CN105177187A

CN105177187A - Preparing method and application of sample for detecting cucumber green mottle mosaic viruses carried by cucurbitaceae seeds

Info

Publication number: CN105177187A
Application number: CN201510661589.4A
Authority: CN
Inventors: 吴建祥; 宋革; 周雪平; 洪健; 宋西娇
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2015-10-14
Filing date: 2015-10-14
Publication date: 2015-12-23
Anticipated expiration: 2035-10-14
Also published as: CN105177187B

Abstract

The invention discloses a preparing method and an application of a sample for detecting cucumber green mottle mosaic viruses (CGMMV) carried by cucurbitaceae seeds. The preparing method includes the steps that starting from treating seed samples, shell-kernel separating treatment is carried out on the cucurbitaceae seeds, seed kernels with the low virus content are removed, seed shells with the high virus content are left to serve as to-be-tested samples, the seed samples obtained after treatment are sufficiently grinded through a full-automatic rapid sample grinding instrument, and the CGMMV in the large quantity of cucurbitaceae seeds can be rapidly detected through the sufficiently-grinded samples with a RT-PCR method and can also be rapidly detected through the sufficiently-grinded samples with a dot enzyme-linked immunosorbent assay (dot-ELISA) method. The preparing method and the application are suitable for virus detection of the common cucurbitaceae seeds such as calabash seeds, cucumber seeds, lagenaria sicerariae seeds, pumpkin seeds, long gourd seeds and watermelon seeds. A detecting method is high in sensitivity, good in specificity, short in consumed time and low in cost, technical supports are provided for large-scale detection and scientific prevention and control of the CGMMV carried by the cucurbitaceae seeds, and references are provided for detection of plant viruses transmitted by other seeds.

Description

Detect sample preparation methods and purposes that Curcurbitaceae seed carries cucumber green mottle mosaic virus

Technical field

Field belonging to the present invention is biological technical field, relates to a kind of process of carrying the Curcurbitaceae seed sample of cucumber green mottle mosaic virus, and serology and molecular biology for detection and application.

Background technology

Cucumber green mottle mosaic virus (Cucumbergreenmottlemosaicvirus, CGMMV) in nineteen thirty-five in Britain's reported first, along with the interchange of ground family crop between various countries is introduced a fine variety day by day frequent, multiple countries and regions such as Germany, Finland, Denmark, Russia, Greece in Europe report the harm occurring this virus in succession subsequently.

Cucumber green mottle mosaic virus is the member of Brassica 2 et 4 section (Tymoviridae) Tobamovirus (Tobamovirus).Mainly infect ground family crop, can cause the yellowing leaf deformities such as cucurbit, cucumber, watermelon, pumpkin, poor growth, result time delay, the yellow of fruit major part bleaches and produces the necrotic plaque of blackish green water scar shape, production declining.CGMMV is the important quarantine virus in the world on the ground family crop of many countries and regions, has caused the destructiveness of land for growing field crops melon crop to lose in some areas, and serious threat ground family crop is kept the safety in production.In recent years, there is the cucumber green mottle mosaic virus of import source property in some areas of China, cause the great attention of the relevant departments such as the Ministry of Agriculture, State Administration of Quality Supervision, Inspection and Quarantine.Port quarantine department of China in 2002 once intercepted this virus from the seedling that Japan introduces, and this virus is mainly distributed in the some areas such as the Hebei Xinle of China, Jiuquan, Pinggu County, beijing, Liaoning Gaizhou City and Guangxi at present.On December 21st, 2006, the Ministry of Agriculture issues No. 788 bulletins, cucumber green mottle mosaic virus is defined as National Agricultural Plant Quarantine harmful organism.

CGMMV host range is narrow, natural infection host mainly ground family crop, can infect Flos Daturae, Chenopodium amaranticolor, elder brother's promise lamb's-quarters, the western cigarette of coral, three lives cigarette and petunia by artificial juice frictional inoculation.CGMMV is a kind of RNA viruses, typical Seed-borne Virus, and band seed culture of viruses is the main source of infection of these viral long-distance communications.On the epidermis that virus particle invests seed and inner seed coat, after plantation, form primary source of infection.Plant biography host and have cucumber, watermelon, muskmelon and bottle gourd etc., it is 8% that fresh cucumber seed passes malicious rate; It is 1% ~ 5% that watermelon seed passes malicious rate, and wherein appearance belt poison amount is 20 times of entocuticle or endosperm; It is 10% ~ 52% that muskmelon kind passes malicious rate; Bottle gourd seed-transmission rate is up to 84%.In addition, the juice of CGMMV also by diseased plant contacts, to pollinate and the mode such as artificial grafting is propagated.

