CN101498724A - Corn bacterial wilting germ biotin-avidin ELISA detection method - Google Patents

Abstract

A biotin-avidin enzymoimmunoassay aiming at the corn bacterial wilt includes the following steps of preparing a polyclonal antibody, purifying the polyclonal antibody and performing biotin labeling; extracting bacterium in a sample and coating the extracting solution of the sample on a micro-perforated plate; then sequentially adding a biotin labeling antibody, an alkaline phosphatase labeled strepen avidin compound and a PNP zymolyte, developing and then reading the value of the absorbency with the wavelength below 405 nm by an enzyme-labeling instrument. The biotin-avidin enzymoimmunoassay aiming at the corn bacterial wilt has the advantages of strong specificity, high detecting sensitivity reaching 1*10<6> cfu/mL, simple and rapid operation, accurate result and high efficiency.

Description

The corn bacterial wilting germ biotin-avidin enzyme-linked immune detection method

Technical field

The present invention relates to a kind of enzyme-linked immune detection method, particularly relate to a kind of corn bacterial wilting germ biotin-avidin enzyme-linked immune detection method and detection kit.

Background technology

Corn (interior state) wilt disease claim again the corn bacterial wilt disease (Goss ' s bacterial wilt and blight of maize, Clavibacter michiganensis subsp.nebraskensis), be a kind of plant disease that can cause serious corn loss.1969, this disease first in the U.S. not the Lars add the gloomy county of state highway and take place, spread to crolla sieve state, Illinois, Iowa, Ken Sasi state, the state of Michigan, the Minnesota State afterwards successively, the Nebraska State, the South Dakota State and the state of Wisconsin have diffused to lake region, Canadian Ontario to 2000.The harmfulness of wilt disease and distributive province extension of a field spread the great attention that causes people, states such as European Union, Canada all set up should disease risk evaluation system.China lists this germ in May, 2007 " People's Republic of China (PRC) enter the territory plant quarantine harmful organism register " of up-to-date revision.

The corn bacterial wilt disease has two kinds of main forms, and one is that blade wilts, and two wither for system.This disease is initially discontinuous water soaked spots on blade, just be dirty-green to black, back browning look, parallel with vein, often there is bacterial ooze on the surface.During systemic infection, vascular bundle variable color, root and the dry rot of subaerial stem stalk or water stain shape brown rot.Corn seedling with become strain all can fall ill.Bacterium is by wound infection, also can directly infect by blade or root and the stem stalk systemic infection of delicate plant cause morbidity.Infect seedling stage and cause that wilting, death, later stage are infected and cause the plant dwarfing that occur canescence or yellow green striped on the blade, stripe edge waveform or irregular shape have black splotch sometimes, and be withered at last.

Enzyme linked immunological adsorption method (enzyme linked immunosorbent assay, ELISA) be to implement biological detection (to relate to and comprise human pathogen, the animals and plants pathogen, biotoxin, agricultural and veterinary chemicals is residual, protein, DNA etc. are multiple/class biosome and compound) one of the most effective and important means.In view of the high-affinity and the high specific of biotin-avidin reaction system, in detecting, ELISA earned widespread respect in recent years.At present, do not see as yet that both at home and abroad corn bacterial wilting germ ELISA detects the report of research, foundation is sensitive, stable corn bacterial wilting germ biotin-avidin ELISA detection method (biotin-avidin ELISA) is an urgent demand of corn quarantine.

Summary of the invention

The objective of the invention is to set up a kind of corn bacterial wilting germ biotin-avidin enzyme-linked immune detection method that satisfies specificity and high-sensitive polyclonal antibody, and develop kit, for detecting, corn bacterial wilting germ provides a kind of efficient, easy technological means.

A kind of anti-corn bacterial wilting germ polyclonal antibody, the tiring of described polyclonal antibody 10 ^-4More than; Described polyclonal antibody adopts indirect elisa method to measure its specificity, (ATCC 27822 for itself and 2 strain corn bacterial wilting germs, ATCC33114) performance kickback, and with other relevant 11 pathogenetic bacteria kinds or subspecies no cross reactions, be 1x10 for trying the bacterium cell concentration ^8-9Cfu/mL.

Anti-corn bacterial wilting germ Polyclonal Antibody Preparation method of the present invention, may further comprise the steps: (1) preparation immunogene: the corn bacterial wilting germ for examination is inoculated on the liquid NBY nutrient culture media, 28 ℃ of following constant-temperature shaking culture are collected thalline in the bacterial growth logarithmic phase; Centrifugal 10 minutes of 12000g removes supernatant, and thalline redissolves with sterile phosphate damping fluid (PBS), and it is outstanding again to add aseptic PBS repeatedly after the eccentric cleaning three times again, and the turbidimetry counting also decide lysosome concentration and is arrived 1x10 ⁸～1x10 ⁹Cfu/mL makes bacterium thalline suspension;

(2) animal immune: get above-mentioned bacteria suspension routinely immunization method gather serum, and preserve down in-20 ℃ after the packing after rabbit carries out immune operation for examination;

(3) antibody purification: from above-mentioned serum, filter out the highest antiserum of performance of tiring and (tire 10 ^-4More than) carry out purifying, antibody concentration is fixed molten to be 1.0mg/mL, promptly gets to resist corn bacterium wilting germ polyclonal antibody.

Anti-corn bacterial wilting germ Polyclonal Antibody Preparation method of the present invention, sero-fast purification process is ammonium sulfate precipitation method and Protein A post affinity chromatography in the described step (3).

A kind of corn bacterial wilting germ biotin-avidin enzyme-linked immune detection method, may further comprise the steps: above-mentioned polyclonal antibody is carried out biotin labeling, preparation sample extracting solution or bacteria suspension, on microwell plate, wrap then by sample extracting solution or bacteria suspension, add biotin labeling antibody, alkaline phosphatase mark Streptavidin and PNP substrate afterwards in succession, the absorbance of wavelength under 405nm read with microplate reader in the colour developing back.

As negative control, the corn bacterial wilting germ suspension carries out ELISA as positive control and detects wherein said detection method with healthy corn flour suspending liquid.The sample absorbance becomes positive correlation with its contained corn bacterial wilting germ content, adopts the criterion of P/N 〉=2.0 as positive findings.

Its detection sensitivity reaches 1x10 ⁶Cfu/mL.

Corn bacterial wilting germ biotin-avidin enzyme-linked immune detection method of the present invention, wherein said antibody carries out biotin labeled method: 1mL antibody (IgG) 4 ℃ of following dialysed overnight of CBS damping fluid of getting 1.0mg/mL, change damping fluid during this time 3 times, add the 100ul labelled reagent and at room temperature react 30min, add 100ul 1M Tris/HCl cessation reaction then, with 4 ℃ of following dialysed overnight of 0.85% NaCl damping fluid, during change damping fluid 3 times, obtain biotin labeled antibody; Wherein used labelled reagent be 1mg biotin and 1mL dimethyl formamide (DMF) or dimethyl sulfoxide (DMSO) (DMSO) fully dissolving obtain.

The preparation method of wherein said sample extracting solution is: get aseptic water washing 3 times of 30g corn kernel sample, soaked overnight in the 300ml carbonic acid buffer is ground fragmentation with seed then, and obtaining concentration is corn tissue's suspending liquid of 0.1g/mL; The preparation method of described bacteria suspension is: the freeze-dried powder of corn bacterial wilting germ and nutrient culture media is prepared into 1x10 with sterilized water ⁹The bacteria suspension of cfu/mL mixes isopyknic this bacteria suspension with above-mentioned corn tissue suspending liquid, form 5 * 10 ⁸The cfu/mL sample liquid is made into 1x10 respectively by doubling dilution again ⁸, 5 * 10 ⁷, 10 ⁷, 5 * 10 ⁶, 10 ⁶, 5 * 10 ⁵, 10 ⁵The bacteria suspension of cfu/mL.

A kind of corn bacterial wilting germ biotin-avidin enzyme-linked immunologic detecting kit of setting up according to described detection method can detect usefulness for 200 holes, makes it comprise following component:

A, detection antibody: with biotin labeled above-mentioned corn bacterial wilting germ polyclonal antibody;

Capacity: 0.125mL working concentration: 0.1mg/mL extension rate: 1:200

Antibody liquid content: 0.5ug/mL antiseptic: 0.05% NaN ₃Storage: 4 ℃ following 6 months

B, enzyme mark Streptavidin compound;

Capacity: 0.05mL (contains 0.05% NaN ₃) extension rate: 1:500 storage: 4 ℃ are following 6 months

After C, 10 times of PBST concentrate: 1:10 dilute, be used for dilution and detect antibody, enzyme mark Streptavidin and detersive enzyme target;

Specification: 60mL/ bottle storage: room temperature

After D, 10 times of carbonic acid buffers (CBS): the 1:10 dilution, be used for sample extraction and preparation confining liquid;

Specification: 60mL/ bottle storage: room temperature

E, confining liquid: with 0.5% (w/v) BSA or 2% (w/v) skimmed milk power of 1 times of CBS preparation;

F, substrate: para-nitro-pheneye phosphate (PNPP) tablet: 5mg/ sheet;

After G, 5 times of substrate buffer solution: 1:5 dilute, be used for preparation colour developing liquid, pH9.8;

Component: 0.5mol/L diethanolamine buffer

Specification: 10mL/ bottle storage: room temperature

H, colour developing liquid: with the PNPP solution of substrate buffer solution preparation, 1mg/mL, now with the current;

I, stop buffer: 2M NaOH solution;

Specification: 50mL/ bottle storage: room temperature

J, positive control: the freeze-dried powder of corn bacterial wilting germ and nutrient culture media;

K, negative control: healthy corn kernel powder.

Corn bacterial wilting germ biotin-avidin enzyme-linked immune detection method provided by the invention and detection kit thereof, wherein Polyclonal Antibody Preparation is to obtain with pathogenetic bacteria injecting immune White Rabbit and through screening, antibody titer is 10 ^-4(1:10000); Antibody and corn bacterial wilting germ have specific reaction, and with for the examination other pathogenetic bacteria kind/subspecies no cross reactions; Carry out biotin labeling behind the antibody purification, set up biotin-avidin indirect ELISA (BA-ELISA), detection sensitivity reaches 1x10 ⁶Cfu/mL.Now common alkaline phosphatase enzyme labelled antibody formula indirect ELISA adopts polyclonal antibody directly as detecting antibody or using polyclonal antibody through alkali phosphatase enzyme mark as detecting antibody, add general alkali phosphorus enzyme mark goat anti-rabbit igg and/or the colour developing of PNP substrate again, improved 5-10 doubly and the detection sensitivity of indirect BA-ELISA provided by the invention compares.

Embodiment

For further specifying the present invention, specifically set forth in conjunction with following example:

1. immunogen preparing

Be inoculated on the liquid NBY nutrient culture media for the examination corn bacterial wilting germ, 28 ℃ of following constant-temperature shaking culture are collected thalline in the bacterial growth logarithmic phase, under the 12000g centrifugal 10 minutes, remove supernatant; The precipitation thalline uses sterile phosphate damping fluid (PBS) to redissolve again, and it is outstanding again to add aseptic PBS repeatedly after the eccentric cleaning three times again, and the turbidimetry counting also decide lysosome concentration and is arrived 1x10 ^8-9Cfu/mL.

The NBY nutrient culture media:

Beef broth 8g

Yeast extract 2g

K ₂HPO ₄ 2g

KH ₂PO ₄， 0.5g

Glucose 5g

MgSO _4·7H ₂O 0.25g

Distilled water (ddH ₂O) 1000mL

PBS damping fluid: 0.01mol/L phosphate buffer, pH7.4

NaCl 8.0g

KCl 0.2g

KH ₂PO ₄ 0.2g

Na ₂HPO ₄·12H ₂O 2.9g

Distilled water (ddH ₂O) 1000mL

2. animal immune

Get 3mL bacteria suspension (1x10 ^8-9Cfu/mL) mixed with isopyknic Freund's complete adjuvant, to carrying out the subcutaneous multi-point injection in back, carry out the ear edge vein exploitating blood first time after two weeks after the emulsification for the examination rabbit.Carry out injecting immune second time (method is same) after taking a blood sample three days for the first time, two to exempt from be to use isopyknic incomplete Freund, the same subcutaneous multi-point injection in back that carries out after the emulsification.Carry out the ear edge vein exploitating blood second time after two weeks, carry out for the third time immunity after three days, three exempt from identical with two schemes of exempting from; Two weeks back execution animal is gathered whole serum, and preserves down in-20 ℃ after the packing again.

3. antibody purification

1) ammonium sulfate precipitation method

Get the standby antiserum of 2mL, the centrifugal 30min of 10000 * g under 4 ℃ keeps supernatant; In serum, slowly add isopyknic 3.9M saturated ammonium sulfate solution while stirring.Solution stirs down at 4 ℃ and spends the night, so that protein fully precipitates.The antiserum suspension is the same centrifugal, and abandoning supernatant keeps precipitation.Sediment redissolves with a small amount of 1xPBS, puts into bag filter then to the PBS dialysed overnight, changes a dislysate every about 6h.Collect the antibody of preliminary purification, wait to be further purified standby.

2) affinity chromatography

Protein A post (specification: 1mL), crosslinked damping fluid (MAPS II binding buffer) and elution buffer (MAPSII elute buffer), all available from Bio-Rad company.

A. balance cylinder: with 0.01mol/L PBS wash-out pillar until baseline;

B. go up sample absorption: above antibody is slowly added the post bed;

C. wash-out foreign protein: with the MAPS II binding buffer flushing chromatographic column of about 5 times of column volumes;

D. desorption and collection: with the MAPS II elute buffer wash-out antibody of 5 times of column volumes, collect on wash-out limit, limit;

E. the cleaning of post and preservation: with about 5-7 times column volume 50% washed with methanol (flow velocity: 3ml/min), with the 0.1N NaOH flushing of about 8 times of column volumes, also be kept at last in 20% ethanol for 2-3 time with deionized water and 20% alcohol flushing then again.

4. antibody cross reaction is measured

The immunoreactivity that adopts common indirect elisa method to test anti-corn bacterial wilting germ polyclonal antibody, result show that this antibody is strong to target corn wilting bacterial reaction, and to other the 11 kinds/subspecies pathogenetic bacteria no cross reaction for examination.

For the examination pathogenetic bacteria: 1. corn bacterial wilting bacterium Clavibacter michiganensis subsp.nebraskensis (No.27822, No.33114, from the ATCC of American Type Culture Collecti), 2. corn bacterial stem rot germ Erwiniacarotovora subsp.carotovora (No.12104, the Chinese Academy of Agricultural Sciences Institute of Plant Protection), 3. corn bacterial brown patch germ Pseudomonas syringae pv.syringae (No.12056, the Chinese Academy of Agricultural Sciences Institute of Plant Protection), 4. corn bacterial leaf blight bacterium Acidovorax avenae subsp.avenae (No.1.1726, China common micro-organisms preservation center), 5. corn bacterial leaf spot fungi Xanthomonas campestris pv.holcicola (No.1.1510, China common micro-organisms preservation center), 6. corn bacterial wilt Pantoea stewartii subsp.stewartti (No.0085, from plant protection system of Agricultural University Of Nanjing), 7. clover bacterial wilting germ Clavibacter michiganensis subsp.insidiosus (No.1.1908, China common micro-organisms preservation center), 8. bacterial canker of tomato Clavibacter michiganensis subsp.michiganensis (No.12103, the Chinese Academy of Agricultural Sciences Institute of Plant Protection), 9. bacterial ring rot o potato bacterium Clavibacter michiganensis subsp.sepedonicus (No.12057, the Chinese Academy of Agricultural Sciences Institute of Plant Protection), 10. wheat clavate bacillus Clavibacter fangii (No.1.1909,1.1999,1.2000, Chinese common micro-organisms preservation center).

The indirect ELISA operation steps:

1) thalline bag quilt: fixed molten with the dilution of CBS damping fluid to 1x10 for the examination pathogenetic bacteria ^8-9Cfu/mL, every hole 100uL joins on the ELISA Plate, and each bacterial strain repeats 3 holes, and the bag quilt spends the night under 4 ℃;

2) sealing: discard the reacting hole supernatant, PBST washes 3 times (3min/ time), and every hole adds 200 μ L, 2% BSA (bovine serum albumin, bovine serum albumin), and 37 ℃ are sealed 1h down;

3) detect antibody: the same processing and flushing, every hole add 100 μ L corn wilting bacterial antibodies, and working concentration is 1.0ug/mL, and 37 ℃ are reacted 1h down;

4) enzyme labelled antibody: the same processing and flushing, add alkaline phosphatase oxidase mark goat anti-rabbit igg (Sigma company), doubly to dilute by 1:5000, every hole adds 100 μ L, and 37 ℃ are reacted 1h down;

5) colour developing: the same processing and flushing, every hole add 100 μ LPNP colour developing liquid, react 30min under the room temperature, with microplate reader read the absorbance of wavelength under 405nm (optical density, OD);

6) criterion: test bacterium OD value/object bacteria OD value, be defined as no cross reaction less than 10%, greater than and equal 10% and shown as weak cross reaction; Can be defined as cross reaction (antibody can not be used this test bacterium) greater than 30%.

CBS damping fluid: 0.05mol/L carbonate buffer solution, pH9.6

Na ₂CO ₃ 10g

NaHCO ₃ 10g

ddH ₂O 1000mL

PBST washing fluid: 1 times of phosphate buffer, pH7.4

NaCl 8.0g

KCl 0.2g

KH ₂PO ₄ 0.2g

Na ₂HPO ₄12H ₂O 2.9g

Polysorbas20 1mL

NaN ₃ 0.2g

Distilled water (ddH ₂O) 1000mL

5. antibody biotin labeling

1) prepares: biotin X-NHS-Biotin (Sigma company)

Solvent: dimethyl formamide (dimethylformamide, DMF, HCON (CH ₃) ₂),

Perhaps dimethyl sulfoxide (DMSO) (dimethylsulfoxide, DMSO, (CH ₃) ₂SO)

Labelled reagent: take by weighing 1mg biotin and 1mL DMF or DMSO and fully dissolve

Crosslinked damping fluid: 10g NaCl, 10g NaHCO ₃, 1000mL H ₂O, pH7.5

1M Tris/HCl:121g Tris+1000ml H ₂O transfers pH7.4 with 2N HCl

Salt solution (saline): 0.85% NaCl

2) mark: get 1mL IgG (1mg/mL) (4 ℃ of dialysed overnight in crosslinked damping fluid, change damping fluid during this time 3 times), add the 100ul labelled reagent then and react 30min down in room temperature (RT), add 100ul 1M Tris/HCl cessation reaction again, (4 ℃ of dialysed overnight in the saline damping fluid at last, change damping fluid during this time 3 times), packing is preserved.

6. sample detection

Get healthy corn kernel aseptic water washing 3 times of 30g, soaked overnight in the 300ml carbonic acid buffer is ground fragmentation with seed then, and finally obtaining concentration is corn tissue's suspending liquid of 1mg/mL.With isopyknic bacteria suspension (1x10 ⁹Cfu/mL) mix with corn tissue suspending liquid, form 5 * 10 ⁸The cfu/mL sample liquid is made into 1x10 respectively by doubling dilution again ⁸, 5 * 10 ⁷, 10 ⁷, 5 * 10 ⁶, 10 ⁶, 5 * 10 ⁵, 10 ⁵The bacteria suspension of cfu/mL.As negative control, the wilting germ suspension carries out ELISA as positive control and detects with healthy corn kernel extract.The result shows that the lowest detectable limit of this BA-ELISA reaches 1 * 10 ⁶Cfu/mL.

Indirect BA-ELISA operation steps:

1) thalline/sample bag quilt: above thallus suspension liquid is added ELISA Plate, the 100uL/ hole, the bag quilt spends the night under 4 ℃;

2) sealing: discard the reacting hole supernatant, PBST washes 3 times (3min/ time), and every hole adds 200 μ L, 2% BSA (bovine serum albumin, bovine serum albumin), and 37 ℃ are sealed 2h down;

3) biotin labeling antibody: after the same processing and the flushing, add biotin labeling antibody (1:500 doubly dilutes), every hole 100 μ L, 37 ℃ are reacted 1h down;

4) enzyme mark Avidin: the same processing and flushing, every hole add 100 μ L alkaline phosphatase mark-Streptavidins (1:5000 doubly dilutes), and 37 ℃ are reacted 1h down;

5) colour developing: the same processing and flushing, every hole add 100 μ L PNPP colour developing liquid, react 30min under the room temperature, with microplate reader read wavelength under 405nm absorbance (optical density, OD).

7. detect the establishment of corn bacterial wilting germ biotin-avidin enzyme linked immunological kit

Set up the corn bacterial wilting germ enzyme-linked immunologic detecting kit, can detect usefulness, make it comprise following component for 200 holes:

A detection antibody (detection antibody, biotin-antibody)

With biotin labeled corn bacterial wilting germ polyclonal antibody

Capacity: 0.125mL antibody liquid content: 0.5ug/mL extension rate: 1:200

Working concentration: 0.1mg/mL antiseptic: 0.05% NaN ₃Storage: 4 ℃ following 6 months

B alkali phosphatase enzyme mark Streptavidin compound (streptavidin-AP conjugate)

Capacity: 0.05mL (contains 0.05% NaN ₃) extension rate: 1:500 storage: 4 ℃ are following 6 months

10 times of PBST concentrates of C (after the 1:10 dilution, be used for dilution and detect antibody, enzyme mark Streptavidin, detersive enzyme target)

Specification: 60mL/ bottle storage: room temperature

10 times of carbonic acid buffers of D (CBS after the 1:10 dilution, is used for sample extraction and preparation confining liquid)

Specification: 60mL/ bottle storage: room temperature

E confining liquid: with the 0.5%BSA or 2% skimmed milk power (wt/vol) of CBS preparation

F substrate: PNPP tablet (p-Nitrophenylphosphat, para-nitro-pheneye phosphate, 5mg/ sheet)

5 times of substrate buffer solutions of G (after the 1:5 dilution, are used for preparation colour developing liquid, pH9.8)

Component: 0.5mol/L diethanolamine buffer

Specification: 10mL/ bottle storage: room temperature

The H liquid that develops the color: with the PNPP solution of substrate buffer solution preparation, 1mg/mL (w/v, now with the current)

1 stop buffer (2M NaOH solution)

Specification: 50mL/ bottle storage: room temperature

The J positive control: the freeze-dried powder of corn bacterial wilting germ and nutrient culture media adds the CBS damping fluid and dissolves standby during use

The K negative control: healthy corn flour, extract with the method sample, it is standby to get supernatant after centrifugal.

The % that relates among the present invention if no special instructions outside, all refer to percentage by weight.

Above-described embodiment is described preferred implementation of the present invention; be not that scope of the present invention is limited; design under the prerequisite of spirit not breaking away from the present invention; various distortion and improvement that the common engineering technical personnel in this area make technical scheme of the present invention all should fall in the definite protection domain of claims of the present invention.

Claims (9)

1, a kind of anti-corn bacterial wilting germ polyclonal antibody, it is characterized in that: described polyclonal antibody is tired at 1 x 10 ^-4More than; The specificity of described polyclonal antibody, itself and 2 strain corn bacterial wilting germs (ATCC 27822, and ATCC 33114) performance kickback, and with other relevant 11 pathogenetic bacteria kinds or subspecies no cross reactions.

2, the described anti-corn bacterial wilting germ preparation method of polyclonal antibody of claim 1 is characterized in that: may further comprise the steps:

(1) preparation immunogene: corn bacterial wilting germ bacterial strain (from the ATCC of American Type Culture Collecti) is inoculated on the liquid NBY nutrient culture media, 28 ℃ of following constant-temperature shaking culture; Collect thalline in the bacterial growth logarithmic phase, centrifugal 10 minutes of 12000g removes supernatant, and thalline redissolves with sterile phosphate damping fluid (PBS), and it is outstanding again to add aseptic PBS repeatedly after the eccentric cleaning three times again; With turbidimetry counting and fixed molten bacterium cell concentration to 1 x 10 ⁸～1 x 10 ⁹Cfu/mL;

(2) animal immune: immunization method is to carrying out immune operation for the examination rabbit routinely to get above-mentioned bacteria suspension, and preservation is standby down regularly to gather serum, and be placed in after packing-20 ℃;

(3) antibody purification: from above-mentioned serum, filter out the highest antiserum of performance of tiring and (tire 10 ^-4More than) carry out purifying, concentration is fixed molten to be 1.0mg/mL, promptly gets to resist the corn bacterial wilting germ polyclonal antibody.

3, anti-corn bacterial wilting germ preparation method of polyclonal antibody according to claim 2 is characterized in that: sero-fast purification process is ammonium sulfate precipitation method and Protein A post affinity chromatography in the described step (3).

4, a kind of corn bacterial wilting germ biotin-avidin enzyme-linked immune detection method is characterized in that: the described polyclonal antibody of claim 1 is carried out biotin labeling; Preparation sample extracting solution or bacterial suspension, and be coated on the microwell plate; Add biotin labeling antibody, alkaline phosphatase mark Streptavidin and PNP substrate afterwards in succession, the absorbance of wavelength under 405nm read with microplate reader in the colour developing back.

