CN106226524B - A kind of detection method of edible mushroom dsRNA viruses - Google Patents
A kind of detection method of edible mushroom dsRNA viruses Download PDFInfo
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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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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Abstract
The invention discloses a kind of detection methods of edible mushroom dsRNA viruses, and edible mushroom dsRNA viruses are detected by dot hybridization using dsRNA monoclonal antibodies J2, the method includes：Edible mushroom detects the preparation of sample, sample spot is on hybond membrane, using non-specific DNA binding sites extra on confining liquid quick closure hybond membrane, hybond membrane develops the color after being incubated respectively with dsRNA monoclonal antibodies J2, sheep anti-mouse igg, and visual color variation determines the malicious situation of edible mushroom band.The present invention can detect multiple samples simultaneously, have many advantages, such as easy to operate, rapid and convenient, without special instrument and equipment, positive signal is clear, easily determines.
Description
Technical field
The present invention relates to the detection methods of Mushroom virus, and in particular to a kind of to be passed through using dsRNA monoclonal antibodies J2
The method that dot hybridization detects edible mushroom dsRNA viruses.
Background technology
With the extensive development of edible fungus culturing, the generation of disease is also more and more common, is caused quite to the producer
Big loss.Virosis is to endanger more important a kind of disease of Edible Fungi.Virus disease is endangered with bacterium, fungal disease
Evil is different, and disease symptom has characteristic of hiding, and can just show symptom until the stage of some in production.Therefore, edible mushroom disease
Poison is to agaricus bisporus (Agaricus bisporus), mushroom (Lentinula edodes), oyster mushroom（Pleurotus
ostreatus）, needle mushroom（Flammulina velutipes）A kind of potential danger is constituted etc. main Edible Fungi
Evil.
Virosis is at present the most effective of prevention Mushroom virus disease using nontoxic strain still without effective control means
Measure, thus quickly, easily viral diagnosis means are particularly important in Edible Fungi.Presently found Mushroom virus with
Based on diplornavirus.Not only kind is numerous for edible mushroom, and a kind of edible mushroom may carry a variety of viruses.Therefore, in practical life
In production operation, can detect a variety of viruses simultaneously by a kind of antibody can not only improve the accuracy of detection, but also overcome
One-to-one limitation, greatly reduces testing cost when conventional antibody immune detection.
DsRNA monoclonal antibodies J2 can identify the spiral structure for being more than 40 bp of double-stranded RNA, and the antibody is to double
The identification of chain RNA is formed independent of the sequence or nucleotide of antigen.J2 antibody will not with rRNA, tRNA, or
Viroid RNA are combined, although these RNA also have partially double stranded structure.Due to the universal and development of round pcr, and with height
The feature of sensitivity and specificity, and have the characteristics that immunity is low using nucleic acid as immunogen, result in the latent of its application
Power could not give full play to over the past several decades, therefore also fewer using the application of nucleic acid antibody immunity test virus.With existing
For the improvement of experimental technique, the immunogenicity and specificity of nucleic acid antibody are improved so that nucleic acid antibody is ground in virology
Become in studying carefully more and more extensive.
Viral diagnosis is the important link of virus-free research and avirulent strains selection and breeding.Currently, common Mushroom virus
Detection method has following several：Electronic Speculum observation, prepared using virion or coat protein antiserum carry out Serologic detection,
DsRNA electrophoresis detections technology and PCR methods.Electron microscopy is cumbersome, costly and need special installation, therefore it is in practical life
Application in production is restricted.Polyclonal or monoclonal antibody, which is prepared, using virion or coat protein carries out serology inspection
It surveys, this method is firstly the need of virus is isolated and purified, and when the virus concentration of extraction is relatively low, the antiserum titre of preparation is not
It is very high, and specificity is not also strong, also limits detect virus by immuning hybridization or ELISA using it to a certain extent.Due to
The genome for the Mushroom virus having now been found that is mostly dsRNA, and isolating and purifying dsRNA by agarose electrophoresis detection is
A kind of most common method of virus is detected in laboratory.PCR methods are usually combined with other detection methods, keep detection more accurate.
Such as PCR and dsRNA detections combine.