Seed transmission is the main path of cucumber green mottle mosaic virus long-distance communications, once the sick seedling of Seed transmission appears in field, just as primary source of infection, can be spread, cause heavy losses by non-amboceptor factor.In addition, cucurbit is China's a lot of watermelon producing region prevents and treats blight main stock for grafting, and cucurbit Seed transmission easily via graft transmission scion watermelon seedling, thus is diffused into land for growing field crops and causes serious consequence.

Set up sound seed virus detection method, be quick, accurate, the highly sensitive qualification of CGMMV and the quarantine detection of master to kind of propagation, strengthen the inspection and quarantine work of inward Curcurbitaceae seed, and prevent the tool that imports into of this virus to be of great significance, also spreading the diffusion controlling this virus from source has great importance.

The early stage detection method of CGMMV mainly relies on biological assay and electron microscopic observation, and along with molecular biological development, the detection technique of this virus develops from traditional detection method to the detection technique based on nucleic acid gradually.Have the detection method of CGMMV at present: biological method, Electron Microscopy, serological technique, Protocols in Molecular Biology etc.Wherein relatively more conventional detection method is serology and molecular Biological Detection.

Although Biological assay is simple, required time is long, and workload is large, and only by visual inspection, stability and credibility are not high.

Utilize Electron Microscopy observable by the structural changes of infected cell and the size and geometric of virion.Need very high hardware requirement because electron microscope technique detects, non-specialised staff has been difficult to, and thus the method is suitable only for assistant identification.

Protocols in Molecular Biology is from viral nucleic acid level to detect virus, and conventional Protocols in Molecular Biology mainly RT-PCR detects, and RT-PCR technology is quick, easy, highly sensitive, high specificity and required sample is few.But due to too high with the lipid content of malicious Curcurbitaceae seed own so too low being not enough to of viral level being difficult to extract high-quality RNA or CGMMV produces enough templates, so use conventional liquid nitrogen grinding method to extract the RNA of the Curcurbitaceae seed of band poison, and detect by RT-PCR method, often can not increase object band.So the Curcurbitaceae seed sample of the present invention to band poison carries out processing the concentration that can improve viral template.

Serological method utilizes specialization antiserum(antisera) to detect this virus, there is highly sensitive, high specificity, simple operation and other advantages, 1 ~ 10ng/mL virus can be detected. Serology test has many forms, and also conventional ELISA detection technique detects the band poison situation of Curcurbitaceae seed at present.

Diffusion controls the prerequisite that this virus imports into by seed trade and remote allocation and transportation to prevent CGMMV, strengthens the detection of ground family crop seed, is to prevent this disease from the capable key of seed source diffusion flow.From spreading of this disease of Sources controlling, significant to the safety in production of protection watermelon, muskmelon industry.

Summary of the invention

The object of the invention is to overcome the deficiencies in the prior art, provide and detect sample preparation methods and the application thereof that Curcurbitaceae seed carries cucumber green mottle mosaic virus.

Detect the sample preparation methods that Curcurbitaceae seed carries cucumber green mottle mosaic virus, comprise the steps:

1) separation of seed sample kind Renhe kind shell: every Curcurbitaceae seed eye scissors is laterally cut open, divests kind of a benevolence part, collect remaining kind of shell;

2) grinding of sample: put into the 1.5-2mleppendorf centrifuge tube putting 3-5 the solid steel ball of diameter 2.5mm as 1 sample using the kind shell that 1 seed is separated, cover centrifuge tube lid, lost freezing treatment in liquid nitrogen after centrifuge tube is carried out mark to pull out after 1 minute, then putting into the clean reliable industry Development Co., Ltd in Shanghai article No. is that the quick beveller of full-automatic sample of Tissuelyser-48 grinds 60 seconds with frequency 60HZ, sample is made to be Powdered, if sample does not repeat grinding once in Powdered;

3) this step comprises the thick extraction of cucumber green mottle mosaic virus or the extraction two kinds process of cucumber green mottle mosaic virus RNA:

The thick extraction step of cucumber green mottle mosaic virus is: add 0.1-1mlpH7.40.01mol/LPBS phosphate buffered saline buffer in ground sample, the VortexGenie2 turbine mixer of ScientificIndustries company mixes 0.5-5 minute, and then 5000rpm gets supernatant liquor and carries out serological method as the crude extract of cucumber green mottle mosaic virus and detect cucumber green mottle mosaic virus after centrifugal 3 minutes;

Or the extraction step of cucumber green mottle mosaic virus RNA is: extract viral RNA by conventional TRIzol reagent method after adding 1mlTRIzol reagent in ground sample, the viral RNA of extraction is used for the Molecular Detection of cucumber green mottle mosaic virus.

