CN105177187B - Detect sample preparation methods and purposes that Curcurbitaceae seed carries cucumber green mottle mosaic virus - Google Patents
Detect sample preparation methods and purposes that Curcurbitaceae seed carries cucumber green mottle mosaic virus Download PDFInfo
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- CN105177187B CN105177187B CN201510661589.4A CN201510661589A CN105177187B CN 105177187 B CN105177187 B CN 105177187B CN 201510661589 A CN201510661589 A CN 201510661589A CN 105177187 B CN105177187 B CN 105177187B
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Abstract
The invention discloses sample preparation methods and purposes that detection Curcurbitaceae seed carries cucumber green mottle mosaic virus (CGMMV).The present invention starts with from processing seed sample, Curcurbitaceae seed is subjected to shell, benevolence separating treatment, remove the lower benevolence part of toxic amount, higher kind of shell parts of toxic amount are left as sample to be tested, treated seed sample is fully ground by the quick beveller of automatic sample, grind sufficient sample both can also can quickly detect the CGMMV in a large amount of Curcurbitaceae seeds by RT PCR methods by dot enzyme-linked immuno absorption method (dot ELISA).The present invention is suitable for the band poison detection of the common Curcurbitaceae seeds such as cucurbit, cucumber, lagenaria sicerariae, pumpkin, long melon, watermelon.The invention detection method high sensitivity, specificity are good, it is short, at low cost to take, and carry the extensive detection of cucumber green mottle virosis for Curcurbitaceae seed and science bridle provides technical support, and are that the detection of other seeds biography plant virus is offered reference.
Description
Technical field
Fields of the present invention are biotechnology, are related to a kind of Curcurbitaceae kind carrying cucumber green mottle mosaic virus
The processing of subsample and serology and molecular biology for detection and application.
Background technology
Cucumber green mottle mosaic virus (Cucumber green mottle mosaic virus, CGMMV) is in nineteen thirty-five
Reported for the first time in Britain, with exchange of the ground family crop between various countries introduce a fine variety increasingly frequently, then Europe Germany, Finland,
There is the harm of the virus in report in succession for multiple countries and regions such as Denmark, Russia, Greece.
Cucumber green mottle mosaic virus is Brassica 2 et 4 section (Tymoviridae) Tobamovirus
(Tobamovirus) member.Ground family crop mainly is infected, the yellowing leafs such as cucurbit, cucumber, watermelon, pumpkin can be caused abnormal
Shape, it is slow-growing, it is as a result delayed, fruit major part yellow bleaches and generates the necrotic plaque of blackish green water scar shape, and yield declines.
CGMMV is the important quarantine virus on many countries and regions ground family crops in the world, and crop field is had resulted in some areas
The crushing loss of melon crop, seriously threatens ground family crop safety in production.In recent years, in China, there is import in some areas
The cucumber green mottle mosaic virus of source property causes the great attention of the relevant departments such as the Ministry of Agriculture, State Administration of Quality Supervision, Inspection and Quarantine.China mouthful in 2002
Bank quarantine departments once from Japan introduce seedling in intercepted the virus, at present the virus be mainly distributed on China Hebei it is new
The some areas such as pleasure, Jiuquan, Pinggu County, beijing, Liaoning Gaizhou City and Guangxi.On December 21st, 2006, Ministry of Agriculture's publication 788
Number bulletin, cucumber green mottle mosaic virus is determined as National Agricultural plant quarantine harmful organism.
CGMMV host ranges are narrow, and natural infection host is mainly ground family crop, pass through artificial juice frictional inoculation
Thorn-Apple, Chenopodium amaranticolor, elder brother's promise lamb's-quarters, the western cigarette of coral, three lives cigarette and petunia can be infected.CGMMV is a kind of RNA virus, typical kind
Virus is passed, is the main source of infection of the virus long-distance communications with seed culture of viruses.Virion invests epidermis and the endotesta of seed
On, primary source of infection is formed after plantation.Kind, which passes host, has cucumber, watermelon, muskmelon and bottle gourd etc., fresh cucumber seed to pass malicious rate and be
8%；It is 1%~5% that watermelon seed, which passes malicious rate, and wherein exocuticle band poison amount is 20 times of endepidermis or endosperm；Muskmelon kind passes poison
Rate is 10%~52%；Bottle gourd seed-transmission rate is up to 84%.In addition, CGMMV also can by the juice of diseased plant contact, pollination and
The modes such as artificial grafting are propagated.