It can be caused after edible mushroom virus infection under slow mycelial growth, the decline of mycelia vigor, fructification deformity, yield
Drop, or even total crop failure.And the bacterial strain Jing Guo detoxification has obvious raising in mycelia vigor, yield etc..Due to edible mushroom
The research of virus is also relatively backward, and there is presently no effectively preventing methods for virosis.Therefore, quick, easy, accurate, efficient
Detection method be carry out virus precaution and control premise, be the guarantee for obtaining disease-free toxic bacterial strain, detoxification bacterial strain has to pass through
It just can apply to produce after detection determination is virus-free.Therefore it establishes and is hybridized by spot immune using dsRNA monoclonal antibodies J2
Detection Mushroom virus can provide a kind of quick, sensitive method for the detection of Mushroom virus, be the screening of avirulent strains
Easily technology platform is provided with selection and breeding.
Invention content
The purpose of the present invention is to provide a kind of detection method of edible mushroom dsRNA viruses, this method utilizes dsRNA Dan Ke
Grand antibody establishes the dot hybridization system of edible mushroom dsRNA viruses, can produce or cultivate each stage to edible in edible fungus species
The progress of bacterium virus is quick, convenient, efficiently detects, and substantially reduces the potential hazard of Edible Fungi process virosis.
To achieve the above object, the present invention adopts the following technical scheme that：
A kind of detection method of edible mushroom dsRNA viruses is the supernatant point after being homogenized edible mushroom on hybond membrane, is added
Enter non-specific DNA binding sites extra in Block buffer close membrane；Then hybond membrane is resisted with dsRNA monoclonals respectively
Body J2, sheep anti-mouse igg, which are incubated and film are placed in developing solution after rinsing, to develop the color, and observes the color change of film, judges that edible mushroom is
No carrying dsRNA viruses.
The detection method of the edible mushroom dsRNA viruses specifically includes following steps：
（1）Detect the preparation of sample：The edible mushroom sample homogenization that fresh or refrigerator freezing to be detected preserves is obtained even
Slurries, the homogenate centrifuge 1-2 min with the rotating speed of 12000-14000 rpm/min, take supernatant for use；
（2）Point film：According to the appropriately sized film of sample size clip, the film be nitrocellulose filter, nylon membrane or
Film is divided into different grids, the position for distinguishing different samples by pvdf membrane with pencil scribing line（Per about 0.5 cm of grid size
×0.5 cm）, take the supernatant 3-5 μ L point samples centrifuged after above-mentioned homogenate on film, film be positioned over baking oven drying or room temperature certainly
It is so dry；
（3）The closing of film：According to the size of film, the envelope of certain volume is added in culture dish or other appropriate containers
Liquid is closed, one jiao of film is clamped with tweezers, film is placed in confining liquid, confining liquid is made to be totally submerged film, is placed on horizontal shaker
Close 10-15min；
（4）DsRNA monoclonal antibodies are incubated：DsRNA monoclonal antibodies J2 is diluted into 2000- using TBST buffer solutions
It 3000 times, takes out the film after closing and exhausts confining liquid with filter paper, be immediately placed in the dsRNA monoclonal antibodies J2 after dilution, room
Warm jog is incubated 1-1.5 hours or is incubated overnight at 4 DEG C；After incubation, recycling antibody is put in -20 DEG C of refrigerators and preserves, and repeats
It utilizes 2-3 times；
（5）Wash film：After incubation, using PBST buffer solutions as cleaning solution, film is put in cleaning solution and slowly shakes washing 5-
15min after exhausting cleaning solution, adds cleaning solution washing 5-15min, washs 3-4 times altogether；
（6）Secondary antibody is incubated：Secondary antibody selects the sheep anti-mouse igg of alkali phosphatase enzyme mark, with reference to the specification of secondary antibody, according to
Proper proportion dilutes the secondary antibody of alkali phosphatase enzyme mark with TBST buffer solutions, film is immediately placed in the secondary antibody after dilution, room temperature
Jog is incubated 1-1.5 hours；After incubation, two antibody of recycling are put in -20 DEG C of refrigerators and preserve, and repeat and utilize 2-3 times；
（7）Wash film：Using PBST buffer solutions as cleaning solution, film is put in cleaning solution and slowly shakes washing 5-15min, is exhausted
After cleaning solution, cleaning solution washing 5-15min is added, is washed 3-4 times altogether；
（8）Colour developing：Film is put in appropriate BCIP/NBT colorbuffers, colorbuffer lid crosses film, color development at room temperature
10-30min observes the color change of film, judges whether edible mushroom sample carries dsRNA viruses.
