CN104513860B - A kind of method of quick diagnosis mulberry fruit scaled-down version sclerotiniose - Google Patents
A kind of method of quick diagnosis mulberry fruit scaled-down version sclerotiniose Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
A kind of method of quick diagnosis mulberry fruit scaled-down version sclerotiniose, by the cause of disease core ground cane bacterium specific conservative's DNA sequence dna found, it is thus achieved that specific SCAR PCR primer, establishes mulberry fruit scaled-down version sclerotiniose SCAR PCR diagnosis detecting method.Highly sensitive (detection sensitivity is 1 pg/ μ L) of the method, high specificity, convenient to use, testing result accurately and reliably, can be used for field diagnosis and the detection inspection of sorosis Carriage of mulberry fruit scaled-down version sclerotiniose；Can be additionally used in disease incubation period (infected sorosis but do not shown the period of disease) diagnosis simultaneously, thus disease is made early prediction, to implement necessary control measure in time, the safely and effectively generation of controlling disease and harm, economic loss is alleviated or avoided.Mulberry fruit scaled-down version sclerotiniose " detection kit " product can be developed according to the method, be widely used in scientific research and the production practices of mulberries related industry.
Description
Technical field
The present invention relates to control of plant disease technical field, particularly relate to one molecular biotechnology quick diagnosis mulberry
The method of mulberry scaled-down version sclerotiniose.
Background technology
The Economic Importance of mulberry fruit scaled-down version sclerotiniose. mulberry fruit scaled-down version sclerotiniose (shrunken-
Fruitsclerotiniose of mulberry) it is one of three kinds of diseases of mulberry fruits, its pathogen is core ground cane bacterium
(Scleromitrula shiraiana) (Schuamacher T, Holst-Jensen A. A synopsis of the
Genus Scleromitrula. Mycoscience, 1997,38 (1): 55-69), it is that the most universal and danger occurs in the world
The diseases of mulberry fruits that evil is the most serious.This disease occurs very universal in China, Jiangsu, Zhejiang, Shanghai, Sichuan, Chongqing, Shaanxi and platform
The main fruit mulberry growing area such as gulf all has occurrence and distribution, and the incidence of disease the most all can reach 30% 60%, the general underproduction 20% 50%；Closely
In the past few years in many areas mulberry sclerotiniose eruption and prevalences, cause large area fruit mulberry ruin product total crop failure (Kuai Yuanzhang, Wu Fuan. mulberry fruit
Sclerotiniose cause of disease and disease prevention techniques summary. silkworm industry science, 2012,38 (6): 1099-1104).Therefore, mulberry fruit sclerotium
Disease is the important disease of harm fruit mulberry crop, is to affect growing of mulberry to grow and the important restriction factor of mulberry fruit yield, and research should
The diagnosis identification technology of disease, popular with controlling disease effectively to predict exactly, stable development is produced for sorosis and has
Significance.
There is the importance of diagnosis in mulberry fruit scaled-down version sclerotiniose. and mulberry hyphal cluster germ is with the sclerotium in Soil of Mulberry Garden that drops
Surviving the winter, next year spring temperature starts after raising to sprout to produce apothecium (mushroom), forms a large amount of ascospore and distributes in air,
Infect flower florescence in mulberry tree, show symptom during fruit maturation, the part or all of little fruit of mulberry fruit is become sclerotium.Reduce
Type sclerotiniose mulberry is reduced significantly, canescence, and quality is hard, and there is the thin spot of crineous on surface, forms the hard sclerotium of black in sick mulberry.Mulberry
The time history that mulberry sclerotiniose is fallen ill from pathogen infection to sorosis in a large number is the shortest, the quick and precisely diagnosis of disease and early prediction
It it is the key of disease control in Instructing manufacture.
The necessity of mulberry fruit scaled-down version sclerotiniose quick Accurate Diagnosis technology. at present, the diagnosis of diseases of mulberry fruits is mainly adopted
By tradition disease screening method, i.e. determine the kind of disease according to symptom and Pathogens.But, traditional disease screening needs
Want disease just can carry out after showing symptom, and need the pure culture of isolated pathogen and pathogen is carried out micro-
Observation of characteristics.On the one hand, the isolated and purified process of pathogen is considerably complicated, and experiment condition and technology require the highest, diagnosis
Accuracy and reliability are difficult to ensure that；And pathogen growth is slow under culture conditions, it is generally required to after 15 20 days
Can observe and identify, it is thus achieved that diagnosis qualification result；On the other hand, disease, once aobvious disease, will develop rapidly, now pay a home visit
Disconnected late, take prophylactico-therapeutic measures can not obtain effective disease-controlling effect according to diagnostic result.Therefore one it is badly in need of
The method of quick diagnosis mulberry fruit scaled-down version sclerotiniose.
