CN1715924A - Method for detecting tobacco mosaic virus using phycoerythrin fluorescent probe - Google Patents
Method for detecting tobacco mosaic virus using phycoerythrin fluorescent probe Download PDFInfo
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- CN1715924A CN1715924A CN 200510044139 CN200510044139A CN1715924A CN 1715924 A CN1715924 A CN 1715924A CN 200510044139 CN200510044139 CN 200510044139 CN 200510044139 A CN200510044139 A CN 200510044139A CN 1715924 A CN1715924 A CN 1715924A
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Abstract
The method of detecting tobacco mosaic virus with phycoerythrin fluorescent probe uses the material comprising phycoerythrin fluorescent probe, nitrocellulose film, TMV polyclonal antibody, negative and positive reference, closing buffering liquid and washing buffering liquid. The phycoerythrin marked antibody and nitrocellulose film coated solid antibody are used mainly. During detection, the solid antibody captures the antigen to be detected via antigen reaction to form immunological compound, and phycoerythrin fluorescent probe is added to react with the immunological compound. After washing, the reaction product is detected under turned fluorescent microscope to judge the result. The direct phycoerythrin immune fluorescence method has the advantages of high specificity, less reagent consumption, simple process, low cost, etc.
Description
Technical field the present invention relates to a kind of fluorescence probe, and utilizes fluorescence probe to detect viral method, particularly a kind of phycoerythrin fluorescence probe, and the method for utilizing phycoerythrin fluorescence probe fast detecting tobacco mosaic virus (TMV).
The background technology plant virus detects enzyme linked immunosorbent assay (ELISA) (ELISA) method commonly used, EIA enzyme immunoassay is the technology that the specificity with the antigen-antibody combination combines with the efficient catalytic of enzyme, have highly sensitive, characteristics such as high specificity, but operation steps is many, length consuming time, and need relatively costly enzyme labelled antibody and 96 hole ELISA Plate.
Fluoroimmunoassay is to utilize a kind of labelled immune analytical approach of various fluorescence as probe molecule, is used widely in the analysis of various micro-bioactivators such as nucleic acid, protein, polypeptide, amino acid, enzyme, hormone, growth factor, cell factor, cell surface marker, tumour specific antigen, acceptor, medicine, the infection sources at present.Traditional fluorescence probe comprises fluorescein, fluorescein isothiocynate (FITC), rhodamine (Rodam) etc.Fluorimetric detection limit value often is subjected to the restriction of background fluorescence in serum and the other biological sample, and in addition, conventional fluorescent probe non-specific adsorption has a strong impact on the susceptibility of detection, hinders the fluorescence immune analysis method development.Phycoerythrin is a kind of novel fluorescence probe, is used widely in numerous areas such as medical diagnosis, Celluar and Molecular Biology.
Yan Fengying etc. delivered literary composition in the 4th the 10th phase of volume of Traditional Chinese Medicine magazine in 2004 and are " fluorescent dye R-phycoerythrin mark mouse anti human CD series monoclonal antibody ", fluorescent reagent with R-phycoerythrin (R-PE) mark mouse anti human CD series monoclonal antibody, be applied to flow cytometry analysis, the antibody specificity that the analysis showed that the R-PE mark remains intact, and fluorescence intensity height, but compatibility Cheng Shuanbiao reagent also.The Iannellli of the U.S. in 1997, D etc. deliver literary composition and are " utilizing flow cytometer to detect cucumber mosaic virus (CMV), tobacco mosaic virus (TMV) (TMV) and marmor upsilon (PVY) simultaneously " on the virological method magazine, the plant tissue juice of PVY, CMV, three kinds of virus infections of TMV is hatched with the latex particle that varies in size respectively, hatch in first antibody and second antibody successively after the washing, wherein second antibody is carried out mark with the dyestuff that these two kinds of phycoerythrin and fluorescein can send out fluorescence different respectively.Judge the kind of virus according to difference that produces fluorescence and the size that captures the latex particle of virus, thereby detect when having realized these three kinds of viruses.
At present, the reagentization of phycoerythrin fluorescence probe and commercialized degree are not high, still untappedly go out popular diagnostic reagent, and the detection research of plant virus is rarely had report especially.
Summary of the invention the objective of the invention is to utilize the good fluorescent characteristic of phycoerythrin, the exploitation phycoerythrin fluorescence probe, and be used for tobacco mosaic virus (TMV) (Tobacco mosaic virus, detection TMV).
