CN105759031A - Method for quickly detecting campylobacter jejuni - Google Patents
Method for quickly detecting campylobacter jejuni Download PDFInfo
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- CN105759031A CN105759031A CN201610156407.2A CN201610156407A CN105759031A CN 105759031 A CN105759031 A CN 105759031A CN 201610156407 A CN201610156407 A CN 201610156407A CN 105759031 A CN105759031 A CN 105759031A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
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- G01N33/531—Production of immunochemical test materials
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Abstract
The invention provides a method for detecting campylobacter jejuni. According to the method, firstly, campylobacter jejuni and coated polyclonal antibodies are combined, then connected with biotinylated monoclonal antibodies and connected with catalase C100 labeled with streptavidin, fluorescence quenching of cadmium telluride quantum dots modified with mercaptopropionic acid is reduced through decomposition of hydrogen peroxide under the catalytic function of catalase, and the concentration of campylobacter jejuni in a sample is judged according to the fluorescence intensity. The method is based on a double-antibody sandwich enzyme immunoassay technique and adopts a biotin-avidin system for amplifying a reaction. More importantly, a novel antibody labeling enzyme (catalase C100) and a more sensitive fluorogenic substrate (cadmium telluride quantum dots) are adopted and matched with an effective reaction condition, accordingly, the detection sensitivity is remarkably improved, the cost is reduced, the detection efficiency is improved, and the method has good popularization prospect.
Description
Technical field
The present invention relates to technical field of microbial detection, further to Antigen Detection Techniques based on ELISA,
It is specifically related to a kind of method for quick for campylobacter jejuni.
Background technology
Campylobacter jejuni (Campylobacter), belongs to spiral Cordycepps, Gram-negative, thalline hettocyrtosis
Like funny point-like, long 1.5～5 μm, wide 0.2～0.8 μm, thalline one or both ends amphitrichous, motion is active.Should
Bacterial classification separated in Feces of Patients With Diarrhea first in 1973, was one of Main Pathogenic Bacteria of causing the mankind to suffer from diarrhoea.
Campylobacter jejuni has endotoxin can attack small intestine and colorectal mucosa causes chordapsus, also can cause breaking out of diarrhoea
Popular or mass food poisoning.Being generally incubation period 3～5 days, the pathogenic position to people is jejunum, ileum and knot
Intestines.Cardinal symptom is diarrhoea and stomachache, sometimes generates heat, and occasionally has vomiting and dehydration.Bacterium can be glued by intestines sometimes
Film enters blood flow and causes septicemia and other organs to infect, such as meningitis, arthritis, pyelonephritis etc..Pregnant woman feels
Contaminate this bacterium and may result in miscarriage, premature labor, and neonate can be made to be contaminted.In this case, set up sensitive, fast
The detection method of speed is the important technology premise effectively preventing and treating campylobacter jejuni.
Prior art is mainly biochemical identification method for campylobacter jejuni detection method, but detection time length,
Reagent cost micro-aerobic characteristic high, especially campylobacter jejuni so that it is be separately cultured increasingly difficult, need extra
Maintain the facility of micro-aerobic condition and reagent, in recent years, along with the development of molecular biology, DNA sequencing
Technology is widely used in the detection of campylobacter jejuni, and this type of method sensitiveness is higher, specific relatively
By force, but defect the most more highlights one side, and it is relatively costly, and operating procedure is relatively cumbersome, the longest in addition,
Therefore the commercial application of this type of method is restricted all the time.In recent years, enzyme linked immunosorbent assay (ELISA)
Receiving increasing attention in the detection of campylobacter jejuni, this type of method is quick, sensitive, special because having
Different, accurate, can be quantitative, easy and simple to handle, without valuable instrument and equipment and less demanding to sample purity, because of
This is particularly well-suited to the detection of batch samples.But, prior art detects the ELISA of campylobacter jejuni
Method generally antibody based on horseradish peroxidase-labeled or antigen catalysis hydrogen peroxide generate hydroxy radical, and then
Aoxidize colourless chemical colour reaction substrate tetramethyl biphenyl diamines (TMB) and form blue product, then use end
Only liquid (2M H2SO4) terminate reaction formed yellow solution at 450nm, record absorbance.Such method
Because its colored intensity is relatively low, therefore detection sensitivity is relatively low, when in testing sample, object content is relatively low
False negative result easily occurs, thus the requirement of actual application cannot be met.
In recent years, some novel signal transduction mechanisms have been reported for substituting the signal conduction of traditional E LISA
Mechanism is for improving the sensitivity of ELISA, as at the bottom of radiommunoassay substrate, chemical luminous substrate, fluorescence
Thing and resonance colloidal gold solution etc..But, the structure of luminescence system needs to take into full account dividing of detected object
Sub-biological property, such as in method aspect, antibody be coated and with the binding ability of antigen, which kind of is selected
Label enzyme and the coupling method with antibody thereof, the selection of chromogenic substrate and concrete luminescent method；At effect layer
Face, should ensure the sensitivity of chromogenic reaction, should meet again the linear relationship of colored intensity and object content.
Therefore, for the ELISA detection method of campylobacter jejuni, especially for the purpose of promoting its detection sensitivity
Method is improved, and has prominent technical difficulty.
Summary of the invention
It is contemplated that for the technological deficiency of prior art, it is provided that a kind of quick detection for campylobacter jejuni
Method, relatively low to solve the campylobacter jejuni ELISA detection method precision of prior art.
Another that the invention solves the problems that technical problem is that what prior art detected for campylobacter jejuni antigen
In ELISA method, the enzyme sensitivity of labelled antibody is relatively low.