Mulberry fruit scaled-down version sclerotiniose molecular diagnostic techniques is not yet set up. and traditional classification of fungi and qualification are based primarily upon shape
State feature, Pathogenicity etc., this method time-consumingly long, sensitivity is low and empirical by force, separate from the plant of morbidity
Take long enough with pathogen identification, it is difficult to adapt to plant disease prevention and the needs of Synthetical prevention practice in modern agricultural production.
(2006) utilizations such as along with the development of molecular biology, various molecular engineerings are applied to the detection of the phytopathy original, Tang Jianhui
RAPD technical transform is SCAR mark, and watermelon anthrax bacteria Colletotrichum orbiculare is entered by design primer RB/RC
Row specific detection.Qin et al (2010) applies Nested PCR Technique, according to ITS sequence, designs primer XJJ21/
XJJ222 carries out specific detection to the Sclerotinia sclerotiorum causing sclerotinia rot of colza.Zhang et al
(2014) the Real-Time PCR quantitation detection technique of paddy rice secret note contracting virosis is established.In a word, current molecular biotechnology is being planted
Thing disease screening identifies that field is the most extensively applied, and the molecular detection technology of the most important plant disease is set up the most.But so far
Till the present, yet there are no the discovery about the core ground special conserved sequence of cane bacterium DNA (or gene) and mulberry fruit scaled-down version sclerotium both at home and abroad
Sick molecule fast diagnoses the research report of authentication method.
Summary of the invention
A kind of method that it is an object of the invention to provide quick diagnosis mulberry fruit scaled-down version sclerotiniose.Applicant is by research
Diseases of mulberry fruits cause of disease core ground cane bacterium DNA, filters out applicable gene C S23 and carries out RAPD amplification, it is thus achieved that the core ground of 590bp
Cane bacterium specific and conserved sequence (SCAR sequence is shown in sequence table and embodiment), thus designs and filters out and can effectively expand for a pair
The specificity SCAR primers (SSF/SSR) of this conserved sequence, then the optimization by several important PCR factors, establish core ground cane
The SCAR-PCR system of bacterium and technology, test the sensitivity of clear and definite technology, and the field sample test gathered by mulberry field is checked
The practicality of technology and reliability.
The method of a kind of molecular biotechnology quick diagnosis mulberry fruit scaled-down version sclerotiniose of the present invention, by following
Step realizes:
(1) take testing sample, use CTAB method to extract STb gene from sample；
(2) it is special that the core ground cane bacterium specific and conserved sequence utilizing 590bp design and screen with obtaining core a pair of cane bacterium
Property SCAR-PCR primer, primer forward sequence SS-F is 5'-AGTAAAGAGAACATCAAACTTCG-3', is positioned at distinguished sequence
8 bp-30 bp；Reverse sequence SS-R is 5'-ACTTCCACCGACCA ATCT-3', is positioned at the 580bp-of distinguished sequence
596bp；
(3) carrying out PCR amplification, amplification system is: 2.5 μ L 10 × PCR Buffer； 2.25μL Mg2+(25mmol/
L)； 2.5μL dNTPs(2.5mmol/L)；0.625 μ L primer SS-F/SS-R (10 μm ol/L)；1 μ L template (10ng/ μ L)；
0.2U rTaq enzyme； ddH2O complements to 25 μ L；Response procedures is: 94 DEG C of denaturations 5min；94 DEG C of sex change 30s；50 DEG C of annealing
30s；72 DEG C extend 45s, circulate 35 times；72 DEG C are supplemented extension 5min；Mulberry fruit scaled-down version sclerotiniose is diagnosed according to amplified band.
Step (1) testing sample is sorosis, mulberry leaf, mulberry stem, Sang Gen and pedotheque etc..