Seed transmission is the main path of cucumber green mottle mosaic virus long-distance communications, once there is Seed transmission in field
Sick seedling, will be spread by non-mediator factor as primary source of infection, cause heavy losses.In addition, cucurbit is China
Many watermelon producing regions are used to graft the main stock of prevention droop, and cucurbit Seed transmission is easy to through graft transmission scion west
Melon seedling causes serious consequence to be diffused into crop field.
Sound seed virus detection method is established, quick, accurate, highly sensitive to the CGMMV based on kind of propagation
Identification and quarantine detection, reinforce the inspection and quarantine work of inward Curcurbitaceae seed, and the incoming of the virus is prevented to have very
Important meaning, also being spread to the diffusion for controlling the virus from source has great importance.
The detection method of CGMMV early stages relies primarily on Biology identification and Electronic Speculum observation, with the development of molecular biology,
The detection technique of the virus is gradually from traditional detection method to the detection technique development based on nucleic acid.At present to the inspection of CGMMV
Survey method has：Biological method, Electron Microscopy, serological technique, Protocols in Molecular Biology etc..It is wherein more commonly used
Detection method is serology and molecular Biological Detection.
Biological assay is although simple and practicable, but required time is grown, heavy workload, and only stablizes by visually observing
Property and it is credible not high.
Using Electron Microscopy observable by the structure change of infected cell and the size of virion and form.
Since electron microscope technique detection needs very high hardware requirement, layman is difficult to complete, thus this method is suitable only for
Auxiliary identification.
Protocols in Molecular Biology is to detect virus from viral nucleic acid level, and common Protocols in Molecular Biology is mainly
RT-PCR detect, RT-PCR technology quickly, simplicity, high sensitivity, high specificity and required sample it is few.But due to the malicious cucurbit of band
Section's seed fat content itself is excessively high so the viral level for being difficult to extract the RNA of high quality or CGMMV is too low is not enough to generate
RT-PCR method is used in combination so using the RNA of conventional Curcurbitaceae seed of the liquid nitrogen grinding method extraction with poison in enough templates
It is detected, tends not to amplification and arrive purpose band.So the present invention can to being handled with malicious Curcurbitaceae seed sample
Improve the concentration of viral template.
Serological method is to detect the virus using specialization antiserum, has high sensitivity, high specificity, operation letter
The advantages that single, can detect 1~10ng/mL virus Serology tests, there are many forms, also commonly use ELISA at present and detect skill
Art detects the band poison situation of Curcurbitaceae seed.
It is to control the incoming premise of the virus to prevent CGMMV from being spread by seed trade and remote allocation and transportation, reinforces calabash
The detection of Lu Ke crop seeds is the key that prevent the disease from seed source diffusion flow row.The climing of the disease is controlled from source
Prolong, to protecting the safety in production of watermelon, muskmelon industry to be of great significance.
Invention content
The purpose of the present invention is overcome the deficiencies of the prior art and provide detection Curcurbitaceae seed to carry cucumber green mottle floral leaf
The sample preparation methods of virus and its application.
A kind of detection Curcurbitaceae seed carries the sample preparation methods of cucumber green mottle mosaic virus, includes the following steps：
1) separation of seed sample kind Renhe kind shell：Every Curcurbitaceae seed is laterally splitted with eye scissors, is divested
Remaining kind of shell is collected in benevolence part；
2) grinding of sample：The kind shell detached using 1 seed is put into as 1 sample that have put 3-5 diameter 2.5mm solid
In the 1.5-2ml eppendorf centrifuge tubes of steel ball, centrifuge tube lid is covered, is lost into liquid after being marked on centrifuge tube
Freezing processing is pulled out after 1 minute in nitrogen, and being then placed in Shanghai, reliable industry Development Co., Ltd article No. is Tissuelyser-48 only
The quick beveller of automatic sample in ground 60 seconds with frequency 60HZ, make sample in powdered, if sample is in powder
The repeatable grinding of shape is primary；
3) step includes the coarse extraction of cucumber green mottle mosaic virus or the extraction two of cucumber green mottle mosaic virus RNA
Kind processing：
The coarse extraction step of cucumber green mottle mosaic virus is：0.1-1ml is added in ground sample
PH7.40.01mol/L PBS phosphate buffers are vortexed in the VortexGenie2 of Scientific Industries companies
Mixing 0.5-5 minutes on mixer, then 5000rpm takes supernatant as cucumber green mottle mosaic virus after centrifuging 3 minutes
Crude extract carries out serological method and detects cucumber green mottle mosaic virus；
Or the extraction step of cucumber green mottle mosaic virus RNA is：After 1mlTRIzol reagents are added in ground sample
Viral RNA is extracted with conventional TRIzol reagents method, the viral RNA of extraction is used for the Molecular Detection of cucumber green mottle mosaic virus.