The invention solves the problems that further technical problem is that what prior art detected for campylobacter jejuni antigen
In ELISA method, Color Appearance System sensitivity is relatively low.
Another technical problem is that the invention solves the problems that ought use catalase C100 curved to jejunum as antibody labeling enzyme
When aspergillus antigen performs ELISA detection, concrete technology method is the most indefinite.
Another technical problem is that the invention solves the problems that works as employing catalase C100 as antibody labeling enzyme, employing pair
The cadmium telluride quantum dot that oxygen water and mercaptopropionic acid are modified performs ELISA as substrate to campylobacter jejuni antigen and examines
During survey, testing result is the best with the linear relationship of antigen concentration.
Another technical problem is that the invention solves the problems that works as employing catalase C100 as antibody labeling enzyme, employing pair
The cadmium telluride quantum dot that oxygen water and mercaptopropionic acid are modified performs ELISA as substrate to campylobacter jejuni antigen and examines
During survey, during clean result the best.
For realizing above technical purpose, the present invention by the following technical solutions:
A kind of method for quick for campylobacter jejuni, the method belongs to double antibodies sandwich ELISA, should
Method is the detection for campylobacter jejuni antigen, and in the method, the enzyme for labelled antibody is catalase C100.
As preferably, in the method, substrate includes the cadmium telluride quantum dot that hydrogen peroxide and mercaptopropionic acid are modified.Wherein
Hydrogen peroxide is as cadmium telluride quantum dot fluorescence quenching, and in course of reaction, hydrogen peroxide decomposes under catalase effect,
Thus reduce fluorescent quenching effect, it is achieved luminous.
As preferably, the method comprises the following steps:
1) it is coated Antibodies of Campylobacter Jejuni；
2) take step 1) be coated after antibody, after mixing with testing sample in 35～39 DEG C of light protected environment react
40～80min, washing；
3) mixing of biotinylated campylobacter jejuni monoclonal antibody is then added, in 35～39 DEG C of light protected environment
Middle reaction 40～80min, washing；
4) then add the catalase C100 mixing of marked by streptavidin, react in 35～39 DEG C of light protected environment
40～80min, washing；
5) the hydrogen peroxide solution mixing that concentration is 8～12 μm ol/L is then added, in 35～39 DEG C of light protected environment
Reaction 20～40min；
6) the cadmium telluride quantum dot mixing that mercaptopropionic acid is modified then is added, anti-in 35～39 DEG C of light protected environment
Answer 10～20min；
7) detecting step 6) fluorescence intensity of product.
This fluorescence intensity is i.e. used for reacting testing sample jejuni content, available known in practical operation
Many groups campylobacter jejuni titer of concentration and distribution gradient draws fluorescence intensity-bacteria concentration by above method
Calibration curve, the fluorescence intensity of recycling testing sample calculates the campylobacter jejuni of testing sample from calibration curve
Content.Concrete operation method can be according to the arbitrary selection of general technology general knowledge of the art.Above-mentioned gradient is divided
Many groups campylobacter jejuni titer of cloth, can select 10 respectively1CFU/mL、102CFU/mL、103CFU/mL、
104CFU/mL、105CFU/mL、106CFU/mL。
In this optimal technical scheme, step 2) for obtaining the antigen antibody complex of solid phase；Step 3) real
Existing connection between biotinylated campylobacter jejuni monoclonal antibody and the antigen antibody complex of solid phase；Step
4) biotin-Streptavidin system is utilized to realize the connection with catalase C100；Step 5) enzymatic dioxygen
Water decomposition；Step 6) realize chromogenic reaction.In this optimal technical scheme: each reagent before use can be first
Can re-use prior to more than equilibrium at room temperature 30min；Step 2) in testing sample addition be preferably
80～120 μ L/ holes, more optimizedly 100 μ L/ hole；Step 3) in biotinylated campylobacter jejuni monoclonal resist
The addition of body is preferably 80～120 μ L/ holes, more optimizedly 100 μ L/ hole；Step 4) in Streptavidin mark
The addition of the catalase C100 of note is preferably 80～120 μ L/ holes, more optimizedly 100 μ L/ hole；Step 5) in
The addition of hydrogen peroxide solution is preferably 80～120 μ L/ holes, more optimizedly 100 μ L/ hole；Step 6) in sulfydryl
The addition of the cadmium telluride quantum dot that propionic acid is modified is preferably 30～70 μ L/ holes, more optimizedly 50 μ L/ hole.
As preferably, step 1) specifically include following operation:
A) on ELISA Plate using 0.04～0.06mol/L, pH9.4～9.8 carbonate buffer solution as being coated liquid,
Dilution campylobacter jejuni polyclonal antibody is to 9～11 μ g/mL；
B) utilize cleaning solution detersive enzyme target after removing the liquid on ELISA Plate, add bovine serum albumin(BSA) envelope
Close liquid, close 1～3h in 35～39 DEG C, then discard confining liquid.
Can perform further following preferably: the concentration of described bovine serum albumin(BSA) is 0.3～0.7%, more excellent
It is 0.5%；The addition of confining liquid is 320～360 μ L/ holes, and more excellent is 340 μ L/ holes；Discard closing
ELISA Plate after liquid is dried at room temperature, then in 2～6 DEG C of preservations.
As preferably, step A) in be coated the addition of liquid be 80～120 μ g/ holes, stand 8～12h after dilution
Perform step B again).