Sensitivity test shows, in 25 μ L systems, specific primer SS-F/SS-R can be 1 from mass concentration
Pg/ μ L and above core ground cane bacterium DNA profiling thereof amplify the specific band of 590 bp, thereby determines that this SCAR-
The detection sensitivity of PCR is 1 pg/ μ L.
In the sample testing experiment of field, use this field, 24 mulberry fields of SCAR-PCR technology for detection sample, only from reducing
The STb gene that type disease sorosis extracts amplifies SCAR-band sequence, from healthy sorosis, mulberry leaf, mulberry stem, Sang Gen and soil-like
Product (note: wherein contain different microorganisms or plant and animal residues) STb gene does not all expand SCAR band, illustrates except reducing
Outside type sorosis, other sample is all without core ground cane bacterium.
The significance of the present invention and value. mulberry fruit scaled-down version sclerotiniose is the important plant disease of harm mulberry tree, is limit
The critical limitation factor of sorosis industry stable development processed.The present invention is by the cause of disease core ground cane bacterium specific conservative's DNA sequence found
Row, it is thus achieved that specific SCAR-PCR primer, establish mulberry fruit scaled-down version sclerotiniose SCAR-PCR diagnosis detection technique.This technology
Highly sensitive, detection sensitivity is 1 pg/ μ L, and high specificity, convenient to use, testing result accurately and reliably, can be used for
The field diagnosis of mulberry fruit scaled-down version sclerotiniose and the detection inspection of sorosis Carriage；Can be additionally used in disease (to invade incubation period simultaneously
Contaminate sorosis but do not show disease) diagnosis, thus disease is made early prediction, to implement necessary control measure in time, safely and effectively
The generation of ground controlling disease and harm, be alleviated or avoided economic loss.Meanwhile, available the technology of the present invention development mulberry fruit reduces
The commercially available reagent box of type sclerotiniose diagnosis, has certain commercial application value.
Accompanying drawing explanation
The present invention comprises the figure of 6 width display inventive result, is respectively as follows:
Fig. 1. the random primer CS23 random amplification result fingerprint image to different strains tested DNA. note: M:DL2 in figure
000 DNA marker；Swimming lane 15: the random amplification fingerprint of core ground cane bacterium different strains DNA segment；Swimming lane 6 14 is respectively
Be: mulberry reality cup cup fungi, caruncula shape cup cup fungi, intend that healthy and free from worry wood is mould, this wood of lid nurse is mould, green trichoderma, worm hairy wood wood mould, hook-shaped is mould,
Sclerotinia sclerotiorum, grey mold DNA segment random amplification fingerprint;It it is the specific band of core ground cane bacterium in white wire frame.
Fig. 2. core ground cane bacterium SCAR-PCR detection architecture optimizes electrophoretogram. note: M:DL2 000 DNA marker;A is
Annealing temperature, swimming lane 17 is respectively 60.0,59.4,58.3,56.3,53.9,52,50.7 and 50.0 DEG C;B is Mg2+Concentration,
Swimming lane 17 is respectively 2.5,2.25,2,1.75,1.5,1.25 and 1 mmol/L；C is dNTPs concentration, and swimming lane 17 is respectively
025,0.225,0.2,0.175,0.15,0.125 and 0.1 mmol/L；D is primer concentration: swimming lane 17 respectively 0.35,
0.3,0.25,0.2,0.15,0.1 and 0.05 μm ol/L；E is rTaq enzyme dosage, swimming lane 17 is respectively 2,1.75,1.5,
1.25,1,0.75 and 0.5U.
Fig. 3. with SCAR primer SS-F/SS-R PCR amplification core ground cane bacterium and Relative Fungi DNA electrophoretogram. note: M:
DL2 000 DNA marker；Swimming lane 15: core ground cane bacterium amplification；Swimming lane 6 14 is respectively: mulberry reality cup cup fungi, caruncula
Shape cup cup fungi, intend that healthy and free from worry wood is mould, this wood of lid nurse is mould, green trichoderma, worm hairy wood wood mould, hook-shaped is mould, Sclerotinia sclerotiorum, grey mold；
Swimming lane 15: negative control.
Fig. 4. primer detection core ground cane bacterium DNA sensitivity technique electrophoretogram. note: M:DL2 000 DNA marker；Swimming lane
18: be to measure containing 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fgDNA in the system of 25 l respectively
Amplification.
Fig. 5. the SCAR-PCR testing result electrophoretogram of mulberry field sample. note: M:DL2 000 DNA marker；Swimming lane 1:
Positive control；Swimming lane 2: negative control；Swimming lane 34: florescence mulberry fruit；Swimming lane 5: loose sick fruit；Swimming lane 6: the sick fruit reduced；
Swimming lane 7: loose sick fruit；Swimming lane 8: Mulberry Roots；Swimming lane 9 10: mulberry stem；Swimming lane 11 12: mulberry leaves；Swimming lane 13 24:
The soil sample of random acquisition；Swimming lane 25 26: core ground cane bacterium does not shows the sorosis of disease after inoculating 2 days.
Fig. 6. with the result electrophoretogram of sample core ground, SCAR-PCR detection mulberry field cane bacterium. note: M:DL2 000 DNA
marker；Swimming lane 1: positive control；Swimming lane 2: negative control；Swimming lane 3 10: the sick fruit reduced；Swimming lane 11 18: loose disease
Really
Detailed description of the invention
Strains tested. for finding strains tested totally 17 strain of SCAR sequence in core ground cane bacterium DNA, its center ground cane bacterium 5
Strain, other diseases of mulberry fruits pathogen 2 strain, diseases of mulberry fruits sclerotium bacterial parasite 5 strain, other corresponding plants pathogenic bacteria 2 strain (table
1).These bacterial strains are isolated and purified and obtain from different types of sclerotiniose disease sorosis respectively.
Table 1 separates mulberry field system and supplies examination fungal bacterial strain for what specific primer screened
Embodiment 1: the extraction of material to be tested DNA. aforementioned strains tested is inoculated into the PDA culture medium posting glassine paper
On, sclerotinite, grey mold and wood are mould in 20 DEG C of dark culturing 2d, and diseases of mulberry fruits pathogen is 25 DEG C of dark culturing 15d and collects
Mycelia.With CTAB method ((Liu Li, Zhang Yongjun, the perhaps Long March, Luo Feng. the CTAB method of a kind of improvement is extracted and is produced polysaccharide fungi
DNA. Chinese biological engineering magazine, 2014,34 (5): 75-79) genome of the plant tissue such as fungi and mulberry fruit is extracted
DNA, pedotheque reference Woodhall etc. (Woodhall J W, Webb K M, Giltrap P M, Adams I P,
Peters J C, Budge G E, Boonham N. A new large scale soil DNA extraction
procedure and real-time PCR assay for the detection of Sclerotium cepivorum
In soil. European Journal of Plant Pathology, 2012,134 (3): 467-473) method carries
Taking, all concentration extracting the DNA sample obtained and quality, by comparing agarose gel electrophoresis band intensity and measurement
260 nm/280 nm extinction ratios detect.The DNA Sample storage detected is standby to 20 DEG C.
Embodiment 2: the acquisition of the core ground cane special conserved segments of bacterium DNA. first with RAPD (random amplified
Polymorphism DNA)-round pcr expand and filter out core ground cane bacterium special Conservative segment of DNA sequence.Used 40
Random primer is by Shanghai raw work synthesis.With reference to (Williams J G K, Kubelik A R, Liv S ak K such as Williams
J, Rafalski J A,Tingey V. DNA polymorphisms amplified by arbitrary primers
are useful as genetic markers. Nucleic Acids Research, 1990, 18(22): 6531-
6535) response procedures, each RAPD amplified reaction PCR cumulative volume is 25 μ L, comprises 2.5 μ L 10 × PCR Buffer；
1.5μL Mg2+(25mmol/L)； 2μL dNTPs(2.5 mmol/L)；1 μ L 10mM random primer, 1 μ L DNA profiling, 0.25
μ L rTaq enzyme (2.5U/ μ L)；Use ddH2O supplies.Amplification program is: 94 DEG C of predeformation 5min；94 DEG C of sex change 30s, 36 DEG C of annealing
30s, 72 DEG C extend 1.5min, 35 circulations；Last 72 DEG C of supplementary 10 min that extend preserve at 4 DEG C.After reaction terminates, take
Electrophoresis 30 min under 6 μ L amplified productions (are previously added Glodview) in 1.5% Ago-Gel 5V/cm voltage conditions,
BIO-RAD gel imaging system is observed, takes pictures.
According to each primer RAPD amplification electrophoresis pattern, the most only primer CS23(5'-CGCAGCCGAGAT-
3') special, stable and band (Fig. 1) clearly can be iteratively produced, and this band can only from pathogenic bacteria S. shiraiana
Amplification out, other participate in the experiment strain gene group DNA and distilled water comparison all can not amplify this band.Primer CS23 is inspection
Survey the special random primer of mulberry fruit scaled-down version hyphal cluster germ, and the specific band amplified is the specific fragment of pathogenic bacteria, should
Specific fragment Genome Size is about about 610 bp, the most based on this design special primer detection mulberry fruit scaled-down version sclerotium
Sick.
Use gel reclaim kit specific fragment that RAPD is filtered out from Ago-Gel isolated and purified out,
Then it is cloned on pMD19-T carrier, then converts to Escherichia coli (Escherichia coli) DH5 α cell.To convert
After E. coli be placed in containing on ampicillin medium, 37 DEG C of overnight incubation, select 20 bacterial plaques at random to added with ammonia
In the LB liquid medium of parasiticin, cultivate to muddy for 37 DEG C.Detect by PCR method, positive colony is sent to the raw work in Shanghai raw
Thing Technology Co., Ltd. checks order.
By primer CS23 produce core ground cane bacteria strain specific fragment reclaim from Ago-Gel be purified and with
Carrier pMD19-T Vector connects, and then converts to E. coli DH5 α cell, is checked order by positive colony, and will survey
Sequence result logs on NCBI(http: //www.ncbi.nlm.nih.gov), it is thus achieved that accession number isKP146145。
The design of embodiment 3:SCAR special primer and screening. according to the sequence of specific fragment, soft with Premier 5.0
SCAR primer, the primer designed are synthesized by part design 20 to by Shanghai Sheng Gong Bioisystech Co., Ltd.By PCR to this 20
Screening primer, from these primers, screening obtains a pair optimal specific primer SS-F/SS-R.Primer forward
Sequence SS-F is 5'-AGTAAAGAGAACATCAAACTTCG-3', is positioned at 8 bp-30 bp of distinguished sequence；Reverse sequence
SS-R is 5'-ACTTCC ACCGACCA ATCT-3', is positioned at the 580bp-596bp of distinguished sequence, and purpose fragment is 590
Bp(is shown in underscore part in sequence table).
Embodiment 4: the optimization of detection architecture. first with grads PCR optimize annealing temperature (Lowes C, Lamb H,
Walter J. Predicting the optimal PCR primer annealing temperature for use
with the Transgenomic Optimase (TM) polymerase. European Journal of Human
Genetics, 2002,10:296) 50 DEG C to 60 DEG C 8 gradients (table 2) are set automatically by PCR.Utilize the suitableeest temperature pair
Each component (Mg in PCR system2+, dNTPs, primer, rTaq enzyme) concentration be optimized, 7 different concentration gradients are set
(table 2).
The setting of each condition in table 2 system
Utilize 8 gradient optimizing annealing temperatures that PCR instrument is arranged automatically.Result shows, 8 temperature all can amplify preferably
Band, the last item is more preferable comparatively speaking, and therefore the suitableeest annealing temperature of this specific primer is defined as 50 DEG C (Fig. 2-A).Mg2+
Gradient test shows, works as Mg2+When concentration is 2.25 mmol/L, PCR expanding effect is preferably (Fig. 2-B)；DNTPs gradient test table
Bright, when dNTPs concentration is 0.25mmol/L, PCR expanding effect is preferably (Fig. 2-C)；Primer gradient test shows, when primer is dense
When degree is 0.25 μm ol/L, PCR expanding effect is preferably (Fig. 2-D)；RTaq enzyme gradient test shows, each rTaq enzyme dosage can
Preferably amplification, for cost-effective, selects minimum rTaq enzyme dosage 0.2U(Fig. 2-E).
The optimization PCR amplification system determining special primer according to the above results is: 2.5 μ L 10 × PCR Buffer；
2.25μL Mg2+(25mmol/L)；2.5μL dNTPs(2.5mmol/L)；0.625 μ L primer SS-F/SS-R (10 μm ol/L)； 1
μ L template (10ng/ μ L)；0.2U rTaq enzyme；ddH2O complements to 25 μ L.Response procedures is: 94 DEG C of denaturations 5min；94 DEG C of changes
Property 30s；50 DEG C of annealing 30s；72 DEG C extend 45s, circulate 35 times；72 DEG C are supplemented extension 5min；Last 4 DEG C of preservations.
Embodiment 5:SCAR special primer specificity verification. with the DNA of all strains testeds as template, draw with special
Thing SS-F/SS-R carries out PCR amplification, with distilled water for comparison.PCR amplification is carried out with the system optimized.After reaction terminates, take
Electrophoresis 30 min under 6 μ L amplified productions (are previously added Glodview) in 1.5% Ago-Gel 5V/cm voltage conditions,
BIO-RAD gel imaging system is observed, takes pictures.
Electrophoresis result shows, the genomic DNA of only 5 core ground cane bacteria strains can amplify a bar being about 590 bp
Band, other strains tested and comparison are all without band generation (Fig. 3), and the primer designed by explanation has the strongest specific, it is possible to
Mulberry fruit scaled-down version hyphal cluster germ is uniquely detected from related strain.
It is separately recovered each band to check order, and the specific fragment sequence filtered out with RAPD in NCBI is carried out
BLAST comparison, comparison result shows that the sequence that the band sequence of all 5 bacterial strains amplifies with RAPD is identical, similitude
It is 100%.Illustrate that special primer SS-F/SS-R is identical with random primer CS23 testing result, prove RAPD mark further
Note successful conversion is more stable SCAR mark, also demonstrates that the SCAR primer designing and screening acquisition in the present invention can be used for
The cause of disease core ground cane bacterium of specific detection mulberry fruit scaled-down version sclerotiniose.
Embodiment 6: the sensitivity technique of specific primer. measure, with nucleic acid concentration analyzer, the mulberry fruit extracted and reduce
Type hyphal cluster germ genomic DNA concentration, and use concentration gradient dilution method (Han Guangtao, Yang Zhihui, Zhu Jiehua, Zhao Dongmei,
Han Yanqing. double PCR technology for detection Potato Ring Rot and the foundation of black shank bacterium method. Scientia Agricultura Sinica,
2011,44 (20): 4199-4206) it is diluted to 1 × 10-5Ng/ μ L, with distilled water for comparison, uses specific primer SS-F/SS-
R is carried out according to the reaction system optimized and program.After reaction terminates, take 6 μ L amplified productions (pre-in 1.5% Ago-Gel
It is initially charged Glodview) electrophoresis 30 min under 5V/cm voltage conditions, observes on BIO-RAD gel imaging system, takes pictures.
By S. shiraiana genomic DNA from 10ng/ μ L start successively 10 times be diluted to 1fg/ μ L, each concentration takes 1 μ L
The system optimized is utilized to expand for template.Result shows in 25 μ L systems, and specific primer SS-F/SS-R can be from
Mass concentration be 1 pg/ more than μ L template in amplify the specific band of 1 treaty 590 bp, and fail in dilution
To the template DNA and comparison of 1 pg/ below μ L, amplification is to specific band (Fig. 4).
Embodiment 7: the Fields detection of detection architecture. utilize set up detection architecture diseases of mulberry fruits is fallen ill fruit,
Healthy fruit, root, stem, leaf and mulberry tree underlying soil detect.Healthy mulberry newly formed after 2 florescences of testing inspection
Mulberry, the sick fruit of 3 different onset forms, 1 Mulberry Roots, 2 mulberry stem, 2 mulberry leaves, the soil sample of 12 parts of random acquisitions, 2
Core ground cane bacterium inoculation after two days but do not show the sorosis sample of disease, with mulberry fruit scaled-down version hyphal cluster germ genomic DNA as positive control,
With water as negative control, expand by newly-established SCAR-PCR detection architecture.Result shows, sick fruit is in the sample reducing shape
Can amplify band, the sorosis sample of 2 inoculation core ground cane bacterium also can amplify same band；And the gene of other sample
Group DNA all can not amplify band (Fig. 5).
Embodiment 8: detection architecture is used for disease screening. the sick fruit sample gathered is observed by Artificial Diagnosis.Due to mulberry
Mulberry hypertrophy type sclerotiniose and granule type sclerotiniose the most all show as loose shape, thus sample can only manually be divided into loose type and
Scaled-down version.Extract sick fruit STb gene respectively, utilize the SCAR-PCR technology for detection set up；Testing result is carried out with Artificial Diagnosis
Relatively.