Step（1）, the edible mushroom sample is edible mushroom mycelium or fruit body of edible fungi.
Step（1）, the concrete operations of the homogenate are：0.5 g of edible mushroom sample is put into lap tool, 200 μ L are added
TE buffer solutions and a small amount of quartz sand, quickly grinding homogenate obtain homogenate.
Step（1）, the concrete operations of the homogenate are：0.1 g of edible mushroom sample is put in containing 34 mm steel balls of diameter
In 2 mL centrifuge tubes, 50 μ L TE buffer solutions are added, is placed in FastPrep nucleic acid extracting instruments and is homogenized, homogenate speed 4.5K（I.e.
4.5 meter per second）, 15 to 30 s of time.
Step（2）, the drying temperature is 40-80 DEG C..
Step（3）, the confining liquid is QuickBlock confining liquids.
Step（8）If observing purple dot after colour developing, judge that edible mushroom sample carries dsRNA viruses.
Edible mushroom of the present invention is other edible mushrooms such as agaricus bisporus, mushroom, oyster mushroom, needle mushroom, straw mushroom.
The method provided by the invention that Mushroom virus is detected by dot hybridization using dsRNA antibody, detected with Electronic Speculum,
DsRNA electrophoresis, PCR methods detection Mushroom virus are compared, and have that detection time is short, detection flux is high, easily determine, need not be big
The advantages that type equipment, general Edible Fungi unit or research unit of base can meet the experimental condition needed for experiment,
Grass-roots unit can will even send to the good unit of experimental condition after the sample packaging after film and complete detection, be conducive to edible mushroom
A wide range of popularization and application in cultivation.
Compared with prior art, particularly advantage of the invention is as follows：
1, when Electronic Speculum detection Mushroom virus it is cumbersome, costly, to need ultracentrifuge and electron microscope etc. special
Different equipment, general production, research unit do not have testing conditions, and the personnel that Electronic Speculum operation needs Special Training to cross could be complete
At being unfavorable for widely promoting and applying.
2, dsRNA electrophoretic techniques is the current detection common detection means of Mushroom virus, since edible mushroom usually contains
A large amount of polysaccharide often influences the extraction quality of dsRNA, and the viral nucleic acid for extracting high quality is relatively difficult, may electricity when detection
Band of swimming is fuzzy, tends not to obtain clearly viral nucleic acid band when viral level is relatively low, to influence the sensitivity of detection.
Although 3, PCR methods detection Mushroom virus has higher sensitivity and reliability, PCR methods detection operation step
It is rapid cumbersome, it needs first to extract edible mushroom total serum IgE or virus dsRNA, then carries out RT-PCR and expand virus-specific band.The party
Method needs the equipment such as PCR instrument, electrophoresis tank, imaging system, liquid nitrogen and consumptive material, limits the popularization and application of base to a certain extent,
And when detect sample it is more when it is cumbersome, time-consuming, be unfavorable for the quick detection of a large amount of samples.
4, the present invention detects Mushroom virus using dsRNA antibody using dot hybridization, and detection time is shorter, needs sample
It measures less, multiple samples can be detected simultaneously, visual result easily determines（It can be judged according to the variation of color after chromogenic reaction, as a result
It is very clear）, Mushroom virus can be detected in each stage in edible fungus species stage or cultivation production, it is right that virus can be greatly reduced
Harm, the also selection and breeding for the nontoxic strain of edible mushroom provide good detection means caused by Edible Fungi, are edible mushroom
High and stable yields lay the foundation.The required experimental condition of detection method provided by the invention is simple, does not need Electronic Speculum, ultracentrifugation
The large-scale instrument and equipments such as machine, PCR instrument can complete the detection of virus, favorably in general production unit and research unit of base
In the popularization and application of the present invention.
Description of the drawings
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail：
Fig. 1 is the chromogenic reaction result of embodiment 1；Wherein, A1 to A5, B1-B5 are the mushroom fruiting body with poison, C1 to C5
For the malicious shiitake mushroom hypha of band, A6, B6 are respectively that be used as negative control, C6 be fragrant for nontoxic mushroom fruiting body and nontoxic shiitake mushroom hypha
DsRNA is as positive control for mushroom virus.
Specific implementation mode
Test method used in following embodiments is conventional method unless otherwise specified.