The present invention realizes by the following method:
One, material
Used main agents: phycoerythrin is the preparation of plant virus research institute of University Of Agriculture and Forestry In Fujian; Cross-linking reagent SPDP, 2-IT and terminator NEM are available from Peirce company; Medium Superdex200HR is the product of Amersham company; Bovine serum albumin(BSA) (BSA) is the Sigma product; Nitrocellulose filter 0.45 μ is an Amersham company product; Employed other conventional medicines and reagent are the general pure or chemically pure reagent of homemade analysis in the test.
1, the preparation of phycoerythrin
Fully grind knuckle algae (Amphiroa ephdraea) or coralgal (Corallinaofficinalis) in pulping machine, spend the night with the lixiviate of 0.01M PBS pH7.0 damping fluid, after three layers of filtered through gauze, 4 ℃ of centrifugal 15min of following 10000rpm get supernatant; Use saturation degree 30%-45% ammonium sulfate precipitation albumen then, get rough phycoerythrin red precipitate; Red precipitate is dissolved in 0.01M PBS pH7.0 damping fluid, is splined on Superdex G-15 desalting column then, desalination; The desalinization liquor of collecting can be splined on has used the good hydroxyapatite column of 0.15M NaCl+0.01M PBS pH7.0 damping fluid balance in advance, continuation is made linear gradient elution with 0.15M NaCl+0.01M～0.2M pH7.0PBS damping fluid after washing 30min with damping fluid again; Eluent collection, concentrated, centrifugal.
2, TMV Polyclonal Antibody Preparation
Get the acetate buffer solution that the 4ml antiserum adds 8ml 0.06M pH4.0, proofread and correct pH value to 4.8, under oscillating condition, slowly add 120 μ l caprylic acids with NaOH, vibration mixes 30min at room temperature, and the centrifugal 15min of 10000rpm in 4 ℃ goes precipitation, supernatant is packed in the bag filter, in the 0.01MpH7.4PBS damping fluid, dialysis 12h slowly adds the equal-volume saturated ammonium sulfate then, leave standstill 30min at 4 ℃, the centrifugal 20min of 12000rpm gets precipitation, and use the dialysis back.
3, the preparation of the positive, negative sample
Positive is the tobacco mosaic virus (TMV) of purifying, and concentration is in 0.1 μ g-100 μ g/ml scope; Negative sample is sealing buffer solution.
4. the preparation of sealing damping fluid and lavation buffer solution
Sealing buffer solution: 20-100mmol/L pH7.5PBS+0.5-3%BSA
Washing buffer solution: 50-200mmol/L pH7.5PBS+0.05-0.3%Tween20
Two, the preparation of phycoerythrin fluorescence probe
(1) crosslinking protein: two kinds of crosslinked albumen are respectively 6mg/ml, the phycoerythrin of 1.25ml and 4mg/ml, and 0.75ml tobacco mosaic virus (TMV) (TMV) polyclonal antibody, the mol ratio of the two is 1: 1～2: 1;
(2) exclusive-OR function reagent activated protein: with exclusive-OR function reagent SPDP activation phycoerythrin, the two mol ratio is 80: 1～200: 1, and the reaction time is 15～60min; Exclusive-OR function reagent 2-IT sulfhydrylation TMV polyclonal antibody, the two mol ratio is 160: 1～320: 1, the reaction time is 0.5～3h;
(3) hybrid reaction: behind the exclusive-OR function reagent priming reaction, the unreacted activating reagent is removed in desalination; Get equal-volume reactant hybrid reaction, 4 ℃, reaction 6h.
(4) stopped reaction: add stop buffer NEM 80mmol/L 100 μ l and end cross-linking reaction, 4 ℃ of reaction 1h pack reactant in the dialysis clamp into, and dialysis concentrates.
(5) purifies and separates crosslinking mixture: with crosslinking mixture be splined on the Superdex200HR gel chromatography lean on (1.6 * 60cm), with the 0.15ml/min flow velocity, with 0.15M NaCL+100mmol/L pH7.0PBS buffer solution elution.
Three, detection method
The material that detects use is by phycoerythrin fluorescence probe, nitrocellulose filter, TMV polyclonal antibody, positive negative control product, sealing buffer solution and washing buffer solution composition.