On above-mentioned sample preparation methods basis, the invention also discloses a kind of purposes of the sample using the method to prepare, be specially the crude extract of cucumber green mottle mosaic virus the method prepared and RNA and be respectively used to the serology of cucumber green mottle mosaic virus in Curcurbitaceae seed and molecular method detects.

Described serological method comprises dot-ELISA, ACP-ELISA, DAS-ELISA and TAS-ELISA.

Described dot-ELISA Serology test, comprises the steps:

1) draw the crude extract 2.5 μ l obtaining cucumber green mottle mosaic virus in claims 1, point sample in nitrocellulose filter, and arranges positive negative control, and room temperature leaves standstill dries 10 minutes;

2) 30 minutes are closed for 37 DEG C during nitrocellulose filter is immersed in containing 5% skim-milk PBST confining liquid;

3) above-mentioned nitrocellulose filter puts into the anti-cucumber green mottle mosaic virus mouse monoclonal antibody doubly diluted containing 1:5000, hatches 1-2 hour for 37 DEG C;

4) nitrocellulose filter PBST damping fluid shake washing 3 times, each 3-5 minute;

5) nitrocellulose filter puts into the Sigma company article No. doubly diluted containing 1:8000 is that the sheep anti-mouse igg two of the alkali phosphatase enzyme mark of A3562 resists, and hatches 1-2 hour for 37 DEG C;

6) nitrocellulose filter PBST damping fluid shake washing 4 times, then wash 1 time with PBS, each 3-5 minute;

7) in the colorbuffer of 10ml, adding Promega company article No. is the BCIP33ul of S3771, NBT66ul, is mixed, and is immersed room temperature lucifuge colour developing 5-15 minute after nitrocellulose filter dries;

8) when negative control does not develop the color until positive presents purple, then the nitrocellulose filter completely that develops the color is moved to rinsing color development stopping reaction in deionized water, record result.

Described molecular method detects and comprises RT-PCR and quantitative RT-PCR.

Described RT-PCR detection method comprises the steps:

1) according to 5 isolate CP gene conserved sequence design primers of the CGMMV reported in GenBank, upstream primer PrimerF:5 '-CTTACAATCCGATCACACCTAG-3 ', downstream primer PrimerR:5 '-CTAAGCTTTCGAGGTGGTAGC-3 ', amplified fragments size is 480bp, and primer is synthesized by Shanghai Ying Jun Bioisystech Co., Ltd;

2) sex change of CGMMVRNA: after 5 minutes, be put in cooled on ice immediately stand-by by obtaining cucumber green mottle mosaic virus RNA thermally denature under 65 DEG C of conditions in claims 1;

3) reverse transcription is the CGMMVRNA of CGMMVRNA:2ul5XRTBuffer, 0.5ulRTEnzymeMix, 0.5ulPrimerR, 6ulNuclease-freeWater, 1ul sex change adding reagent that TOYOBO company article No. is 451100 test kits, primer and sex change in the PCR pipe of the 200ul of 33713383 at AXYGEN company article No., in PCR instrument, 37 DEG C of 15 minutes reverse transcription reactions are carried out after mixing, 98 DEG C of 5 minutes enzyme deactivations reactions, the product of generation is CGMMVcDNA;

4) PCR reaction: add following reacted constituent in the PCR pipe of 200ul:

12.5ulBioteke company article No. is each 0.5ul, 1ul reverse transcription of 2XPowerTaqPCRMasterMix, CGMMVPrimerR and PrimerF primer CGMMVcDNA, 10.5ulddH2O of 0020150709, after mixing, centrifugal drying once, at the enterprising performing PCR of PCR instrument, reaction parameter is 94 DEG C of denaturations 5 minutes; 94 DEG C of sex change 30 seconds, 52 DEG C of annealing 30 seconds, 72 DEG C extend 30 seconds, circulate 30 times; Last 72 DEG C 10 minutes;

5) interpretation of result: amplified production through 1% agarose gel electrophoresis, to amplified production and the DNA molecular amount Marker of sample be detected and be with the positive control of malicious CGMMV to compare, the sample with the band in the same size with positive control is that detected result is positive, there is no the sample of specific band for negative, and Taking Pictures recording detected result.