Culture medium used in the present invention, reagent are as follows：（All chemical reagent are that analysis is pure）
PDA plate：200 g of potato, 20 g of glucose, 20 g of agar, water 1 L, pH are naturally, 121 DEG C of high pressure sterilizations 30
min；
DsRNA monoclonal antibodies J2 is purchased from English and Scientific Consulting, Hungary companies；
The sheep anti-mouse igg of alkali phosphatase enzyme mark, BCIP/NBT developing solutions are limited purchased from Shanghai life work bioengineering share
Company.
QuickBlock confining liquids (TBSTw) are purchased from the green skies Bioisystech Co., Ltd in Shanghai.
A kind of detection method of edible mushroom dsRNA viruses, specifically includes following steps：
（1）Detect the preparation of sample：The edible mushroom sample that fresh or refrigerator freezing to be detected is preserved（Mycelium or
Fructification）Homogenate obtains homogenate, and the homogenate centrifuges 1-2 min with the rotating speed of 12000-14000 rpm/min, takes supernatant
Liquid is for use；
The concrete operations of the homogenate are：0.5 g of edible mushroom sample is put into lap tool, 200 μ L TE buffer solutions are added
And a small amount of quartz sand, quickly grinding homogenate obtain homogenate.
Or 0.1 g of edible mushroom sample is put in the 2 mL centrifuge tubes containing 34 mm steel balls of diameter, 50 μ L TE are added
Buffer solution is placed in FastPrep nucleic acid extracting instruments and is homogenized, and is homogenized speed 4.5K, 15 to 30 s of time.
（2）Point film：According to the appropriately sized film of sample size clip, the film be nitrocellulose filter, nylon membrane or
Film is divided into different grids, the position for distinguishing different samples by pvdf membrane with pencil scribing line（Per about 0.5 cm of grid size
×0.5 cm）, take the supernatant 3-5 μ L point samples centrifuged after above-mentioned homogenate on film, by film be positioned over 40-80 DEG C of baking oven drying or
Natural drying at room temperature；
（3）The closing of film：According to the size of film, certain volume is added in culture dish or other appropriate containers
QuickBlock confining liquids (TBSTw) clamp one jiao of film with tweezers, film are placed in confining liquid, keeps confining liquid complete
Film is submerged, is placed on horizontal shaker and closes 10-15min；
（4）DsRNA monoclonal antibodies are incubated：DsRNA monoclonal antibodies J2 is diluted into 2000- using TBST buffer solutions
It 3000 times, takes out the film after closing and exhausts confining liquid with filter paper, be immediately placed in the dsRNA monoclonal antibodies J2 after dilution, room
Warm jog is incubated 1-1.5 hours or is incubated overnight at 4 DEG C；After incubation, recycling antibody is put in -20 DEG C of refrigerators and preserves, and repeats
It utilizes 2-3 times；
（5）Wash film：After incubation, using PBST buffer solutions as cleaning solution, film is put in cleaning solution and slowly shakes washing 5-
15min after exhausting cleaning solution, adds cleaning solution washing 5-15min, washs 3-4 times altogether；
（6）Secondary antibody is incubated：Secondary antibody selects the sheep anti-mouse igg of alkali phosphatase enzyme mark, with reference to the specification of secondary antibody, according to
Proper proportion dilutes the secondary antibody of alkali phosphatase enzyme mark with TBST buffer solutions, film is immediately placed in the secondary antibody after dilution, room temperature
Jog is incubated 1-1.5 hours；After incubation, two antibody of recycling are put in -20 DEG C of refrigerators and preserve, and repeat and utilize 2-3 times；
（7）Wash film：Using PBST buffer solutions as cleaning solution, film is put in cleaning solution and slowly shakes washing 5-15min, is exhausted
After cleaning solution, cleaning solution washing 5-15min is added, is washed 3-4 times altogether；
（8）Colour developing：Film is put in appropriate BCIP/NBT colorbuffers, colorbuffer lid crosses film, color development at room temperature 10-
30min observes the color change of film, judges whether edible mushroom sample carries dsRNA viruses.As edible mushroom sample carries double-strand
RNA virus, then chromogenic reaction purple dot can be observed.
Edible mushroom of the present invention is other edible mushrooms such as agaricus bisporus, mushroom, oyster mushroom, needle mushroom, straw mushroom.