On the basis of above-mentioned sample preparation methods, the invention also discloses a kind of use of the sample prepared using this method
On the way, specially the crude extract of the cucumber green mottle mosaic virus of this method preparation and RNA are respectively used in Curcurbitaceae seed
The serology and molecular method of cucumber green mottle mosaic virus detect.
The serological method includes dot-ELISA, ACP-ELISA, DAS-ELISA and TAS-ELISA.
The dot-ELISA Serology tests, include the following steps：
1) the 2.5 μ l of crude extract that cucumber green mottle mosaic virus is obtained in claims 1 are drawn, point sample is in nitric acid fibre
The plain film of dimension, and positive negative control is set, it is stored at room temperature and dries 10 minutes；
2) nitrocellulose filter is immersed in the PBST confining liquids containing 5% skimmed milk power and closes 30 minutes for 37 DEG C；
3) above-mentioned nitrocellulose filter is put into containing 1:In 5000 times of diluted anti-cucumber green mottle mosaic virus mouse monoclonal antibodies, 37
DEG C be incubated 1-2 hours；
4) nitrocellulose filter shakes washing 3 times with PBST buffer solutions, 3-5 minutes each；
5) nitrocellulose filter is put into containing 1:The alkaline phosphatase mark that 8000 times of diluted Sigma companies article No.s are A3562
In the sheep anti-mouse igg secondary antibody of note, 37 DEG C are incubated 1-2 hours；
6) nitrocellulose filter shakes washing 4 times with PBST buffer solutions, then is washed 1 time with PBS, 3-5 minutes each；
7) BCIP 33ul, the NBT 66ul that Promega companies article No. is S3771 are added in the colorbuffer of 10ml,
By its mixing, nitrocellulose filter is immersed room temperature and is protected from light colour developing 5-15 minutes after drying；
8) when purple is presented in positive and negative control does not develop the color, then the complete nitrocellulose filter that will develop the color moves to
Color development stopping reaction is rinsed in deionized water, records result.
The molecular method detection includes RT-PCR and quantitative RT-PCR.
The RT-PCR detection method includes the following steps：
1) according to the 5 of the CGMMV reported in GenBank isolate CP gene conserved sequence design primers, sense primer
Primer F:5 '-CTTACAATCCGATCACACCTAG-3 ', downstream primer Primer R:5’-
CTAAGCTTTCGAGGTGGTAGC-3 ', amplified fragments size are 480bp, and primer is closed by Shanghai Ying Jun Bioisystech Co., Ltd
At；
2) denaturation of CGMMV RNA：Cucumber green mottle mosaic virus RNA will be obtained in claims 1 in 65 DEG C of conditions
Lower thermal denaturation after five minutes, it is for use to be immediately placed in cooled on ice；
3) TOYOBO companies article No. is added in reverse transcription in AXYGEN companies article No. is the PCR pipe of 33713383 200ul
For the reagent of 451100 kits, primer and the CGMMV of denaturation RNA:2ul5X RT Buffer、0.5ul RT Enzyme
The CGMMV RNA that Mix, 0.5ul Primer R, 6ul Nuclease-free Water, 1ul have been denaturalized, in PCR instrument after mixing
37 DEG C of 15 minutes reverse transcription reactions of upper progress, the product of 98 DEG C of 5 minutes enzyme inactivation reactions, generation is CGMMV cDNA；
4) PCR reacts：Following reacted constituent is added in the PCR pipe of 200ul:
2X Power Taq PCR Master Mix, the CGMMV that 12.5ul Bioteke companies article No. is 0020150709
Each 0.5ul, 1ul reverse transcription CGMMV cDNA of Primer R and Primer F primers, 10.5ul ddH2O, centrifugal drying one after mixing
Under, carry out PCR in PCR instrument, response parameter is 94 DEG C of pre-degenerations 5 minutes；94 DEG C are denaturalized 30 seconds, and 52 DEG C are annealed 30 seconds, 72 DEG C
Extend 30 seconds, recycles 30 times；Last 72 DEG C 10 minutes；
5) interpretation of result：Amplified production divides through 1% agarose gel electrophoresis, by the amplified production for detecting sample with DNA
Son amount Marker and the positive control with malicious CGMMV are compared, and it is inspection to have with the sample of positive control band in the same size
It is positive to survey result, the sample of specific band is not feminine gender, and photographs to record testing result.
