coating buffer, dilution jejunum) are used as using the carbonate buffer solution of 0.04~0.06mol/L, pH9.4~9.8 on ELISA Plate
Campylobacter spp 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 of the non-disease diagnostic purpose according to claim 3 for campylobacter jejuni, its feature
Be step A) in the addition of coating buffer be 80~120 μ g/ holes, 8~12h is stood after dilution and performs step B again).
5. the quick determination method of the non-disease diagnostic purpose according to claim 2 for campylobacter jejuni, its feature
It is that step 3) the biotinylated campylobacter jejuni monoclonal antibody is prepared by the following method：Prepare containing 1~
3mg/mL campylobacter jejunis monoclonal antibody, the PBS solution of 0.07~0.08mg/mL biotins, react 30 under the conditions of lucifuge
~60min, the concentration of the PBS solution is 0.005~0.02mol/L, and then dialysis removes biotin, that is, obtains the biology
The campylobacter jejuni monoclonal antibody of elementization.
6. the quick determination method of the non-disease diagnostic purpose according to claim 2 for campylobacter jejuni, its feature
It is that the catalase C100 of the step 4) marked by streptavidin is prepared by the following method：Preparation contains 0.5~2mg/
The PBS solution of mL Streptavidins, the concentration of the PBS solution is 0.005~0.02mol/L, then adds SM (PEG)24Instead
30~60min is answered, then gel column purification, collect filtrate and add catalase C100 thereto to 1~3mg/mL of final concentration, that is, obtain
The catalase C100 of the marked by streptavidin.
7. the quick determination method of the non-disease diagnostic purpose according to claim 2 for campylobacter jejuni, its feature
It is that the cadmium telluride quantum dot of step 6) the 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, described
The pH of precursor solution is 11~11.5, by the precursor solution heating water bath to 93~97 DEG C, that is, obtains the mercaptopropionic acid and repaiies
The cadmium telluride quantum dot of decorations.
8. the quick determination method of the non-disease diagnostic purpose according to claim 2 for campylobacter jejuni, its feature
It is that the detection of fluorescence intensity in step 7) is realized using microplate reader, excitation wavelength 310nm, launch wavelength 590nm.
9. according to claim 2~8 any one of them for the quick detection side of the non-disease diagnostic purpose of campylobacter jejuni
Method, it is characterised in that the washing is rinsed or soaked using cleaning solution, and the cleaning solution is spat containing 0.3~0.7% (v/v)
The PBST solution of temperature -20, the concentration of the PBST solution be 0.005~0.02mol/L, the pH of the PBST solution is 7.0~
7.5。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US5529910A (en) * | 1989-07-18 | 1996-06-25 | Shimadzu Corporation | Method for testing causative microorganisms of food poisioning and reagents therefor |
| CN103792361A (en) * | 2014-01-22 | 2014-05-14 | 上海理工大学 | Enzyme-linked immunosorbent assay kit of enterohemorrhagic E.col O157:H7 |
| CN204832212U (en) * | 2015-07-21 | 2015-12-02 | 东莞出入境检验检疫局检验检疫综合技术中心 | Crooked fungus ELISA kit of jejunum |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US5529910A (en) * | 1989-07-18 | 1996-06-25 | Shimadzu Corporation | Method for testing causative microorganisms of food poisioning and reagents therefor |
| CN103792361A (en) * | 2014-01-22 | 2014-05-14 | 上海理工大学 | Enzyme-linked immunosorbent assay kit of enterohemorrhagic E.col O157:H7 |
| CN204832212U (en) * | 2015-07-21 | 2015-12-02 | 东莞出入境检验检疫局检验检疫综合技术中心 | Crooked fungus ELISA kit of jejunum |
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