CN107478833A

CN107478833A - A kind of sensitive probe and preparation method and the method using its detection Bacterium enteritidis

Info

Publication number: CN107478833A
Application number: CN201710733861.4A
Authority: CN
Inventors: 王建龙; 张道宏; 补彤; 闫灵芝; 黄琼; 窦磊娜; 赵兵欣; 赵东阳
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2017-08-22
Filing date: 2017-08-22
Publication date: 2017-12-15
Anticipated expiration: 2037-08-22
Also published as: CN107478833B

Abstract

A kind of sensitive probe and preparation method and the method using its detection Bacterium enteritidis, it is related to the application of a kind of probe and preparation method and probe.It is expensive, unstable the invention aims to solve existing probe, the easy inactivation and the problem of poor sensitivity of antibody.A kind of sensitive probe is prepared by antibiotic and signal vehicle, and its preparation method is：First, yellowish-brown emulsion is prepared；2nd, the immunomagnetic beads of carboxylated is prepared；3rd, the magnetic microsphere of activation is prepared；4th, re-suspension liquid is added to preserve；Utilize the method for sensitive probe detection Bacterium enteritidis：First, the immuno-chromatographic test paper strip of Bacterium enteritidis is prepared；2nd, testing liquid mixes incubation with sensitive probe, in the sample pad for the immuno-chromatographic test paper strip for adding dropwise Bacterium enteritidis, reacts 10 minutes, reads result.The present invention only has a kind of antibody stroke directly to be detected on nitrocellulose filter, greatlys save cost, simply, convenient.The present invention is applied to detection Bacterium enteritidis.

Description

A kind of sensitive probe and preparation method and the method using its detection Bacterium enteritidis

Technical field

The present invention relates to the application of a kind of probe and preparation method and probe.

Background technology

Death toll caused by bacterial infection disease accounts for 1/3rd of global death toll, and this is always one important Public health problem.Therefore, the infection of EARLY RECOGNITION bacterium is by detecting (POC) to water immediately, food and clinical diagnosis Safety is most important.Immuno-chromatographic test paper strip, due to its preparation and it is easy to operate, cost is low, disposable, detection time It is short, the advantages that visual result is reliable, it is not necessary to special equipment, be most common checkout and diagnosis instrument immediately, for each hatching egg The detection of white matter, antibiotic, metal ion, toxin etc..Typical sandwich test strips are using having specific biotic component, such as Antibody, based on antigen and antibody specific combination principle, the gold labeling antibody probe reaction shape on target molecule and pad in sample Into target molecule-gold mark probe complex, this compound again with the antibody for another pairing tested on nitrocellulose (NC) film Zygotic induction goes out macroscopic color change.

Although the operation principle of test strips is very simple, the inspection of food borne bacteria in actual sample is not directly applicable Survey, because the concentration of bacterium is very low in food samples, most of is all swill.Can be by using magnetic nanoparticle To solve this problem, this causes target bacteria to be captured from a large amount of sample solutions, and the solution for separating and being condensed into small size is carried out Detection.However, because the activity of antibody is easily affected by environment, in different samples, can be inactivated in being extracted such as organic sample, such as material Expect pH, salt etc. in labeling process, all have an impact to the activity of antibody；For sandwich-format, it is highly difficult to prepare antibody itself, Screening can identify that the different epitopes of same target can form the antibody of pairing and be more difficult to, and cost is high, poor sensitivity, to enteritis The lowest detection of salmonella is limited to 400CFU/mL~10⁵CFU/mL。

The content of the invention

It is expensive, unstable the invention aims to solve existing probe, the easy inactivation and the problem of poor sensitivity of antibody, And a kind of sensitive probe and preparation method are provided and detect the method for Bacterium enteritidis using it.

A kind of sensitive probe is prepared by antibiotic and signal vehicle；Described antibiotic is ampicillin；It is described Signal vehicle be carboxylated immunomagnetic beads.

A kind of preparation method of sensitive probe, is specifically realized by the following steps：

First, by FeCl₃·6H₂O and two citric acid monohydrate trisodiums are added in ethylene glycol, then temperature be 36 DEG C~37 DEG C It is dissolving 30min~60min in 200r/min~300r/min gas bath shaking table with rotating speed, adds sodium acetate, then in temperature To dissolve 30min~60min in 36 DEG C~37 DEG C gas bath shaking tables for being 200r/min~300r/min with rotating speed, yellowish-brown is obtained Emulsion；

FeCl described in step 1₃·6H₂O quality and the volume ratio of ethylene glycol are (0.5g~1g):(10mL~ 30mL)；

FeCl described in step 1₃·6H₂O and the mass ratio of two citric acid monohydrate trisodiums are (0.5~1):(0.1~ 0.4)；

FeCl described in step 1₃·6H₂The mass ratio of O and sodium acetate is (0.5~1):(1~2)；

2nd, the yellowish-brown emulsion obtained in step 1 is transferred in the reactor of polytetrafluoroethylene liner, then will be poly- The reactor of four fluorine ethylene liners places 8h~12h in the baking oven that temperature is 190 DEG C~210 DEG C, then naturally cools to room temperature, Obtain dark brown product；First by absolute ethyl alcohol to dark brown product cleaning 3 times~5 times, ultra-pure water is reused to dark brown Product cleaning 3 times~5 times, dried at being finally 60 DEG C~70 DEG C in temperature, obtain the immunomagnetic beads of carboxylated；

The particle diameter of the immunomagnetic beads of carboxylated described in step 2 is 150nm~200nm；

3rd, the immunomagnetic beads of carboxylated is added in distilled water, adds 1- ethyls-(3- dimethylaminopropyls) carbon Acyl diimmonium salt hydrochlorate and n-hydroxysuccinimide, then it is 36 DEG C~37 DEG C in temperature and rotating speed is 200r/min~300r/ 20min~30min is reacted in min gas bath shaking table, then carries out Magneto separate, reject solvent, obtains reactant I；Use distilled water Clean reactant I 3 times~5 times, the magnetic microsphere activated；

The quality of the immunomagnetic beads of carboxylated described in step 3 and the volume ratio of distilled water are (0.8mg~1.5mg): 1mL；

The quality and distilled water of 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate described in step 3 Volume ratio be (0.8mg~1.5mg):1mL；

The quality of n-hydroxysuccinimide described in step 3 and the volume ratio of distilled water are (0.5mg~1mg): 1mL；

4th, 1., by the magnetic microsphere of activation it is added in the borate buffer that pH value is 6.6, adds ampicillin, 50min~60min is reacted in the gas bath shaking table that temperature is 36 DEG C~37 DEG C and rotating speed is 200r/min~300r/min again, then Magneto separate is carried out, abandoning supernatant, obtains reactant II；2., into reactant II add the pH containing 1% bovine serum albumin(BSA) It is worth in the borate buffer for 6.6, continues in the gas bath that temperature is 36 DEG C~37 DEG C and rotating speed is 200r/min~300r/min 50min~60min is reacted in shaking table, then carries out Magneto separate, abandoning supernatant, obtains sensitive probe；It is 6.6 to reuse pH value Borate buffer cleans 3 times~5 times to sensitive probe, adds re-suspension liquid, obtains the resuspension that sensitive probe concentration is 1mg/mL Liquid；

Step 4 1. described in activation magnetic microsphere quality be 6.6 with pH value the volume ratio of borate buffer be 1mg:(1mL~2mL)；

Step 4 1. described in activation magnetic microsphere and ampicillin mass ratio be 1:(2~5)；

Step 4 2. described in reactant II quality and the pH value containing 1% bovine serum albumin(BSA) be 6.6 boric acid The volume ratio of buffer solution is 1mg:(1mL~2mL).

