CN104198713B - A kind of method of tomato spotted wilf virus in quick detection thrips body - Google Patents
A kind of method of tomato spotted wilf virus in quick detection thrips body Download PDFInfo
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- CN104198713B CN104198713B CN201410450554.1A CN201410450554A CN104198713B CN 104198713 B CN104198713 B CN 104198713B CN 201410450554 A CN201410450554 A CN 201410450554A CN 104198713 B CN104198713 B CN 104198713B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
Abstract
The invention discloses the method for tomato spotted wilf virus in a kind of quick detection thrips body, to be measured 1 or several thrips being placed into finishes on the nitrocellulose filter of separation, add 2 μ l sample treatment buffer, with liquid transfer gun head, thrips is ground in damping fluid, process different polypide sample successively, the same absorption 2 μ l positive and negative controls point are on nitrocellulose filter; After film drying, close 1h with 5% skimmed milk power of PBST buffer, then the primary antibodie adding confining liquid dilution cultivates 1h, washes film afterwards 3 times; Add two anti-cultivation 1h of confining liquid dilution, then wash film 3 times; Add nitrite ion colour developing 15min, discard nitrite ion and add the reaction of distilled water color development stopping, can interpretation of result be carried out.The present invention is easy to operate, quick, measurement result has good stability, reappearance and accuracy, can meet the detection needs of tomato spotted wilf virus in thrips body, lays a good foundation for avoiding the harm of tomato spotted wilf virus as far as possible.
Description
Technical field
The invention belongs to detection technique field, belong to insect bodies inner virus detection technique field further, be specifically related to the method for tomato spotted wilf virus in a kind of quick detection thrips body.
Background technology
Tomato spotted wilf virus is a kind of harmful organism, because of first catch an illness on tomato find and name, the widely dispersed of tomato spotted wilf virus, can encroach on 160 kinds of dicotyledons and 10 kinds of monocotyledons, the crop diseases occurred because infecting this virus is called spotted wilt.
Tomato spotted wilf virus (Tomatospottedwiltvirus, TSWV) is a kind of important virus endangering agricultural production.Its host range is very wide, can encroach on 160 kinds of dicotyledons and 10 kinds of monocotyledons, especially the Activities of Some Plants of Solanaceae, composite family and pulse family.Extensively distribute in multiple countries and regions such as Europe, North America, South America, asia and ocenias, all there is generation the torrid zone, subtropics, Temperate Region in China.Belong to Europe and Mediterranean Plant Protection Organization (EPPO) A2 class quarantine harmful organisms, be also the inward plant quarantine harmful organism of China simultaneously.
The change of tomato spotted wilf virus symptom greatly, catch an illness, and spire becomes in coppery to be rolled up by seedling stage, the many little blackspots of rear formation, leaf back is along the pulse in purple, and some growing points are died, the downright bad streak of stem end formation brown, diseased plant only one side of something grows or downgrades completely or fall leaves in wilting shape, the not result of falling ill morning.Catch an illness after bearing fruit, fruit occurs chlorisis ring spot, green fruit is slightly protruding, and wheel line is not obvious, and Chinese olive produces brown necrotic plaque, in strumae, fruit easily comes off.Ripening fruits catches an illness wheel line obviously, reddish yellow or red and white, and chlorisis spot is obvious in the panchromatic phase, serious full fruit deadlock contracting, and umbilical region symptom is similar to blossom-end rot, but this sick fruit surface browning is downright bad other in blossom-end rot.
Tomato spotted wilf virus route of transmission: juice can be inoculated, seed also can infect, and onion thrips (Thripstabaci), beans thrips (T.Setosus), thrips (Frankliniellaschutzei), tobacco brown thrip (F.Fusca) and alfalfa thrips (F.Occidentatis) etc. all can carry out persistence biography poison in addition.
Thrips can only obtain virus in larval phase.Pass poison to need to breed in vivo, onion thrips could pass poison after becoming adult through 5 ~ 10 days, and onion thrips is the shortest, and to obtain the poison phase be 15 ~ 30 minutes, and beans thrips needs 30 minutes, and the time, long Virus spreading rate raised, and thrips is once be with malicious, and biography poison reaches 20 days, and tool passes malicious ability throughout one's life.Virus inoculation is many to be obtained when the shallow epidermis of tomato leaf table cell is sucked, and generally namely falls ill through 4 days incobations.The exosper such as tomato, cineraria band poison, does not enter embryo.
This virus can be survived the winter by some wild plant species.But viral prevalence depends on insect density.The adult of topmost amboceptor insect thrips is ensconced in soil, and after soil moisture rising in spring, insect can move on weeds.Therefore, develop a kind of method that can detect tomato spotted wilf virus in thrips body to be fast very important.
Summary of the invention
The object of the present invention is to provide the method for tomato spotted wilf virus in a kind of quick detection thrips body.
The object of the present invention is achieved like this, comprises pre-treatment, detection, interpretation of result step, specifically comprise:
A, pre-treatment: be placed on nitrocellulose filter by thrips to be measured, add sample preparation PBS damping fluid, grind, with same method process band poison thrips and nontoxic thrips, and the position of record each sample harmonizing yinyang contrast;
B, detect: after the nitrocellulose filter drying after pre-treatment, be placed in container, add confining liquid submergence 0.5 ~ 1.5h, discard confining liquid, add the tomato spotted wilf virus monoclonal with confining liquid dilution or polyclonal antibody, reaction 0.5 ~ 1.5h, discard tomato spotted wilf virus monoclonal or polyclonal antibody, with PBST buffer solution cellulose nitrate 3 ~ 4 times, add the second antibody of the alkali phosphatase enzyme mark with confining liquid dilution again, reaction 0.5 ~ 1.5h, discard the second antibody of alkali phosphatase enzyme mark, with PBST buffer solution nitrocellulose filter 3 ~ 4 times, add colorbuffer submergence 5 ~ 6min, discard colorbuffer, add nitrite ion reaction 15 ~ 30min, discard nitrite ion and add the reaction of distilled water color development stopping,
C, interpretation of result: can carry out interpretation of result after color development stopping reaction, positive control sample spot that sample solution is put manifests pitchy or redness, when negative control sample spot that sample solution is put changes without color, show that detection normally completes; Detect sample spot that sample solution is put and manifest pitchy or redness, show that this detection sample belt surveys virus to some extent, namely the thrips band in this detection sample surveys virus to some extent, detect sample spot that sample solution is put to change without color, show this detection sample without survey virus, the thrips namely in this detection sample without survey virus.
The present invention is easy to operate, quick, measurement result has good stability, reappearance and accuracy, can meet the detection needs of tomato spotted wilf virus in thrips body, lays a good foundation for avoiding the harm of tomato spotted wilf virus as far as possible.
Accompanying drawing explanation
Fig. 1 is TSWV(tomato spotted wilf virus in single head thrips body of the present invention) testing result schematic diagram;
Fig. 2 is thrips TSWV testing result figure on Kunming periphery tomato, capsicum and weeds.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated, but limited the present invention never in any form, and any conversion done based on training centre of the present invention or replacement, all belong to protection scope of the present invention.
The method of tomato spotted wilf virus in quick detection thrips body of the present invention, comprises pre-treatment, detection, interpretation of result step, specifically comprises:
A, pre-treatment: thrips to be measured is placed on nitrocellulose filter, (PBS damping fluid includes the Proteinase K of 0.01% polysorbas20,0.01 ~ 0.02% polyvinylpyrrolidone, 0.01-0.1M to add sample treatment buffer, pH7.0), grind, with same method process band poison thrips and nontoxic thrips, the position of record each sample harmonizing yinyang contrast;
B, detect: after the nitrocellulose filter drying after pre-treatment, be placed in container, add confining liquid submergence 0.5 ~ 1.5h, discard confining liquid, add the tomato spotted wilf virus monoclonal with confining liquid dilution or polyclonal antibody, reaction 0.5 ~ 1.5h, discard tomato spotted wilf virus monoclonal or polyclonal antibody, with PBST buffer solution cellulose nitrate 3 ~ 4 times, add the second antibody of the tomato spotted wilf virus alkali phosphatase enzyme mark with confining liquid dilution again, reaction 0.5 ~ 1.5h, discard the second antibody of alkali phosphatase enzyme mark, with PBST buffer solution nitrocellulose filter 3 ~ 4 times, add colorbuffer submergence 5 ~ 6min, discard colorbuffer, add nitrite ion reaction 15 ~ 30min, discard nitrite ion and add the reaction of distilled water color development stopping,
C, interpretation of result: can carry out interpretation of result after color development stopping reaction, positive control sample spot that sample solution is put manifests pitchy or redness, when negative control sample spot that sample solution is put changes without color, show that detection normally completes; Detect sample spot that sample solution is put and manifest pitchy or redness, show that this detection sample belt surveys virus to some extent, namely the thrips band in this detection sample surveys virus to some extent, detect sample spot that sample solution is put to change without color, show this detection sample without survey virus, the thrips namely in this detection sample without survey virus.