The present invention passes through the detection to seed different parts viral level, it is determined that the kind shell parts toxic amount of seed is most
Then Curcurbitaceae seed to be measured is carried out separation of hull from kernel processing by height, collect higher kind of shell parts of toxic amount.Then it uses entirely certainly
Dynamic grinding instrument replaces traditional liquid nitrogen grinding mode, and quickly, efficiently, labour-saving has been prepared can both be examined with RT-PCR technology
It surveys, and can be used for the Curcurbitaceae seed sample of dot-ELISA detections.The invention can carry out the band poison situation of Curcurbitaceae seed
Investigation reduces the virus using seed as the remote diffusion in source prevalence, can be applied to Curcurbitaceae Seed transmission situation and quickly examine
It surveys.The detection method result is reliable, rapidly and efficiently, it is sensitive special, high-throughput and easy to operate, be cucumber green mottle mosaic virus
Quick diagnosis, port quarantine and the prevention and control of disease provide substance and technical support, and the detection for other Seed-borne Virus seeds provides
It uses for reference, lays the foundation for the prevention and control of Curcurbitaceae Seed-borne Virus.
The present invention has the advantage that compared with prior art：
(1) the present invention provides a kind of samples carrying cucumber green mottle mosaic virus specifically for detection Curcurbitaceae seed
Preparation method remains the higher part of toxic amount in seed, reduces by carrying out shell, benevolence separating treatment to Curcurbitaceae seed
Present in the benevolence high fat content the problems such as due to missing inspection situation that occurs, be also RT-PCR detections, dot-ELISA detection
Provide the sample to be tested of high quality.
(2) present invention saves manpower than traditional by full-automatic grinding instrument with liquid nitrogen hand lapping, improves speed
Rate shortens detection cycle.Grinding sufficient sample both can be by RT-PCR method, can also be quick by dot-ELISA
The CGMMV in Curcurbitaceae seed is detected, a large amount of Curcurbitaceae seed sample can be detected simultaneously, it is high-throughput and time saving, suitable for quarantine
Personnel promote and apply in seed trade, remote allocation and transportation and port quarantine；
(3) Serologic detection is combined by the present invention with molecular Biological Detection, and testing result is true and reliable, can be accurate
The band poison rate for calculating Curcurbitaceae seed carries extensive detection and the science bridle of cucumber green mottle virosis for Curcurbitaceae seed
Technical support is provided, and the detection for other seeds biography plant virus is offered reference.To controlling the climing of the disease from seed source
Prolong, ensure the safety in production of the industries such as watermelon, muskmelon, pumpkin, it is quick-fried to prevent prevalence of the cucumber green mottle mosaic virus disease in field
Send out significant.
Description of the drawings
Fig. 1 cucurbit seed-transmission rate dot-ELISA testing results；
Fig. 2 cucurbit seed-transmission rate RT-PCR testing results.
Specific implementation mode
A kind of detection Curcurbitaceae seed carries the sample preparation methods of cucumber green mottle mosaic virus, includes the following steps：
1) separation of seed sample kind Renhe kind shell：Every Curcurbitaceae seed is laterally splitted with the small scissors of ophthalmology, is shelled
Except benevolence part, remaining kind of shell is collected；
2) grinding of sample：The kind shell detached using 1 seed is put into as 1 sample that have put 3-5 diameter 2.5mm solid
In the 1.5-2ml eppendorf centrifuge tubes of small steel ball, cover centrifuge tube lid, lost after being marked on centrifuge tube into
Freezing processing is pulled out after 1 minute in liquid nitrogen, and being then placed in Shanghai, reliable industry Development Co., Ltd article No. is Tissuelyser- only
It is ground 60 seconds with frequency 60HZ in the 48 quick beveller of automatic sample, it is in powdered to make sample, if sample is in powder
The repeatable grinding of last shape is primary；
3) step includes the coarse extraction of cucumber green mottle mosaic virus or the extraction two of cucumber green mottle mosaic virus RNA
Kind processing：
The coarse extraction step of cucumber green mottle mosaic virus is：0.1-1ml will be added in the ground sample of step 2)
PH7.40.01mol/L PBS phosphate buffers are vortexed in the VortexGenie2 of Scientific Industries companies
Mixing 0.5-5 minutes on mixer, then 5000rpm takes supernatant as cucumber green mottle mosaic virus after centrifuging 3 minutes
Crude extract carries out serological method and detects cucumber green mottle mosaic virus；
Or the extraction step of cucumber green mottle mosaic virus RNA is：It will be added in the ground sample of step 2)
Viral RNA is extracted with routine TRIzol reagent methods after 1mlTRIzol reagents, the viral RNA of extraction is used for cucumber green mottle floral leaf
The Molecular Detection of virus.