Detection method is as follows:
(1) bag quilt: on nitrocellulose filter, drip 2-6 μ lTMV antibody, the distance of the 1-2cm of being separated by between each point places 4 ℃, absorption 0.5-3h;
(2) sealing: nitrocellulose filter is directly immersed in the 20-100mmol/L pH7.5PBS+0.5-3%BSA sealing damping fluid 37 ℃ of incubation 0.5-3h;
(3) washing A: prepare the lavation buffer solution of 50-200mmol/L pH7.5PBS+0.05-0.3%Tween20, and clean nitrocellulose filter at least three times with lavation buffer solution, standing and drying;
(4) point sample: drip positive and negative sample and testing sample, every hole 2-6 μ l;
(5) washing B: treat that sample liquid infiltrates nitrocellulose filter fully, room temperature leaves standstill 10-30min, with described lavation buffer solution cleaning diaphragm at least three times, and standing and drying;
(6) fluorescence labeling spike: add phycoerythrin fluorescence probe 2-6 μ l, at 37 ℃ of incubation 0.5-3h;
(7) washing C: clean nitrocellulose filter at least three times with described lavation buffer solution, take out diaphragm and remove the remaining liquid in surface with clean thieving paper;
(8) detect judgement: nitrocellulose filter is tiled on the clean glass sheet, under inverted fluorescence microscope, detects and observe result of determination.
The phycoerythrin fluorescence probe immunofluorescence detects two kinds of antibody of main utilization, the insolubilized antibody of phycoerythrin labelled antibody and nitrocellulose filter bag quilt.Insolubilized antibody is caught determined antigen by the antigen-reactive specificity during detection, forms immune complex, adds phycoerythrin fluorescence probe, after fully reacting with immune complex, removes non-specific material through washing, can detect result of determination.
Detection effect for check phycoerythrin fluorescence probe direct immunofluorescence (DFIA) has detected 55 parts of TMV samples with this method, testing result compare with the testing result of ELISA (seeing attached list 1).
The comparison of subordinate list 1 phycoerythrin direct immunofluorescence and ELISA testing result
| ELISA(+) | ELISA(+/-) | ELISA(-) | Add up to | |
| DFIA (+) DFIA (+/-) DFIA (-) total | 41 0 0 41 | 2 6 0 8 | 0 1 5 6 | 43 7 5 55 |
Annotate: "+" represents positive; "+/-" represent false positive; "-" represents negative.
41 parts of samples by the ELISA test positive, direct immunofluorescence detects also all positive;
8 parts are detected by ELISA and to be that 6 parts that false-positive sample, direct immunofluorescence detect wherein be false positive, other 2 parts positive;
6 parts are detected negative sample by ELISA, and 5 parts of detecting wherein of direct immunofluorescence are negative, and 1 part is false positive in addition;
Result from above-mentioned detection, the phycoerythrin direct immunofluorescence detects has specificity preferably, compare with the ELISA testing result, positive recall rate is 100%, false-positive recall rate is 75%, negative recall rate is 83.3%, overall recall rate is 94.55%, illustrates that the NC film is that the phycoerythrin direct immunofluorescence and the ELISA of solid phase carrier has high degree of conformity.
Utilize the phycoerythrin fluorescence probe direct immunofluorescence to detect tobacco mosaic virus (TMV), have that selectivity is strong, sample size is few and advantage such as method is easy, the detection method ELISA commonly used with present plant virus compares, the direct immunofluorescence detection method is carrier with the nitrocellulose filter, the membrane-like carrier operation is simple, convenient, step is few, it is short to detect the used time, does not need the enzyme labelled antibody of comparison costliness simultaneously, relatively economical.In addition, phycoerythrin is from natural seaweed, safe and harmless as fluorescence probe, overcome the synthetic harm that is brought of the synthetic fluoresceins fluorescence probe of traditional chemical, the fluorescence intensity height of albumen own, long preservation does not have the fluorescent characteristic of obvious decay, and this phycoerythrin that all indicates is used on detecting as fluorescence probe has great potential.
Embodiment now is illustrated in conjunction with the embodiments in order fully to disclose a kind of method of utilizing phycoerythrin fluorescence probe to detect tobacco mosaic virus (TMV) of the present invention.
Embodiment: a kind of method of utilizing phycoerythrin fluorescence probe to detect tobacco mosaic virus (TMV) may further comprise the steps:
1. bag quilt: drip 3 μ l TMV antibody on nitrocellulose filter, the 1cm of being separated by between each point puts 4 ℃, absorption 2h.
2. sealing: nitrocellulose filter is directly immersed in the 50mmol/L pH7.5PBS+2%BSA confining liquid, 37 ℃, incubation 1h.
3. wash A: prepare the lavation buffer solution of 100mmol/L pH7.5PBS+0.1%Tween20, and clean nitrocellulose filter at least three times with lavation buffer solution, standing and drying.
4. point sample: drip positive and negative and testing sample, each point 3 μ l.、
5. washing B: treat that sample liquid infiltrates nitrocellulose filter fully, room temperature leaves standstill 15min, with described lavation buffer solution cleaning diaphragm at least three times, and standing and drying.
6. fluorescent tracing: add phycoerythrin fluorescence probe 3 μ l, at 37 ℃ of incubation 1h.
7. wash C: clean nitrocellulose filter at least three times with described lavation buffer solution, take out diaphragm and remove remaining liquid with clean thieving paper.
8. detect and judge: under inverted fluorescence microscope, observe result of determination on the detecting instrument.
Fluorescence immunoassay detecting instrument and criterion:
With Leica inversion type fluorescent microscope is detecting instrument, detects by an unaided eye when judging, fluorescence signal intensity is judged by following standard:
The no fluorescence of "-" expression;
"-+" expression fluorescence a little less than;
"+" expression fluorescence is high-visible;
Obviously as seen " ++ " represent fluorescence, and intensity is big
" ++ +～++ ++ " represent that fluorescence is very bright;
"-" is decided to be feminine gender, and "+" is decided to be false positive, and the result that "+" and "+" is above all is decided to be the positive.
Testing sample qualitatively judges the positive and negative of analytic sample testing result by above standard.
According to said method, dispose used corresponding reagent and material, also can be assembled into the kit that detects tobacco mosaic virus (TMV), the convenient use.
Claims (5)
1, a kind of phycoerythrin fluorescence probe that utilizes detects tobacco mosaic virus (TMV) (Tobacco mosaicvirus, TMV) method is characterized in that the material that uses is made up of phycoerythrin fluorescence probe, nitrocellulose filter, TMV polyclonal antibody, positive negative control product, sealing damping fluid and lavation buffer solution; Detection method is as follows:
(1) bag quilt: on nitrocellulose filter, drip 2-6 μ lTMV antibody, the distance of the 1-2cm of being separated by between each point places 4 ℃, absorption 0.5-3h;
(2) sealing: nitrocellulose filter is directly immersed in the 20-100mmol/L pH7.5PBS+0.5-3%BSA sealing damping fluid 37 ℃ of incubation 0.5-3h;
(3) washing A: prepare the lavation buffer solution of 50-200mmol/L pH7.5PBS+0.05-0.3%Tween20, and clean nitrocellulose filter at least three times with lavation buffer solution, standing and drying;
(4) point sample: drip positive and negative sample and testing sample, every hole 2-6 μ l;
(5) washing B: treat that sample liquid infiltrates nitrocellulose filter fully, room temperature leaves standstill 10-30min, with described lavation buffer solution cleaning diaphragm at least three times, and standing and drying;
(6) fluorescence labeling spike: add phycoerythrin fluorescence probe 2-6 μ l, at 37 ℃ of incubation 0.5-3h;
(7) washing C: clean nitrocellulose filter at least three times with described lavation buffer solution, take out diaphragm and remove the remaining liquid in surface with clean thieving paper;
(8) detect judgement: nitrocellulose filter is tiled on the clean glass sheet, under inverted fluorescence microscope, detects and observe result of determination.
2, a kind of method of utilizing phycoerythrin fluorescence probe to detect tobacco mosaic virus (TMV) according to claim 1 is characterized in that the preparation method of phycoerythrin fluorescence probe is as follows:
(1) crosslinking protein: two kinds of crosslinked albumen are respectively 6mg/ml, the phycoerythrin of 1.25ml and 4mg/ml, and 0.75ml tobacco mosaic virus (TMV) (TMV) polyclonal antibody, the mol ratio of the two is 1: 1～2: 1;
(2) exclusive-OR function reagent activated protein: with exclusive-OR function reagent SPDP activation phycoerythrin, the two mol ratio is 80: 1～200: 1, and the reaction time is 15～60min; Exclusive-OR function reagent 2-IT sulfhydrylation TMV polyclonal antibody, the two mol ratio is 160: 1～320: 1, the reaction time is 0.5～3h;
(3) hybrid reaction: behind the exclusive-OR function reagent priming reaction, the unreacted activating reagent is removed in desalination; Get equal-volume reactant hybrid reaction, 4 ℃, reaction 6h;
(4) stopped reaction: add stop buffer NEM 80mmol/L 100 μ l and end cross-linking reaction, 4 ℃ of reaction 1h pack reactant in the dialysis clamp into, and dialysis concentrates;
(5) purifies and separates crosslinking mixture: with crosslinking mixture be splined on the Superdex200HR gel chromatography lean on (1.6 * 60cm), with the 0.15ml/min flow velocity, with 0.15M NaCL+100mmol/L pH7.0PBS buffer solution elution.