Described nitrocellulose filter is the nitrocellulose filter of 0.45 μm.
Sample treatment buffer described in step A is the Proteinase K that PBS damping fluid includes 0.01% polysorbas20,0.01 ~ 0.02% polyvinylpyrrolidone, 0.01-0.1M, pH7.0.
Confining liquid described in step B is the PBST damping fluid of mass volume ratio 1 ~ 10% skimmed milk power.
The confining liquid submergence 0.5 ~ 1.5h that adds described in step B adds confining liquid 5 ~ 20ml to flood nitrocellulose filter, then is placed in the incubator 0.5 ~ 1.5h of 28 ~ 37 DEG C.
The tomato spotted wilf virus monoclonal with confining liquid dilution described in step B or polyclonal antibody refer to confining liquid 500 ~ 100000 times of tomato spotted wilf virus monoclonals diluted or polyclonal antibody by volume; The second antibody of the described alkali phosphatase enzyme mark with confining liquid dilution refers to the second antibody with the confining liquid alkali phosphatase enzyme marks of 500 ~ 100000 times of dilutions by volume.
Tomato spotted wilf virus monoclonal or polyclonal antibody that adding described in step B dilutes with confining liquid, reaction 0.5 ~ 1.5h refers to the tomato spotted wilf virus monoclonal or polyclonal antibody that add the dilution of 5 ~ 20ml confining liquid, flood nitrocellulose filter, be placed in 28 ~ 37 DEG C of incubator reaction 0.5 ~ 1.5h; The described second antibody adding the alkali phosphatase enzyme mark with confining liquid dilution, reaction 0.5 ~ 1.5h refers to the second antibody of the alkali phosphatase enzyme mark adding the dilution of 5 ~ 20ml confining liquid, flood nitrocellulose filter, be placed in 28 ~ 37 DEG C of incubator reaction 0.8 ~ 1.2h.
Referring to for 3 ~ 4 times with PBST buffer solution cellulose nitrate described in step B adds PBST damping fluid in the container that nitrocellulose filter is housed, and is placed in 5 ~ 6min that horizontal shaker vibrates, carries out 3 ~ 4 times altogether.
Colorbuffer described in step B is alkaline phosphatase buffer, and its formula is by the NaCl solution 10ml of Tris-HCl damping fluid 10ml, 1mol/L of the pH9.5 of 1mol/L and the MgCl of 1mol/L
2solution 0.5ml, is settled to 100ml with distilled water and obtains; Described nitrite ion is that the NBT getting in 10ml colorbuffer BCIP and the 66 μ l adding 33 μ l stirs and evenly mixs and obtains.
Described NBT solution prepares in dimethyl formamide 0.5gNBT being dissolved in 10ml70%; Described BCIP solution prepares in dimethyl formamide 0.5gBCIP being dissolved in 10ml100%.
The present invention detects the method for tomato spotted wilf virus in 1 or several thrips bodies fast, comprises and detects sample and the preparation of control sample sample solution, detection and interpretation of result operation, specifically comprise:
A, thrips to be detected 1 or several is placed into finish separation (0.3*0.3) nitrocellulose filter on, (PBS damping fluid includes the Proteinase K of 0.01% polysorbas20,0.01 ~ 0.02% polyvinylpyrrolidone, 0.01-0.1M to add 2 μ l sample treatment buffer, pH7.0), with liquid transfer gun head, thrips is ground, process different polypide sample successively; With method process band poison thrips or nontoxic thrips, the position of record each sample harmonizing yinyang contrast.
B, detection: after the nitrocellulose filter drying of above process, be placed in container, add confining liquid submergence 0.5 ~ 1.5h, discard confining liquid, add the tomato spotted wilf virus monoclonal with confining liquid dilution or polyclonal antibody, reaction 0.5 ~ 1.5h, discard tomato spotted wilf virus monoclonal or polyclonal antibody, with PBST buffer solution nitrocellulose filter 3 ~ 4 times, add the second antibody of the alkali phosphatase enzyme mark with confining liquid dilution again, reaction 0.5 ~ 1.5h, discards the second antibody of alkali phosphatase enzyme mark, with PBST buffer solution nitrocellulose filter 3 ~ 4 times.
