CN106226524A - A kind of detection method of edible fungi dsRNA virus - Google Patents
A kind of detection method of edible fungi dsRNA virus Download PDFInfo
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- CN106226524A CN106226524A CN201610531148.7A CN201610531148A CN106226524A CN 106226524 A CN106226524 A CN 106226524A CN 201610531148 A CN201610531148 A CN 201610531148A CN 106226524 A CN106226524 A CN 106226524A
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Abstract
The invention discloses the detection method of a kind of edible fungi dsRNA virus, utilize dsRNA monoclonal antibody J2 by dot blot hybridization detection edible fungi dsRNA virus, described method includes: the preparation of edible fungi detection sample, sample spot is on hybond membrane, utilize non-specific DNA binding site unnecessary on confining liquid quick closure hybond membrane, hybond membrane develops the color after hatching with dsRNA monoclonal antibody J2, sheep anti-mouse igg respectively, and visual color change determines edible cingula poison situation.The present invention can detect multiple sample simultaneously, has easy to operate, rapid and convenient, without special instrument and equipment, the advantage such as positive signal is clear, easily determine.
Description
Technical field
The present invention relates to the detection method of Mushroom virus, be specifically related to one and utilize dsRNA monoclonal antibody J2 to pass through
The method of dot blot hybridization detection edible fungi dsRNA virus.
Background technology
Along with the extensive development of edible fungus culturing, the generation of disease is more universal, causes quite to Producer
Big loss.Virosis is a most important class disease of harm Edible Fungi.Virus disease and antibacterial, fungal disease danger
Evil difference, its disease symptom has characteristic of hiding, and can just show symptom until certain stage in producing.Therefore, edible fungi is sick
Poison is to Agaricus bisporus (Agaricus bisporus), Lentinus Edodes (Lentinula edodes), Pleurotus ostreatus (Pleurotus
Ostreatus), the main Edible Fungi such as Flammulina velutiper (Fr.) Sing (Flammulina velutipes) constitutes a kind of potential danger
Evil.
Virosis does not the most still have effective control device, and using nontoxic strain is the most effective of preventing and treating Mushroom virus disease
Measure, the most quickly, easily Viral diagnosis means particularly important in Edible Fungi.The Mushroom virus having now been found that with
Diplornavirus is main.Edible fungi not only kind is numerous, and a kind of edible fungi may carry multiple virus.Therefore, raw in reality
Produce in operation, multiple virus can be detected by a kind of antibody simultaneously and be possible not only to improve the accuracy of detection, and overcome
During conventional antibody immune detection, man-to-man limitation, greatly reduces testing cost.
DsRNA monoclonal antibody J2 can identify the spiral structure more than 40 bp of double-stranded RNA, and this antibody is to double
The identification of chain RNA does not relies on sequence or the nucleotide composition of antigen.J2 antibody will not be with ribosomal RNA, tRNA, or
Viroid RNA combines, although these RNA also have partially double stranded structure.Due to the universal of round pcr and development, and there is height
Susceptiveness and specific feature, and utilize nucleic acid to have the advantages that immunity is low as immunogenic, result in the latent of its application
Power could not give full play at past many decades, and the application hence with nucleic acid antibody immunity test virus is the most fewer.Along with existing
For the improvement of experimental technique, immunogenicity and the specificity of nucleic acid antibody are improved so that nucleic acid antibody grinds in virusology
Become more and more extensive in studying carefully.
Viral diagnosis is virus-free research and the important step of avirulent strains selection-breeding.At present, conventional Mushroom virus
Detection method has following several: electron microscopic observation, utilize virion or coat protein prepare antiserum carry out Serologic detection,
DsRNA electrophoresis detection technology and PCR method.Electron microscopy complex operation, costly and need special installation, therefore it is raw in reality
Application in product is restricted.Utilize virion or coat protein to prepare polyclone or monoclonal antibody carries out serology inspection
Surveying, this method is firstly the need of isolated and purified virus, when the virus concentration ratio extracted is time relatively low, and the antiserum titre of preparation is not
The highest, and specificity is not the strongest, also limit to a certain extent and utilizes it to pass through immuning hybridization or ELISA detection virus.Due to
The genome of the Mushroom virus having now been found that is mostly dsRNA, detects isolated and purified dsRNA by sepharose electrophoresis and is
A kind of method that in laboratory, detection virus is the most frequently used.PCR method generally combines with other detection method, makes detection more accurate.