On the basis of above-mentioned sample preparation methods, the invention also discloses a kind of use of the sample prepared using this method
On the way, specially the crude extract of the cucumber green mottle mosaic virus of this method preparation and RNA are respectively used in Curcurbitaceae seed
The serology and molecular method of cucumber green mottle mosaic virus detect.
The serological method includes dot-ELISA, ACP-ELISA, DAS-ELISA and TAS-ELISA.
The dot-ELISA Serology tests, include the following steps：
1) the 2.5 μ l of crude extract that cucumber green mottle mosaic virus is obtained in claims 1 are drawn, point sample is in nitric acid fibre
The plain film of dimension, and positive negative control is set, it is stored at room temperature and dries 10 minutes；
2) nitrocellulose filter is immersed in the PBST confining liquids containing 5% skimmed milk power and closes 30 minutes for 37 DEG C；
3) above-mentioned nitrocellulose filter is put into containing 1:In 5000 times of diluted anti-cucumber green mottle mosaic virus mouse monoclonal antibodies, 37
DEG C be incubated 1-2 hours；
4) nitrocellulose filter shakes washing 3 times with PBST buffer solutions, 3-5 minutes each；
5) nitrocellulose filter is put into containing 1:The alkaline phosphatase mark that 8000 times of diluted Sigma companies article No.s are A3562
In the sheep anti-mouse igg secondary antibody of note, 37 DEG C are incubated 1-2 hours；
6) nitrocellulose filter shakes washing 4 times with PBST buffer solutions, then is washed 1 time with PBS, 3-5 minutes each；
7) BCIP 33ul, the NBT 66ul that Promega companies article No. is S3771 are added in the colorbuffer of 10ml,
By its mixing, nitrocellulose filter is immersed room temperature and is protected from light colour developing 5-15 minutes after drying；
8) when purple is presented in positive and negative control does not develop the color, then the complete nitrocellulose filter that will develop the color moves to
Color development stopping reaction is rinsed in deionized water, records result.
The molecular method detection includes RT-PCR and quantitative RT-PCR.
The RT-PCR detection method includes the following steps：
1) according to the 5 of the CGMMV reported in GenBank isolate CP gene conserved sequence design primers, sense primer
Primer F:5 '-CTTACAATCCGATCACACCTAG-3 ', downstream primer Primer R:5’-
CTAAGCTTTCGAGGTGGTAGC-3 ', amplified fragments size are 480bp, and primer is closed by Shanghai Ying Jun Bioisystech Co., Ltd
At；
2) denaturation of CGMMV RNA：Cucumber green mottle mosaic virus RNA will be obtained in claims 1 in 65 DEG C of conditions
Lower thermal denaturation after five minutes, it is for use to be immediately placed in cooled on ice；
3) TOYOBO companies goods is added in reverse transcription in AXYGEN companies article No. is the PCR tubule of 33713383 200ul
Number be 451100 kits reagent, primer and the CGMMV of denaturation RNA:2ul5X RT Buffer、0.5ul RT Enzyme
The CGMMV RNA that Mix, 0.5ul Primer R, 6ul Nuclease-free Water, 1ul have been denaturalized, in PCR instrument after mixing
37 DEG C of 15 minutes reverse transcription reactions of upper progress, the product of 98 DEG C of 5 minutes enzyme inactivation reactions, generation is CGMMV cDNA；
4) PCR reacts：Following reacted constituent is added in the PCR tubules of 200ul:
2X Power Taq PCR Master Mix, the CGMMV that 12.5ul Bioteke companies article No. is 0020150709
Each 0.5ul, 1ul reverse transcription CGMMV cDNA of Primer R and Primer F primers, 10.5ul ddH2O, centrifugal drying one after mixing
Under, carry out PCR in PCR instrument, response parameter is 94 DEG C of pre-degenerations 5 minutes；94 DEG C are denaturalized 30 seconds, and 52 DEG C are annealed 30 seconds, 72 DEG C
Extend 30 seconds, recycles 30 times；Last 72 DEG C 10 minutes；
5) interpretation of result：Amplified production divides through 1% agarose gel electrophoresis, by the amplified production for detecting sample with DNA
Son amount Marker and the positive control with malicious CGMMV are compared, and it is inspection to have with the sample of positive control band in the same size
It is positive to survey result, the sample of specific band is not feminine gender, and photographs to record testing result.
With reference to embodiment and attached drawing, the invention will be further described.
The ACP-ELISA detections of embodiment one, Curcurbitaceae seed different parts viral level
20, cucurbit seed for taking the band poison of laboratory preservation, is laterally splitted seed with the small scissors of ophthalmology, by benevolence
Part and kind shell parts are separated.The kind shell and benevolence of same grain seed place them into mortar use respectively as one group
Liquid nitrogen is fully ground.Ground sample is stored at room temperature, dilute with 30 times of 0.05M carbonic acid buffers (PH9.6) after band liquid nitrogen volatilization
It releases, then 5000rpm, centrifugation takes the kind shell and benevolence grinding supernatant of same grain seed after 3 minutes, left and right is adjacent to be separately added into
Positive, negative control is arranged per hole 100ul in elisa plate.4 DEG C of coatings are added the PBS containing 3% skimmed milk power after overnight and close
Liquid is closed 30 minutes for 37 DEG C per hole 250ul, and elisa plate is patted dry on gauze.5000 times of diluted CGMMV 8E3 mouse monoclonal antibodies
As primary antibody elisa plate is added per hole 100ul in ascites, 37 DEG C be incubated 1 hour after with PBST buffer solutions wash 3 times 3 minutes every time,
Elisa plate is patted dry on gauze.The sheep anti-mouse igg of 8000 times of dilution alkali phosphatase enzyme marks is that secondary antibody addition elisa plate is every
Hole 100ul, 37 DEG C be incubated 1 hour after with PBST buffer solutions wash 4 times 3 minutes every time, elisa plate is patted dry on gauze.With
Para-nitro-pheneye phosphate (PNPP) substrate develops the color, per hole 100ul.OD is read with 680 microplate reader of BIO-RAD405Value, P/N>3.0 being
Positive criterion is (with specific reference to Shang et al.Virology Journal 2011,8:228 documents).Test result is aobvious
Show that the kind shell lapping liquid of 20 seeds is all positive, and the benevolence lapping liquid of only 12 seeds is positive, and these
The OD of benevolence405Value is all than the kind shell OD of same grain seed405It is worth much lower.According to test result it can be found that carrying CGMMV
The kind shell of Curcurbitaceae seed show stronger positive reaction than benevolence, illustrate that the kind shell of Curcurbitaceae seed is more suitable for doing than benevolence
It detects in spite of illness.
Embodiment two, the observation of the Electronic Speculum of Curcurbitaceae seed different parts viral level
20, cucurbit seed for taking the band poison of laboratory preservation, is laterally splitted seed with the small scissors of ophthalmology, by benevolence
Part and kind shell parts are separated.The kind shell and benevolence of same grain seed place them into mortar use respectively as one group
Liquid nitrogen is fully ground.Ground sample is stored at room temperature, and after band liquid nitrogen volatilization, is diluted for 10 times with distilled water, then 5000rpm,
It is CGMMV virus crude extracts to centrifuge 3 minutes supernatants.Virus purification liquid through 2% phosphotungstic acid (phosphotungstic acid,
PTA) (pH 6.7) is dyed and is observed particle shape under postposition JEOLJEM-1200EX Electronic Speculum (JEOL, Japan), Electronic Speculum as a result, it has been found that
The elongated rod shape CGMMV virion of some long 300nm is can see in the kind shell sample crude extract of same grain seed, and is being planted
Minority is can only see in the sample crude extract of benevolence or does not observe the particle of these elongated rod shapes.Test result illustrates Curcurbitaceae kind
The kind shell parts carrying CGMMV of son is more compared with benevolence part, this result also obtained with embodiment one matches.