Embodiment 1
For detecting Lentinula edodes Virus, using dsRNA monoclonal antibody nexus Dot hybridizations the step of is as follows：
1, the mycelial culture of mushroom strain and collection
In superclean bench by mushroom strain switching in 9cm PDA plates, 25 DEG C of constant temperature incubations scrape perfume after 15 days
Mushroom silk, is directly used in the malicious situation detection of band or -20 DEG C save backup.
2, dot hybridization detects
（1）Detect the preparation of sample：0.1 g of shiitake mushroom hypha of the generation Lentinula edodes Virus disease of freezen protective is put in containing 3
In 2 mL centrifuge tubes of 4 mm steel balls of a diameter, 50 μ L TE buffer solutions are added and are homogenized in FastPrep instrument for extracting nucleic acid（Speed
4.5K, 15 to 30 s of time）, 12000 rpm/min centrifuge 1 min, take supernatant for use.
It takes 0.5 g mushroom fruiting bodies to be put into grinding, 200 μ L TE buffer solutions and a small amount of quartz sand is added, quickly grinds
Homogenate is shifted in homogenate to 1.5 mL centrifuge tubes, and 12000 rpm/min centrifuge 1 min, take supernatant for use.
（2）Point film：Film is divided into different grids with pencil scribing line, is used for by the appropriately sized nitrocellulose filter of clip
The position for distinguishing different samples takes the 5 μ L point samples of supernatant centrifuged after homogenate on film, film is positioned over 80 DEG C of baking oven drying.
（3）The closing of film：The QuickBlock confining liquids (TBSTw) of 15 mL are added in culture dish, are pressed from both sides with tweezers
Firmly one jiao of film, film is placed in confining liquid, confining liquid is made to be totally submerged film, is placed on horizontal shaker and is closed about 10 minutes,
Close non-specific DNA binding sites extra on hybond membrane.
（4）DsRNA monoclonal antibodies are incubated：DsRNA monoclonal antibodies J2 is diluted 3000 first with TBST buffer solutions
Times, it takes out the film after closing and exhausts confining liquid with filter paper, be immediately placed in the antibody diluted, room temperature jog is incubated 1 hour.
（5）Wash film：Film is put in PBST buffer solutions and slowly shakes washing 5 minutes, after exhausting cleaning solution, adds and washes
It washs liquid to wash 5 minutes, wash 3 times altogether.
（6）Secondary antibody is incubated：Secondary antibody selects the sheep anti-mouse igg of alkali phosphatase enzyme mark, dilutes 2000 times with TBST, film is stood
It is put into the secondary antibody diluted, room temperature jog is incubated 1 hour.
（7）Wash film：Film is put in PBST buffer solutions and slowly shakes washing 5 minutes, after exhausting cleaning solution, adds washing
Liquid washs 5 minutes, washs 3 times altogether.
（8）Colour developing：Film is put in 15 mL BCIP/NBT colorbuffers, color development at room temperature 20 minutes, deionization washing
Reaction is terminated after washing, and observes the variation of color.
The results are shown in Figure 1 for chromogenic reaction, is centrifuged after the homogenate of as can be seen from Figure 1 ground and FastPre instruments
With malicious mushroom crude extract, total serum IgE, positive control with malicious mushroom（The Lentinula edodes Virus dsRNA of purifying）In show purple spot
Point, and do not show purple dot without the crude extract and total serum IgE of malicious mushroom.The present invention utilizes dsRNA antibody combination spots
The method whether hybridization check edible mushroom carries dsRNA viruses, detection time is short, and high sensitivity easily determines.
Embodiment 2
For detecting Dual Mushroom mushroom virus, using dsRNA monoclonal antibody nexus Dot hybridizations the step of is as follows：
（1）Detect the preparation of sample：Fresh 0.5 g of Dual Mushroom massee fruiting bodies to be detected is put into lap tool, is added
200 μ L TE buffer solutions and a small amount of quartz sand, quickly grinding homogenate obtain homogenate, and the homogenate is with 14000 rpm/min
Rotating speed centrifuge 2 min, take supernatant for use.
（2）Point film：According to the appropriately sized nylon membrane of sample size clip, film is divided into different sides with pencil scribing line
Lattice, the position for distinguishing different samples（Per about 0.5 cm of cm × 0.5 of grid size）, take the supernatant centrifuged after above-mentioned homogenate
Film is positioned over 40 DEG C of baking oven drying by 3 μ L point samples of liquid on film.