As preferably, step 3) described biotinylated campylobacter jejuni monoclonal antibody is by the following method
Preparation: preparation is containing 1～3mg/mL campylobacter jejuni monoclonal antibody, 0.07～0.08mg/mL biotin
PBS solution, under the conditions of lucifuge react 30～60min, the concentration of described PBS solution is
0.005～0.02mol/L, biotin is removed in then dialysis, i.e. obtains described biotinylated campylobacter jejuni Dan Ke
Grand antibody.Can perform further following preferably: described biotin is vivaciously to be esterified biotin；Described dialysis
Time is 66～78h, more optimizedly 72h.
As preferably, step 4) the catalase C100 of described marked by streptavidin is to be prepared by the following method
: the preparation PBS solution containing 0.5～2mg/mL Streptavidin, the concentration of described PBS solution is
0.005～0.02mol/L, then add SM (PEG)24Reaction 30～60min, then gel column purifies, and receives
Collection filtrate is added thereto to catalase C100 to final concentration 1～3mg/mL, i.e. obtains described marked by streptavidin
Catalase C100.In this optimal technical scheme: gel column purifies link for removing unnecessary crosslinking agent；
Collect the filtrate that obtains and can first detect wherein protein content add after being mixed by the solution containing albumen tactile
Enzyme C100.
As preferably, step 6) cadmium telluride quantum dot modified of described mercaptopropionic acid is to be prepared by the following method
: preparation is containing 8～12mmol/L cadmium nitrates, 20～28mmol/L mercaptopropionic acid, 3～7mmol/L hydrogen telluride
The solution received is precursor solution, and the pH of described precursor solution is 11～11.5, by described precursor solution water-bath
It is heated to 93～97 DEG C, i.e. obtains the cadmium telluride quantum dot that described mercaptopropionic acid is modified.
As preferably, step 7) in the detection of fluorescence intensity utilize ELIASA to realize, excitation wavelength is
310nm, a length of 590nm of transmitted wave.
On the basis of any of the above technical scheme preferably, described washing is to utilize cleaning solution to rinse or soak, institute
Stating cleaning solution is that the concentration of described PBST solution is containing 0.3～the PBST solution of 0.7% (v/v) Tween-20
0.005～0.02mol/L, the pH of described PBST solution is 7.0～7.5.
In above technical scheme, catalase (Catalase) is also called catalase, used in the present invention
Catalase C100 refer exclusively to be produced by Sigma-Aldrich, model be touching of cat.No.C100
Enzyme.Described biotinylated campylobacter jejuni monoclonal antibody, refers to biotin is covalently attached to jejunum campylobacter
The compound obtained after bacterium monoclonal antibody molecule.The catalase C100 of described marked by streptavidin, refer to by
Streptavidin and catalase C100 carry out the compound of covalent bond gained.
In above technical scheme, ELISA Plate can select 96 hole black fluorescent ELISA Plates.96 hole black fluorescent enzymes
Target, biotin, anti-campylobacter jejuni monoclonal antibody, anti-campylobacter jejuni polyclonal antibody, strepto-is affine
Element, catalase C100 all can buy in market.
As preferably, described washing is the cleaning solution adding 320～360 μ L/ holes in enzyme mark hole, washs 3～4
Secondary, every minor tick 10～30s.
As preferably, described testing sample can be vegetables, when testing sample is vegetables, first carries out following behaviour
Detect again: the vegetables bought are weighed 1mg and are put in sterile test tube by (1), sterilizing 1h in super-clean bench,
Smash to pieces again；(2) 9mL PBS (pH 7.4,0.01mol/L) and the bacterium solution of 1mL are added, acutely concussion 30 minutes；
(3) taking supernatant and adding immunomagnetic beads enrichment method is that 1mL is standby.
The present invention uses enzyme-linked immunologic adsorption test method to detect.For detecting the novel fluorescence of campylobacter jejuni
The principle of ELISA detection method is: be combined with coated polyclonal antibody by campylobacter jejuni, then plus raw
The monoclonal antibody of thing element, adds SA@CAT (the catalase C100 of marked by streptavidin, lower same),
It is catalyzed decomposing hydrogen dioxide solution by catalase, reduces the fluorescent quenching to the cadmium telluride quantum dot that mercaptopropionic acid is modified, root
The concentration of judgement sample jejuni is carried out according to the height of fluorescence intensity.If the campylobacter jejuni in sample is dense
Degree height, fluorescence intensity is high；Otherwise, then fluorescence intensity is low.I.e. the height of fluorescence intensity and the bacterium in sample is dense
Spend into positive correlation.The method can be directly used for detecting the campylobacter jejuni in romaine lettuce.The detection method behaviour of the present invention
Make simplicity, detect sensitive, accurate, quick, it is adaptable to the detection of batch samples.
Employing technical solution of the present invention has the advantages that
1, the inventive method uses the sensitiveest novel catalase to replace the horseradish mistake in conventional ELISA method
Oxide enzyme, can be greatly saved cost.
2, the inventive method uses the sensitiveest novel fluorogenic substrate (the cadmium telluride amount that mercaptopropionic acid is modified
Sub-point) replace the chemical colour reaction substrate in conventional ELISA method, its detection sensitivity can be greatly improved,
The 2-3 order of magnitude is at least improve relative to traditional E LISA.
Accompanying drawing explanation
Fig. 1 is detection method with conventional using horseradish peroxidase as the detection of antibody labeling thing enzyme
The principle comparison diagram of method.
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.
Detailed description of the invention
The detailed description of the invention of the present invention will be described in detail below.In order to avoid the most unnecessary details,
In the examples below to belonging to known structure or function will not be described in detail.
Approximating language used in following example can be used for quantitative expression, shows do not changing basic function
In the case of quantity can be allowed to have certain variation.Therefore, revised with the language such as " about ", " left and right "
Numerical value be not limited to this exact value itself.In certain embodiments, number of its correction of permission " about " is represented
Value changes in the range of positive and negative 10 (10%), and such as, what " about 100 " represented can be 90
Any numerical value between 110.Additionally, in the statement of " the about first numerical value is to second value ", about
Revise two numerical value of the first and second numerical value simultaneously.In some cases, approximating language may be with measuring instrument
Precision relevant.
In addition to being defined, technology used in following example and scientific terminology have and art skill of the present invention
The identical meanings that art personnel are commonly understood by.
Test reagent consumptive material used in following example, if no special instructions, is routine biochemistry reagent；Institute
State experimental technique, if no special instructions, be conventional method；Quantitative test in following example, is respectively provided with
Repeat experiment, results averaged for three times；% in following example, if no special instructions, is quality hundred
Divide content.
In following example, the campylobacter jejuni monoclonal antibody of Anti-Biotin and polyclonal antibody are bought in nothing
Tin 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)
All buy in Sigma-Aldrich.Described catalase C100 i.e. refers to catalase (cat.No.C100).
(present invention detects the novel fluorescence ELISA detection method of campylobacter jejuni in detection romaine lettuce to embodiment 1
Application in jejunum campylobacter bacterial content)
Novel fluorescence ELISA detection method of the present invention is in time detecting the jejunum campylobacter bacterial content in romaine lettuce, logical
Cross following steps to implement: sample pre-treatments, with detection method carry out detecting, analysis result.
(1) sample pre-treatments
Take the romaine lettuce sample 1g handled well, join in the aseptic PBS of 9ml, add 1ml bacterium solution, acutely
Shake 30 minutes；Taking supernatant and adding immunomagnetic beads and carry out enrichment to concentrate is 1mL liquid, takes out standby.
(2) carry out detecting above-mentioned sample jejuni content by detection method
Take the ELISA Plate being coated with anti-jejunum campylobacter bacterial content polyclonal antibody, add standard items/sample 100 μ L/ hole
In corresponding micropore；Take the ELISA Plate being coated with anti-campylobacter jejuni polyclonal antibody, add sample 100 μ L/
Hole is in corresponding micropore, and mixing of vibrating gently, with placing reaction in 37 DEG C of light protected environment after cover plate membrane cover plate
60min；Carefully open cover plate film, liquid in hole is dried, with wash operating solution 340 μ L/ hole, fully wash
Wash 4 times, every minor tick 10s, pat dry with blotting paper that (bubble not being eliminated after patting dry can be with original
Rifle head is poked), add biotinylation campylobacter jejuni monoclonal antibody 100 μ L/ hole, mixing of vibrating gently,
With placing after cover plate membrane cover plate, 37 DEG C of light protected environment react 60min；Carefully open cover plate film, by liquid in hole
Body dries, and with wash operating solution 340 μ L/ hole, fully washing 3 times, every minor tick 10s, clap with blotting paper
Dry (bubble not being eliminated after patting dry can be poked with original rifle head), adds SA CAT, shakes gently
Swing mixing, with placing after cover plate membrane cover plate, 37 DEG C of light protected environment react 60min；Carefully open cover plate film,
Being dried by liquid in hole, with wash operating solution 340 μ L/ hole, fully washing 3 times, every minor tick 10s, use
Blotting paper pats dry (bubble not being eliminated after patting dry can be poked) with original rifle head.Add substrate solution A double
Oxygen water (10 μm ol/L) dilution, 100 μ L/ holes, mixing of vibrating gently, keeps away with rearmounted 37 DEG C of cover plate membrane cover plate
Luminous environment reacts 30min；Add the cadmium telluride quantum dot substrate solution that fluorogenic substrate liquid B mercaptopropionic acid is modified
50 μ L/ holes, mixing of vibrating gently, with the rearmounted 37 DEG C of light protected environment of cover plate membrane cover plate react 15min, set
Fluorescence microplate reader (U.S.'s Thermo Varioskan Flash multi-functional ELIASA of all-wave length) in excitation wavelength is
310nm, detects at a length of 590nm of transmitted wave, measures every hole fluorescent value (please running through data in 5min)；
Drawing calibration curve with the fluorescent value of standard items test with the log concentration value of standard items, reference standard curve calculates
The content of sample jejuni.
The preparation of the coated 96 hole black fluorescent ELISA Plates of described anti-campylobacter jejuni polyclonal antibody is to use
Carbonate (CBS) buffer solution of 0.05mol/L pH 9.6 is as being coated liquid, by campylobacter jejuni polyclonal antibody
(buying in Wuxi Zhongde Bore Bioisystech Co., Ltd) is interpreted into 10 μ g/mL, 100 μ L/ holes, 4 DEG C of placements
Overnight, take out ELISA Plate and get rid of liquid in plate, with the concentrated cleaning solution 340 μ L/ hole after dilution, wash plate,
30s/ time；Be subsequently adding 0.5% bovine serum albumin(BSA) (BSA, buys in Sigma-Aldrich,
Cat.No.A4737) close, 340 μ L/ holes, place 2h, discard confining liquid, the ELISA Plate after patting dry for 37 DEG C
Place (25 DEG C) between constant temperature to dry；Inspect by random samples qualified after ELISA Plate vacuum is sealed and preserves at rearmounted 4 DEG C.