Embodiment three, detection Curcurbitaceae seed carry the sample preparation methods Curcurbitaceae seed of cucumber green mottle mosaic virus
The preparation of sample includes the following steps：
1) separation of seed sample kind Renhe kind shell：Every Curcurbitaceae seed is laterally splitted with the small scissors of ophthalmology, is shelled
Except benevolence part, remaining kind of shell is collected；
2) grinding of sample：The kind shell detached using 1 seed is put into as 1 sample that have put 3-5 diameter 2.5mm solid
In the 1.5-2ml eppendorf centrifuge tubes of small steel ball, cover centrifuge tube lid, lost after being marked on centrifuge tube into
Freezing processing is pulled out rapidly after 1 minute in liquid nitrogen, and being then placed in Shanghai, reliable industry Development Co., Ltd article No. is only
Frequency 60HZ is ground 60 seconds in the quick beveller of automatic sample of Tissuelyser-48, and it is in powdered to make sample, if sample
Product are not primary in powdered repeatable grinding；
3) coarse extraction of cucumber green mottle mosaic virus：0.1-1mlpH7.40.01mol/L is added in ground sample
PBS phosphate buffers shake mixing on the VortexGenie2 turbine mixers of Scientific Industries companies
0.5-5 minutes, then 5000rpm, centrifugation took supernatant to carry out serological method detection cucumber green mottle mosaic virus after 3 minutes；
Or the extraction of cucumber green mottle mosaic virus RNA：Routine is used after 1mlTRIzol reagents are added in ground sample
TRIzol reagent methods extract viral RNA, and the viral RNA of extraction is used for the Molecular Detection of cucumber green mottle mosaic virus.
Example IV, detection Curcurbitaceae seed carry the dot-ELISA serological methods of cucumber green mottle mosaic virus
Detecting step is as follows：
1) 22, cucurbit seed is taken to prepare Curcurbitaceae seed sample with three the method for embodiment；
2) the 2.5 μ l of crude extract of cucumber green mottle mosaic virus are drawn, sun is arranged in nitrocellulose filter in point sample
Property, negative control, are stored at room temperature and dry 10 minutes；
3) nitrocellulose filter is immersed in the PBST confining liquids containing 5% skimmed milk power and closes 30 minutes for 37 DEG C；
4) above-mentioned nitrocellulose filter is put into containing 1:In 5000 times of diluted anti-CGMMV 8E3 mouse monoclonal antibodies, 37 DEG C
It is incubated 1-2 hours；
5) nitrocellulose filter shakes washing 3 times with PBST buffer solutions, 3-5 minutes each；
6) nitrocellulose filter is put into containing 1:The alkaline phosphatase mark that 8000 times of diluted Sigma companies article No.s are A3562
In the sheep anti-mouse igg secondary antibody of note, 37 DEG C are incubated 1-2 hours；
7) nitrocellulose filter shakes washing 4 times with PBST buffer solutions, then is washed 1 time with PBS, 3-5 minutes each；
8) BCIP 33ul, the NBT 66ul that Promega companies article No. is S3771 are added in the colorbuffer of 10ml,
By its mixing, nitrocellulose filter is immersed room temperature and is protected from light colour developing 5-15 minutes after drying；
9) when positive is presented purple and negative control and does not develop the color, then by the complete nitrocellulose filter of chromogenic reaction
It moves in deionized water and rinses color development stopping reaction, record result.
By the early elegant kind 50g of the cucurbit seed of Hangzhou plant quarantine station inspection, 22 are therefrom randomly selected.Use embodiment
Three the methods prepare Curcurbitaceae seed sample；The band poison rate (malicious rate=number positive/total number of samples of band of seed is calculated according to result
× 100%).In 22 cucurbit seeds of testing result display detection, there are 20 to be positive (Fig. 1), be computed, this 22
The band poison rate of cucurbit seed is 90.91%.
Embodiment five, detection Curcurbitaceae seed carry the RT-PCR molecular methods of cucumber green mottle mosaic virus
It is as follows：
1) according to the 5 of the CGMMV reported in GenBank isolate CP gene conserved sequence design primers, sense primer
Primer F:5 '-CTTACAATCCGATCACACCTAG-3, downstream primer Primer R:5’-
CTAAGCTTTCGAGGTGGTAGC-3, amplified fragments size are 480bp, and primer is closed by Shanghai Ying Jun Bioisystech Co., Ltd
At；
2) with 22 Curcurbitaceae seeds in example IV Curcurbitaceae seed sample is prepared according to three the method for embodiment；
3) denaturation of RNA will obtain cucumber green mottle mosaic virus RNA under the conditions of 65 DEG C thermal denaturation after five minutes, immediately
It is for use to be put in cooled on ice；
4) TOYOBO companies goods is added in reverse transcription in AXYGEN companies article No. is the PCR tubule of 33713383 200ul
Number be 451100 following reaction reagent：
Above-mentioned system is carried out to 37 DEG C of 15 minutes reverse transcription reactions in PCR instrument, 98 DEG C of 5 minutes enzyme inactivation reactions generate
Product be CGMMV cDNA；
5) following reaction system is added in the PCR tubules of 200ul in PCR reactions, and positive, negative control is arranged
Wherein 2X Power Taq PCR Master Mix are 0020150709, CGMMV purchased from Bioteke companies article No.