（3）The closing of film：According to the size of film, the QuickBlock confining liquids of certain volume are added in culture dish
(TBSTw), one jiao of film is clamped with tweezers, film is placed in confining liquid, confining liquid is made to be totally submerged film, is placed in horizontal shaker
Upper closing 15min.
（4）DsRNA monoclonal antibodies are incubated：DsRNA monoclonal antibodies J2 is diluted 2000 times using TBST buffer solutions,
It takes out the film after closing and exhausts confining liquid with filter paper, be immediately placed in the dsRNA monoclonal antibodies J2 after dilution, room temperature jog is incubated
It educates 1.5 hours.After incubation, recycling antibody is put in -20 DEG C of refrigerators and preserves, and repeats and utilizes 2-3 times.
（5）Wash film：After incubation, using PBST buffer solutions as cleaning solution, film is put in cleaning solution slowly to shake and is washed
10min after exhausting cleaning solution, adds cleaning solution washing 10min, washs 3-4 times altogether.
（6）Secondary antibody is incubated：Secondary antibody selects the sheep anti-mouse igg of alkali phosphatase enzyme mark, with reference to the specification of secondary antibody, according to
Proper proportion dilutes the secondary antibody of alkali phosphatase enzyme mark with TBST buffer solutions, film is immediately placed in the secondary antibody after dilution, room temperature
Jog is incubated 1.5 hours；After incubation, two antibody of recycling are put in -20 DEG C of refrigerators and preserve, and repeat and utilize 2-3 times.
（7）Wash film：Using PBST buffer solutions as cleaning solution, film is put in cleaning solution and slowly shakes washing 10min, exhaustion is washed
After washing liquid, cleaning solution washing 10min is added, is washed 4 times altogether；
（8）Colour developing：Film is put in appropriate BCIP/NBT colorbuffers, colorbuffer lid crosses film, color development at room temperature
10min observes the color change of film, judges whether edible mushroom sample carries dsRNA viruses.As edible mushroom sample carries double-strand
RNA virus, then chromogenic reaction purple dot can be observed.
Embodiment 3
For detecting needle mushroom virus, using dsRNA monoclonal antibody nexus Dot hybridizations the step of is as follows：
（1）Detect the preparation of sample：0.1 g of golden mushroom mycelium that refrigerator freezing to be detected preserves is put in containing 3
In 2 mL centrifuge tubes of 4 mm steel balls of diameter, 50 μ L TE buffer solutions are added, is placed in FastPrep nucleic acid extracting instruments and is homogenized,
It is homogenized speed 4.5K, 15 to 30 s of time, gained homogenate centrifuges 2 min with the rotating speed of 12000 rpm/min, takes supernatant
For use；
（2）Point film：According to the appropriately sized pvdf membrane of sample size clip, film is divided into different sides with pencil scribing line
Lattice, the position for distinguishing different samples（Per about 0.5 cm of cm × 0.5 of grid size）, take the supernatant centrifuged after above-mentioned homogenate
4 μ L point samples of liquid are on film, by film natural drying at room temperature；
（3）The closing of film：According to the size of film, the QuickBlock confining liquids of certain volume are added in culture dish
(TBSTw), one jiao of film is clamped with tweezers, film is placed in confining liquid, confining liquid is made to be totally submerged film, is placed in horizontal shaker
Upper closing 10min；
（4）DsRNA monoclonal antibodies are incubated：DsRNA monoclonal antibodies J2 is diluted 3000 times using TBST buffer solutions,
It takes out the film after closing and exhausts confining liquid with filter paper, be immediately placed in the dsRNA monoclonal antibodies J2 after dilution, 4 DEG C were incubated
Night；After incubation, recycling antibody is put in -20 DEG C of refrigerators and preserves, and repeats and utilizes 2-3 times；
（5）Wash film：After incubation, using PBST buffer solutions as cleaning solution, film is put in cleaning solution slowly to shake and is washed
15min after exhausting cleaning solution, adds cleaning solution washing 15min, washs 3 times altogether；
（6）Secondary antibody is incubated：Secondary antibody selects the sheep anti-mouse igg of alkali phosphatase enzyme mark, with reference to the specification of secondary antibody, according to
Proper proportion dilutes the secondary antibody of alkali phosphatase enzyme mark with TBST buffer solutions, film is immediately placed in the secondary antibody after dilution, room temperature
Jog is incubated 1.5 hours；After incubation, two antibody of recycling are put in -20 DEG C of refrigerators and preserve, and repeat and utilize 2-3 times；
（7）Wash film：Using PBST buffer solutions as cleaning solution, film is put in cleaning solution and slowly shakes washing 15min, exhaustion is washed
After washing liquid, cleaning solution washing 15min is added, is washed 3 times altogether；
（8）Colour developing：Film is put in appropriate BCIP/NBT colorbuffers, colorbuffer lid crosses film, color development at room temperature
30min observes the color change of film, judges whether edible mushroom sample carries dsRNA viruses.As edible mushroom sample carries double-strand
RNA virus, then chromogenic reaction purple dot can be observed.