The present invention is by the detection to seed different sites viral level, and the kind shell part toxic amount determining seed is the highest, then Curcurbitaceae seed to be measured is carried out separation of hull from kernel process, collects the kind shell part that toxic amount is higher.Then replace traditional liquid nitrogen grinding mode with full-automatic grinding instrument, fast, efficiently, labour-saving has been prepared and both can have been detected by RT-PCR technology, can be used for again the Curcurbitaceae seed sample that dot-ELISA detects.This invention can be investigated the band of Curcurbitaceae seed poison situation, and reducing this virus is that the remote diffusion in source is popular with seed, can be applicable to Curcurbitaceae Seed transmission situation rapid detection.This detection method reliable results, rapidly and efficiently, sensitive special, high-throughput is easy and simple to handle again, for the quick diagnosis of cucumber green mottle mosaic virus disease, port quarantine and prevention and control provide material and technical support, and offer reference, for the prevention and control of Curcurbitaceae Seed-borne Virus lay the foundation for the detection of other Seed-borne Virus seed.

The beneficial effect that the present invention compared with prior art has:

(1) the invention provides a kind of sample preparation methods carrying cucumber green mottle mosaic virus specially for detection Curcurbitaceae seed, by carrying out shell, benevolence separating treatment to Curcurbitaceae seed, remain the part that toxic amount in seed is higher, reduce the problems such as the high fat content because existing in kind of benevolence and the undetected situation that occurs, also for RT-PCR detect, dot-ELISA detects and provides high-quality sample to be tested.

(2) the present invention saves manpower than traditional with liquid nitrogen hand lapping by full-automatic grinding instrument, improves speed, shortens sense cycle.Grind sufficient sample and both can pass through RT-PCR method, also can by the CGMMV in dot-ELISA rapid detection Curcurbitaceae seed, a large amount of Curcurbitaceae seed samples can be detected simultaneously, high-throughput saves time again, and suitable health officer applies when seed trade, at a distance allocation and transportation and port quarantine;

(3) Serologic detection combines with molecular Biological Detection by the present invention, detected result is true and reliable, can accurate calculation Curcurbitaceae seed band poison rate, carry the extensive detection of cucumber green mottle virus disease for Curcurbitaceae seed and science bridle provides technical support, and the detection passing plant virus for other seeds is offered reference.To spreading from this disease of seed Sources controlling, ensure the safety in production of the industries such as watermelon, muskmelon, pumpkin, prevent cucumber green mottle mosaic virus sick significant in the popular outburst in field.

Accompanying drawing explanation

Fig. 1 cucurbit seed-transmission rate dot-ELISA detected result;

Fig. 2 cucurbit seed-transmission rate RT-PCR detected result.

Embodiment

1) separation of seed sample kind Renhe kind shell: every little scissors of Curcurbitaceae seed ophthalmology is laterally cut open, divests kind of a benevolence part, collect remaining kind of shell;

2) grinding of sample: put into the 1.5-2mleppendorf centrifuge tube putting 3-5 the solid little steel ball of diameter 2.5mm as 1 sample using the kind shell that 1 seed is separated, cover centrifuge tube lid, lost freezing treatment in liquid nitrogen after centrifuge tube is carried out mark to pull out after 1 minute, then putting into the clean reliable industry Development Co., Ltd in Shanghai article No. is that the quick beveller of full-automatic sample of Tissuelyser-48 grinds 60 seconds with frequency 60HZ, sample is made to be Powdered, if sample does not repeat grinding once in Powdered;

The thick extraction step of cucumber green mottle mosaic virus is: by step 2) add 0.1-1mlpH7.40.01mol/LPBS phosphate buffered saline buffer in ground sample, the VortexGenie2 turbine mixer of ScientificIndustries company mixes 0.5-5 minute, and then 5000rpm gets supernatant liquor and carries out serological method as the crude extract of cucumber green mottle mosaic virus and detect cucumber green mottle mosaic virus after centrifugal 3 minutes;

Or the extraction step of cucumber green mottle mosaic virus RNA is: by step 2) add 1mlTRIzol reagent in ground sample after extract viral RNA by conventional TRIzol reagent method, the viral RNA of extraction is used for the Molecular Detection of cucumber green mottle mosaic virus.