Such as PCR and dsRNA detects combination.
Edible fungi infect can cause after virus mycelial growth slowly, mycelia vigor declines, under sporophore deformity, yield
Fall, even has no harvest.And have significantly raising through the bacterial strain of detoxification at aspects such as mycelia vigor, yield.Due to edible fungi
The research of virus is the most relatively backward, and virosis there is presently no effectively preventing method.Therefore, quickly, easy, accurately, efficiently
Detection method be by the premise of virus precaution and control, be the guarantee obtaining virus-free bacterial strain, detoxification bacterial strain has to pass through
Detection determine virus-free after just can apply to produce.Hence set up and utilize dsRNA monoclonal antibody J2 to be hybridized by spot immune
Detection Mushroom virus can be that the detection of Mushroom virus provides a kind of quick, sensitive method, for the screening of avirulent strains
Technology platform easily is provided with selection-breeding.
Summary of the invention
It is an object of the invention to provide the detection method of a kind of edible fungi dsRNA virus, the method utilizes dsRNA Dan Ke
Grand antibody, sets up the dot blot hybridization system of edible fungi dsRNA virus, can produce or cultivate each stage to edible at edible fungus species
Bacterium virus carry out quick, convenient, detect efficiently, be substantially reduced the potential hazard of Edible Fungi process virosis.
For achieving the above object, the present invention adopts the following technical scheme that
A kind of edible fungi dsRNA virus detection method, be by edible fungi be homogenized after supernatant point on hybond membrane, add envelope
Close non-specific DNA binding site unnecessary on buffer blind film；Then by hybond membrane respectively with dsRNA monoclonal antibody
Film is placed in nitrite ion colour developing after hatching and rinsing by J2, sheep anti-mouse igg, observes the color change of film, it is judged that edible fungi whether
Carry dsRNA virus.
The detection method of described edible fungi dsRNA virus specifically includes following steps:
(1) preparation of sample is detected: the edible fungi sample homogenization that fresh or refrigerator freezing to be detected preserves be homogenized
Liquid, described homogenate is centrifuged 1-2 min with the rotating speed of 12000-14000 rpm/min, takes supernatant stand-by；
(2) some film: according to the film that sample size clip is appropriately sized, described film is nitrocellulose filter, nylon membrane or PVDF
Film, is divided into different grids with pencil line by film, for distinguish different sample position (every grid size about 0.5 cm ×
0.5 cm), film, on film, is positioned over oven for drying or room temperature is natural by supernatant 3-5 μ L point sample centrifugal after taking above-mentioned homogenate
It is dried；
(3) closing of film: according to the size of film, add the confining liquid of certain volume in culture dish or other appropriate containers,
Clamp film with tweezers one jiao, is placed on film in confining liquid, makes confining liquid be totally submerged film, is placed on horizontal shaker closing
10-15min；
(4) dsRNA monoclonal antibody is hatched: utilize TBST buffer that dsRNA monoclonal antibody J2 is diluted 2000-3000 times,
Taking out the film filter paper after closing and exhaust confining liquid, be immediately placed in dsRNA monoclonal antibody J2 after dilution, room temperature jog is incubated
Educate 1-1.5 hour or 4 DEG C of overnight incubation；After hatching, reclaim antibody and be put in-20 DEG C of Refrigerator stores, repeatable utilize 2-3
Secondary；
(5) wash film: after hatching, with PBST buffer as cleaning mixture, film be put in cleaning mixture slowly shake washing 5-15min,
After exhausting cleaning mixture, add cleaning mixture washing 5-15min, washing 3-4 time altogether；
(6) two anti-hatch: the sheep anti-mouse igg of two anti-selection alkali phosphatase enzyme marks, with reference to two anti-description, according to suitably
Ratio resists with the two of TBST buffer dilution alkali phosphatase enzyme mark, film is immediately placed in two after dilution anti-in, room temperature jog
Hatch 1-1.5 hour；After hatching, reclaim two antibody and be put in-20 DEG C of Refrigerator stores, repeatable utilize 2-3 time；
(7) wash film: with PBST buffer as cleaning mixture, film is put in cleaning mixture slowly shake washing 5-15min, exhausts washing
After liquid, add cleaning mixture washing 5-15min, washing 3-4 time altogether；
(8) colour developing: being put in by film in appropriate BCIP/NBT colorbuffer, colorbuffer lid crosses film, color development at room temperature 10-
30min, observes the color change of film, it is judged that whether edible fungi sample carries dsRNA virus.
Step (1), described edible fungi sample is edible fungi mycelium or fruit body of edible fungi.
Step (1), the concrete operations of described homogenate are: put in lap tool by edible fungi sample 0.5 g, add 200 μ L
TE buffer and a small amount of quartz sand, quickly grind homogenate and obtain homogenate.
Step (1), the concrete operations of described homogenate are: be put in edible fungi sample 0.1 g containing 3 diameter 4 mm steel balls
In 2 mL centrifuge tubes, adding 50 μ L TE buffer, be placed in FastPrep nucleic acid extracting instrument homogenate, homogenate speed 4.5K(is i.e.
4.5 meter per seconds), time 15 to 30 s.
Step (2), described drying temperature is 40-80 DEG C..
Step (3), described confining liquid is QuickBlock confining liquid.
Step (8), if observing purple dot after Xian Se, then judges that edible fungi sample carries dsRNA virus.
Edible fungi of the present invention is other edible fungi such as Agaricus bisporus, Lentinus Edodes, Pleurotus ostreatus, Flammulina velutiper (Fr.) Sing, Volvariella volvacea (Bull.Ex Franch.) Singer．.
What the present invention provided utilizes dsRNA antibody to pass through the method that dot blot hybridization detects Mushroom virus, detect with Electronic Speculum,
DsRNA electrophoresis, PCR method detection Mushroom virus is compared, have that the detection time is short, detection flux is high, easily determine, need not big
The advantages such as type equipment, general Edible Fungi unit or research unit of basic unit can meet the experimental condition needed for experiment,
Grass-roots unit even can complete detection, beneficially edible fungi by delivering to the good unit of experimental condition after the sample packaging after a film
Popularization and application on a large scale in cultivation.
Compared with prior art, particularly advantage of the invention is as follows:
1, complex operation during Electronic Speculum detection Mushroom virus, costly, need that supercentrifuge and ultramicroscope etc. are special to be set
Standby, general production, research unit do not possess testing conditions, and the personnel that Electronic Speculum operation needs Special Training to cross just can complete,
It is unfavorable for popularization and application widely.
2, dsRNA electrophoretic techniques is the detection means that detection Mushroom virus is conventional at present, owing to edible fungi usually contains
Substantial amounts of polysaccharide, often affects the extraction quality of dsRNA, extracts high-quality viral nucleic acid relatively difficult, may electricity during detection
Swimming band obscures, and tends not to obtain viral nucleic acid band clearly, thus affect the sensitivity of detection when viral level is relatively low.
Although 3, PCR method detection Mushroom virus has higher sensitivity and reliability, but the detection operation of PCR method walks
The most loaded down with trivial details, need first to extract edible fungi total serum IgE or virus dsRNA, then carry out RT-PCR amplicon virus specific band.The party
Method needs equipment and the consumptive materials such as PCR instrument, electrophoresis tank, imaging system, liquid nitrogen, limits the popularization and application of basic unit to a certain extent,
And when detecting sample and being more complex operation, the longest, be unfavorable for the quick detection of a large amount of sample.
4, the present invention utilizes dsRNA antibody to utilize dot blot hybridization to detect Mushroom virus, and the detection time is shorter, need sample
Amount less, multiple sample can be detected simultaneously, visual result easily determine (can according to after chromogenic reaction color change judge, result
Very clear), Mushroom virus can be detected in each stage of edible fungus species stage or cultivation and production, virus can be greatly reduced right
The harm that Edible Fungi causes, also the selection-breeding for the nontoxic strain of edible fungi provides good detection means, for edible fungi
High and stable yields lay the foundation.Experimental condition required for the detection method that the present invention provides is simple, it is not necessary to Electronic Speculum, ultracentrifugation
The large-scale instrument and equipment such as machine, PCR instrument, all can complete the detection of virus, favorably at general production unit and research unit of basic unit
Popularization and application in the present invention.
Accompanying drawing explanation
Below in conjunction with the drawings and specific embodiments, the present invention is further detailed explanation:
Fig. 1 is the chromogenic reaction result of embodiment 1；Wherein, A1 to A5, B1-B5 are the mushroom fruiting body of band poison, and C1 to C5 is band
Poison shiitake mushroom hypha, A6, B6 be respectively nontoxic mushroom fruiting body and nontoxic shiitake mushroom hypha as negative control, and C6 is that Lentinus Edodes is sick
Poison dsRNA is as positive control.
Detailed description of the invention
Test method used in following embodiment if no special instructions, is conventional method.
Culture medium used in the present invention, reagent are as follows: (all chemical reagent are analytical pure)
PDA plate: Rhizoma Solani tuber osi 200 g, glucose 20 g, agar 20 g, water 1 L, pH are natural, 121 DEG C of autoclaving 30 min；
DsRNA monoclonal antibody J2 is purchased from English and Scientific Consulting, Hungary company；
The sheep anti-mouse igg of alkali phosphatase enzyme mark, BCIP/NBT nitrite ion is purchased from Shanghai Sheng Gong biological engineering limited company.
QuickBlock confining liquid (TBSTw) is purchased from the green skies, Shanghai Bioisystech Co., Ltd.
The detection method of a kind of edible fungi dsRNA virus, specifically includes following steps:
(1) preparation of sample is detected: (mycelium or son are real for the edible fungi sample preserved by fresh or refrigerator freezing to be detected
Body) homogenate obtain homogenate, described homogenate is centrifuged 1-2 min with the rotating speed of 12000-14000 rpm/min, takes supernatant and treats
With；
The concrete operations of described homogenate are: put in lap tool by edible fungi sample 0.5 g, add 200 μ L TE buffer and lack
Amount quartz sand, quickly grinds homogenate and obtains homogenate.
Or edible fungi sample 0.1 g is put in the 2 mL centrifuge tubes containing 3 diameter 4 mm steel balls, adds 50 μ L TE
Buffer, is placed in FastPrep nucleic acid extracting instrument homogenate, is homogenized speed 4.5K, time 15 to 30 s.
(2) some film: according to the film that sample size clip is appropriately sized, described film be nitrocellulose filter, nylon membrane or
Pvdf membrane, is divided into different grids with pencil line by film, for distinguishing position (every grid size about 0.5 cm of different sample
× 0.5 cm), take supernatant 3-5 μ L point sample centrifugal after above-mentioned homogenate on film, film is positioned over 40-80 DEG C of oven for drying or
Natural drying at room temperature；
(3) closing of film: according to the size of film, adds certain volume in culture dish or other appropriate containers
QuickBlock confining liquid (TBSTw), clamp film with tweezers one jiao, film is placed in confining liquid, makes confining liquid complete
Submergence film, is placed on horizontal shaker closing 10-15min；
(4) dsRNA monoclonal antibody is hatched: utilize TBST buffer that dsRNA monoclonal antibody J2 is diluted 2000-3000 times,
Taking out the film filter paper after closing and exhaust confining liquid, be immediately placed in dsRNA monoclonal antibody J2 after dilution, room temperature jog is incubated
Educate 1-1.5 hour or 4 DEG C of overnight incubation；After hatching, reclaim antibody and be put in-20 DEG C of Refrigerator stores, repeatable utilize 2-3
Secondary；
(5) wash film: after hatching, with PBST buffer as cleaning mixture, film be put in cleaning mixture slowly shake washing 5-15min,
After exhausting cleaning mixture, add cleaning mixture washing 5-15min, washing 3-4 time altogether；
(6) two anti-hatch: the sheep anti-mouse igg of two anti-selection alkali phosphatase enzyme marks, with reference to two anti-description, according to suitably
Ratio resists with the two of TBST buffer dilution alkali phosphatase enzyme mark, film is immediately placed in two after dilution anti-in, room temperature jog
Hatch 1-1.5 hour；After hatching, reclaim two antibody and be put in-20 DEG C of Refrigerator stores, repeatable utilize 2-3 time；
(7) wash film: with PBST buffer as cleaning mixture, film is put in cleaning mixture slowly shake washing 5-15min, exhausts washing
After liquid, add cleaning mixture washing 5-15min, washing 3-4 time altogether；
(8) colour developing: being put in by film in appropriate BCIP/NBT colorbuffer, colorbuffer lid crosses film, color development at room temperature 10-
30min, observes the color change of film, it is judged that whether edible fungi sample carries dsRNA virus.As edible fungi sample carries double-strand
RNA viruses, then chromogenic reaction can be observed purple dot.
Edible fungi of the present invention is other edible fungi such as Agaricus bisporus, Lentinus Edodes, Pleurotus ostreatus, Flammulina velutiper (Fr.) Sing, Volvariella volvacea (Bull.Ex Franch.) Singer．.
Embodiment 1
As a example by detection Lentinula edodes Virus, the step utilizing dsRNA monoclonal antibody nexus Dot hybridization is as follows:
1, the mycelial cultivation of mushroom strain and collection
Being transferred by mushroom strain in 9cm PDA plate in superclean bench, 25 DEG C of constant temperature culture scrape Lentinus Edodes fungus after 15 days
Silk, be directly used in band poison situation detection or-20 DEG C save backup.
2, dot blot hybridization detection
(1) preparation of sample is detected: be put in containing 3 straight by shiitake mushroom hypha 0.1 g occurring Lentinula edodes Virus sick of freezen protective
In 2 mL centrifuge tubes of footpath 4 mm steel ball, add 50 μ L TE buffer in FastPrep instrument for extracting nucleic acid, be homogenized (speed
4.5K, the time 15 to 30, s) 12000 rpm/min were centrifuged 1 min, take supernatant stand-by.
Take 0.5 g mushroom fruiting body and put in grinding, add 200 μ L TE buffer and a small amount of quartz sand, quickly grind
Homogenate, in transfer homogenate to 1.5 mL centrifuge tubes, 12000 rpm/min are centrifuged 1 min, take supernatant stand-by.
(2) some film: the nitrocellulose filter that clip is appropriately sized, is divided into different grids with pencil line by film, is used for
Distinguishing the position of different sample, film, on film, is positioned over 80 DEG C of oven for drying by supernatant 5 μ L point sample centrifugal after taking homogenate.
(3) closing of film: add the QuickBlock confining liquid (TBSTw) of 15 mL in culture dish, press from both sides with tweezers
Live film one jiao, is placed on film in confining liquid, makes confining liquid be totally submerged film, be placed on horizontal shaker and close about 10 minutes,
Close non-specific DNA binding site unnecessary on hybond membrane.
(4) dsRNA monoclonal antibody is hatched: first with TBST buffer, dsRNA monoclonal antibody J2 is diluted 3000
Times, taking out the film filter paper after closing and exhaust confining liquid, be immediately placed in the antibody diluted, room temperature jog hatches 1 hour.
(5) film is washed: film is put in PBST buffer slowly shake washing 5 minutes, after exhausting cleaning mixture, adds and wash
Wash liquid to wash 5 minutes, washing 3 times altogether.
(6) two anti-hatch: the sheep anti-mouse igg of two anti-selection alkali phosphatase enzyme marks, dilute 2000 times with TBST, are stood by film
I.e. put into two diluted anti-in, room temperature jog hatches 1 hour.
(7) film is washed: film is put in PBST buffer slowly shake washing 5 minutes, after exhausting cleaning mixture, adds washing
Liquid washs 5 minutes, washing 3 times altogether.
(8) colour developing: be put in by film in 15 mL BCIP/NBT colorbuffer, color development at room temperature 20 minutes, deionization is washed
Terminate reaction after washing, observe the change of color.
Chromogenic reaction result is as it is shown in figure 1, the most ground and FastPre instrument is centrifugal after being homogenized
Band poison Lentinus Edodes crude extract, the total serum IgE of band poison Lentinus Edodes, positive control (the Lentinula edodes Virus dsRNA of purification) all show purple speckle
Point, and the crude extract and total serum IgE without poison Lentinus Edodes does not all show purple dot.The present invention utilizes dsRNA antibodies speckle
Whether hybridization check edible fungi carries the method for dsRNA virus, and its detection time is short, highly sensitive, easily determines.
Embodiment 2
As a example by detection Dual Mushroom mushroom virus, the step utilizing dsRNA monoclonal antibody nexus Dot hybridization is as follows:
(1) detect the preparation of sample: put in lap tool by fresh Dual Mushroom massee fruiting bodies 0.5 g to be detected, add 200
μ L TE buffer and a small amount of quartz sand, quickly grind homogenate and obtain homogenate, described homogenate turning with 14000 rpm/min
Centrifugal 2 min of speed, take supernatant stand-by.
(2) some film: according to the nylon membrane that sample size clip is appropriately sized, rules with pencil and film is divided into different sides
Lattice, for distinguishing the position (every grid size about 0.5 cm × 0.5 cm) of different sample, supernatant centrifugal after taking above-mentioned homogenate
Film, on film, is positioned over 40 DEG C of oven for drying by liquid 3 μ L point sample.
(3) closing of film: according to the size of film, add the QuickBlock confining liquid of certain volume in culture dish
(TBSTw), clamp film with tweezers one jiao, film is placed in confining liquid, makes confining liquid be totally submerged film, be placed in horizontal shaker
Upper closing 15min.
(4) dsRNA monoclonal antibody is hatched: utilize TBST buffer that dsRNA monoclonal antibody J2 is diluted 2000 times,
Taking out the film filter paper after closing and exhaust confining liquid, be immediately placed in dsRNA monoclonal antibody J2 after dilution, room temperature jog is incubated
Educate 1.5 hours.After hatching, reclaim antibody and be put in-20 DEG C of Refrigerator stores, repeatable utilize 2-3 time.
(5) wash film: after hatching, with PBST buffer as cleaning mixture, film is put in cleaning mixture and slowly shakes washing
10min, after exhausting cleaning mixture, adds cleaning mixture washing 10min, washing 3-4 time altogether.
(6) two anti-hatch: the sheep anti-mouse igg of two anti-selection alkali phosphatase enzyme marks, with reference to two anti-description, according to
Proper proportion resists with the two of TBST buffer dilution alkali phosphatase enzyme mark, film is immediately placed in two after dilution anti-in, room temperature
Jog hatches 1.5 hours；After hatching, reclaim two antibody and be put in-20 DEG C of Refrigerator stores, repeatable utilize 2-3 time.
(7) washing film: with PBST buffer as cleaning mixture, film is put in cleaning mixture slowly shake washing 10min, exhaustion is washed
After washing liquid, add cleaning mixture washing 10min, washing 4 times altogether；
(8) colour developing: film is put in appropriate BCIP/NBT colorbuffer, colorbuffer lid crosses film, color development at room temperature 10min,
Observe the color change of film, it is judged that whether edible fungi sample carries dsRNA virus.As edible fungi sample carries diplornavirus,
Then chromogenic reaction can be observed purple dot.
Embodiment 3
As a example by detection Flammulina velutiper (Fr.) Sing virus, the step utilizing dsRNA monoclonal antibody nexus Dot hybridization is as follows:
(1) preparation of sample is detected: be put in golden mushroom mycelium 0.1 g that refrigerator freezing to be detected preserves containing 3 diameters
In 2 mL centrifuge tubes of 4 mm steel balls, add 50 μ L TE buffer, be placed in FastPrep nucleic acid extracting instrument homogenate, homogenate
Speed 4.5K, time 15 to 30 s, gained homogenate is centrifuged 2 min with the rotating speed of 12000 rpm/min, takes supernatant stand-by；
(2) some film: according to the pvdf membrane that sample size clip is appropriately sized, with pencil line, film is divided into different grids, uses
In the position (every grid size about 0.5 cm × 0.5 cm) of the different sample of differentiation, supernatant 4 μ L centrifugal after taking above-mentioned homogenate
Point sample is on film, by film natural drying at room temperature；
(3) closing of film: according to the size of film, add the QuickBlock confining liquid of certain volume in culture dish
(TBSTw), clamp film with tweezers one jiao, film is placed in confining liquid, makes confining liquid be totally submerged film, be placed in horizontal shaker
Upper closing 10min；
(4) dsRNA monoclonal antibody is hatched: utilizes TBST buffer that dsRNA monoclonal antibody J2 is diluted 3000 times, takes out
Film filter paper after closing exhausts confining liquid, is immediately placed in dsRNA monoclonal antibody J2 after dilution, 4 DEG C of overnight incubation；
After hatching, reclaim antibody and be put in-20 DEG C of Refrigerator stores, repeatable utilize 2-3 time；
(5) wash film: after hatching, with PBST buffer as cleaning mixture, film is put in cleaning mixture slowly shake washing 15min, inhales
To the greatest extent after cleaning mixture, add cleaning mixture washing 15min, washing 3 times altogether；
(6) two anti-hatch: the sheep anti-mouse igg of two anti-selection alkali phosphatase enzyme marks, with reference to two anti-description, according to suitably
Ratio resists with the two of TBST buffer dilution alkali phosphatase enzyme mark, film is immediately placed in two after dilution anti-in, room temperature jog
Hatch 1.5 hours；After hatching, reclaim two antibody and be put in-20 DEG C of Refrigerator stores, repeatable utilize 2-3 time；
(7) wash film: with PBST buffer as cleaning mixture, film is put in cleaning mixture slowly shake washing 15min, exhausts cleaning mixture
After, add cleaning mixture washing 15min, washing 3 times altogether；
(8) colour developing: film is put in appropriate BCIP/NBT colorbuffer, colorbuffer lid crosses film, color development at room temperature 30min,
Observe the color change of film, it is judged that whether edible fungi sample carries dsRNA virus.As edible fungi sample carries diplornavirus,
Then chromogenic reaction can be observed purple dot.
Claims (10)
1. the detection method of an edible fungi dsRNA virus, it is characterised in that: the supernatant point after being homogenized by edible fungi is in hybridization
On film, add non-specific DNA binding site unnecessary on Block buffer closing membrane；Then by hybond membrane respectively with dsRNA
Film is placed in nitrite ion colour developing after hatching and rinsing by monoclonal antibody J2, sheep anti-mouse igg, observes the color change of film, it is judged that
Whether edible fungi carries dsRNA virus.
The detection method of a kind of edible fungi dsRNA the most according to claim 1 virus, it is characterised in that: described detection side
Method specifically includes following steps:
(1) preparation of sample is detected: edible fungi sample homogenization to be detected is obtained homogenate, and described homogenate is with 12000-
The rotating speed of 14000 rpm/min is centrifuged 1-2 min, takes supernatant stand-by；
(2) some film: according to the film that sample size clip is appropriately sized, described film is nitrocellulose filter, nylon membrane or PVDF
Film, is divided into different grids with pencil line by film, for distinguishing the position of different sample, and supernatant centrifugal after taking above-mentioned homogenate
Film, on film, is positioned over oven for drying or natural drying at room temperature by liquid 3-5 μ L point sample；
(3) closing of film: according to the size of film, add the confining liquid of certain volume in a reservoir, film is placed on confining liquid
In, make confining liquid be totally submerged film, be placed on horizontal shaker closing 10-15min；
(4) dsRNA monoclonal antibody is hatched: utilize TBST buffer that dsRNA monoclonal antibody J2 is diluted 2000-3000 times,
Taking out the film filter paper after closing and exhaust confining liquid, be immediately placed in dsRNA monoclonal antibody J2 after dilution, room temperature jog is incubated
Educate 1-1.5 hour or 4 DEG C of overnight incubation；
(5) wash film: after hatching, with PBST buffer as cleaning mixture, film be put in cleaning mixture slowly shake washing 5-15min,
After exhausting cleaning mixture, add cleaning mixture washing 5-15min, washing 3-4 time altogether.
(6) two anti-hatch: the sheep anti-mouse igg of two anti-selection alkali phosphatase enzyme marks, with reference to two anti-description, according to suitably
Ratio resists with the two of TBST buffer dilution alkali phosphatase enzyme mark, film is put into two after dilution anti-in, room temperature jog is hatched
1-1.5 hour；
(7) wash film: with PBST buffer as cleaning mixture, film is put in cleaning mixture slowly shake washing 5-15min, exhausts washing
After liquid, add cleaning mixture washing 5-15min, washing 3-4 time altogether；
(8) colour developing: being put in by film in appropriate BCIP/NBT colorbuffer, colorbuffer lid crosses film, color development at room temperature 10-
30min, observes the color change of film, it is judged that whether edible fungi sample carries dsRNA virus.
The detection method of a kind of edible fungi dsRNA the most according to claim 1 virus, it is characterised in that: step (1), institute
Stating edible fungi sample is edible fungi mycelium or fruit body of edible fungi, and described edible fungi is Agaricus bisporus, Lentinus Edodes, Pleurotus ostreatus, acupuncture needle
Mushroom or Volvariella volvacea (Bull.Ex Franch.) Singer．.
The detection method of a kind of edible fungi dsRNA the most according to claim 1 virus, it is characterised in that: step (1), institute
The concrete operations stating homogenate are: put in lap tool by edible fungi sample, add TE buffer and quartz sand, quickly grind and are homogenized
To homogenate.
The detection method of a kind of edible fungi dsRNA the most according to claim 4 virus, it is characterised in that: described edible fungi
Sample is 0.5g:200 μ L with the mass volume ratio of TE buffer.
The detection method of a kind of edible fungi dsRNA the most according to claim 1 virus, it is characterised in that: step (1), institute
The concrete operations stating homogenate are: be put in by edible fungi sample in the 2 mL centrifuge tubes containing 3 diameter 4 mm steel balls, add TE buffering
Liquid, is placed in FastPrep nucleic acid extracting instrument homogenate, is homogenized speed 4.5K, time 15 to 30 s.
The detection method of a kind of edible fungi dsRNA the most according to claim 6 virus, it is characterised in that: described edible fungi
Sample is 0.1g:50 μ L with the mass volume ratio of TE buffer.
The detection method of a kind of edible fungi dsRNA the most according to claim 1 virus, it is characterised in that: step (2), institute
State drying temperature and be 40-80 DEG C..
The detection method of a kind of edible fungi dsRNA the most according to claim 1 virus, it is characterised in that: step (3), institute
Stating confining liquid is QuickBlock confining liquid.
The detection method of a kind of edible fungi dsRNA the most according to claim 1 virus, it is characterised in that: step (8), as
Observe purple dot after fruit colour developing, then judge that edible fungi sample carries dsRNA virus.
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