Using the method for sensitive probe detection Bacterium enteritidis, it is specifically realized by the following steps：

First, the immuno-chromatographic test paper strip of Bacterium enteritidis is prepared：

The immuno-chromatographic test paper strip of Bacterium enteritidis includes liner plate, and nitrocellulose filter, cellulose nitrate are posted on liner plate One end covering adsorptive pads of plain film, the other end of nitrocellulose filter cover sample pad and pad, nitrocellulose filter successively Non-covered face on detection line is transversely set；Detection line is coated with Bacterium enteritidis monoclonal antibody, pad and sample Pad is respectively through confining liquid Seal treatment；

The preparation method of the immuno-chromatographic test paper strip of described Bacterium enteritidis is as follows：

1., the preparation of detection line：

Bacterium enteritidis monoclonal antibody is dissolved in coating buffer, obtaining Bacterium enteritidis MAb concentration is 1mg/mL solution；With hatched manner by Bacterium enteritidis MAb concentration be 1mg/mL solution with 1 μ L/cm speed Degree is laterally coated in away from along 25mm~30mm position, detection line is obtained on nitrocellulose filter, then in 36 DEG C~37 DEG C Under the conditions of dry 20min~30min, obtain the nitrocellulose filter containing detection line；

2., the preparation of sample pad：

Glass fibre membrane is cut out and grows into 13mm~18mm, a width of 2mm~4mm, places into confining liquid and soaks, is taken out 10h~16h is dried at being afterwards 36 DEG C~37 DEG C in temperature, obtains sample pad；

3., the preparation of pad：

Glass fibre membrane is cut out and grows into 7mm~9mm, wide 2mm~4mm, places into confining liquid and soaks, after taking-up Temperature is that 10h~16h is dried at 36 DEG C~37 DEG C, obtains pad；

4., the preparation of adsorptive pads：

Blotting paper is cut out and grows into 16mm~20mm, a width of 2mm~4mm, obtains adsorptive pads；

5., assembling：

Cardboard one side from top to bottom successively paste adsorptive pads, the nitrocellulose filter containing detection line, pad and Sample pad, adjacent each pad overlap connection in junction, and overlapping length is 1~3mm, obtains the immunity-chromatography test of Bacterium enteritidis Paper slip；

2nd, 100 μ L liquid to be detected are taken, it is 1mg/mL's that 5 μ L sensitive probes concentration are added into 100 μ L liquid to be detected Re-suspension liquid, then it is sufficiently mixed incubation in the shaking table that temperature is 35~37 DEG C and rotating speed is 200r/min~300r/min 20min~40min, then Magneto separate is carried out, abandoning supernatant, obtain the compound of magnetic and material to be detected；To magnetic and to be detected The phosphate buffer that 100 μ L distilled water or 100 μ L 0.01mol/L are added in the compound of material is resuspended, and obtains sample Solution；Sample solution is added drop-wise to dropwise in the sample pad of the immuno-chromatographic test paper strip of Bacterium enteritidis, as detection Test strips, while take 100 μ L distilled water or 100 μ L 0.01mol/L phosphate buffer to be dripped dropwise as negative controls It is added in the sample pad of the immuno-chromatographic test paper strip of another Bacterium enteritidis, as control stripes bar, is read after 10min As a result；

Testing result：(1) it is positive：When the detection line of the immuno-chromatographic test paper strip of Bacterium enteritidis shows yellow line When, the positive is judged to, shows that the concentration of the Bacterium enteritidis in liquid to be detected is greater than or equal to 80CFU/mL；(2)：It is negative： When the detection line not display color of the immuno-chromatographic test paper strip of Bacterium enteritidis, negative findings is judged to, shows liquid to be detected Bacterium enteritidis in body is less than 80CFU/mL.

Compared with prior art, advantages of the present invention is with good effect：

First, the present invention is prepared for sensitive probe to be enriched with to solve this problem of immunochromatography detection muting sensitivity Determinand, detection antibody is instead of, both reduce cost, improve the sensitivity of analysis system again, this is for monitoring meat, eggs and milk Bacterium enteritidis tool in based food has very important significance and application value；By the structure, Bacterium enteritidis can Effectively captured by the capture antibody on T lines, it has been broken tradition while using test antibody and the sandwich test paper of capture antibody The form of bar, a kind of antibody is only used, cost is greatly reduced and realizes super-sensitive detection.In addition, this easy biography Sensor enormously simplify the early stage preparation process of immunoassays correlation analysis reagent, such as：Preload the preparation process of probe.This sets Standby to have very high sensitivity and very strong specificity, simple in construction, cost benefit is high, it is not necessary to equipment, when quickly analyzing Between and portability, there is great application potential to meet the requirement of POC diagnostic assays.This method has been successfully applied in milk The detection of salmonella, demonstrate its practicality and applicability.Therefore a kind of antibody is only needed, this method can serve as general inspection Platform is surveyed to go to detect institute's pathogen；

2nd, the immunomagnetic beads of carboxylated prepared by the present invention is easy to as sensitive signal carrier, the immunomagnetic beads of carboxylated Prepare, good optical characteristics, not only with appropriate size (150-200nm), good uniformity, dispersiveness and stably Property, and a large amount of carboxyl functional groups are contained on surface, in favor of activating and modifying, so imparting the nanowire signal load of its superelevation Ability.Compared with other large scale material such as nano silicons, single-walled carbon nanotube, graphene etc., carboxylated it is immune Magnetic bead synthesis is simple, stable, disperses, and is easy to activation modification, avoids the chemical synthesis of complexity, and this, which can simplify, produces and ensure Uniformity between uniformity and batch；The ampicillin that the present invention selects goes to capture bacterium, instead of the effect of antibody, ammonia Amino is contained on parasiticin surface, can be combined with the carboxyl on the immunomagnetic beads surface of carboxylated, both can firmly act on bacterium Surface and bacterium can be killed, cost is low, is a kind of very promising signal vehicle；

3rd, traditional sandwich-type detection procedures have been broken：The present invention is only drawn on nitrocellulose filter directly with a kind of antibody Detected, broken tradition while using the sandwich-type detection procedures of two kinds of antibody, greatlyd save cost, solve with confrontation The difficulty of body, therefore, the present invention are simpler, convenient and novel；

4th, sensitive probe：The present invention is exempted from immuno-chromatographic test paper strip detection with ampicillin and carboxylated first Epidemic disease magnetic bead builds probe, instead of the detection antibody of costliness, and this work detects Bacterium enteritidis for actual samples such as milk Develop cheap, sensitive, the portable and analysis system that quickly reads；

5th, high sensitivity：Test strips provided by the invention are limited to 80CFU/mL to the lowest detection of Bacterium enteritidis, its Detected value of the value less than prior art report；

6th, good practical application：The present invention can detect the Bacterium enteritidis in milk, and sensitivity and standard bacteria Strain concentration is the same, can reach 80CFU/mL, have good application prospect, can be detected as universal test method all Pathogen；

7th, compared with traditional sandwich-type detection procedures, cost reduces the method for Autonomous test Bacterium enteritidis of the present invention 40%~60%.