Described campylobacter jejuni debita spissitudo gradient is respectively 100CFU/mL、101CFU/mL、102CFU/mL、
103CFU/mL、104CFU/mL、105CFU/mL。
Described biotinylated campylobacter jejuni monoclonal antibody obtains in the following manner: by curved for 1mg jejunum
Aspergillus monoclonal antibody PBS (pH 8.6,0.01mol/L) dilutes, and adds 50 μ L and is vivaciously esterified biology
Element (0.76mg/mL) makes the final concentration of 2mg/mL of antibody, room temperature lucifuge reaction 45min.Product
Dialyse in the PBS solution of 0.01mol/L 72h, removes free biotin；After dialysis terminates, by sample
Product freeze-drying obtains biotinylated campylobacter jejuni monoclonal antibody, packing ,-20 DEG C of preservations.
Described SA@CAT obtains in the following manner: by molten for SA PBS (pH 8.6,0.01mol/L)
Solving is 1mg/mL, adds 10 μ L SM (PEG)24(Thermo:22104,82.8mg/mL) reacts
45min, reaction is isolated and purified with gel column (Thermo:43230) after terminating, and goes out unnecessary crosslinking agent；
The liquid collected Nanodrop measures, and by there being mixing with solution of albumen, is then added to 5mg catalase solution
In (final concentration 2mg/mL).The sample freeze-drying that will obtain, packing ,-20 DEG C of preservations.
The preparation of described biotinylation campylobacter jejuni monoclonal antibody working solution: use active esterification biotin with
Campylobacter jejuni monoclonal antibody coupling obtains, and is diluted to 1:230 with PBS (0.01M, pH7.4).
The preparation of described SA@CAT working solution: use Streptavidin and catalase coupling to obtain, use PBS
(0.01M, pH7.4) is diluted to 1:300.
The preparation of described substrate solution A hydrogen peroxide: be diluted to 10 μm ol/L with PBS (0.01mol/L, pH7.4).
The preparation of cadmium telluride quantum dot fluorogenic substrate liquid that described mercaptopropionic acid is modified: method, with PBS (0.01mol/L,
PH7.4) it is diluted to 1:400.
Described concentrated cleaning solution is 10 times of concentrated cleaning solutions, and it comprises 0.5% Tween-20,0.01mol/L's
PBST, between pH value range 7.0-7.5.
(3) analysis result
Bacterium solution 10 with 6 campylobacter jejuni variable concentrations of above-mentioned preparation1CFU/mL、102CFU/mL、
103CFU/mL、104CFU/mL、105CFU/mL、106CFU/mL.It is 310nm in excitation wavelength, sends out
Fluorescence intensity at a length of 590nm of ejected wave.
The calculating of cancellation percentage fluorescence rate, the cancellation percentage fluorescence rate of standard items or sample is equal to first standard
The fluorescence intensity level of (0 standard) deducts the mean value (diplopore) of the fluorescence intensity level of standard items or sample, then removes
In first standard (0 standard), i.e. percentage fluorescence rate (%)=(F0-F)/F0× 100%, wherein F0It is
The fluorescence intensity level of one standard (0 standard), F is that the mean value of the fluorescence intensity level of standard items or sample is (double
Hole).
With cancellation percentage fluorescence rate as ordinate, draw mark with campylobacter jejuni bacteria concentration (CFU/mL) abscissa
Directrix curve, obtains linear equation.Calibration curve is y=-2.822Log (x)+78.031, R2=0.9808, see
Accompanying drawing 2.The LDL of the method is defined as the fluorescent value of 0CFU/mL plus three standard deviations.Logical
Crossing this calibration curve and calculating lowest detection line is 5*101CFU/mL.When carrying out actual sample detection, will
The fluorescent value ((F of sample0-F)/F0× 100%), during value substitutes into calibration curve, from calibration curve, institute is read
The concentration of corresponding sample, the extension rate being multiplied by its correspondence is the actual concentrations of sample jejuni.
Embodiment 2 (with horseradish peroxidase be antibody labeling enzyme, using TMB as the jejunum campylobacter of chromogenic substrate
Bacterium ELISA detection method)
Traditional E LISA detection method is in time detecting the jejunum campylobacter bacterial content in romaine lettuce and beef items, logical
Cross following steps to implement: sample pre-treatments, with traditional E LISA detection method carry out detecting, analysis result.
(1) sample pre-treatments
Take the romaine lettuce sample 1g handled well, join in the aseptic PBS of 9ml, add 1ml bacterium solution, acutely shake
30 minutes；Taking supernatant and adding immunomagnetic beads and carry out enrichment to concentrate is 1mL liquid, takes out standby.
(2) carry out detecting above-mentioned sample jejuni content by traditional E LISA detection method
Take the ELISA Plate being coated with anti-jejunum campylobacter bacterial content polyclonal antibody, add standard items/sample 100 μ L/ hole
In corresponding micropore；Take the ELISA Plate being coated with anti-campylobacter jejuni polyclonal antibody, add sample 100 μ L/
Hole is in corresponding micropore, and mixing of vibrating gently, with placing reaction in 37 DEG C of light protected environment after cover plate membrane cover plate
60min；Carefully open cover plate film, liquid in hole is dried, with wash operating solution 340 μ L/ hole, fully wash
Wash 4 times, every minor tick 10s, pat dry with blotting paper that (bubble not being eliminated after patting dry can be with original
Rifle head is poked), add biotinylation campylobacter jejuni monoclonal antibody 100 μ L/ hole, mixing of vibrating gently,
With placing after cover plate membrane cover plate, 37 DEG C of light protected environment react 60min；Carefully open cover plate film, by liquid in hole
Body dries, and with wash operating solution 340 μ L/ hole, fully washing 3 times, every minor tick 10s, clap with blotting paper
Dry (bubble not being eliminated after patting dry can be poked with original rifle head), adds SA HRP, vibrates gently
Mixing, reacts 60min with placing after cover plate membrane cover plate in 37 DEG C of light protected environment；Carefully open cover plate film, will
In hole, liquid dries, and with wash operating solution 340 μ L/ hole, fully washing 3 times, every minor tick 10s, with water suction
Paper pats dry (bubble not being eliminated after patting dry can be poked) with original rifle head；Add TMB nitrite ion,
100 μ L/ holes, mixing of vibrating gently, with reacting 15min in the rearmounted 37 DEG C of light protected environment of cover plate membrane cover plate；Add
Stop buffer 50 μ L/ hole, mixing of vibrating gently, set ELIASA and examine at 450nm or at dual wavelength 450nm
Survey, measure every hole absorbance (please running through data in 5min)；Contrast testing sample and the suction of standard items
Shading value size, the residual quantity of the campylobacter jejuni in quantitative analysis testing sample.