Described dot-ELISA Serology test, comprises the steps:

Described RT-PCR detection method comprises the steps:

3) reverse transcription is the CGMMVRNA of CGMMVRNA:2ul5XRTBuffer, 0.5ulRTEnzymeMix, 0.5ulPrimerR, 6ulNuclease-freeWater, 1ul sex change adding reagent that TOYOBO company article No. is 451100 test kits, primer and sex change in the PCR tubule of the 200ul of 33713383 at AXYGEN company article No., in PCR instrument, 37 DEG C of 15 minutes reverse transcription reactions are carried out after mixing, 98 DEG C of 5 minutes enzyme deactivations reactions, the product of generation is CGMMVcDNA;

4) PCR reaction: add following reacted constituent in the PCR tubule of 200ul:

Below in conjunction with embodiment and accompanying drawing, the invention will be further described.

The ACP-ELISA of embodiment one, Curcurbitaceae seed different sites viral level detects

Get 20, the cucurbit seed of the band poison that laboratory is preserved, with ophthalmology little scissors, seed is laterally cut open, kind of benevolence part and kind shell part are separated.They, as one group, are put into mortar liquid nitrogen and fully grind by the kind shell of same grain seed and kind benevolence respectively.Ground sample chamber is gentle and quiet puts, after the volatilization of band liquid nitrogen, with 0.05M carbonic acid buffer (PH9.6) 30 times dilution, then 5000rpm, get the kind shell of same grain seed after centrifugal 3 minutes and plant benevolence grinding supernatant, left and right is adjacent adds the every hole 100ul of elisa plate respectively, and arranges the positive, negative control.4 DEG C of bags are closed 30 minutes by the every hole 250ul37 of PBS confining liquid DEG C of adding after spending the night containing 3% skim-milk, are patted dry by elisa plate on gauze.The CGMMV8E3 mouse odd contradictive hydroperitoneums of 5000 times of dilutions add the every hole 100ul of elisa plate as primary antibodie, hatch after 1 hour each 3 minutes with PBST buffer solution 3 times, are patted dry by elisa plate on gauze for 37 DEG C.The sheep anti-mouse igg of 8000 times of dilution alkali phosphatase enzyme marks is two anti-add the every hole 100ul of elisa plate, hatches after 1 hour each 3 minutes with PBST buffer solution 4 times, is patted dry by elisa plate on gauze for 37 DEG C.With the colour developing of para-nitro-pheneye phosphate (PNPP) substrate, every hole 100ul.OD is read by BIO-RAD680 microplate reader ₄₀₅value, P/N>3.0 is positive judging criterion (concrete with reference to Shangetal.VirologyJournal2011,8:228 document).The kind shell lapping liquid that test-results shows 20 seeds is all positive, and only has the kind benevolence lapping liquid of 12 seeds to be positive, and the OD of these kind of benevolence ₄₀₅value is all than the kind shell OD of same grain seed ₄₀₅value is much lower.Can find that according to test-results the kind shell of the Curcurbitaceae seed carrying CGMMV is than kind of the positive reaction that benevolence performance is stronger, illustrate that the kind shell of Curcurbitaceae seed is more suitable for doing than kind of benevolence and detect in spite of illness.

The electron microscopic observation of embodiment two, Curcurbitaceae seed different sites viral level

Get 20, the cucurbit seed of the band poison that laboratory is preserved, with ophthalmology little scissors, seed is laterally cut open, kind of benevolence part and kind shell part are separated.They, as one group, are put into mortar liquid nitrogen and fully grind by the kind shell of same grain seed and kind benevolence respectively.Ground sample chamber is gentle and quiet puts, and after the volatilization of band liquid nitrogen, with distilled water 10 times dilution, then 5000rpm, within centrifugal 3 minutes, supernatant is CGMMV virus crude extract.Virus purification liquid is through 2% phospho-wolframic acid (phosphotungsticacid, PTA) (pH6.7) dyes rearmounted JEOLJEM-1200EX Electronic Speculum (JEOL, Japan) under observe particle shape, Electronic Speculum found that the elongated rod shape CGMMV virus particle can seeing some long 300nm in the kind shell sample crude extract of same grain seed, and can only see minority or do not observe the particle of these elongated rod shape in the sample crude extract of kind of benevolence.It is many compared with planting benevolence part that test-results illustrates that the kind shell part of Curcurbitaceae seed carries CGMMV, and this result also drawn with embodiment one matches.