8th, the present invention only has a kind of antibody to draw and directly detected on nitrocellulose filter, has broken tradition while has adopted With the sandwich-type detection procedures of two kinds of antibody, cost is greatlyd save, the difficulty for solving pairing antibody is more simply, convenient, newly Grain husk.The present invention is applied to detection Bacterium enteritidis.

The present invention is applied to detection Bacterium enteritidis.

Brief description of the drawings

Fig. 1 is the schematic diagram of sensitive probe prepared by embodiment one, in Fig. 11 be carboxylated immunomagnetic beads, 2 be ammonia benzyl Penicillin, 3 be sensitive probe, and EDC is 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate, and NHS is N- hydroxyls Succinimide；

The structural representation of the immuno-chromatographic test paper strip for the Bacterium enteritidis that Fig. 2 is prepared for the step 1 of embodiment two, Fig. 2 In 1 be sample pad, 2 be pad, and 3 be detection line, and 4 be nitrocellulose filter, and 5 be adsorptive pads；

Fig. 3 is the immuno-chromatographic test paper strip detection principle diagram of Bacterium enteritidis prepared by embodiment two, and 1 is treats in Fig. 3 Liquid is detected, 2 be sensitive probe, and 3 are resuspended to add phosphate buffer, and 4 be Bacterium enteritidis monoclonal antibody, 5 To react 10min；

Fig. 4 is the sensitivity of the immuno-chromatographic test paper strip of Bacterium enteritidis prepared by embodiment two；

Fig. 5 is the specificity of the immuno-chromatographic test paper strip of Bacterium enteritidis prepared by embodiment two, and 1 is to be checked in Fig. 5 Survey liquid is Bacterium enteritidis bacterium solution, and 2 be that liquid to be detected is salmonella typhimurium bacterium solution, and 3 be that liquid to be detected is human relations Honest salmonella bacterium solution, 4 be that liquid to be detected is Salmonella hadar bacterium solution, and 5 be that liquid to be detected is B-mode salmonella Bacterium solution, 6 be that liquid to be detected is Escherichia coli bacteria liquid, and 7 be that liquid to be detected is staphylococcus aureus bacterium solution, and 8 be to be detected Liquid is campylobacter bacterium solution, and 9 be that liquid to be detected is Listeria bacterium solution, and 10 be that liquid to be detected is Enterobacter sakazakii bacterium Liquid, 11 be that liquid to be detected is Candida albicans bacterium solution；

Fig. 6 is the concentration results that the sensitive probe prepared using embodiment one determines Bacterium enteritidis in milk；

Fig. 7 is that the SEM of Bacterium enteritidis schemes；

Fig. 8 is the SEM figures of the immunomagnetic beads for the carboxylated that the step 2 of embodiment one obtains；

Fig. 9 is sensitive probe capture 10 in the step 2 of embodiment three²The SEM of Bacterium enteritidis in CFU/mL testing liquids Figure；

Figure 10 is different material to the inhibition zone of Bacterium enteritidis, and AMP is ampicillin in Figure 10, sterile Water is sterilized water, and MNPs is the immunomagnetic beads for the carboxylated that the step 2 of embodiment one obtains, and AMP-MNPs is the step of embodiment one Rapid four obtained sensitive probes；

Figure 11 is saturation magnetization curve, in Figure 11 1 be the carboxylated that the step 2 of embodiment one obtains immunomagnetic beads Saturation magnetization curve, 2 be the saturation magnetization curve of sensitive probes that the step 4 of embodiment one obtains；

Figure 12 is infrared spectrogram, in Figure 12 1 be ampicillin infrared spectrum curve, 2 be the step 2 of embodiment one The infrared spectrum curve of the immunomagnetic beads of obtained carboxylated, 3 be the infrared light for the sensitive probe that the step 4 of embodiment one obtains Spectral curve；

Figure 13 is uv absorption spectra, in Figure 13 1 be ampicillin ultra-violet absorption spectrum curve, 2 be embodiment The ultra-violet absorption spectrum curve of the immunomagnetic beads for the carboxylated that one step 2 obtains, 3 be the step 4 of embodiment one obtain it is sensitive The ultra-violet absorption spectrum curve of probe.

Embodiment

Embodiment one：Present embodiment is a kind of sensitive probe, and the probe is prepared by antibiotic and signal vehicle Form；Described antibiotic is ampicillin；Described signal vehicle is the immunomagnetic beads of carboxylated.

Embodiment two：Present embodiment is with the difference of embodiment one：Described carboxylated is immunized The particle diameter of magnetic bead is 150nm~200nm.Other steps are identical with embodiment one.

Embodiment three：Present embodiment is with one of embodiment one or two difference：Described carboxyl What the immunomagnetic beads of change was specifically prepared according to the following steps：

2nd, the yellowish-brown emulsion obtained in step 1 is transferred in the reactor of polytetrafluoroethylene liner, then will be poly- The reactor of four fluorine ethylene liners places 8h~12h in the baking oven that temperature is 190 DEG C~210 DEG C, then naturally cools to room temperature, Obtain dark brown product；First by absolute ethyl alcohol to dark brown product cleaning 3 times~5 times, ultra-pure water is reused to dark brown Product cleaning 3 times~5 times, dried at being finally 60 DEG C~70 DEG C in temperature, obtain the immunomagnetic beads of carboxylated.Other steps with Embodiment one or two is identical.

Embodiment four：Present embodiment is with one of embodiment one to three difference：Will in step 2 The yellowish-brown emulsion obtained in step 1 is transferred in the reactor of polytetrafluoroethylene liner, then by polytetrafluoroethylene liner Reactor places 8h~10h in the baking oven that temperature is 190 DEG C~200 DEG C, then naturally cools to room temperature, obtains dark brown production Thing；First by absolute ethyl alcohol to dark brown product cleaning 3 times~4 times, reuse ultra-pure water to dark brown product cleaning 3 times~ 4 times, dried at being finally 60 DEG C~65 DEG C in temperature, obtain the immunomagnetic beads of carboxylated.Other steps and embodiment One to three is identical.

Embodiment five：Present embodiment is that a kind of preparation method of sensitive probe is specifically realized by the following steps 's：

Embodiment six：The difference of present embodiment and embodiment five is：Weight described in step 4 Suspension is the mixed liquor of borate buffer solution, sucrose, trehalose, sodium azide, bovine serum albumin(BSA) and polyvinylpyrrolidone； The mass ratio of sucrose and borate buffer solution is 5 in described re-suspension liquid:100, the mass ratio of trehalose and borate buffer solution For 1:100, the mass ratio of sodium azide and borate buffer solution is 0.5:100, bovine serum albumin(BSA) and borate buffer solution Mass ratio is 1:100, the mass ratio of polyvinylpyrrolidone and borate buffer solution is 1:100；Described borate buffer solution PH value be 9, ion concentration 0.02mol/L.Other steps are identical with embodiment five.