(3) analysis result
Bacterium solution 10 with 6 campylobacter jejuni variable concentrations of above-mentioned preparation1CFU/mL、102CFU/mL、
103CFU/mL、104CFU/mL、105CFU/mL、106CFU/mL.The calculating of percentage absorptance, standard
The percent absorption of product or sample deducts first equal to the photon absorbing intensity mean value (diplopore) of standard items or sample
The photon absorbing intensity value of standard (0 standard), then remove in the photon absorbing intensity mean value (diplopore) of standard items or sample,
I.e. percentage absorptance (%)=(B B0)/B × 100%, wherein B is the photon absorbing intensity of standard items or sample
Mean value (diplopore), B0It it is the photon absorbing intensity value of first standard (0 standard).
With standard items percentage absorptance as ordinate, it is that abscissa is drawn with jejunum campylobacter bacteria concentration (CFU/mL)
Calibration curve, obtains linear equation.Calibration curve is y=9.9009Log (x)-115.74, R2=0.9738, see
Accompanying drawing 3.The lowest detection line of the method is defined as the percentage absorptance absorptance more than 0CFU/mL and adds
Three standard deviations.Calculate lowest detection by this calibration curve and be limited to 5*105CFU/mL.When carrying out reality
During the pattern detection of border, by percentage fluorescence rate ((the B B of sample0)/B × 100%) value substitutes in calibration curve,
Reading the concentration of corresponding sample from calibration curve, being multiplied by the extension rate of its correspondence, to be jejunum in sample curved
The actual concentrations of aspergillus.
By the contrast of embodiment 1 and embodiment 2 it is found that the new E LISA method sensitivity of the present invention
Up to 10000 times of classic ELISA method, also demonstrate that the new E LISA method of the present invention is applicable simultaneously
In the material that can detect with any conventional ELISA method of high-sensitivity detection.
Embodiment 3
A kind of method for quick for campylobacter jejuni, the method belongs to double antibodies sandwich ELISA, should
Method is the detection for campylobacter jejuni antigen, and in the method, the enzyme for labelled antibody is catalase C100.
On the basis of above technical scheme, meet following condition:
In the method, substrate includes the cadmium telluride quantum dot that hydrogen peroxide and mercaptopropionic acid are modified.
Concrete detection method comprises the following steps:
1) it is coated Antibodies of Campylobacter Jejuni；
2) take step 1) be coated after antibody, after mixing with testing sample in 35 DEG C of light protected environment react
40min, washing；
3) mixing of biotinylated campylobacter jejuni monoclonal antibody is then added, anti-in 35 DEG C of light protected environment
Answer 40min, washing；
4) then add the catalase C100 mixing of marked by streptavidin, react in 35 DEG C of light protected environment
40min, washing；
5) then add the hydrogen peroxide solution mixing that concentration is 8 μm ol/L, react in 35 DEG C of light protected environment
20min；
6) then add the cadmium telluride quantum dot mixing that mercaptopropionic acid is modified, react in 35 DEG C of light protected environment
10min；
7) detecting step 6) fluorescence intensity of product.
Wherein step 1) specifically include following operation:
A) using the carbonate buffer solution of 0.04mol/L, pH9.4 as being coated liquid on ELISA Plate, jejunum is diluted
Campylobacter spp polyclonal antibody is to 9 μ g/mL, and the addition being coated liquid is 80 μ g/ holes, stands 8h after dilution；
B) utilize cleaning solution detersive enzyme target after removing the liquid on ELISA Plate, add bovine serum albumin(BSA) envelope
Close liquid, close 1h in 35 DEG C, then discard confining liquid.
Step 3) described biotinylated campylobacter jejuni monoclonal antibody is prepared by the following method: joins
System is containing 1mg/mL campylobacter jejuni monoclonal antibody, the PBS solution of 0.07mg/mL biotin, in keeping away
Reacting 30min under optical condition, the concentration of described PBS solution is 0.005mol/L, and biotin is removed in then dialysis,
I.e. obtain described biotinylated campylobacter jejuni monoclonal antibody.
Step 4) the catalase C100 of described marked by streptavidin is prepared by the following method: preparation contains
Having the PBS solution of 0.5mg/mL Streptavidin, the concentration of described PBS solution is 0.005mol/L, and
Rear addition SM (PEG)24Reaction 30min, then gel column purifies, and collects filtrate and is added thereto to catalase
C100 to final concentration 1mg/mL, i.e. obtains the catalase C100 of described marked by streptavidin.
Step 6) described mercaptopropionic acid modify cadmium telluride quantum dot be prepared by the following method: preparation contain
It is molten that the solution having 8mmol/L cadmium nitrate, 20mmol/L mercaptopropionic acid, 3mmol/L hydrogen telluride to receive is precursor
Liquid, the pH of described precursor solution is 11, by described precursor solution heating water bath to 93 DEG C, i.e. obtains described
The cadmium telluride quantum dot that mercaptopropionic acid is modified.
Step 7) in the detection of fluorescence intensity utilize ELIASA to realize, excitation wavelength is 310nm, launches
Wavelength is 590nm.
Described washing is to utilize cleaning solution to rinse or soak, and described cleaning solution is containing 0.3% (v/v) Tween-20
PBST solution, the concentration of described PBST solution is 0.005mol/L, and the pH of described PBST solution is 7.0.
Embodiment 4
A kind of method for quick for campylobacter jejuni, the method belongs to double antibodies sandwich ELISA, should
Method is the detection for campylobacter jejuni antigen, and in the method, the enzyme for labelled antibody is catalase C100.
On the basis of above technical scheme, meet following condition:
In the method, substrate includes the cadmium telluride quantum dot that hydrogen peroxide and mercaptopropionic acid are modified.
Concrete detection method comprises the following steps:
1) it is coated Antibodies of Campylobacter Jejuni；
2) take step 1) be coated after antibody, after mixing with testing sample in 39 DEG C of light protected environment react
80min, washing；
3) mixing of biotinylated campylobacter jejuni monoclonal antibody is then added, anti-in 39 DEG C of light protected environment
Answer 80min, washing；
4) then add the catalase C100 mixing of marked by streptavidin, react in 39 DEG C of light protected environment
80min, washing；
5) then add the hydrogen peroxide solution mixing that concentration is 12 μm ol/L, react in 39 DEG C of light protected environment
40min；
6) then add the cadmium telluride quantum dot mixing that mercaptopropionic acid is modified, react in 39 DEG C of light protected environment
20min；
7) detecting step 6) fluorescence intensity of product.
Wherein step 1) specifically include following operation:
A) using the carbonate buffer solution of 0.06mol/L, pH9.8 as being coated liquid on ELISA Plate, jejunum is diluted
Campylobacter spp polyclonal antibody is to 11 μ g/mL, and the addition being coated liquid is 120 μ g/ holes, stands 12h after dilution；
B) utilize cleaning solution detersive enzyme target after removing the liquid on ELISA Plate, add bovine serum albumin(BSA) envelope
Close liquid, close 3h in 39 DEG C, then discard confining liquid.
Step 3) described biotinylated campylobacter jejuni monoclonal antibody is prepared by the following method: joins
System is containing 3mg/mL campylobacter jejuni monoclonal antibody, the PBS solution of 0.08mg/mL biotin, in keeping away
Reacting 60min under optical condition, the concentration of described PBS solution is 0.02mol/L, and biotin is removed in then dialysis,
I.e. obtain described biotinylated campylobacter jejuni monoclonal antibody.
Step 4) the catalase C100 of described marked by streptavidin is prepared by the following method: preparation contains
Having the PBS solution of 2mg/mL Streptavidin, the concentration of described PBS solution is 0.02mol/L, then adds
Enter SM (PEG)24Reaction 60min, then gel column purifies, and collects filtrate and is added thereto to catalase C100
To final concentration 3mg/mL, i.e. obtain the catalase C100 of described marked by streptavidin.
Step 6) described mercaptopropionic acid modify cadmium telluride quantum dot be prepared by the following method: preparation contain
The solution having 12mmol/L cadmium nitrate, 28mmol/L mercaptopropionic acid, 7mmol/L hydrogen telluride to receive is precursor
Solution, the pH of described precursor solution is 11.5, by described precursor solution heating water bath to 97 DEG C, i.e. obtains institute
State the cadmium telluride quantum dot that mercaptopropionic acid is modified.
Step 7) in the detection of fluorescence intensity utilize ELIASA to realize, excitation wavelength is 310nm, launches
Wavelength is 590nm.
Described washing is to utilize cleaning solution to rinse or soak, and described cleaning solution is containing 0.7% (v/v) Tween-20
PBST solution, the concentration of described PBST solution is 0.02mol/L, and the pH of described PBST solution is 7.5.
Embodiment 5
A kind of method for quick for campylobacter jejuni, the method belongs to double antibodies sandwich ELISA, should
Method is the detection for campylobacter jejuni antigen, and in the method, the enzyme for labelled antibody is catalase C100.
On the basis of above technical scheme, meet following condition:
In the method, substrate includes the cadmium telluride quantum dot that hydrogen peroxide and mercaptopropionic acid are modified.
Concrete detection method comprises the following steps:
1) it is coated Antibodies of Campylobacter Jejuni；
2) take step 1) be coated after antibody, after mixing with testing sample in 37 DEG C of light protected environment react
60min, washing；
3) mixing of biotinylated campylobacter jejuni monoclonal antibody is then added, anti-in 37 DEG C of light protected environment
Answer 60min, washing；
4) then add the catalase C100 mixing of marked by streptavidin, react in 37 DEG C of light protected environment
60min, washing；
5) then add the hydrogen peroxide solution mixing that concentration is 10 μm ol/L, react in 37 DEG C of light protected environment
30min；
6) then add the cadmium telluride quantum dot mixing that mercaptopropionic acid is modified, react in 37 DEG C of light protected environment
15min；
7) detecting step 6) fluorescence intensity of product.