Embodiment three, detection Curcurbitaceae seed carry the sample preparation methods Curcurbitaceae seed of cucumber green mottle mosaic virus

Comprising the steps: of the preparation of sample

2) grinding of sample: put into the 1.5-2mleppendorf centrifuge tube putting 3-5 the solid little steel ball of diameter 2.5mm as 1 sample using the kind shell that 1 seed is separated, cover centrifuge tube lid, lost freezing treatment in liquid nitrogen after centrifuge tube is carried out mark to pull out rapidly after 1 minute, then putting into the clean reliable industry Development Co., Ltd in Shanghai article No. is that the full-automatic sample quick beveller medium frequency 60HZ of Tissuelyser-48 grinds 60 seconds, sample is made to be Powdered, if sample does not repeat grinding once in Powdered;

3) the thick extraction of cucumber green mottle mosaic virus: add 0.1-1mlpH7.40.01mol/LPBS phosphate buffered saline buffer in ground sample, concussion mixing 0.5-5 minute on the VortexGenie2 turbine mixer of ScientificIndustries company, then 5000rpm, gets supernatant and carries out serological method detection cucumber green mottle mosaic virus after centrifugal 3 minutes;

Or the extraction of cucumber green mottle mosaic virus RNA: extract viral RNA by conventional TRIzol reagent method after adding 1mlTRIzol reagent in ground sample, the viral RNA of extraction is used for the Molecular Detection of cucumber green mottle mosaic virus.

Embodiment four, detection Curcurbitaceae seed carry the dot-ELISA serological method of cucumber green mottle mosaic virus

Detecting step is as follows:

1) get 22, cucurbit seed method described in embodiment three and prepare Curcurbitaceae seed sample;

2) draw the crude extract 2.5 μ l of cucumber green mottle mosaic virus, point sample in nitrocellulose filter, and arranges the positive, negative control, and room temperature leaves standstill dries 10 minutes;

3) 30 minutes are closed for 37 DEG C during nitrocellulose filter is immersed in containing 5% skim-milk PBST confining liquid;

4) above-mentioned nitrocellulose filter puts into the anti-CGMMV8E3 mouse monoclonal antibody doubly diluted containing 1:5000, hatches 1-2 hour for 37 DEG C;

5) nitrocellulose filter PBST damping fluid shake washing 3 times, each 3-5 minute;

6) nitrocellulose filter puts into the Sigma company article No. doubly diluted containing 1:8000 is that the sheep anti-mouse igg two of the alkali phosphatase enzyme mark of A3562 resists, and hatches 1-2 hour for 37 DEG C;

7) nitrocellulose filter PBST damping fluid shake washing 4 times, then wash 1 time with PBS, each 3-5 minute;

8) in the colorbuffer of 10ml, adding Promega company article No. is the BCIP33ul of S3771, NBT66ul, is mixed, and is immersed room temperature lucifuge colour developing 5-15 minute after nitrocellulose filter dries;

9) when until positive presents purple, negative control does not develop the color, then by color reaction completely nitrocellulose filter to move in deionized water the reaction of rinsing color development stopping, record result.

By the cucurbit seed early elegant kind 50g of Hangzhou Plant Quarantine station censorship, therefrom randomly draw 22.Curcurbitaceae seed sample is prepared by method described in embodiment three; Band poison rate (being with malicious rate=number positive/total number of samples × 100%) of seed is calculated according to result.In 22 cucurbit seeds that detected result display detects, have 20 be positive (Fig. 1), as calculated, the band poison rate of these 22 cucurbit seeds is 90.91%.