Embodiment seven：Present embodiment is using the method for sensitive probe detection Bacterium enteritidis, is specifically Complete according to the following steps：

1., the preparation of detection line：

2., the preparation of sample pad：

3., the preparation of pad：

4., the preparation of adsorptive pads：

5., assembling：

Present embodiment step 1 1. described in Bacterium enteritidis monoclonal antibody be by Zhang Daohong et al. In publication《Analytical Chimica Acta》The 63-69 pages of volume 635, entitled " Production of ultrasensitive generic monoclonal antibodies against major aflatoxins using a Prepared by the method that modified two-step screening procedure " are recorded.

Compared with prior art, it is the advantages of present embodiment with good effect：

First, present embodiment is prepared for sensitive probe to solve this problem of immunochromatography detection muting sensitivity Determinand is enriched with, detection antibody is instead of, both reduces cost, improve the sensitivity of analysis system again, this is for monitoring meat Bacterium enteritidis tool in egg-milk food has very important significance and application value；Pass through the structure, Bacterium enteritidis Can effectively it be captured by the capture antibody on T lines, it has been broken tradition while has used test antibody and capture the sandwich of antibody The form of test strips, a kind of antibody is only used, cost is greatly reduced and realizes super-sensitive detection.In addition, this simplicity Sensor enormously simplify the early stage preparation process of immunoassays correlation analysis reagent, such as：Preload the preparation process of probe. The equipment has very high sensitivity and very strong specificity, simple in construction, and cost benefit is high, it is not necessary to equipment, quickly divides Time and portability are analysed, there is great application potential to meet the requirement of POC diagnostic assays.This method has been successfully applied to ox The detection of salmonella in milk, demonstrates its practicality and applicability.Therefore a kind of antibody is only needed, this method can serve as leading to Gone to detect institute's pathogen with detection platform；

2nd, the immunomagnetic beads of carboxylated prepared by present embodiment is as sensitive signal carrier, the immunomagnetic beads of carboxylated Easily prepared, good optical characteristics is not only dispersed and steady with appropriate size (150-200nm), good uniformity It is qualitative, and a large amount of carboxyl functional groups are contained on surface, in favor of activating and modifying, so the nanowire signal for imparting its superelevation is born Loading capability.Compared to other large scale material such as nano silicons, single-walled carbon nanotube, graphene etc., carboxylated is exempted from The synthesis of epidemic disease magnetic bead is simple, stable, disperses, and is easy to activation modification, avoids the chemical synthesis of complexity, and this can simplify production and true The uniformity protected between uniformity and batch；The ampicillin that present embodiment is selected goes to capture bacterium, instead of antibody Effect, ampicillin surface contains amino, can be combined with the carboxyl on the immunomagnetic beads surface of carboxylated, both can firmly acted on In bacterium surface and bacterium can be killed, cost is low, is a kind of very promising signal vehicle；

3rd, traditional sandwich-type detection procedures have been broken：Present embodiment is only drawn on nitrocellulose filter with a kind of antibody Directly detected, broken tradition while using the sandwich-type detection procedures of two kinds of antibody, greatlyd save cost, solve and match somebody with somebody To the difficulty of antibody, therefore, the present invention is simpler, convenient and novel；

4th, sensitive probe：Present embodiment is first with ampicillin and carboxylated in immuno-chromatographic test paper strip detection Immunomagnetic beads structure probe, instead of the detection antibody of costliness, this work detects enteritis sramana for the actual sample such as milk Salmonella develops cheaply, sensitive, the portable and analysis system that quickly reads；

5th, high sensitivity：The test strips that present embodiment provides are limited to 80CFU/ to the lowest detection of Bacterium enteritidis ML, its value are less than the detected value of prior art report；

6th, good practical application：Present embodiment can detect the Bacterium enteritidis in milk, and sensitivity and mark Quasi- bacterial strain concentration is the same, can reach 80CFU/mL, has good application prospect, can be used as universal test method detection institute Some pathogens；

7th, the method for present embodiment Autonomous test Bacterium enteritidis is compared with traditional sandwich-type detection procedures, cost drop Low 40%~60%.

8th, present embodiment only has a kind of antibody to draw and directly detected on nitrocellulose filter, has broken traditional same The sandwich-type detection procedures of two kinds of antibody of Shi Caiyong, greatly save cost, solve the difficulty of pairing antibody, more simply, convenient, It is novel.The present invention is applied to detection Bacterium enteritidis.

Embodiment eight：Present embodiment is with one of embodiment one to seven difference：Step 1 1. institute The coating buffer stated configures obtain by the following method：By 0.02g~0.04g sodium azide, 0.8g~1.0g sodium chloride, 0.29g~0.35g disodium hydrogen phosphates, 0.02g~0.04g potassium chloride and 0.02g~0.04g potassium dihydrogen phosphates, add water It is settled to obtained by 100mL.Other steps are identical with embodiment one to seven.

Embodiment nine：Present embodiment is with one of embodiment one to eight difference：Step 1 2. in Described confining liquid configures obtain by the following method：By 1.5g~2.5g bovine serum albumin(BSA)s, 0.02g~0.04g nitrine Change sodium, 0.8g~1.0g sodium chloride, 0.29g~0.35g disodium hydrogen phosphates, 0.02g~0.04g potassium chloride and 0.02g ~0.04g potassium dihydrogen phosphates, water is added to be settled to obtained by 100mL.Other steps are identical with embodiment one to eight.

Embodiment ten：Present embodiment is with one of embodiment one to nine difference：Step 1 3. in Described confining liquid configures obtain by the following method：By 1.5g~2.5g bovine serum albumin(BSA)s, 0.02g~0.04g nitrine Change sodium, 0.8g~1.0g sodium chloride, 0.29g~0.35g disodium hydrogen phosphates, 0.02g~0.04g potassium chloride and 0.02g ~0.04g potassium dihydrogen phosphates, water is added to be settled to obtained by 100mL.Other steps are identical with embodiment one to nine.

Beneficial effects of the present invention are verified using following examples：

Embodiment one：A kind of preparation method of sensitive probe, is specifically realized by the following steps：

First, by 0.675g FeCl₃·6H₂O and 0.245g two citric acid monohydrate trisodiums are added in 20mL ethylene glycol, then 40min is dissolved in the gas bath shaking tables that temperature is 37 DEG C and rotating speed is 220r/min, adds 1.2g sodium acetates, then in temperature is 37 DEG C and rotating speed be 220r/min gas bath shaking table in dissolve 40min, obtain yellowish-brown emulsion；

2nd, the yellowish-brown emulsion obtained in step 1 is transferred in the reactor of polytetrafluoroethylene liner, then will be poly- The reactor of four fluorine ethylene liners places 10h in the baking oven that temperature is 200 DEG C, then naturally cools to room temperature, obtains dark brown Product；First by absolute ethyl alcohol to dark brown product cleaning 5 times, ultra-pure water is reused to dark brown product cleaning 5 times, finally Dried at being 60 DEG C in temperature, obtain the immunomagnetic beads of carboxylated；

3rd, the immunomagnetic beads of 1mg carboxylated is added in 1mL distilled water, adds 1mg 1- ethyls-(3- dimethyl Aminopropyl) phosphinylidyne diimmonium salt hydrochlorate and 0.6mg n-hydroxysuccinimides, then be 37 DEG C in temperature and rotating speed is 220r/ 30min is reacted in min gas bath shaking table, then carries out Magneto separate, reject solvent, obtains reactant I；Cleaned and reacted using distilled water Thing I 5 times, the magnetic microsphere activated；

4th, 1., by the 1mg magnetic microspheres activated it is added in the borate buffer that 1mL pH value is 6.6, adds 2mg Ampicillin, then 60min is reacted in the gas bath shaking table that temperature is 37 DEG C and rotating speed is 220r/min, then Magneto separate is carried out, Abandoning supernatant, obtain reactant II；2., add the 2mL pH value that contains 1% bovine serum albumin(BSA) into 1mg reactants II and be In 6.6 borate buffer, continue to react 60min in the gas bath shaking table that temperature is 37 DEG C and rotating speed is 220r/min, then enter Row Magneto separate, abandoning supernatant, obtains sensitive probe；Reuse the borate buffer that pH value is 6.6 and 5 are cleaned to sensitive probe It is secondary, re-suspension liquid is added, obtains the re-suspension liquid that sensitive probe concentration is 1mg/mL；

Re-suspension liquid described in step 4 is borate buffer solution, sucrose, trehalose, sodium azide, bovine serum albumin(BSA) With the mixed liquor of polyvinylpyrrolidone；The mass ratio of sucrose and borate buffer solution is 5 in described re-suspension liquid:100, marine alga The mass ratio of sugar and borate buffer solution is 1:100, the mass ratio of sodium azide and borate buffer solution is 0.5:100, ox blood The mass ratio of pure albumen and borate buffer solution is 1:100, the mass ratio of polyvinylpyrrolidone and borate buffer solution is 1:100；The pH value of described borate buffer solution is 9, ion concentration 0.02mol/L.

Embodiment two：Using embodiment one prepare sensitive probe detection Bacterium enteritidis method, specifically press with What lower step was completed：

1., the preparation of detection line：

Bacterium enteritidis monoclonal antibody is dissolved in coating buffer, obtaining Bacterium enteritidis MAb concentration is 1mg/mL solution；With hatched manner by Bacterium enteritidis MAb concentration be 1mg/mL solution with 1 μ L/cm speed Degree is laterally coated in away from along 30mm position, detection line is obtained on nitrocellulose filter, is then dried under the conditions of 37 DEG C 30min, obtain the nitrocellulose filter containing detection line；

1. described coating buffer configures obtain by the following method step 1：By 0.02g sodium azide, 0.8g chlorinations Sodium, 0.29g disodium hydrogen phosphates, 0.02g potassium chloride and 0.02g potassium dihydrogen phosphates, water is added to be settled to obtained by 100mL；

2., the preparation of sample pad：

Glass fibre membrane is cut out and grows into 15mm, a width of 3mm, places into confining liquid and soaks, be in temperature after taking-up 13h is dried at 37 DEG C, obtains sample pad；

Step 1 2. described in confining liquid configure obtain by the following method：2g bovine serum albumin(BSA)s, 0.02g are folded Sodium nitride, 0.8g sodium chloride, 0.29g disodium hydrogen phosphates, 0.02g potassium chloride and 0.02g potassium dihydrogen phosphates, add water constant volume Obtained by 100mL；

3., the preparation of pad：

Glass fibre membrane is cut out and grows into 8mm, wide 3mm, places into confining liquid and soaks, after taking-up temperature be 37 DEG C Lower dry 13h, obtains pad；

Step 1 3. described in confining liquid configure obtain by the following method：2g bovine serum albumin(BSA)s, 0.02g are folded Sodium nitride, 0.8g sodium chloride, 0.29g disodium hydrogen phosphates, 0.02g potassium chloride and 0.02g potassium dihydrogen phosphates, add water constant volume Obtained by 100mL；

4., the preparation of adsorptive pads：

Blotting paper is cut out and grows into 18mm, a width of 3mm, obtains adsorptive pads；

5., assembling：

Cardboard one side from top to bottom successively paste adsorptive pads, the nitrocellulose filter containing detection line, pad and Sample pad, adjacent each pad overlap connection in junction, overlapping length 2mm, obtain the immune chromatography test paper of Bacterium enteritidis Bar；

2nd, 100 μ L liquid to be detected are taken, the sensitive probe of 5 μ L embodiments one preparation is added into 100 μ L liquid to be detected Concentration is 1mg/mL re-suspension liquid, is then sufficiently mixed incubation in the shaking table that temperature is 37 DEG C and rotating speed is 220r/min 30min, then Magneto separate is carried out, abandoning supernatant, obtain the compound of magnetic and material to be detected；To answering for magnetic and material to be detected The phosphate buffer that 100 μ L 0.01mol/L are added in compound is resuspended, and obtains sample solution；Sample solution is dripped dropwise It is added in the sample pad of the immuno-chromatographic test paper strip of Bacterium enteritidis, as test strip, while takes 100 μ L 0.01mol/L phosphate buffer is added drop-wise to the immunity-chromatography test of another Bacterium enteritidis as negative controls dropwise In the sample pad of paper slip, as control stripes bar, result is read after 10min；

The step 1 of embodiment two 1. described in Bacterium enteritidis monoclonal antibody be to exist by Zhang Daohong et al. Publication《Analytical Chimica Acta》The 63-69 pages of volume 635, entitled " Production of ultrasensitive generic monoclonal antibodies against major aflatoxins using a Prepared by the method that modified two-step screening procedure " are recorded.

Embodiment three：Bacterium enteritidis prepared by the sensitive probe measure embodiment two prepared using embodiment one is exempted from What the sensitivity of epidemic disease chromatograph test strip was specifically realized by the following steps：

First, the culture of microorganism：

Bacterium enteritidis (Salmonella Enteritidis) is inoculated in LB culture mediums, the quiescent culture at 37 DEG C 24 hours；Picking single bacterium colony is inoculated in 250mL LB broth bouillons, then is trained under conditions of 150r/min shaking tables and 37 DEG C Support 24 hours, then bacterium solution is centrifuged into 15min with 4000r/min centrifugal speed, collect thalline；Be 7.4 with pH value, concentration be 0.01mol/L phosphate buffer solution washing thalline 3 times, then with 10mL 0.01mol/L phosphate buffer solution weight It is outstanding, then add the formalin solutions of 50 μ L 0.5%, place 24 hours and inactivate at room temperature；After inactivation terminates, use 0.01mol/L's After phosphate buffer solution is washed 3 times, then with 0.01mol/L PBS it is adjusted to suitable concn, adjustment antigen concentration point Wei 10⁷CFU/mL、10⁶CFU/mL、10⁵CFU/mL、10⁴CFU/mL、10³CFU/mL、10²CFU/mL、80CFU/mL、40CFU/ ML and 0CFU/mL, is saved backup in -20 DEG C；Each concentration takes 100 μ L as liquid to be detected respectively；

2nd, the liquid to be detected of 100 each concentration of μ L is taken, 5 μ L realities are separately added into the liquid to be detected of 100 each concentration of μ L The sensitive probe concentration for applying the preparation of example one is 1mg/mL re-suspension liquid, is then 37 DEG C in temperature respectively and rotating speed is 220r/min Shaking table in be sufficiently mixed and be incubated 30min, then carry out Magneto separate respectively, abandoning supernatant, obtain the magnetic of various concentrations and to be checked Survey the compound of material；100 μ L 0.01mol/ are separately added into the compound of the magnetic to various concentrations and material to be detected respectively L phosphate buffer is resuspended, and obtains the sample solution of various concentrations；The sample solution of various concentrations is dripped dropwise respectively It is added in the sample pad of the immuno-chromatographic test paper strip of 9 Bacterium enteritidis, as 9 test strips, while takes 100 μ L 0.01mol/L phosphate buffer is added drop-wise to the immunochromatography of another Bacterium enteritidis as negative controls dropwise In the sample pad of test strips, as control stripes bar, result is read after 10min；

Testing result is as shown in Figure 4.

Bacterium enteritidis (Salmonella Enteritidis) described in embodiment three is bought from the micro- life of Chinese industrial Thing culture presevation administrative center.

As can be seen from Figure 4, with the reduction of Bacterium enteritidis in testing liquid, the immune chromatography test paper of Bacterium enteritidis The yellow of bar T lines is more and more shallow, and macroscopic concentration is 80CFU/mL, therefore, Bacterium enteritidis prepared by embodiment two Immuno-chromatographic test paper strip can detect that Bacterium enteritidis least concentration is 80CFU/mL.Energy high-sensitivity detection of the invention Bacterium enteritidis, and universal method can be used as only to need a kind of antibody and simple dyeing just detectable, rapid and convenient.

Example IV：Bacterium enteritidis prepared by the sensitive probe measure embodiment two prepared using embodiment one is exempted from The specificity of epidemic disease chromatograph test strip：

First, respectively by Bacterium enteritidis (Salmonella Enteritidis), Salmonella hadar (Salmonella Hadar), salmonella london (Salmonella London), salmonella typhimurium (Salmonella Typhimurium), B-mode salmonella (Salmonella Paratyphi B), Escherichia coli (Escherichia coli), Staphylococcus aureus (Staphylococcus aureus), Listeria (Listeria monocytogenes), bent stick Bacterium (Campylobacter coli), Enterobacter sakazakii (Enterobacter sakazakii) and Candida albicans (Candida Albicans bacterium solution) is diluted to 10 with 0.01mol/L phosphate buffer⁷CFU/mL concentration, each bacterium solution take 100 respectively For μ L solution as survey liquid to be detected, the sensitive probe concentration with the preparation of 5 μ L embodiments one is 1mg/mL re-suspension liquid at 37 DEG C Incubation 30 minutes is sufficiently mixed in shaking table, then carries out Magneto separate, abandoning supernatant, obtains the compound of 11 magnetic and material to be detected Thing；The phosphate buffer that 100 μ L 0.01mol/L are separately added into the compound of 11 magnetic and material to be detected carries out weight It is outstanding, obtain 11 sample solutions；11 sample solutions are added drop-wise to the immunity-chromatography test of 11 Bacterium enteritidis dropwise respectively In the sample pad of paper slip, as 11 test strips, while 100 μ L 0.01mol/L phosphate buffer conduct is taken Negative controls, in the sample pad for the immuno-chromatographic test paper strip for being added drop-wise to another Bacterium enteritidis dropwise, as control Test strips, result is read after 10min；

Testing result：(1) it is positive：When the detection line of the immuno-chromatographic test paper strip of Bacterium enteritidis shows yellow line When, it is judged to the positive；(2) it is negative：When the detection line not display color of the immuno-chromatographic test paper strip of Bacterium enteritidis, the moon is judged to Property result.

Testing result is as shown in Figure 5；

Bacterium enteritidis (Salmonella Enteritidis) described in example IV, Salmonella hadar (Salmonella Hadar), salmonella london (Salmonella London), salmonella typhimurium (Salmonella Typhimurium), B-mode salmonella (Salmonella Paratyphi B), Escherichia coli (Escherichia coli), Staphylococcus aureus (Staphylococcus aureus), Listeria (Listeria monocytogenes), bent stick Bacterium (Campylobacter coli), Enterobacter sakazakii (Enterobacter sakazakii) and Candida albicans (Candida Albicans) buy from Chinese industrial Microbiological Culture Collection administrative center.

As can be seen from Figure 5, except the test strips T line for detecting Bacterium enteritidis bacterium solution has macroscopic bright yellow Outside, the test strips T line for detecting other bacteriums does not all have color, illustrates the immunochromatography of Bacterium enteritidis prepared by embodiment two Test strips energy high degree of specificity identifies Bacterium enteritidis, there is extra high specificity.

Embodiment five：The concentration of Bacterium enteritidis in the sensitive probe measure milk prepared using embodiment one：

The immuno-chromatographic test paper strip of Bacterium enteritidis (Salmonella Enteritidis) includes liner plate, is pasted on liner plate There are nitrocellulose filter, one end covering adsorptive pads of nitrocellulose filter, the other end of nitrocellulose filter covers sample successively Pad and pad, detection line is transversely set on the non-covered face of nitrocellulose filter；Detection line is coated with Bacterium enteritidis Monoclonal antibody, pad and sample pad are respectively through confining liquid Seal treatment；

1., the preparation of detection line：

Bacterium enteritidis monoclonal antibody is dissolved in coating buffer, obtaining Bacterium enteritidis MAb concentration is 1mg/mL solution；With hatched manner by Bacterium enteritidis MAb concentration be 1mg/mL solution with 1 μ L/cm speed Degree is laterally coated in away from along 30mm position, detection line is obtained on nitrocellulose filter, is then dried under the conditions of 37 DEG C 30min, obtain the nitrocellulose filter containing detection line；；

2., the preparation of sample pad：

3., the preparation of pad：

4., the preparation of adsorptive pads：

5., assembling：

2nd, Bacterium enteritidis bacterium solution is added in skim milk and is diluted to 10⁷CFU/mL、10⁶CFU/mL、10⁵CFU/ mL、10⁴CFU/mL、10³CFU/mL、10²CFU/mL, 80CFU/mL, 40CFU/mL and 0CFU/mL, the 100 above-mentioned bacterium solutions of μ L of difference As liquid to be detected, the sensitive probe concentration for adding the preparation of 5 μ L embodiments one into 9 100 μ L liquid to be detected respectively is 1mg/mL re-suspension liquid, then it is sufficiently mixed in the shaking table that temperature is 37 DEG C and rotating speed is 220r/min and is incubated 30min, then is entered Row Magneto separate, abandoning supernatant, obtain the compound of 9 magnetic and material to be detected；To 9 magnetic and the compound of material to be detected In be separately added into 100 μ L 0.01mol/L phosphate buffer and be resuspended, obtain 9 sample solutions；By 9 sample solutions It is added drop-wise to dropwise in the sample pad of immuno-chromatographic test paper strip of Bacterium enteritidis respectively, as test strip, simultaneously 100 μ L 0.01mol/L phosphate buffer is taken to be added drop-wise to exempting from for another Bacterium enteritidis dropwise as negative controls In the sample pad of epidemic disease chromatograph test strip, as control stripes bar, result is read after 10min；

Testing result：(1) it is positive：When the detection line of the immuno-chromatographic test paper strip of Bacterium enteritidis shows yellow line When, the positive is judged to, shows that the concentration of the Bacterium enteritidis in testing liquid is greater than or equal to 80CFU/mL；(2)：It is negative：When The detection line of the immuno-chromatographic test paper strip of Bacterium enteritidis not display color when, be judged to negative findings, show in testing liquid Bacterium enteritidis be less than 80CFU/mL.

Testing result is as shown in Figure 6.

Bacterium enteritidis (Salmonella Enteritidis) described in embodiment five is bought from the micro- life of Chinese industrial Thing culture presevation administrative center.

As can be seen from Figure 6, reduced with the concentration of Bacterium enteritidis in liquid to be detected, yellow is increasingly in test strips It is shallow, there is the visible concentration of eye to can reach 80CFU/mL, therefore, the Bacterium enteritidis that the present embodiment can be detected in milk is minimum dense 80CFU/mL is spent, reflects its good actual application value.

In order to verify that the immunomagnetic beads of carboxylated and ampicillin can combine well, tests below has been done；

Fig. 7 is that the SEM of Bacterium enteritidis schemes；

As can be seen from Figure 7, Bacterium enteritidis is bar-shaped that bacterium shape is complete, about 1 μm~2 μm；

As it can be observed in the picture that the immunomagnetic beads for the carboxylated that the step 2 of embodiment one obtains to be spherical, about 150nm~ 200nm。

As can be seen from Figure 9, many sensitive probes of Bacterium enteritidis surface attachment, and bacterium crushes, it is imperfect, illustrate sensitive Probe can both capture Bacterium enteritidis, can kill Bacterium enteritidis again.

As can be seen from Figure 10, sterilized water and the immunomagnetic beads of carboxylated do not have inhibition zone, illustrate that they will not produce shadow to bacterium Ring, ampicillin has inhibition zone, can kill bacterium, and sensitive probe is also evident that inhibition zone, illustrates carboxylated Immunomagnetic beads has been combined with ampicillin sensitive probe.

As can be seen from Figure 11, the hysteresis curve of the immunomagnetic beads of carboxylated is typical S types curve, the immunomagnetic beads of carboxylated Magnetic induction intensity increase with the increase of externally-applied magnetic field, and progressively reach saturation after 2000 (Oe), its saturated magnetization Intensity is 43.54emg/g.The saturation magnetization of sensitive probe is decreased, but it is not very big to influence, and sensitive probe is satisfied It is 39.41emg/g with the intensity of magnetization.

As can be seen from Figure 12, the immunomagnetic beads for the carboxylated that the step 2 of embodiment one obtains is in 1633cm^-1And 1388cm^-1Place There is obvious absorption, illustrate that the immunomagnetic beads surface of carboxylated has the carboxyl of a large amount of scalable vibrations.590cm^-1The absorption gone out The stretching vibration for the Fe-O that peak is, 3436cm^-1It is the stretching vibration from O-H.And sensitive probe has obvious suction at 1780 Receive, the absworption peak is the characteristic absorption peak of ampicillin beta-lactam nucleus, illustrates that ampicillin pass flag has arrived carboxyl On the immunomagnetic beads surface of change.

As can be seen from Figure 13, the immunomagnetic beads of carboxylated has individual small peak at 400nm, and the characteristic peak of ampicillin exists 280nm, the characteristic absorption peak of ampicillin are high-visible on the absworption peak of sensitive probe, it was demonstrated that ampicillin success exists Combined on the immunomagnetic beads of carboxylated.

Claims

1. a kind of sensitive probe, it is characterised in that the probe is prepared by antibiotic and signal vehicle；Described antibiotic is Ampicillin；Described signal vehicle is the immunomagnetic beads of carboxylated.

A kind of 2. sensitive probe according to claim 1, it is characterised in that the particle diameter of the immunomagnetic beads of described carboxylated For 150nm~200nm.

3. a kind of sensitive probe according to claim 1, it is characterised in that the immunomagnetic beads of described carboxylated is specifically Prepare according to the following steps：

First, by FeCl₃·6H₂O and two citric acid monohydrate trisodiums are added in ethylene glycol, then for 36 DEG C~37 DEG C and are turned in temperature Speed adds sodium acetate to dissolve 30min~60min in 200r/min~300r/min gas bath shaking table, then in temperature is 36 DEG C~37 DEG C and rotating speed be 200r/min~300r/min gas bath shaking table in dissolve 30min~60min, obtain yellowish-brown emulsus Liquid；

FeCl described in step 1₃·6H₂O quality and the volume ratio of ethylene glycol are (0.5g~1g):(10mL~30mL)；

FeCl described in step 1₃·6H₂O and the mass ratio of two citric acid monohydrate trisodiums are (0.5~1):(0.1~0.4)；

2nd, the yellowish-brown emulsion obtained in step 1 is transferred in the reactor of polytetrafluoroethylene liner, then by polytetrafluoro The reactor of the dilute liner of second places 8h~12h in the baking oven that temperature is 190 DEG C~210 DEG C, then naturally cools to room temperature, obtains Dark brown product；First by absolute ethyl alcohol to dark brown product cleaning 3 times~5 times, ultra-pure water is reused to dark brown product Cleaning 3 times~5 times, dried at being finally 60 DEG C~70 DEG C in temperature, obtain the immunomagnetic beads of carboxylated.

4. a kind of sensitive probe according to claim 3, it is characterised in that yellowish-brown by what is obtained in step 1 in step 2 Color emulsion is transferred in the reactor of polytetrafluoroethylene liner, then by the reactor of polytetrafluoroethylene liner temperature be 190 DEG C~200 DEG C of baking oven in place 8h~10h, then naturally cool to room temperature, obtain dark brown product；First by absolute ethyl alcohol To dark brown product cleaning 3 times~4 times, ultra-pure water is reused to dark brown product cleaning 3 times~4 times, is finally 60 in temperature DEG C~65 DEG C at dry, obtain the immunomagnetic beads of carboxylated.

A kind of a kind of 5. preparation method of sensitive probe as claimed in claim 1, it is characterised in that preparation side of sensitive probe What method was specifically realized by the following steps：

2nd, the yellowish-brown emulsion obtained in step 1 is transferred in the reactor of polytetrafluoroethylene liner, then by polytetrafluoro The reactor of the dilute liner of second places 8h~12h in the baking oven that temperature is 190 DEG C~210 DEG C, then naturally cools to room temperature, obtains Dark brown product；First by absolute ethyl alcohol to dark brown product cleaning 3 times~5 times, ultra-pure water is reused to dark brown product Cleaning 3 times~5 times, dried at being finally 60 DEG C~70 DEG C in temperature, obtain the immunomagnetic beads of carboxylated；

3rd, the immunomagnetic beads of carboxylated is added in distilled water, adds 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne two Inferior amine salt hydrochlorate and n-hydroxysuccinimide, then it is 36 DEG C~37 DEG C in temperature and rotating speed is 200r/min~300r/min's 20min~30min is reacted in gas bath shaking table, then carries out Magneto separate, reject solvent, obtains reactant I；It is anti-using distilled water cleaning Answer thing I 3 times~5 times, the magnetic microsphere activated；

The quality of the immunomagnetic beads of carboxylated described in step 3 and the volume ratio of distilled water are (0.8mg~1.5mg):1mL；

1- ethyls-quality of (3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate and the body of distilled water described in step 3 Product ratio is (0.8mg~1.5mg):1mL；

The quality of n-hydroxysuccinimide described in step 3 and the volume ratio of distilled water are (0.5mg~1mg):1mL；

4th, 1., by the magnetic microsphere of activation it is added in the borate buffer that pH value is 6.6, adds ampicillin, then 50min~60min is reacted in the gas bath shaking table that temperature is 36 DEG C~37 DEG C and rotating speed is 200r/min~300r/min, then is carried out Magneto separate, abandoning supernatant, obtain reactant II；2., into reactant II add the pH value containing 1% bovine serum albumin(BSA) be In 6.6 borate buffer, continue in the gas bath shaking table that temperature is 36 DEG C~37 DEG C and rotating speed is 200r/min~300r/min Middle reaction 50min~60min, then Magneto separate is carried out, abandoning supernatant, obtain sensitive probe；Reuse the boric acid that pH value is 6.6 Buffer solution cleans 3 times~5 times to sensitive probe, adds re-suspension liquid, obtains the re-suspension liquid that sensitive probe concentration is 1mg/mL；

Step 4 1. described in activation magnetic microsphere quality and pH value be 6.6 the volume ratio of borate buffer be 1mg: (1mL~2mL)；

Step 4 2. described in reactant II quality and the pH value containing 1% bovine serum albumin(BSA) be 6.6 borate buffer The volume ratio of liquid is 1mg:(1mL~2mL).

A kind of 6. preparation method of sensitive probe according to claim 5, it is characterised in that the resuspension described in step 4 Liquid is the mixed liquor of borate buffer solution, sucrose, trehalose, sodium azide, bovine serum albumin(BSA) and polyvinylpyrrolidone；Institute The mass ratio of sucrose and borate buffer solution is 5 in the re-suspension liquid stated:100, the mass ratio of trehalose and borate buffer solution is 1:100, the mass ratio of sodium azide and borate buffer solution is 0.5:100, the matter of bovine serum albumin(BSA) and borate buffer solution Amount is than being 1:100, the mass ratio of polyvinylpyrrolidone and borate buffer solution is 1:100；Described borate buffer solution PH value is 9, ion concentration 0.02mol/L.

7. utilize a kind of method of sensitive probe detection Bacterium enteritidis as claimed in claim 1, it is characterised in that utilize What the method for sensitive probe detection Bacterium enteritidis was specifically realized by the following steps：

The immuno-chromatographic test paper strip of Bacterium enteritidis includes liner plate, and nitrocellulose filter, nitrocellulose filter are posted on liner plate One end covering adsorptive pads, the other end of nitrocellulose filter covers sample pad and pad successively, nitrocellulose filter it is non- Detection line is transversely set on coverage rate；Detection line is coated with Bacterium enteritidis monoclonal antibody, pad and sample pad point Not through confining liquid Seal treatment；

1., the preparation of detection line：

Bacterium enteritidis monoclonal antibody is dissolved in coating buffer, it is 1mg/ to obtain Bacterium enteritidis MAb concentration ML solution；With hatched manner by Bacterium enteritidis MAb concentration be 1mg/mL solution it is horizontal with 1 μ L/cm speed To being coated in away from along 25mm~30mm position, detection line is obtained on nitrocellulose filter, then in 36 DEG C~37 DEG C conditions Lower dry 20min~30min, obtains the nitrocellulose filter containing detection line；

2., the preparation of sample pad：

Glass fibre membrane is cut out and grows into 13mm~18mm, a width of 2mm~4mm, places into confining liquid and soaks, after taking-up Temperature is that 10h~16h is dried at 36 DEG C~37 DEG C, obtains sample pad；

3., the preparation of pad：

Glass fibre membrane is cut out and grows into 7mm~9mm, wide 2mm~4mm, places into confining liquid and soaks, in temperature after taking-up To dry 10h~16h at 36 DEG C~37 DEG C, pad is obtained；

4., the preparation of adsorptive pads：

5., assembling：

Adsorptive pads, the nitrocellulose filter containing detection line, pad and sample are pasted successively from top to bottom in the one side of cardboard Pad, adjacent each pad overlap connection in junction, and overlapping length is 1~3mm, obtains the immune chromatography test paper of Bacterium enteritidis Bar；

2nd, 100 μ L liquid to be detected are taken, the resuspension that 5 μ L sensitive probes concentration are 1mg/mL is added into 100 μ L liquid to be detected Liquid, be then sufficiently mixed in the shaking tables that temperature is 35~37 DEG C and rotating speed is 200r/min~300r/min be incubated 20min~ 40min, then Magneto separate is carried out, abandoning supernatant, obtain the compound of magnetic and material to be detected；To answering for magnetic and material to be detected The phosphate buffer that 100 μ L distilled water or 100 μ L 0.01mol/L are added in compound is resuspended, and obtains sample solution；Will Sample solution is added drop-wise in the sample pad of the immuno-chromatographic test paper strip of Bacterium enteritidis dropwise, as test strip, 100 μ L distilled water or 100 μ L 0.01mol/L phosphate buffer is taken to be added drop-wise to dropwise another as negative controls simultaneously In the sample pad of the immuno-chromatographic test paper strip of Bacterium enteritidis, as control stripes bar, result is read after 10min；

Testing result：(1) it is positive：When the detection line of the immuno-chromatographic test paper strip of Bacterium enteritidis shows yellow line, The positive is judged to, shows that the concentration of the Bacterium enteritidis in liquid to be detected is greater than or equal to 80CFU/mL；(2)：It is negative：Work as intestines The detection line of the immuno-chromatographic test paper strip of scorching salmonella not display color when, be judged to negative findings, show in liquid to be detected Bacterium enteritidis be less than 80CFU/mL.

A kind of 8. method of sensitive probe detection Bacterium enteritidis according to claim 7, it is characterised in that step 1 1. described coating buffer configures obtain by the following method：By 0.02g~0.04g sodium azide, 0.8g~1.0g sodium chloride, 0.29g~0.35g disodium hydrogen phosphates, 0.02g~0.04g potassium chloride and 0.02g~0.04g potassium dihydrogen phosphates, add water It is settled to obtained by 100mL.

A kind of 9. method of sensitive probe detection Bacterium enteritidis according to claim 7, it is characterised in that step 1 2. confining liquid described in configures by the following method to be obtained：By 1.5g~2.5g bovine serum albumin(BSA)s, 0.02g~0.04g Sodium azide, 0.8g~1.0g sodium chloride, 0.29g~0.35g disodium hydrogen phosphates, 0.02g~0.04g potassium chloride and 0.02g~0.04g potassium dihydrogen phosphates, water is added to be settled to obtained by 100mL.

A kind of 10. method of sensitive probe detection Bacterium enteritidis according to claim 7, it is characterised in that step 1 3. confining liquid described in configures by the following method to be obtained：By 1.5g~2.5g bovine serum albumin(BSA)s, 0.02g~0.04g Sodium azide, 0.8g~1.0g sodium chloride, 0.29g~0.35g disodium hydrogen phosphates, 0.02g~0.04g potassium chloride and 0.02g~0.04g potassium dihydrogen phosphates, water is added to be settled to obtained by 100mL.