5, corn bacterial wilting germ biotin-avidin enzyme-linked immune detection method according to claim 4, set up feminine gender and positive control separately, it is characterized in that: as negative control, the corn bacterial wilting germ suspension carries out ELISA as positive control and detects with healthy corn tissue suspending liquid.

6, corn bacterial wilting germ biotin-avidin enzyme-linked immune detection method according to claim 5 is characterized in that: its detection sensitivity reaches 1 x 10 ⁶Cfu/mL.

7, corn bacterial wilting germ biotin-avidin enzyme-linked immune detection method according to claim 6, it is characterized in that: described antibody carries out biotin labeled method and is: get 1mL antibody (IgG, 1.0mg/mL) with 4 ℃ of following dialysed overnight of crosslinked damping fluid, during change damping fluid 3 times; Add the 100ul labelled reagent and at room temperature react 30min, add the 100ul1MTris/HCl cessation reaction; Use 4 ℃ of following dialysed overnight of 0.85%NaCl damping fluid then, during change damping fluid 3 times, obtain the good biotin antibody of mark; Wherein used labelled reagent be 1mg biotin and 1mL dimethyl formamide (DMF) or dimethyl sulfoxide (DMSO) (DMSO) fully dissolving obtain.

8, corn bacterial wilting germ biotin-avidin enzyme-linked immune detection method according to claim 7, it is characterized in that: the preparation method of described sample extracting solution is: get aseptic water washing 3 times of 30g corn kernel sample, soaked overnight in 300ml carbonic acid buffer (CBS), then seed is ground fragmentation, obtaining concentration is corn tissue's suspending liquid of 0.1g/mL; The preparation method of described bacteria suspension is: the freeze-dried powder of corn bacterial wilting germ and nutrient culture media is prepared into 1 x 10 with sterilized water ^8-9The bacteria suspension of cfu/mL mixes isopyknic this bacteria suspension with above-mentioned corn tissue suspending liquid, form 5 * 10 ⁸The cfu/mL sample liquid is made into 1 x 10 respectively by doubling dilution again ⁸, 5 * 10 ⁷, 10 ⁷, 5 * 10 ⁶, 10 ⁶, 5 * 10 ⁵, 10 ⁵The bacteria suspension of cfu/mL.

9, a kind of corn bacterial wilting germ biotin-avidin enzyme-linked immunologic detecting kit of detection method establishment according to claim 4 is characterized in that: detect usefulness for 200 holes, make it comprise following component:

A, detection antibody: claim 1 is described through biotin labeled corn bacterial wilting germ polyclonal antibody;

Capacity: 0.125mL working concentration: 0.1mg/mL extension rate: 1:200

Content: 0.5ug/mL antiseptic: 0.05%NaN ₃Storage: 4 ℃ following 6 months

B, enzyme mark Streptavidin compound;

Capacity: 0.05mL (contains 0.05%NaN ₃) extension rate: 1:500 storage: 4 ℃ are following 6 months

After C, 10 times of PBST concentrate: 1:10 dilute, be used for dilution and detect antibody, enzyme mark Streptavidin and detersive enzyme target;

Specification: 60mL/ bottle storage: room temperature

After D, 10 times of carbonate buffer solutions (CBS): the 1:10 dilution, be used for the preparation of sample extraction and confining liquid;

Specification: 60mL/ bottle storage: room temperature

E, confining liquid: with 0.5% (w/v) BSA or 2% (w/v) skimmed milk power of CBS preparation;

F, substrate: para-nitro-pheneye phosphate (PNPP) tablet: 5mg/ sheet;

After G, 5 times of substrate buffer solution: 1:5 dilute, be used for preparation colour developing liquid, pH9.8;

Component: 0.5mol/L diethanolamine buffer

Specification: 10mL/ bottle storage: room temperature

H, colour developing liquid: with the PNPP solution of substrate buffer solution preparation, 1mg/mL, now with the current;

I, stop buffer: 2M NaOH solution;

Specification: 50mL/ bottle storage: room temperature

J, positive control: the freeze-dried powder of corn bacterial wilting germ and nutrient culture media;

K, negative control: healthy corn kernel powder.