Embodiment 6
A kind of method for quick for campylobacter jejuni, the method belongs to double antibodies sandwich ELISA, should
Method is the detection for campylobacter jejuni antigen, and in the method, the enzyme for labelled antibody is catalase C100,
In the method, substrate includes the cadmium telluride quantum dot that hydrogen peroxide and mercaptopropionic acid are modified.
Embodiment 7
A kind of method for quick for campylobacter jejuni, the method belongs to double antibodies sandwich ELISA, should
Method is the detection for campylobacter jejuni antigen, and in the method, the enzyme for labelled antibody is catalase C100.
Above embodiments of the invention are described in detail, but described content has been only the preferable enforcement of the present invention
Example, not in order to limit the present invention.All made in the application range of the present invention any amendment, equivalent
With improvement etc., should be included within the scope of the present invention.
Claims (10)
1., for a method for quick for campylobacter jejuni, the method belongs to double antibodies sandwich ELISA,
The method is the detection for campylobacter jejuni antigen, and in the method, the enzyme for labelled antibody is catalase C100.
Method for quick for campylobacter jejuni the most according to claim 1, it is characterised in that should
In method, substrate includes the cadmium telluride quantum dot that hydrogen peroxide and mercaptopropionic acid are modified.
Method for quick for campylobacter jejuni the most according to claim 2, it is characterised in that bag
Include following steps:
1) it is coated Antibodies of Campylobacter Jejuni；
2) take step 1) be coated after antibody, after mixing with testing sample in 35～39 DEG C of light protected environment react
40～80min, washing；
3) mixing of biotinylated campylobacter jejuni monoclonal antibody is then added, in 35～39 DEG C of light protected environment
Middle reaction 40～80min, washing；
4) then add the catalase C100 mixing of marked by streptavidin, react in 35～39 DEG C of light protected environment
40～80min, washing；
5) the hydrogen peroxide solution mixing that concentration is 8～12 μm ol/L is then added, in 35～39 DEG C of light protected environment
Reaction 20～40min；
6) the cadmium telluride quantum dot mixing that mercaptopropionic acid is modified then is added, anti-in 35～39 DEG C of light protected environment
Answer 10～20min；
7) detecting step 6) fluorescence intensity of product.
Method for quick for campylobacter jejuni the most according to claim 3, it is characterised in that step
Rapid 1) following operation is specifically included:
A) on ELISA Plate using 0.04～0.06mol/L, pH9.4～9.8 carbonate buffer solution as being coated liquid,
Dilution campylobacter jejuni polyclonal antibody is to 9～11 μ g/mL；
B) utilize cleaning solution detersive enzyme target after removing the liquid on ELISA Plate, add bovine serum albumin(BSA) envelope
Close liquid, close 1～3h in 35～39 DEG C, then discard confining liquid.
Method for quick for campylobacter jejuni the most according to claim 4, it is characterised in that step
Rapid A) in be coated the addition of liquid be 80～120 μ g/ holes, stand 8～12h after dilution and perform step B again).
Method for quick for campylobacter jejuni the most according to claim 3, it is characterised in that step
Rapid 3) described biotinylated campylobacter jejuni monoclonal antibody is prepared by the following method: preparation contains
1～3mg/mL campylobacter jejuni monoclonal antibody, 0.07～the PBS solution of 0.08mg/mL biotin, in keeping away
Reacting 30～60min under optical condition, the concentration of described PBS solution is 0.005～0.02mol/L, and then dialysis is gone
Except biotin, i.e. obtain described biotinylated campylobacter jejuni monoclonal antibody.
Method for quick for campylobacter jejuni the most according to claim 3, it is characterised in that step
Rapid 4) the catalase C100 of described marked by streptavidin is prepared by the following method: preparation contains
The PBS solution of 0.5～2mg/mL Streptavidin, the concentration of described PBS solution is 0.005～0.02mol/L,
Then add SM (PEG)24Reaction 30～60min, then gel column purifies, and collects filtrate and is added thereto to
Catalase C100, to final concentration 1～3mg/mL, i.e. obtains the catalase C100 of described marked by streptavidin.
Method for quick for campylobacter jejuni the most according to claim 3, it is characterised in that step
Rapid 6) cadmium telluride quantum dot that described mercaptopropionic acid is modified is prepared by the following method: preparation contains
The solution that 8～12mmol/L cadmium nitrates, 20～28mmol/L mercaptopropionic acid, 3～7mmol/L hydrogen telluride are received is
Precursor solution, the pH of described precursor solution is 11～11.5, by described precursor solution heating water bath to 93～97 DEG C,
I.e. obtain the cadmium telluride quantum dot that described mercaptopropionic acid is modified.
Method for quick for campylobacter jejuni the most according to claim 3, it is characterised in that step
Rapid 7) in, the detection of fluorescence intensity utilizes ELIASA to realize, and excitation wavelength is 310nm, and transmitted wave is a length of
590nm。
10., according to the method for quick for campylobacter jejuni described in any one of claim 3～9, it is special
Levying and be that described washing is to utilize cleaning solution to rinse or soak, described cleaning solution is containing 0.3～0.7% (v/v) tween
The PBST solution of-20, the concentration of described PBST solution is 0.005～0.02mol/L, described PBST solution
PH be 7.0～7.5.
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| CN106556698A (en) * | 2016-12-05 | 2017-04-05 | 百奥森（江苏）食品安全科技有限公司 | A kind of campylobacter jejuni detection method |
Citations (3)
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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 |
|---|---|---|---|---|
| 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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| CN106556698A (en) * | 2016-12-05 | 2017-04-05 | 百奥森（江苏）食品安全科技有限公司 | A kind of campylobacter jejuni detection method |
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