Claims (4)
1. a kind of detection method of edible mushroom dsRNA viruses, it is characterised in that：The detection method specifically includes following steps：
（1）Detect the preparation of sample：Edible mushroom sample homogenization to be detected is obtained into homogenate, the homogenate is with 12000-
The rotating speed of 14000 rpm centrifuges 1-2 min, takes supernatant for use；
Wherein, the concrete operations of homogenate are：Edible mushroom sample is put into lap tool, TE buffer solutions and quartz sand, the food is added
Mass volume ratio with bacterium sample and TE buffer solutions is 0.5g：200 μ L, quickly grinding homogenate obtain homogenate；Alternatively, will food
It is put in the 2 mL centrifuge tubes containing 34 mm steel balls of diameter with bacterium sample, TE buffer solutions, edible mushroom sample and TE buffer solutions is added
Mass volume ratio be 0.1g：50 μ L, are placed in FastPrep nucleic acid extracting instruments and are homogenized, and are homogenized speed 4.5K, time 15
To 30 s；
（2）Point film：According to the appropriately sized film of sample size clip, the film is nitrocellulose filter, nylon membrane or pvdf membrane,
Film is divided into different grids with pencil scribing line, the position for distinguishing different samples takes the supernatant centrifuged after above-mentioned homogenate
Film is positioned over baking oven drying or natural drying at room temperature by 3-5 μ L point samples on film；
（3）The closing of film：According to the size of film, the QuickBlock confining liquids of certain volume are added in a reservoir, film is put
It sets in confining liquid, confining liquid is made to be totally submerged film, be placed on horizontal shaker and close 10-15min；
（4）DsRNA monoclonal antibodies are incubated：DsRNA monoclonal antibodies J2 is diluted 2000-3000 times using TBST buffer solutions,
It takes out the film after closing and exhausts confining liquid with filter paper, be immediately placed in the dsRNA monoclonal antibodies J2 after dilution, room temperature jog is incubated
It educates 1-1.5 hours or is incubated overnight at 4 DEG C；
（5）Wash film：After incubation, using PBST buffer solution as cleaning solution, film is put in cleaning solution and slowly shakes washing 5-15min,
After exhausting cleaning solution, cleaning solution washing 5-15min is added, is washed 3-4 times altogether；
（6）Secondary antibody is incubated：Secondary antibody selects the sheep anti-mouse igg of alkali phosphatase enzyme mark, with reference to the specification of secondary antibody, according to appropriate ratio
The secondary antibody of example TBST buffer solutions dilution alkali phosphatase enzyme mark, film is put into the secondary antibody after dilution, room temperature jog is incubated 1-
1.5 hour；
（7）Wash film：Using PBST buffer solutions as cleaning solution, film is put in cleaning solution and slowly shakes washing 5-15min, exhausts washing
After liquid, cleaning solution washing 5-15min is added, is washed 3-4 times altogether；
（8）Colour developing：Film is put in appropriate BCIP/NBT colorbuffers, colorbuffer lid crosses film, color development at room temperature 10-
30min observes the color change of film, judges whether edible mushroom sample carries dsRNA viruses.
2. a kind of detection method of edible mushroom dsRNA viruses according to claim 1, it is characterised in that：Step（1）, institute
It is edible mushroom mycelium or fruit body of edible fungi to state edible mushroom sample, and the edible mushroom is agaricus bisporus, mushroom, oyster mushroom, acupuncture needle
Mushroom or straw mushroom.
3. a kind of detection method of edible mushroom dsRNA viruses according to claim 1, it is characterised in that：Step（2）, institute
It is 40-80 DEG C to state drying temperature.
4. a kind of detection method of edible mushroom dsRNA viruses according to claim 1, it is characterised in that：Step（8）, such as
Purple dot is observed after fruit colour developing, then judges that edible mushroom sample carries dsRNA viruses.
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