3, a kind of method of utilizing phycoerythrin fluorescence probe to detect tobacco mosaic virus (TMV) according to claim 1 and 2, the preparation method who it is characterized in that phycoerythrin fully grinds marine alga in pulping machine, spend the night with the lixiviate of 0.01M PBS pH7.0 damping fluid, after three layers of filtered through gauze, 4 ℃ of centrifugal 15min of following 10000rpm get supernatant; Use saturation degree 30%-45% ammonium sulfate precipitation albumen then, get rough phycoerythrin red precipitate; Red precipitate is dissolved in 0.01M PBS pH7.0 damping fluid, is splined on Superdex G-15 desalting column then, desalination; The desalinization liquor of collecting can be splined on has used the good hydroxyapatite column of 0.15MNaCl+0.01M PBS pH7.0 damping fluid balance in advance, continuation is made linear gradient elution with 0.15M NaCl+0.01M～0.2M pH7.0PBS damping fluid after washing 30min with damping fluid again; Eluent collection, concentrated, centrifugal.
4, a kind of method of utilizing phycoerythrin fluorescence probe to detect tobacco mosaic virus (TMV) according to claim 1 is characterized in that described sealing, is that nitrocellulose filter is directly immersed in the 50mmol/LpH7.5PBS+2%BSA confining liquid, 37 ℃, and incubation 1h.
5, a kind of method of utilizing phycoerythrin fluorescence probe to detect tobacco mosaic virus (TMV) according to claim 1 is characterized in that described washing, is the lavation buffer solution with 100mmol/L pH7.5PBS+0.1%Tween20.
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Cited By (6)
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| CN101750482A (en) * | 2008-12-17 | 2010-06-23 | 上海海洋大学 | Method for preparing porphyra yezoensis R-phycoerythrin fluorescence probe |
| CN101846625B (en) * | 2010-05-13 | 2012-08-01 | 西北工业大学 | Method for measuring concentration of protein in solution by using glutaraldehyde to induce infrared fluorescence |
| CN102680674A (en) * | 2012-05-17 | 2012-09-19 | 集美大学 | Fish parvalbumin fluorescent mark antibody and preparation method and application thereof |
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| CN108508214A (en) * | 2018-05-31 | 2018-09-07 | 福州大学 | A kind of phycoerythrin fluorescence probe and its method quickly detected for aflatoxin B1 |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101750482A (en) * | 2008-12-17 | 2010-06-23 | 上海海洋大学 | Method for preparing porphyra yezoensis R-phycoerythrin fluorescence probe |
| CN101846625B (en) * | 2010-05-13 | 2012-08-01 | 西北工业大学 | Method for measuring concentration of protein in solution by using glutaraldehyde to induce infrared fluorescence |
| CN102680674A (en) * | 2012-05-17 | 2012-09-19 | 集美大学 | Fish parvalbumin fluorescent mark antibody and preparation method and application thereof |
| CN107219201A (en) * | 2017-04-20 | 2017-09-29 | 浙江工商大学 | Utilize the immune rear bullfrog antibody of fluorescence microscope citrinin and affine method |
| CN107219201B (en) * | 2017-04-20 | 2019-11-12 | 浙江工商大学 | Utilize the immune rear bullfrog antibody of fluorescence microscope citrinin and affine method |
| CN108508214A (en) * | 2018-05-31 | 2018-09-07 | 福州大学 | A kind of phycoerythrin fluorescence probe and its method quickly detected for aflatoxin B1 |
| CN112098640A (en) * | 2020-09-16 | 2020-12-18 | 浙江正熙生物医药有限公司 | Fluorescent protein and/or coupled protein monoclonal antibody marking method and kit thereof |
| CN112098640B (en) * | 2020-09-16 | 2021-12-14 | 浙江正熙生物技术股份有限公司 | Fluorescent protein and/or coupled protein monoclonal antibody marking method and kit thereof |
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