Primer R and CGMMV Primer F are synthesized by Shanghai Ying Jun Bioisystech Co., Ltd, and CGMMV is positive, and feminine gender is that Zhejiang is big
It learns biotechnology research institute and preserves sample；
Reaction cycle parameter：94 DEG C of pre-degenerations 5 minutes；94 DEG C are denaturalized 30 seconds, and 52 DEG C are annealed 30 seconds, and 72 DEG C extend 30 seconds,
Cycle 30 times；Last 72 DEG C 10 minutes.
By the early elegant kind 50g of the cucurbit seed of Hangzhou plant quarantine station inspection, 22 are therefrom randomly selected.Use embodiment
Three the methods prepare the RNA of Curcurbitaceae seed sample；Amplified production is detached through 1% agarose gel electrophoresis, will detect sample
The positive negative comparison of the band of product and setting, record is as a result, calculate seed-transmission rate, (with malicious rate=number positive/total number of samples
× 100%).Testing result is shown in cucurbit seed, has 20 samples to have positive band at 480bp, has 2 (to scheme without band
2).It is computed, the band poison rate of this 22 cucurbit seeds is 90.91%.By the RT-PCR positive rates detected and example IV
The positive rate that middle dot-ELISA detections obtain is it was found that the two matches.
Claims (4)
1. a kind of detection Curcurbitaceae seed carries the sample preparation methods of cucumber green mottle mosaic virus, it is characterised in that including such as
Lower step：
1）The separation of seed sample kind Renhe kind shell：Every Curcurbitaceae seed is laterally splitted with eye scissors, divests benevolence
Remaining kind of shell is collected in part；
2）The grinding of sample：The kind shell detached using 1 seed is put into as 1 sample has put the 3-5 solid steel ball of diameter 2.5mm
1.5-2ml eppendorf centrifuge tubes in, cover centrifuge tube lid, lost in liquid nitrogen after being marked on centrifuge tube
Freezing processing is pulled out after 1 minute, and being then placed in Shanghai, reliable industry Development Co., Ltd article No. is the complete of Tissuelyser-48 only
Ground 60 seconds with frequency 60HZ in the quick beveller of automatic sample, make sample in powdered, if sample not in it is powdered can
It repeats to grind primary；
3）The coarse extraction of cucumber green mottle mosaic virus：It is 7.4 and a concentration of that 0.1-1ml pH are added in ground sample
0.01 mol/L PBS phosphate buffers, in the VortexGenie2 vortex mixeds of Scientific Industries companies
Mixing 0.5-5 minutes on device, then 5000rpm takes supernatant to be carried as the thick of cucumber green mottle mosaic virus after centrifuging 3 minutes
Liquid is taken to carry out serological method detection cucumber green mottle mosaic virus；
Or the extraction of cucumber green mottle mosaic virus RNA：Routine is used after 1mlTRIzol reagents are added in ground sample
TRIzol reagent methods extract viral RNA, and the viral RNA of extraction is used for the Molecular Detection of cucumber green mottle mosaic virus.
2. a kind of purposes of sample prepared by method as described in claim 1, it is characterised in that cucumber green statin prepared by this method
It refutes the crude extract of mosaic virus and RNA is respectively used to the serology and molecule of cucumber green mottle mosaic virus in Curcurbitaceae seed
Method detects.
3. purposes as claimed in claim 2, it is characterised in that the serological method includes dot-ELISA, ACP-
ELISA, DAS-ELISA and TAS-ELISA.
4. purposes as claimed in claim 2, it is characterised in that the molecular method detection includes quantitative RT-PCR.
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| CN107515302A (en) * | 2017-08-10 | 2017-12-26 | 江苏省农业科学院 | A kind of cucumber green mottle mosaic virus double crush syndrome detection kit |
| CN110438250B (en) * | 2018-05-04 | 2022-08-30 | 浙江省农业科学院 | Application of LsH3 as internal reference gene in analysis of cucumber green mottle mosaic virus infection of cucumber by using bottle gourd |
| CN115029484A (en) * | 2022-06-14 | 2022-09-09 | 云南省农业科学院生物技术与种质资源研究所 | RT-PCR detection method for papaya ringspot virus PRSV carried by cucurbitaceae seeds |
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