From the sample gathered, randomly select 8 apparent " scaled-down version " sick fruit and 8 apparent " loose type " sick fruits, carry respectively
Take STb gene；With mulberry fruit scaled-down version hyphal cluster germ genomic DNA as positive control, with sterile purified water water as negative control, with inspection
Survey system expands.Result shows, has 5 to detected S. shiraiana in 8 scaled-down version disease fruits, and 3 are not detected
Go out；Having 1 to detected S. shiraiana in 8 loose type disease fruits, remaining 7 all do not detect S. shiraiana
(Fig. 6).As can be seen here, there is certain deviation in artificial diagnostic result, and SCAR-PCR detection architecture diagnostic result is the most accurate
Reliably.
Ground cane bacterium (Scleromitrula shiraiana) the SCAR DNA sequence table of mulberry fruit scaled-down version sclerotiniose cause of disease core
001-CCACAGCAGT AAAGAGAACA TCAAACTTCG CACTCTCAAG AAAACCCAAT TCGACAGCAA-060
061-ACATCTAAAT GTTATCACAG GCCGTCTACA ACTCTGGATT AAGAAAGCAG AAAAGAGTCC-120
121-CGAGGCCACA ATGCCTGGAA TGAAATCCTC ACTAGATCGC GCACCCACTT CCTTATCAAC-180
181-CATGAGCCGT ACCTCATTTG CTTCAACCGC TACCATCAAA GCTCAACAAT CAATTCCAGG-240
241-CGCACAACTC ACAGAAATAG ATGATGGTGA TGGTTTGAGA GTTGAGAAGC CTGTGGCACC-300
301-GGTCTTGGTT ATGTTTGTCG AGAGCTCATC GAGGGATAAA GATATTTTGC AGGGTCATAT-360
361-TTTGGCTTTT AAGAGTAAGT CTTGCTCGCT GGAATGTGTC AAATTTGGTA GAAAGGTTTC-420
421-TCGTGGTTGG ATCTTGCTTG CTGAGTTGAC TGTTTACTAA TGAAATATGG TATTTAGTCG-480
481-ACCAAAATGT GAATATTGAT CGTAAAAAAT GTAAATGCGA TGATAAGAAG GCAAAATGTG-540
541-CGAGATGTTT TATTATTCAT CCCGTGCGTG TGGATCTTGA GATTGGTCGG TGGAAGTATC-600
601- CTACTGCTGT GG-660
Core ground cane bacterium specific and conserved sequence (SCAR sequence) of note: 590bp, logs in after order-checking into NCBI GenBank
In, it is thus achieved that its accession number (Access Number)KP146145；In sequence the fragment of underscore designate respectively up/down trip draw
Thing (SS-F/SS-R) position (only position, the not sequence of downstream primer itself).
Claims (1)
1. the method for a quick diagnosis mulberry fruit scaled-down version sclerotiniose, it is characterised in that realized by following steps:
(1) take testing sample, use CTAB method to extract STb gene from sample；
(2) it is specific that the core ground cane bacterium specific and conserved sequence utilizing 590bp design and screen with obtaining core a pair of cane bacterium
SCAR-PCR primer, primer forward sequence SS-F is 5'-AGTAAAGAGAACATCAAACTTCG-3', is positioned at distinguished sequence
8 bp-30 bp；Reverse sequence SS-R is 5'-ACTTCCACCGACCA ATCT-3', is positioned at the 580bp-596 of distinguished sequence
bp；
(3) carrying out PCR amplification, amplification system is: 2.5 μ L 10 × PCR Buffer； 2.25μL Mg2+, concentration is 25mmol/
L；2.5 μ L dNTPs, concentration is 2.5mmol/L；0.625 μ L primer SS-F/SS-R, concentration is 10 μm ol/L；1 μ L template, dense
Degree is 10ng/ μ L；0.2U rTaq enzyme； ddH2O complements to 25 μ L；Response procedures is: 94 DEG C of denaturations 5min；94 DEG C of sex change
30s；50 DEG C of annealing 30s；72 DEG C extend 45s, circulate 35 times；72 DEG C are supplemented extension 5min；According to amplified band diagnosis mulberry fruit contracting
Bulbil is sick.
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