C, interpretation of result: after nitrocellulose filter has washed, add colorbuffer submergence 5 ~ 6min, discard colorbuffer, add the nitrite ion prepared with colorbuffer, colour developing 15 ~ 30min, discards nitrite ion and add the reaction of distilled water color development stopping; Can carry out interpretation of result after color development stopping reaction, positive control sample spot that sample solution is put manifests pitchy or redness, when negative control sample spot that sample solution is put changes without color, shows that detection normally completes; Detect sample spot that sample solution is put and manifest pitchy or redness, show that this detection sample belt surveys virus to some extent, namely the thrips band in this detection sample surveys virus to some extent, detect sample spot that sample solution is put to change without color, show this detection sample without survey virus, the thrips namely in this detection sample without survey virus.
Thrips polypide described in steps A detects pre-treating method; Polypide sample buffer is the PBS(pH7.0 adding 0.01% polysorbas20, the polyvinylpyrrolidone of 0.01-0.02%, the Proteinase K of 0.01M).Polypide is smashed to pieces on the nitrocellulose filter finishing separation with the 10 μ l pipettor gun heads through sterilizing and dry process.
Nitrocellulose filter described in step B is the nitrocellulose filter of 0.45 μm, and confining liquid is be the skimmed milk power of 4 ~ 6% by the mass percent of buffer.
Described in step B nitrocellulose filter is dry, refers to and nitrocellulose filter is placed in natural drying under room temperature, or it is dry nitrocellulose filter to be placed in 28-37 DEG C of incubator.
Described in step B by after nitrocellulose filter drying, be placed in container, add confining liquid submergence, refer to and nitrocellulose filter is placed in container, add 5 ~ 20ml confining liquid, flood nitrocellulose filter, then container is placed in the closed 0.5 ~ 1.5h of 28-37 DEG C of incubator.
The tomato spotted wilf virus monoclonal with confining liquid dilution described in step B or polyclonal antibody, refer to the tomato spotted wilf virus monoclonal with confining liquid 1:500 ~ 100000 times dilution by volume or polyclonal antibody, the second antibody of the described alkali phosphatase enzyme mark with confining liquid dilution, refers to the second antibody of the alkali phosphatase enzyme mark with confining liquid 1:500 ~ 100000 times dilution by volume.
Tomato spotted wilf virus monoclonal or polyclonal antibody that adding described in step B dilutes with confining liquid, reaction 0.5 ~ 1.5h, refer to the tomato spotted wilf virus monoclonal or polyclonal antibody that add the dilution of 5 ~ 20ml confining liquid, flood nitrocellulose filter, be placed in 37 DEG C of incubator reaction 0.5 ~ 1.5h; The described second antibody adding the alkali phosphatase enzyme mark with confining liquid dilution, reaction 0.5 ~ 1.5h, refer to the second antibody of the alkali phosphatase enzyme mark adding the dilution of 5 ~ 20ml confining liquid, flood nitrocellulose filter, be placed in 37 DEG C of incubator reaction 0.5 ~ 1.5h.
Use buffer solution nitrocellulose filter described in step B 3 ~ 4 times, refers to and add appropriate amount of buffer solution in the container that nitrocellulose filter is housed, and is placed in vibration 5 ~ 6min on horizontal shaker, carries out 3 ~ 4 times altogether.
Colorbuffer described in step C refers to alkaline phosphatase buffer, and its formula is 1mol/LTris-Hcl (pH9.5) 10ml, 1mol/LNacl10ml, 1mol/LMgcl
20.5ml, distilled water polishing is to 100ml; Nitrite ion is formulated as in 10ml colorbuffer and adds 33 μ lBCIP and 66 μ lNBT stir and evenly mix.
The compound method of BCIP and NBT used in the nitrite ion preparation described in step C is as follows:
NBT solution: 0.5gNBT is dissolved in the dimethyl formamide of 10mL70%.
BCIP solution: 0.5gBCIP is dissolved in the dimethyl formamide of 10mL100%.
Embodiment 1
Tomato spotted wilf virus in single head thrips body (
tomatospottedwiltvirus, TSWV) detection:
Agents useful for same is as follows:
Sample treatment buffer: PBS damping fluid includes the Proteinase K of 0.01% polysorbas20,0.01 ~ 0.02% polyvinylpyrrolidone, 0.01-0.1M, pH7.0
Confining liquid: be the skimmed milk power of 5% by the mass percent of buffer.
PBST damping fluid: the PBS containing 0.01-0.1%, pH7.0
Primary antibodie: TSWV rabbit source polyclonal antibody or mouse resource monoclonal antibody
Two resist: viral rabbit source antibody uses the goat-anti rabbit of alkali phosphatase enzyme mark, and viral mouse source antibody uses the sheep anti mouse of alkali phosphatase enzyme mark
Colorbuffer: 1mol/LTris-Hcl(pH9.5)) 10ml, 1mol/LNacl10ml, 1mol/LMgcl
20.5ml, distilled water polishing is to 100ml.
Nitrite ion: add 33 μ lBCIP in 10ml colorbuffer and 66 μ lNBT stir and evenly mix.
NBT solution: 0.5gNBT is dissolved in the dimethyl formamide of 10mL70%.
BCIP solution: 0.5gBCIP is dissolved in the dimethyl formamide of 10mL100%.
A, thrips to be detected 1 or several is placed into finish separation (0.3*0.3) nitrocellulose filter on, add 2 μ l sample treatment buffer, with liquid transfer gun head, thrips ground, process different polypide sample successively; With method process band poison thrips or nontoxic thrips, the position of record each sample harmonizing yinyang contrast.
B, detection: the nitrocellulose filter of above process
Be placed in 37 DEG C of incubator finish-dryings, be then placed in the double dish that diameter is 6cm, add 5ml confining liquid, then double dish is placed in the closed 1.0h of 37 DEG C of incubators.Discard confining liquid, add tomato spotted wilf virus monoclonal or polyclonal antibody that the rare confining liquid of 5ml dilutes, be placed in 37 DEG C of incubators and react 1.0h.Discard tomato spotted wilf virus monoclonal or polyclonal antibody, with buffer solution nitrocellulose filter 3 times, be all placed in vibration washing 5min on horizontal shaker at every turn.Then add the second antibody of the alkali phosphatase enzyme mark of 5ml confining liquid dilution, be placed in 37 DEG C of incubators and react 1.0h.Discard the second antibody of alkali phosphatase enzyme mark, with buffer solution nitrocellulose filter 3 times, be all placed in vibration washing 5min on horizontal shaker at every turn.
C, interpretation of result: after nitrocellulose filter has washed, add colorbuffer submergence 5min, discard colorbuffer, add the nitrite ion prepared with colorbuffer, colour developing 15min, discards nitrite ion and add the reaction of distilled water color development stopping; Can carry out interpretation of result after color development stopping reaction, positive control sample spot that sample solution is put manifests pitchy or redness, when negative control sample spot that sample solution is put changes without color, shows that detection normally completes; Detect sample spot that sample solution is put and manifest pitchy or redness, show that this detection sample belt surveys virus to some extent, namely the thrips band in this detection sample surveys virus to some extent, detect sample spot that sample solution is put to change without color, show this detection sample without survey virus, the thrips namely in this detection sample without survey virus.Testing result is shown in Fig. 1, and detection record and testing result are respectively in table 1 and table 2.
Table 1 detects TSWV sample record table in single head thrips body
| 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| 8 | 9 | 10 | 11 | 12 | Positive control | Negative control |
| 8 | 9 | 10 | 11 | 12 | Positive control | Negative control |
TSWV testing result table in table 2 single head thrips body
| Sample number into spectrum | Testing result |
| 1 | - |
| 2 | + |
| 3 | - |
| 4 | - |
| 5 | - |
| 6 | - |
| 7 | - |
| 8 | + |
| 9 | - |
| 10 | + |
| 11 | - |
| 12 | + |
| Positive control | + |
| Negative control | - |
Note: in table, "+" represents positive, namely detects in sample to be with and surveys virus to some extent; "-" represents negative, namely detect in sample without survey virus.
Test example 1
On Kunming periphery tomato, capsicum and weeds, detect TSWV to collection in thrips and tomato leaf, result shows its detection sensitivity, accuracy and repeatability all better (Fig. 2).
Claims (4)
1. detect a method for tomato spotted wilf virus in thrips body fast, it is characterized in that comprising pre-treatment, detection, interpretation of result step, specifically comprise:
A, pre-treatment: thrips to be measured is placed on nitrocellulose filter, adds sample treatment buffer, grind, with same method process band poison thrips and nontoxic thrips, the position of record each sample harmonizing yinyang contrast; Sample treatment buffer is PBS damping fluid, includes the Proteinase K of 0.01% polysorbas20,0.01 ~ 0.02% polyvinylpyrrolidone, 0.01 ~ 0.1M, pH7.0;
B, detection: after the nitrocellulose filter drying after pre-treatment, be placed in container, add confining liquid 5 ~ 20ml and flood nitrocellulose filter, confining liquid is the PBST damping fluid of mass volume ratio 1 ~ 10% skimmed milk power, PBST damping fluid contains PBS, pH7.0 of 0.01-0.1%, be placed in the incubator 0.5 ~ 1.5h of 28 ~ 37 DEG C, discard confining liquid; Add tomato spotted wilf virus monoclonal or the polyclonal antibody of the dilution of 5 ~ 20ml confining liquid, the tomato spotted wilf virus monoclonal of confining liquid 500 ~ 100000 times of dilutions by volume or polyclonal antibody, flood nitrocellulose filter, be placed in 28 ~ 37 DEG C of incubator reaction 0.5 ~ 1.5h, discard tomato spotted wilf virus monoclonal or polyclonal antibody, with PBST buffer solution nitrocellulose filter 3 ~ 4 times; Add the second antibody of the alkali phosphatase enzyme mark of 5 ~ 20ml confining liquid dilution again, the second antibody of alkali phosphatase enzyme marks of confining liquid 500 ~ 100000 times of dilutions by volume, flood nitrocellulose filter, be placed in 28 ~ 37 DEG C of incubator reaction 0.8 ~ 1.2h, discard the second antibody of alkali phosphatase enzyme mark, with PBST buffer solution nitrocellulose filter 3 ~ 4 times; Add colorbuffer submergence 5 ~ 6min, discard colorbuffer, add nitrite ion reaction 15 ~ 30min, discard nitrite ion and add the reaction of distilled water color development stopping; Colorbuffer is alkaline phosphatase buffer, and its formula is by the NaCl solution 10ml of Tris-HCl damping fluid 10ml, 1mol/L of the pH9.5 of 1mol/L and the MgCl of 1mol/L
2solution 0.5ml, is settled to 100ml with distilled water and obtains; Described nitrite ion is that the NBT getting in 10ml colorbuffer BCIP and the 66 μ l adding 33 μ l stirs and evenly mixs and obtains;
C, interpretation of result: can carry out interpretation of result after color development stopping reaction, positive control sample spot that sample solution is put manifests pitchy or redness, when negative control sample spot that sample solution is put changes without color, show that detection normally completes; Detect sample spot that sample solution is put and manifest pitchy or redness, show that this detection sample belt surveys virus to some extent, namely the thrips band in this detection sample surveys virus to some extent, detect sample spot that sample solution is put to change without color, show this detection sample without survey virus, the thrips namely in this detection sample without survey virus.
2. the method for tomato spotted wilf virus in quick detection thrips body according to claim 1, is characterized in that described nitrocellulose filter is the nitrocellulose filter of 0.45 μm.
3. the method for tomato spotted wilf virus in quick detection thrips body according to claim 1, it is characterized in that referring to for 3 ~ 4 times with PBST buffer solution nitrocellulose filter described in step B adds PBST damping fluid in the container that nitrocellulose filter is housed, be placed in vibration 5 ~ 6min on horizontal shaker, carry out 3 ~ 4 times altogether.
4. the method for tomato spotted wilf virus in quick detection thrips body according to claim 1, is characterized in that described NBT solution prepares in dimethyl formamide 0.5gNBT being dissolved in 10ml70%; Described BCIP solution prepares in dimethyl formamide 0.5gBCIP being dissolved in 10ml100%.
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| CN104498632A (en) * | 2014-12-25 | 2015-04-08 | 山东出入境检验检疫局检验检疫技术中心 | Method for detecting tomato spotted wilf virus based on loop-mediated isothermal amplification technology |
| CN105548544A (en) * | 2016-02-04 | 2016-05-04 | 云南省农业科学院生物技术与种质资源研究所 | Detection kit of hippeastrum chlorotic ringspot viruses (HCRV) as well as detection method and application of detection kit |
| CN105548543A (en) * | 2016-02-04 | 2016-05-04 | 云南省农业科学院生物技术与种质资源研究所 | Kit for detecting yellow spotting virus of peanuts, as well as detection method and application thereof |
| CN107918012A (en) * | 2016-10-11 | 2018-04-17 | 上海市农业科学院 | A kind of immunity detection reagent of muskmelon macula lutea virus and its application |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003102246A1 (en) * | 2002-05-31 | 2003-12-11 | Penn State Research Foundation | Dot-elisa for the detection of animal viruses |
| WO2008103824A1 (en) * | 2007-02-23 | 2008-08-28 | Chinese Academy Of Inspection And Quarantine (Caiq) | Sensitivity-enhanced dot-antibody linked immunogold assay for virus detection |
| CN101363851A (en) * | 2008-09-27 | 2009-02-11 | 云南省农业科学院甘蔗研究所 | Method for detecting sugarcane yellow leaf virus |
| CN101430335A (en) * | 2008-11-13 | 2009-05-13 | 江苏省农业科学院 | Immunity detection reagent kit for rice black-streaked dwarf fijivirus in single-head small brown rice planthopper |
| CN101852807A (en) * | 2010-05-14 | 2010-10-06 | 云南省农业科学院生物技术与种质资源研究所 | Sorghum mosaic virus tri-anti sandwich enzyme-linked immuno sorbent assay kit |
| CN102520185A (en) * | 2011-11-18 | 2012-06-27 | 河南省农业科学院 | Serologic detecting kit for potyvirus on sweet potato and detecting method thereof |
| CN102928598A (en) * | 2012-10-30 | 2013-02-13 | 浙江大学 | Dot-ELISA (dot Enzyme-Linked Immunosorbent Assay) method and tissue printing ELISA method for detecting presence of tomato yellow leaf curl virus in plant as well as reagent kit and application thereof |
| CN102937652A (en) * | 2012-10-30 | 2013-02-20 | 浙江大学 | Dot enzyme-linked immunosorbent assay (ELISA) method for detecting bemisia tabaci carrying tomato yellow leaf curl virus and application of method |
| CN103760346A (en) * | 2014-01-25 | 2014-04-30 | 云南省农业科学院生物技术与种质资源研究所 | Dot fluorescence immunoassay method for quantitatively detecting plant virus |
-
2014
- 2014-09-05 CN CN201410450554.1A patent/CN104198713B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003102246A1 (en) * | 2002-05-31 | 2003-12-11 | Penn State Research Foundation | Dot-elisa for the detection of animal viruses |
| WO2008103824A1 (en) * | 2007-02-23 | 2008-08-28 | Chinese Academy Of Inspection And Quarantine (Caiq) | Sensitivity-enhanced dot-antibody linked immunogold assay for virus detection |
| CN101363851A (en) * | 2008-09-27 | 2009-02-11 | 云南省农业科学院甘蔗研究所 | Method for detecting sugarcane yellow leaf virus |
| CN101430335A (en) * | 2008-11-13 | 2009-05-13 | 江苏省农业科学院 | Immunity detection reagent kit for rice black-streaked dwarf fijivirus in single-head small brown rice planthopper |
| CN101852807A (en) * | 2010-05-14 | 2010-10-06 | 云南省农业科学院生物技术与种质资源研究所 | Sorghum mosaic virus tri-anti sandwich enzyme-linked immuno sorbent assay kit |
| CN102520185A (en) * | 2011-11-18 | 2012-06-27 | 河南省农业科学院 | Serologic detecting kit for potyvirus on sweet potato and detecting method thereof |
| CN102928598A (en) * | 2012-10-30 | 2013-02-13 | 浙江大学 | Dot-ELISA (dot Enzyme-Linked Immunosorbent Assay) method and tissue printing ELISA method for detecting presence of tomato yellow leaf curl virus in plant as well as reagent kit and application thereof |
| CN102937652A (en) * | 2012-10-30 | 2013-02-20 | 浙江大学 | Dot enzyme-linked immunosorbent assay (ELISA) method for detecting bemisia tabaci carrying tomato yellow leaf curl virus and application of method |
| CN103760346A (en) * | 2014-01-25 | 2014-04-30 | 云南省农业科学院生物技术与种质资源研究所 | Dot fluorescence immunoassay method for quantitatively detecting plant virus |
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