Embodiment five, detection Curcurbitaceae seed carry the RT-PCR molecular method of cucumber green mottle mosaic virus

Concrete steps are as follows:

1) according to 5 isolate CP gene conserved sequence design primers of the CGMMV reported in GenBank, upstream primer PrimerF:5 '-CTTACAATCCGATCACACCTAG-3, downstream primer PrimerR:5 '-CTAAGCTTTCGAGGTGGTAGC-3, amplified fragments size is 480bp, and primer is synthesized by Shanghai Ying Jun Bioisystech Co., Ltd;

2) Curcurbitaceae seed sample is prepared with the Curcurbitaceae seed of 22 in embodiment four according to method described in embodiment three;

3) sex change of RNA will obtain cucumber green mottle mosaic virus RNA thermally denature under 65 DEG C of conditions and, after 5 minutes, be put in cooled on ice immediately stand-by;

4) reverse transcription is add the following reaction reagent that TOYOBO company article No. is 451100 in the PCR tubule of the 200ul of 33713383 at AXYGEN company article No.:

Above-mentioned system is carried out in PCR instrument 37 DEG C of 15 minutes reverse transcription reactions, 98 DEG C of 5 minutes enzyme deactivations reactions, the product of generation is CGMMVcDNA;

5) PCR reaction adds following reaction system in the PCR tubule of 200ul, and arranges the positive, negative control

Wherein 2XPowerTaqPCRMasterMix is 0020150709 purchased from Bioteke company article No., CGMMVPrimerR and CGMMVPrimerF is synthesized by Shanghai Ying Jun Bioisystech Co., Ltd, CGMMV is positive, is negatively preserved sample by Zhejiang University's biotechnology research;

Reaction cycle parameter: 94 DEG C of denaturations 5 minutes; 94 DEG C of sex change 30 seconds, 52 DEG C of annealing 30 seconds, 72 DEG C extend 30 seconds, circulate 30 times; Last 72 DEG C 10 minutes.

By the cucurbit seed early elegant kind 50g of Hangzhou Plant Quarantine station censorship, therefrom randomly draw 22.The RNA of Curcurbitaceae seed sample is prepared by method described in embodiment three; Amplified production is separated through the agarose gel electrophoresis of 1%, and the positive feminine gender of the band and setting that detect sample contrasted, record result, calculates seed-transmission rate, (being with malicious rate=number positive/total number of samples × 100%).In detected result display cucurbit seed, there are 20 samples to have positive band at 480bp place, have 2 without band (Fig. 2).As calculated, the band poison rate of these 22 cucurbit seeds is 90.91%.RT-PCR is detected the positive rate that obtains and dot-ELISA in embodiment four to detect the positive rate drawn and compare both discoveries and match.

Claims

1. detect the sample preparation methods that Curcurbitaceae seed carries cucumber green mottle mosaic virus, it is characterized in that comprising the steps:

3) the thick extraction of cucumber green mottle mosaic virus: add 0.1-1mlpH7.40.01mol/LPBS phosphate buffered saline buffer in ground sample, the VortexGenie2 turbine mixer of ScientificIndustries company mixes 0.5-5 minute, and then 5000rpm gets supernatant liquor and carries out serological method as the crude extract of cucumber green mottle mosaic virus and detect cucumber green mottle mosaic virus after centrifugal 3 minutes;

2. the purposes of sample prepared of method as claimed in claim 1, is characterized in that the crude extract of cucumber green mottle mosaic virus prepared by the method and RNA are respectively used to the serology of cucumber green mottle mosaic virus in Curcurbitaceae seed and molecular method detects.

3. purposes as claimed in claim 2, is characterized in that described serological method comprises dot-ELISA, ACP-ELISA, DAS-ELISA and TAS-ELISA.

4. purposes as claimed in claim 3, it is characterized in that, described dot-ELISA Serology test comprises the steps:

5. purposes as claimed in claim 2, is characterized in that described molecular method detects and comprises RT-PCR and quantitative RT-PCR.

6. purposes as claimed in claim 5, is characterized in that described RT-PCR detection method comprises the steps:

3) reverse transcription: be the CGMMVRNA of CGMMVRNA:2ul5XRTBuffer, 0.5ulRTEnzymeMix, 0.5ulPrimerR, 6ulNuclease-freeWater, 1ul sex change adding reagent that TOYOBO company article No. is 451100 test kits, primer and sex change in the PCR pipe of the 200ul of 33713383 at AXYGEN company article No., in PCR instrument, 37 DEG C of 15 minutes reverse transcription reactions are carried out after mixing, 98 DEG C of 5 minutes enzyme deactivations reactions, the product of generation is CGMMVcDNA;

4) PCR reaction: add following reacted constituent in the PCR pipe of 200ul: