CN106596935A - Preparation method for multiplex immunocolloidal gold test strip used for apple latent viruses - Google Patents

Abstract

The invention discloses a preparation method for a multiplex immunocolloidal gold test strip used for apple latent viruses. Compared with the prior art, the invention provides a novel means for rapid, specific and simple detection of main apple latent viruses. Research achievements in the invention allow the molecular variation, invasion prevalence and virulence status of latent viruses of apples produced in Shanxi Province to be determined; and the multiplex immunocolloidal gold test strip developed on the basis of the research achievements can accurately and rapidly diagnose whether a nursery stock or a rootstock carries a virus, so propagation and prevalence of a disease caused by the virus can be effectively controlled, and the output and quality of produced apples can be improved.

Description

The preparation method of apple latent virus Multiple immunizations colloidal gold colloidal gold detection test paper strip

Technical field

The present invention relates to a kind of research of plant disease method, more particularly to a kind of apple latent virus Multiple immunizations gold colloidal The preparation method of test strip.

Background technology

Apple chlorotic leaf spot virus (Apple chlorotic leaf spot virus, ACLSV), apple stem pitting virus (Apple stem pitting virus, ASPV) and apple stem grooving virus (Apple stem grooving virus, ASGV) Impact to apple production and quality is extremely serious, and this 3 class cryptovirus, distributed areas are extremely wide, produces consequence serious.Using biography System serology, biology and molecular biology etc. these viruses of technology for detection, complex operation, professional technical is strong, needs specialty Instrument and equipment, it is impossible to meet land for growing field crops popularization and application.

The content of the invention

The purpose of the present invention is that and provide a kind of apple latent virus Multiple immunizations colloid in order to solve the above problems The preparation method of golden test strip.

The present invention is achieved through the following technical solutions above-mentioned purpose：

The present invention is comprised the following steps：

(1) vessel cleaning：

All glass drying ovens wash clean under flowing water, is placed in acid solution (potassium dichromate 100g, concentrated sulphuric acid 50ml, plus distilled water is to 1000ml) immersion 24h, washed away repeatedly with tap water, then using distilled water immersion 24h, use ultra-pure water Washing three times, reuses ultra-pure water immersion 72h, standby after being dried in baking box；Must keep in reagent preparation strict Pure, all reagents will use ddH₂0 prepares；

(2) prepared by gold colloidal：

1% aqueous solution of chloraurate 1ml, plus 99ml ultra-pure waters are taken, boiling is heated to, is stirred while disposable fast drop 1% trisodium citrate aqueous solution of the fresh configurations of 1.8ml, solution colour from Huang gradually switch to it is purplish red, then in boiling water heating 3～ 5 minutes, room temperature, plus ultra-pure water are cooled to original volume, save backup under 4 DEG C of environment, it is to avoid illumination；

(3) preparation of gold labeling antibody：

4 DEG C of dialysed overnights in NaCl solution by antibody to be marked in advance 0.005mol/lpH7.0, it is unnecessary so as to remove Salt ion, 10000rpm4 DEG C be centrifuged 30 minutes, remove polymer；

Determine antibody optimum dose to be marked：With the K of 0.1mol/l₂CO₃Solution adjusts the pH value of colloidal gold solution to 8.5, point Fill 10 pipes, often pipe 1ml, respectively the μ l of Deca 2,4 μ l, 6 μ l, 8 μ l, 10 μ l, 20 μ l antibody, be sufficiently mixed it is uniform after every pipe Deca NaCl solution 0.1ml of 10% (m/v)；Separately set the NaCl solution for only adding 0.1ml10% in control tube, i.e. 1ml gold colloidals, mixing Place 2 hours after uniform, observe result；With the K of 0.1mol/l₂CO₃The pH value of colloidal gold solution is adjusted to into 8.5；Agitation Under, the antibody of Deca optimum mark content into aurosol is uniformly stirred 2～3 minutes；The BSA of Deca 10% as stabilizer, Make its final concentration of 1%, continue stir 15 minutes, 4 DEG C save backup；

Gold labeling antibody solution is centrifuged into 10 minutes so as to remove colloid metal/polymer therein in 1200rpm, 4 DEG C, is taken Clear liquid, then be centrifuged 50 minutes with 14000rpm, 4 DEG C, it is seen that centrifugation thing is divided into 3 layers, anti-containing what is be also not bound with supernatant Body, the colloid gold particle polymer being not bound with forms atrouss speckle in the bottom, and middle part aubergine flocculent deposit is good With reference to gold colloidal-antibody complex；Supernatant discarded, aubergine flocculent deposit be dissolved in 1/10 original volume containing 1%BSA's In PBS (0.01mol/l, pH8.2), 4 DEG C save backup；

(4) assembling of test strips：

The glass fibre membrane for cutting is put in the PBS of the 0.01mol/l containing 1%BSA and closes 1h, treat that nature dries Afterwards, by uniform Deca after the different gold labeling antibody equal-volumes for suitably diluting mixing in glass fibre element film up to saturation, 37 DEG C Dry for standby in baking oven；

Take the antibody and goat anti-rabbit antibody of purified mistake respectively carries out certain dilution with PBST buffer, respectively in nitre Deca sample after room temperature is dried, in being placed on lock solution, carries out 1h and incubates on T lines and C lines in 37 DEG C of environment on acid cellulose film Change and cultivate, washed 2 times with PBST buffer, 5 minutes every time, in normal domestic room temp preservation is dried；

Antibody solid phase nitrocellulose filter, filter paper, gold labeling antibody combine release pad, sample pad and are positioned on PVC board in order On, the overlap of 1～2mm is stayed each other, firmly compress, it is to avoid produce bubble；4 DEG C of lucifuge kept dry；

(5) ELISA test strip：

The Apple Leaves of the RT-PCR that learns from else's experience authenticated infection virus, during clean mortar is placed on after weighing, according to 1:10(m/ V) ratio Deca PBST, is ground and becomes homogenate, and 2000rpm is centrifuged 10 minutes, takes supernatant and use respectively as sample Test strips are checked, then same amount of sample mix homogeneously, by test strips inspection.

The beneficial effects of the present invention is：

The present invention is a kind of preparation method of apple latent virus Multiple immunizations colloidal gold colloidal gold detection test paper strip, with prior art Compare, it is contemplated that initiative is a kind of can be to the brand-new handss of quick, special, the easy main cryptovirus of detection Fructus Mali pumilae Section.Occurrence injury, molecular variant and genome structure first to the 3 kinds of latent virus in Shanxi the major apple production area carries out system and grinds Study carefully；Next to that obtaining coat protein (coat protein, the CP) gene of 3 kinds of cryptoviruses using RT-PCR technology, will pass through Purpose fragment is obtained after enzyme action it is inserted in carrier to build prokaryotic expression carrier, then converts escherichia coli, and using IPTG handss Section abduction delivering coat protein.It is the experimental rabbit of antigen immune to 3 kinds of virus capsid proteins of prokaryotic expression, produces used Antiserum, and purify immunoglobulin, association colloid gold labelling technique and immunochromatography principle finally develop quick inspection The latent virus multiple immunity colloidal gold test paper strip of 3 kinds of Fructus Mali pumilae.The project achievement in research not only can specify Shanxi Province's Fructus Mali pumilae and dive The molecular variant of cryptovirus, prevalence, virulence situation are infected, and the Multiple immunizations colloidal gold strip based on above-mentioned development can be accurate Really, whether band is malicious quickly to diagnose nursery stock, stock, by the spread and epidemic of such disease initiation viral disease of effective control, improves Fructus Mali pumilae life Produce quantity and product quality.

Description of the drawings

RT-PCR testing results of Fig. 1 ASPV in 11, Shanxi Fructus Mali pumilae producing region；

RT-PCR testing results of Fig. 2 ACLSV in 11, Shanxi Main Apple Varieties producing region；

RT-PCR testing results of Fig. 3 ASGV in 11, Shanxi Main Apple Varieties producing region；

The Apple Leaves total serum IgE that Fig. 4 is extracted；

Fig. 5 is the expression of SDS-PAGE electrophoresis detection BL-PE-ASGV-CP albumen；

Fig. 6 is SDS-PAGE electrophoresis detection ASPV-CP protein expressions；

Fig. 7 is the expression of SDS-PAGE electrophoresis detection ACLSC-CP；

Fig. 8 is SDS-PAGE detection ACLSV-CP, ASPV-CP and ASGV-CP purification effect figures；

Fig. 9 is specific detection result figure.

In Fig. 5：1：Without the sample that IPTG is induced；2-4：Respectively Jing IPTG induce the sample of 2h, 4h, 6h；M：Albumen Marker；

In Fig. 6：1：Without the sample that IPTG is induced；2-4：Respectively Jing IPTG induce the sample of 2h, 4h, 6h；M：Albumen Marker；

In Fig. 7：Without the sample that IPTG is induced；2-4：Respectively Jing IPTG induce the sample of 2h, 4h, 6h；M：Albumen Marker；

In Fig. 8：1-3：The non-induced samples of ACLSV-CP, after purification induced samples, sample；4-6：ASPV-CP does not induce sample Product, after purification induced samples, sample；7-9：The non-induced samples of ASGV-CP, after purification induced samples, sample；M：Albumen Marker；

Specific embodiment

Below in conjunction with the accompanying drawings the invention will be further described：

The present invention is comprised the following steps：

(1) vessel cleaning：

All glass drying ovens wash clean under flowing water, is placed in acid solution (potassium dichromate 100g, concentrated sulphuric acid 50ml, plus distilled water is to 1000ml) immersion 24h, washed away repeatedly with tap water, then using distilled water immersion 24h, use ultra-pure water Washing three times, reuses ultra-pure water immersion 72h, standby after being dried in baking box；Must keep in reagent preparation strict Pure, all reagents will use ddH₂0 prepares；

(2) prepared by gold colloidal：

1% aqueous solution of chloraurate 1ml, plus 99ml ultra-pure waters are taken, boiling is heated to, is stirred while disposable fast drop 1% trisodium citrate aqueous solution of the fresh configurations of 1.8ml, solution colour from Huang gradually switch to it is purplish red, then in boiling water heating 3～ 5 minutes, room temperature, plus ultra-pure water are cooled to original volume, save backup under 4 DEG C of environment, it is to avoid illumination；

(3) preparation of gold labeling antibody：

4 DEG C of dialysed overnights in NaCl solution by antibody to be marked in advance 0.005mol/lpH7.0, it is unnecessary so as to remove Salt ion, 10000rpm4 DEG C be centrifuged 30 minutes, remove polymer；

Determine antibody optimum dose to be marked：With the K of 0.1mol/l₂CO₃Solution adjusts the pH value of colloidal gold solution to 8.5, point Fill 10 pipes, often pipe 1ml, respectively the μ l of Deca 2,4 μ l, 6 μ l, 8 μ l, 10 μ l, 20 μ l antibody, be sufficiently mixed it is uniform after every pipe Deca NaCl solution 0.1ml of 10% (m/v)；Separately set the NaCl solution for only adding 0.1ml10% in control tube, i.e. 1ml gold colloidals, mixing Place 2 hours after uniform, observe result；With the K of 0.1mol/l₂CO₃The pH value of colloidal gold solution is adjusted to into 8.5；Agitation Under, the antibody of Deca optimum mark content into aurosol is uniformly stirred 2～3 minutes；The BSA of Deca 10% as stabilizer, Make its final concentration of 1%, continue stir 15 minutes, 4 DEG C save backup；

Gold labeling antibody solution is centrifuged into 10 minutes so as to remove colloid metal/polymer therein in 1200rpm, 4 DEG C, is taken Clear liquid, then be centrifuged 50 minutes with 14000rpm, 4 DEG C, it is seen that centrifugation thing is divided into 3 layers, anti-containing what is be also not bound with supernatant Body, the colloid gold particle polymer being not bound with forms atrouss speckle in the bottom, and middle part aubergine flocculent deposit is good With reference to gold colloidal-antibody complex；Supernatant discarded, aubergine flocculent deposit be dissolved in 1/10 original volume containing 1%BSA's In PBS (0.01mol/l, pH8.2), 4 DEG C save backup；

(4) assembling of test strips：

The glass fibre membrane for cutting is put in the PBS of the 0.01mol/l containing 1%BSA and closes 1h, treat that nature dries Afterwards, by uniform Deca after the different gold labeling antibody equal-volumes for suitably diluting mixing in glass fibre element film up to saturation, 37 DEG C Dry for standby in baking oven；

Take the antibody and goat anti-rabbit antibody of purified mistake respectively carries out certain dilution with PBST buffer, respectively in nitre Deca sample after room temperature is dried, in being placed on lock solution, carries out 1h and incubates on T lines and C lines in 37 DEG C of environment on acid cellulose film Change and cultivate, washed 2 times with PBST buffer, 5 minutes every time, in normal domestic room temp preservation is dried；

Antibody solid phase nitrocellulose filter, filter paper, gold labeling antibody combine release pad, sample pad and are positioned on PVC board in order On, the overlap of 1～2mm is stayed each other, firmly compress, it is to avoid produce bubble；4 DEG C of lucifuge kept dry；

(5) ELISA test strip：

The Apple Leaves of the RT-PCR that learns from else's experience authenticated infection virus, during clean mortar is placed on after weighing, according to 1:10(m/ V) ratio Deca PBST, is ground and becomes homogenate, and 2000rpm is centrifuged 10 minutes, takes supernatant and use respectively as sample Test strips are checked, then same amount of sample mix homogeneously, by test strips inspection.

Research material and method

Sample collecting

4-the June of 2015 to 2016, carry out sample collecting.Specifically acquisition method is：A kind of each regional age of tree 4 orchards of collection, each area gathers altogether 12 orchards, and each orchard randomly selects 4 apple trees, random on every apple tree A branch is chosen, whole blades are plucked, code insurance is divided into two parts and is existed in plastic bag, the age of tree sampled well with marking pen labelling, Place and time, -80 DEG C are stored in, portion is used for Serologic detection, and portion is used for molecule experiments.

Serologic detection

Serum is taken using double antibody sandwich method (DAS-ELISA) virus (ACLSV/ASPV/ASGV) latent to three classes Learn inspection.Wherein, the ELSIA test kits of ACLSV and ASGV are bought from An Dezhen biotechnology (Beijing) company limited, ASPV's ELISA kit is bought from ACD companies of the U.S..

DAS-ELISA need reagent and the formula used

(1) coated antibody：Using the coated antibody of the corresponding virus provided in test kit.

(2) enzyme labelled antibody：Using the enzyme labelled antibody of the corresponding virus provided in test kit.

(3) substrate：4-NPP

(4) it is coated with buffer (50ml)：Sodium carbonate Na₂CO₃, 0.075g；Sodium bicarbonate NaCHO₃, 0.1465g；Distilled water It is settled to 50ml, 4 DEG C of storages.

(5) sample extraction buffer：Sodium sulfite Na₂SO₃, 1.3g；Polyvinylpyrrolidone PVP, 20g；It is dissolved in 1000ml In lavation buffer solution (PBST).

(6) lavation buffer solution (PBST)：Sodium chloride nacl, 8.0g；Disodium hydrogen phosphate (Shi Ershui) Na₂HPO₄·12H₂O, 2.9g；Potassium dihydrogen phosphate KH₂PO₄, 0.2g；Potassium chloride (KCl), 0.2g；Tween20,0.5ml；In being dissolved in 1000ml distilled water.

(7) enzyme mark dilution buffer：Deca bovine serum albumin (BSA) 0.1g in the PBST of 50ml；PVP 1.0g.

(8) substrate buffer solution：By in the distilled water of 0.005g 4-NPP Deca 40ml, pH is adjusted to arrive using HCl 9.8, distilled water constant volume to 50ml is deposited under 4 DEG C of environment.

DAS-ELISA are detected

(1) coated antibody

According to as directed, diluted with coating buffer, in being added drop-wise to elisa plate aperture, 100 holes of μ l mono-, plus upper cover, Carry out 2 hours being incubated under 37 DEG C of environment, empty solution in hole, 3 washings are carried out using PBST, wash 3 minutes each time.

(2) sample preparation

According to 1：1 (weight：Volume) testing sample is added the buffer of extracting, sample is ground until into slurry Liquid, 2000rpm is centrifuged 10 minutes, and supernatant is the detection sample being successfully prepared.With adaptable processing method or according to saying Bright book carries out feminine gender/positive control.

(3) it is loaded

Detection sample that Deca is successfully prepared, feminine gender (with the positive) are compareed.Per the μ l of hole Deca 100, close the lid, be placed on 4 Cultivate in DEG C refrigerator, and thoroughly cleaning is clean elisa plate, then with distilled water flushing 3 times, carry out 1 time by PBST and rinse, it is each It is secondary to carry out 3 minutes.

(4) enzyme labelled antibody is added

Deca has diluted enzyme labelled antibody in elisa plate, per the μ l of hole Deca 100, plus upper cover, 2 are carried out under 37 DEG C of environment Hour or be incubated within 4 hours, with tap water cleaning down elisa plate, then wash with distilled water and PBST 1 time and 3 times respectively, often Once washing 3 minutes.

(5) substrate is added

The substrate solution of gained is dripped in substrate buffer solution so that final concentration becomes 1mg/ml and (notes：Should now with existing With), when weighing substrate and dissolving substrate with substrate buffer solution, lucifuge as far as possible.According to the holes of 100 μ l mono-, in being added drop-wise to elisa plate, Covered with masking foil, lucifuge hot-house culture.

(6) reading

In several different times：30 minutes, 1h, 2h and longer time, with reading OD values, or use at enzyme-linked instrument Human eye observation's colour developing result.

Data processing and result judge

(1) OD₄₀₅Value is compareed (buffering fluid apertures, feminine gender/Positive control wells), as a result should be in quality monitoring.

A buffers fluid apertures and negative control hole measures OD₄₀₅Value is more than 0.05.If negative control hole determines OD₄₀₅Value During less than 0.05, calculate according to 0.05.

B Positive control wells survey OD₄₀₅/ negative control hole surveys OD₄₀₅Income value is more than 510.

C holes it is repeated basically identical.

(2) after reaching above-mentioned prescription, as a result can be according to following judgement：

A samples survey OD₄₀₅Value/negative control surveys OD₄₀₅As a result 2 are obviously greater than, then result is exactly positive.

B samples survey OD₄₀₅/ negative control surveys OD₄₀₅Result be located at threshold value or so, it may be determined that be suspicious specimen, Should try again, or with different means testing identity in addition.

C sample OD₄₀₅/ negative control OD₄₀₅It is significantly less than, it may be determined that for feminine gender.

(3) if above-mentioned prescription can not be reached, then result can not be carried out with 2 and judged.

Apple Leaves total serum IgE and RT-PCR

Phenol chloroform method extracts Apple Leaves total serum IgE

(1) the 1.5mL centrifuge tubes without RNAnase are taken, the μ L RNA extracts of Deca 500,250 μ L chloroforms is distinguished in pipe： Isoamyl alcohol (24:1), the full phenol (pH=7.5) that closes of 250 μ L water covers centrifugation lid, is kept on ice standby；

(2) -80 DEG C of refrigerators take out a certain amount of sample, put cold good mortar into, appropriate liquid nitrogen are put, by pestle Quick ground sample；

(3) with the spoon of pre-cooling the powder in mortar is added drop-wise in the centrifuge tube of step (1) preparation rapidly, covers centrifugation Lid, quickly rocks centrifuge tube, and is placed on whirlpool in whirlpool concussion instrument and shake 1 minute, is stored at room temperature 5 minutes；

(4) in being put into the centrifuge of advance K cryogenic treatment, 4 DEG C of 1,2000rpm are centrifuged 15 minutes；

(5) the μ L of Aspirate supernatant 400 divide 4 in without RNAnase 1.5mL centrifuge tubes by the liquid-transfering gun of 100 μ L ranges Secondary extraction, wants fast and steady when Aspirate supernatant, it is to avoid suck the impurity of lower floor, the μ L chloroforms of Deca 250：Isoamyl alcohol (24: 1), the full phenol (pH=7.5) that closes of 250 μ L water covers centrifugation lid；30s is acutely shaken in whirlpool concussion instrument, 5 are stood in indoor temperature Minute, in being put into advance K cryogenic treatment centrifuge, rotating speed 1,2000 is centrifuged 15 minutes in 4 DEG C of environment；

(6) using the liquid-transfering gun Aspirate supernatant of 50 μ L, points 4 times, totally 200 μ L be added drop-wise to the 1.5mL without RNAnase from In heart pipe, the μ L isopropanols of Deca 200 gently rock centrifuge tube, when the transparent state of liquid in pipe, centrifuge tube is placed on- 40 DEG C precipitate 30 minutes；Or -20 DEG C of precipitation 2h；

(7) after precipitation terminates, centrifuge tube is placed on 4 DEG C of centrifuges, rotating speed 1,2000 is centrifuged 15 minutes；

(8) supernatant is outwelled, 70% ethanol put with ice, 200 μ L washing precipitations；Subsequent 4 DEG C, 1,2000rpm 5 points of centrifugation Clock；

(9) repeat step is once.

(10) supernatant is outwelled, with the pipettor of 10 μ L the liquid of residual is carefully drawn, subsequently by the open placement of centrifuge tube In superclean bench, blow under air vent mode 5 minutes, disperse unnecessary ethanol.

(11) after drying up, the μ L of DEPC water 30～50 of Deca inactivation, are directly used in subsequent experimental or preservation in centrifuge tube It is standby in -80 DEG C of refrigerators.

Post transcription cloning

SuperRT cDNA the first chain synthetic agent box that this experiment is used is that Beijing health is the limited public affairs of century biotechnology What department produced carries out external post transcription cloning to the Apple Leaves sample RNA for extracting for template, has

Body operating procedure is as follows：

According to according to shown in table 1 below, Reverse Transcription rapid Deca 200 centrifugations of the μ L without RNAnase is centrifuged,

Table 1：Reverse transcription system is mixed.

The centrifuge tube without RNAnase for adding reagent is placed on 70 DEG C of environment to carry out 5 minutes being incubated, then carries out 5 minutes ice Bath.

To distinguishing the following reagent of Deca in above-mentioned centrifuge tube：5 × RT Buffer, 5 μ L；SuperRT(200U/μL)1μL； Using the inhibitor of 1 μ LRNA enzymes, the short time is quickly centrifuged mixing, and once describing program according to photograph carries out reverse transcription reaction：42℃ Incubation 1h, 70 DEG C 10 minutes, 10 DEG C of for ever.

After reaction terminates, product directly pcr amplification reaction using or be stored under -20 DEG C of environment and preserve.

PCR expands the coat protein of three kinds of Apple virus

PCR-MIX Beijing CoWin Bioscience Co., Ltd. production that this experiment is used, to obtaining gathering sample CDNA carry out pcr amplification reaction, reaction system and response procedures are as shown in table 2：

Table 2：PCR amplification system

PCR reaction program be：95 DEG C, 3 minutes；94 DEG C, 30s；56 DEG C of annealing temperature, 30s；72 DEG C, 30；35 are followed Ring, 72 DEG C extend 10 minutes；10 DEG C, 10 minutes.After PCR reactions terminate, with the μ L of PCR primer 3, enter in 1 × TAE buffer Row electrophoresis is checked, and voltage is 130V, constant pressure；Electrophoresis time is about 25 minutes, and agarose gel is put into EB after the completion of electrophoresis Dye 5 minutes in dye liquor, in being placed on uviol lamp environment.Observation electrophoresis result and preservation of taking a picture.

The Coat protein gene sequence of three kinds of Apple virus is determined

Positive PCR primer is reclaimed

Using the agarose gel QIAquick Gel Extraction Kit of the Tyke Bioisystech Co., Ltd (Bioteke) of Beijing hundred production to Jing Agarose gel electrophoresiies are accredited as the PCR primer of the positive and are reclaimed, and detailed operating procedure is as follows：

(1) target stripe is taken out using blade in uviol lamp environment, is placed in the 1.5mL centrifuge tubes for claiming weight；Claim Weight, calculates the weight for reclaiming blob of viscose；

(2) according to shining 1:1(g:ML) liquid is combined to Deca colloidal sol in centrifuge tube, 65 DEG C of water-baths 3～5 minutes, period is repeatedly Centrifuge tube is stirred up and down, so that blob of viscose melts speed accelerating；

(3) when blob of viscose all melts, solution is moved in adsorption tube, 700 μ L rinsings is put toward in the centrifugal adsorbing column Liquid (plus dehydrated alcohol), rotating speed 1 in normal domestic room temp, 2000 centrifugations 1 minute；Outwell waste liquid；

(4) repeat step is once；

(5) room temperature 1,2000rpm, sky centrifugation 2 minutes, outwells waste liquid；

(6) adsorption column is placed in 1.5mL centrifuge tubes, is dried 5 minutes；

(7) the μ LEB eluents of Deca in adsorption column 35,2 minutes are stood under Interior Temperature Environment, rotating speed 1,2000, centrifugation 1 minute.Centrifuge tube bottom liquid is target product.

The connection and conversion of recovery product

The connection of recovery product

The connection test kit for using is precious biological engineering (Dalian) company limited, and recovery product can be attached instead Should, linked system is as shown in table 3 below：

Table 3：Linked system

4 DEG C overnight connect.

The conversion of recovery product

By the product that completes of connection according to being transferred in escherichia coli according to following steps：

(1) by -80 DEG C of competent cells are stored in, it is placed in ice bag environment and melts；

(2) melt competent cell connecting product and it is added drop-wise to, inhale by pipettor and beat mix homogeneously, placement in ice chest 30 minutes；

(3) water-bath that mixed liquor is placed on 42 DEG C is carried out into heat shock 90s, ice bath 5 minutes；

(4) by aseptic 1.5mL centrifuge tubes, mixed liquor is proceeded to wherein, the μ L LB fluid mediums of Deca 200,37 DEG C 180rpm shaking table cultures 60～90 minutes；

(5) by 50 μ L conversional solution, LB solid wares (ammonia benzyl resistance) are spread evenly across, are stood overnight in 37 DEG C of incubators.

The screening of converted product

The product that completes of conversion is to filter out positive bacterium colony, operating procedure as follows：

(1) Deca LB fluid medium and antibiotic (ammonia benzyl) and according to shining 1 successively in aseptic 1.5mL centrifuge tubes:1000 Ratio；

(2) bacterial plaque of uniform size is chosen by aseptic toothpick, by the above-mentioned aseptic 1.5mL of the white colony Deca of picking Centrifuge tube, in a centrifuge tube bacterium colony is cultivated；37 DEG C are shaken bacterium, 220rpm, incubated overnight.

(4) PCR checkings are shaken.

Sequencing

The bacterium solution of Jing PCR test positive, according to shining bacterium solution：50% glycerol=1:The 1 1.5ml centrifuge tubes for being placed on sterilizing In, sequencing.

The abduction delivering of genes of interest

The expression of testing goal albumen

According to 1:It is in 0.1%10ml culture medium, to be shaken in 37 DEG C of environment that 100 pass to Kan containing expression vector liquid Swing activation act 3h；1.5ml bacterium solutions are taken as not inducing matched group, the IPTG of the μ l 0.1mol/L of Deca 30 in remaining bacterium solution, after Persistent oscillation, 1.5ml was just drawn every 2 hours to centrifuge tube, and 3 times are carried altogether.Rotating speed 12000 extracts again precipitation after being centrifuged 2 minutes, drips Plus 75 μ l ultra-pure waters and same amount of 2 × sample-loading buffer, boiling 10 minutes, 12000rpm is centrifuged 3 minutes, and taking supernatant is carried out SDS-PAGE。

SDS-PAGE uses discontinuous system, and resolving gel concentration is 10%, and concentration gum concentration is 5%, the μ l of applied sample amount 10；Electricity The voltage stabilizing that glue is concentrated during swimming is 80V, separation gel voltage stabilizing 150V；When sample front is away from glue bottom 1cm, stops electrophoresis and shell glue, With 0.05% coomassie brilliant blue staining 30 minutes, boil in clear water 3 times, soak the decolouring of a whole night.

1.4.2 whether testing goal albumen forms inclusion body

With 1：It is that in 0.1%LB culture medium, 37 DEG C are shaken that 100 ratio will pass to Kan contents containing expression vector BL21 liquid The IPTG of the 0.1mol/L of Deca respective amount after activation 3h is swung, continues shaken cultivation 6h；Taking 2ml induces liquid in centrifuge tube, turns The centrifugation of speed 5,000 10 minutes, removes supernatant, and precipitation is suspended from 200 μ L PBS again；Ultrasonic degradation rotating speed 12000 after 12 minutes Centrifugation 30 minutes, precipitation separation and supernatant, wherein precipitation is resuspended in 10 points of ebuillition of heated in 200 μ 1 × sample-loading buffers of l Clock, supernatant is according to shining 1:3 are dissolved in 1 × sample-loading buffer, and SDS-PAGE is carried out respectively.

The purification of destination protein

(1) condition express express target protein in 100mlLB culture medium is explored according to 1.4.1；

(2) take induction liquid rotating speed 5000 and to be centrifuged 10 minutes in same 50ml centrifuge tubes twice, remove supernatant, precipitation The middle resuspended rear ultrasonications of Deca 10ml PBS 12 minutes, 12000rpm is centrifuged 10 minutes, abandons supernatant；

(3) Deca 20ml buffer B, shake 45 minutes under room temperature, and to most of dissolving is precipitated, rotating speed 12000 is centrifuged 30 minutes, be protein sample after filtration with 0.22 μm of filter membrane using supernatant, is prepared with AKTA protein purifications chromatography system Nickel ion affinity chromatograph post of uniting carries out purification；

(4) with the buffer B balance affinity columns of 20 times of column volumes；

(5) loading；

(6) with buffer E and buffer B according to 1:Foreign protein is cleaned after 24 mixing；

(7) with buffer E eluting destination proteins；

(8) electrodialysiss：Bag filter is cut into the suitable length segment of 10～20cm, in 2% (w/v) NaHCO₃And 1mmol/ Boil in L EDTA (pH8.0) equivalent mixed liquor 10 minutes, it is net using distilled water cleaning, enter in 1mmol/L EDTA (pH8.0) Row is heated for 10 minutes, keeps submerged state to be stored in 4 DEG C after temperature drop, by front filling ddH₂O is cleaned, and bag filter is while clamp Afterwards Deca waits the protein solution for going to be dialysed, and clamps another side, is immersed in and is placed with 0.1mol/L PBSs In Horizontal electrophoresis tank, 45V constant pressures carry out 45 minutes reacting, immediately back action 45sec；

(9) the OD values of target protein solution are detected, the concentration of purifying protein is determined.

(10) SDS-PAGE detects the effect of protein purification, and method is same..

(11) -20 DEG C of frozen destination proteins are standby.

The preparation of polyclonal antibody

Sero-fast preparation

Target protein solution 1ml (0.5～1.2mg) is taken, initial immunity Deca volume identical Freund's complete adjuvant is strengthened The same amount of incomplete Freund's adjuvant of immune Deca, whirlpool concussion fully mixes emulsifying, until rotating speed 1000 is centrifuged 1 minute liquid Till there is no longer multilamellar appearance, rabbit dorsal sc multi-point injection.Carry out 3 booster immunizations within 30 days after initial immunity, every time between Every seven days, last immunity carried out cardiac blood sample collection after 1 week, 37 DEG C of placement 1h, and 4 DEG C overnight, and 3000rpm centrifugations are obtained for 5 minutes Obtain antiserum, Deca NaN₃To 0.1% (w/v), -20 DEG C of preservations.

1.5.2 agar double immunodiffusion method determines antiserum titre

Agar powder high-temperature digestion, pour in culture dish and cool down, using card punch " quincunx " aperture (7 is got Group), each bore dia 0.3cm, two pitchs of holes 0.4cm.The protein solution of purification is added drop-wise in interstitial hole, in holes around respectively The antiserum of Deca different multiples, per the μ l of hole 50, is placed on 37 DEG C in moisture preservation box, 24h, checks that precipitation line judges antiserum titre.

The purification of polyclonal antibody

(1) antiserum binding buffer are according to 1:1 dilution；

(2 supernatant are filtered through 0.22 μm of filter membrane, to facilitate loading to cross protein A post；

(4) use A) dilution after 4 DEG C of sample, 10000rpm be centrifuged 10 minutes；

(3) KTA Protein purification chromatographic systems are carried out with protein A post；

(5) by the binding buffer balance affinity columns of 20 times of column volumes；

(6) loading；

(7) foreign protein is cleaned with the binding buffer of 10 times of column volumes；

(8) with eluting buffer eluting destination proteins, purpose antibody is collected, determines the concentration that OD is worth to antibody purification.

As a result with analysis

A situation arises for three kinds of latent viruses of 11 the major apple production area in Shanxi

(1) a situation arises for ASPV

The Analysis of test results of RT-PCR is shown, ASPV in Pingyao, the incidence rate highest in the two producing regions of Linfen urban district, All more than 50%；The incidence rate in this 5 producing regions of Yuncheng urban district, Huozhou, Xiangfen, Xinjiang and Quwo is placed in the middle, 40% -50% it Between；The incidence rate in this 4 producing regions of Wanrong, Ji County, Pinglu and Linyi is minimum, below 40% (Fig. 1).

(2) a situation arises for ACLSV

The Analysis of test results of RT-PCR is shown, incidence rates of the ACLSV in the 11 Main Apple Varieties producing regions in Shanxi all compares Height, wherein, ACLSV this 6 areas of Yuncheng urban district, Wanrong, Huozhou, Quwo, Ji County and Linyi incidence rate all 60% with On；In Xiangfen, Xinjiang and Pinglu, this 3 regional incidence rates are between 50% -60%；In Pingyao and this 2 areas of Linfen, The incidence rate of ACLSV is minimum, less than 50% (Fig. 2).

(3) a situation arises for ASGV

The Analysis of test results of RT-PCR is shown, incidence rates of the ASGV in the 11 Main Apple Varieties producing regions in Shanxi generally compares Height, incidence rate is all on 50%.Wherein, the incidence rate highest in Yuncheng urban district, Huozhou, Xiangfen, Pingyao and Linfen, all 70% On；The incidence rate of Xinjiang, Quwo, Ji County and Linyi is between 60% -70%；The ASGV in Wanrong and this 2 producing regions of Pinglu sends out Raw rate is 56.53% and 51.38% (Fig. 3) under 60%, respectively.

Plant total serum IgE electrophoresis detection result

Apple Leaves total serum IgE is extracted using the method for Deca β in extracting solution-mercaptoethanol, in RNA precipitate, -40 is adopted DEG C precipitation 30 minutes and -20 DEG C precipitation 2h respectively to extract RNA precipitate, in detected through gel electrophoresis figure, can be clearly See：Tri- bands of 28S, 18S and 5S, wherein 28S band 2 times of 18S (Fig. 4) of brightness.

The abduction delivering and purification result of target protein

3h will be activated containing expression vector bacterial strain BL-PE-ASGV-CP, BL-PE-ASPV-CP and BL-PE-ACLSV-CP Afterwards, IPTG inducing culture 6h, the sampling per 2h, centrifuged supernatant Jing SDS-PAGE analyses, the bacterial strain of induction is generated respectively about The target protein of 32kD (Fig. 5), 37kD (Fig. 6) and 22kD (Fig. 7), without the bacterial strain of induction this band is not produced.

The albumen of great expression AKTA Protein purification chromatographic system Jing Ni ion affinity chromatographies are purified, and purified product is gone forward side by side Dialysis is gone, Jing SDS-PAGE confirm as the albumen (Fig. 8) of purification, and the concentration of final purifying protein is：ASGV-CP is 1.1mg/ ml；ACLSV-CP is 0.5mg/ml；ASPV-CP is 1.4mg/ml.Sero-fast preparation, titration and antibody purification result

Respectively with each immune rabbit of tri- kinds of albumen of ACLSV-CP, ASGV-CP, ASPV-CP of abduction delivering and purification, And blood is taken Jing after 3 booster immunizations, obtain antiserum.3 parts of potency, the wherein resistance of ASGV are determined with agar double immunodiffusion method There is not any sealed Belt with purifying protein in serum, and potency is that 0, ASPV antiserums and ACLSV antiserum titres are 8.

Protein A column purification ASPV antiserums and the sero-fast antibody of ACLSV are crossed using AKTA protein purification systems, purification is produced Thing determines OD is worth its concentration, and wherein ASPV antibody is 0.7mg/ml, and ACLSV antibody is 0.6mg/ml.

ELISA test strip result

Gold colloidal is prepared for according to the method according to discipline tinkling of pieces of jades (2008), gold colloidal is resisted respectively with ASPV antibody and ACLSV Body is combined and is prepared for gold labeling antibody, has been assembled into ACLSV/ASPV Multiple detection test strips, the inspection of ASPV/ASGV Multiple detections Test paper slip, ACLSV/ASGV Multiple detection test strips.ELISA test strip land for growing field crops diseased plant juice, healthy Folium Mali pumilae are used respectively Piece juice shows obvious specific band (Fig. 9) as control, diseased plant juice correspondence test strip.1st article of detection Test strips show that sample contains respectively ACLSV and ASPV, is that hybrid viruses infect sample；2nd article test strip shows, sample Product contain respectively ASPV and ASGV, are that hybrid viruses infect sample；3rd article test strip shows that sample only contains respectively ASGV, is single virus infection sample；4th article test strip shows that healthy Apple Leaves do not carry any virus.

Discuss and conclusion

1ACLSV, ASPV and ASGV have generation in Shanxi Fructus Mali pumilae producing region

150 parts of Apple Leaves samples are detected, as a result table by the method combined by DAS-ELISA and RT-PCR Bright these three latent viruses are all distributed in 12 the major apple production area in Shanxi, but their incidence rate is not presented bright Aobvious Area distribution.Single to infect in situation, apple stem grooving virus (ASGV) incidence rate highest, is 67.6%.In mixed infection In, three kinds of incidence rate highests being infected simultaneously, are 17.8%.In the sample of the different age of trees, the age of tree is bigger, three kinds That a situation arises is more serious for latent virus.

The development of ACLSV, ASPV and ASGV multiple detection test paper bar

If preparing high-quality immune serum, it is necessary first to suitable antigen-like material.At present, obtaining the method for antigen mainly has Two kinds, i.e., using purified virus particle as antigen and using with the albumen of molecular biology method expression and purification as antigen.

Purified virus particle is to prepare sero-fast traditional means as antigen, but due to the virus quantity in pin main body compared with It is low, need enough malicious sources just to obtain enough materials, and requirement of the separating-purifying to experimental apparatus is very high.The most key It is that, with the substantial amounts of antigen of the method acquisition it is difficult to ensure that it has very high purity, often the albumen containing host plant, mixed with this Antiserum prepared by hop protein carries out immune detection, and false-positive risk occur can be very big.

Antigen protein used herein is the virus capsid protein subunit obtained with prokaryotic expression system.With the side of prokaryotic expression Method obtains antigen protein, it is only necessary to which a small amount of virus genome RNA recycles conventional molecular biology side as original material Method can be completed, and it has advantage short the time required to high production quantity, low cost used, production, is particularly suitable for laboratory operation. In addition, after Prokaryotic expression vector construction success with persistence, and induction table can be carried out in any experimental unit at any time Reach.But, it should also be noted that arrive, the virus capsid protein subunit of prokaryotic expression and purification can not be equal to whole virion Protein coat, therefore, its immunity perhaps has differences with the virion under naturalness, can bring false the moon to immune detection The risk of property.

The ultimate principle and principal character and advantages of the present invention of the present invention has been shown and described above.The technology of the industry Personnel it should be appreciated that the present invention is not restricted to the described embodiments, the simply explanation described in above-described embodiment and description this The principle of invention, without departing from the spirit and scope of the present invention, the present invention also has various changes and modifications, these changes Change and improvement is both fallen within scope of the claimed invention.The claimed scope of the invention by appending claims and its Equivalent thereof.

Claims (1)

1. a kind of preparation method of apple latent virus Multiple immunizations colloidal gold colloidal gold detection test paper strip, it is characterised in that including following Step：

(1) vessel cleaning：

All glass drying ovens wash clean under flowing water, be placed in acid solution (potassium dichromate 100g, concentrated sulphuric acid 50ml, plus Distilled water is to 1000ml) immersion 24h, is washed away, then using distilled water immersion 24h, with milli-Q water three repeatedly with tap water It is secondary, ultra-pure water immersion 72h is reused, it is standby after being dried in baking box；Strict pure, institute must be kept in reagent preparation There is reagent to use ddH₂0 prepares；

(2) prepared by gold colloidal：

1% aqueous solution of chloraurate 1ml, plus 99ml ultra-pure waters are taken, boiling is heated to, is stirred while disposable fast drop 1% trisodium citrate aqueous solution of the fresh configurations of 1.8ml, solution colour from Huang gradually switch to it is purplish red, then in boiling water heating 3～ 5 minutes, room temperature, plus ultra-pure water are cooled to original volume, save backup under 4 DEG C of environment, it is to avoid illumination；

(3) preparation of gold labeling antibody：

4 DEG C of dialysed overnights in NaCl solution by antibody to be marked in advance 0.005mol/lpH7.0, so as to remove unnecessary salt Ion, 10000rpm4 DEG C is centrifuged 30 minutes, removes polymer；

Determine antibody optimum dose to be marked：With the K of 0.1mol/l₂CO₃Solution adjusts the pH value of colloidal gold solution to 8.5, subpackage 10 Pipe, every pipe 1ml, the μ l of Deca 2,4 μ l, 6 μ l, 8 μ l, 10 μ l, 20 μ l antibody respectively, be sufficiently mixed it is uniform after every pipe Deca 10% (m/v) NaCl solution 0.1ml；Separately set the NaCl solution for only adding 0.1ml10% in control tube, i.e. 1ml gold colloidals, mix homogeneously Place 2 hours afterwards, observe result；With the K of 0.1mol/l₂CO₃The pH value of colloidal gold solution is adjusted to into 8.5；Under agitation, to The antibody of Deca optimum mark content in aurosol, uniformly stirs 2～3 minutes；The BSA of Deca 10% is used as stabilizer so as to Final concentration of 1%, continue to stir 15 minutes, 4 DEG C save backup；

Gold labeling antibody solution is centrifuged into 10 minutes so as to remove colloid metal/polymer therein in 1200rpm, 4 DEG C, supernatant is taken, It is centrifuged 50 minutes with 14000rpm, 4 DEG C again, it is seen that centrifugation thing is divided into 3 layers, containing the antibody being also not bound with supernatant, does not have The colloid gold particle polymer for having combination forms atrouss speckle in the bottom, and middle part aubergine flocculent deposit is good combination Gold colloidal-antibody complex；Supernatant discarded, aubergine flocculent deposit the bufferings of the PBS containing 1%BSA of 1/10 original volume are dissolved in In liquid (0.01mol/l, pH8.2), 4 DEG C save backup；

(4) assembling of test strips：

The glass fibre membrane for cutting is put in the PBS of the 0.01mol/l containing 1%BSA and closes 1h, after drying naturally, will Uniform Deca is in glass fibre element film up to saturation after the different gold labeling antibody equal-volumes mixing for suitably diluting, in 37 DEG C of baking ovens Dry for standby；

Take the antibody and goat anti-rabbit antibody of purified mistake respectively carries out certain dilution with PBST buffer, fine in nitric acid respectively Deca sample after room temperature is dried, in being placed on lock solution, carries out 1h hatching trainings on T lines and C lines on the plain film of dimension in 37 DEG C of environment Educate, washed 2 times with PBST buffer, 5 minutes every time, in normal domestic room temp preservation is dried；

Antibody solid phase nitrocellulose filter, filter paper, gold labeling antibody combine release pad, sample pad in order in PVC board, phase The overlap of 1～2mm is stayed between mutually, is firmly compressed, it is to avoid produce bubble；4 DEG C of lucifuge kept dry；

(5) ELISA test strip：

The Apple Leaves of the RT-PCR that learns from else's experience authenticated infection virus, during clean mortar is placed on after weighing, according to 1:10 (m/v's) Ratio Deca PBST, is ground and becomes homogenate, and 2000rpm is centrifuged 10 minutes, takes supernatant and use reagent paper respectively as sample Bar is checked, then same amount of sample mix homogeneously, by test strips inspection.