CN106868176A - The specific PCR amplimer and detection architecture of F.graminearum schw and application - Google Patents
The specific PCR amplimer and detection architecture of F.graminearum schw and application Download PDFInfo
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Abstract
The present invention provides the specific PCR amplimer and detection architecture of F.graminearum schw, and the primer includes Fg18TF and Fg18TR (Seq ID No:2) and a pair of nido outer primer HW18TF/HW18TR (Seq ID No 1 and:3 and 4).The present invention is based on F.graminearum schw and difference of other fusarium kinds in β tubulin gene orders, design a series of primer of detection F.graminearum schws, therefrom filter out the most strong primers F g18TF/Fg18TR of specificity, and improve its detection sensitivity by designing a pair of nido outer primer HW18TF/HW18TR, PCR detection architectures are set up on this basis, and pathogen environment that can be complicated from morbidity plant tissue and soil quickly and accurately detects F.graminearum schw.It is easy to operate according to the detection kit that the method builds, specificity is good, sensitivity is high, and the brood body of the various forms of F.graminearum schw such as mycelia, conidium etc. can be detected, the aspect such as cause of disease monitoring of early warning, epidemic-stricken area to F.graminearum schw epidemic situation is significant.
Description
Technical field
The present invention relates to molecular Biological Detection technology, specifically, it is related to F.graminearum schw (Fusarium
Graminearum specific PCR amplimer) and detection architecture, the kit containing the primer and its application.
Background technology
Corn is one of most important cereal crops of China, with food industry and the development of feed industry, corn conduct
The status of Main Foods and forage crop also increasingly rises.Ear rot is the important disease in Maize Production, true by various cause of diseases
Bacterium causes, and not only directly contributes corn yield loss, and pathogenic bacteria in seed can also produce various mycotoxins, such as single
The mould alkene of end spore, fumonisin and zearalenone etc., cause corn quality to decline, and great safety is brought to food and feed
Hidden danger, threatens human and livestock health.
Fusariumsp (Fusarium spp.) is one of disease fungus monoid common in Maize Production, can be in corn
Trigger serious disease on root, stem, leaf, fruit, cause huge economic loss.Numerous studies show that Fusariumsp is to cause corn
The most important pathogen of ear rot, additionally, can also cause the stem rot of corn, sheath maize ear rot, top rot, root rot, seedling blight etc.
Disease.Principal causative fusarium kind includes that F.graminearum schw aggregate species, plan wheel branch fusarium, layer go out fusarium, sharp fusarium aggregate species, yellow sickle
Spore, scouring rush's fusarium etc..The corncob on the ground such as research discovery, the U.S., Canada, Germany, Austria, Nepal, Brazil, Argentina
Maize ear rot fusarium of causing a disease is mainly F.graminearum schw aggregate species, and Canada, Germany, Brazil, Argentina, France, Italy, Ni Luodi
The pathogenic fusarium on the ground such as Asia, Nepal, South Africa, Iran is mainly plan wheel branch fusarium, and layer goes out fusarium and is then mainly distributed on Germany, bar
West, Nepal and the Argentinian northwestward.In China, F.graminearum schw aggregate species be distributed mainly on Shanxi, Shaanxi, Gansu, Yunnan,
Guizhou, northeast and Huang-Huai Area, intend wheel branch fusarium and are distributed mainly on northeast, Beijing, Hebei, Henan, Shandong, Anhui, Sichuan, lake
The ground such as south, Hubei, other pathogenic kinds also have a small amount of distribution.As can be seen here, F.graminearum schw Distribution Area is wider, is that many areas are beautiful
The Prominent pathogen of rice ear rot.
Be distributed widely in nature due to Fusariumsp, species is more, morphological variation is big, and traditional Fusariumsp separate with
Difference division group on morphology, plant, using the pathogenicity difference of different hosts or different cultivars as specialized form or biological strain
Identification of means, identification and appraisal to Fusariumsp bring certain difficulty.In recent years, with Protocols in Molecular Biology
Fast development, round pcr and its correlation molecule detection technique be widely used in the Molecular Detection of phytopathogen growth and decline state with
Early warning, the concept for fusarium kind there occurs great changes on concept, and Fusariumsp classification is closed closer to natural relationship
System, therefore, the different species specific molecular labelings of fusarium are developed, there is larger application value for precise Identification fusarium kind.
Beta tubulin (β-tubulin) gene is widely present in eucaryote, there is no designed for the target gene at present
Specific F.graminearum schw detection primer relevant report.
The content of the invention
It is an object of the invention to provide a kind of specific PCR amplimer that can quickly and accurately detect F.graminearum schw and
Detection architecture and the kit containing the primer.
It is a further object of the present invention to provide the application of above-mentioned primer and kit in F.graminearum schw is detected.
It is of the invention from Fusarium Center's database at Penn State numbers in order to realize the object of the invention
According to beta tubulin (β-tubulin) gene order (KM373925.1) that F.graminearum schw (F.graminearum) is downloaded in storehouse,
Analyze F.graminearum schw and difference (Fig. 1) of other fusarium kinds in β-tubulin gene orders, it was found that several high specials
And exist only in the site in F.graminearum schw, gone out using the Software for Design of Primer 5 a series of for specific detection F.graminearum schw
Primer, therefrom filters out the most strong primers F g18TF/Fg18TR of specificity, and devise nido outer primer HW18TF/HW18TR
To improve sensitivity.The PCR reaction systems of detection F.graminearum schw are established on this basis.
The F.graminearum schw specific PCR amplimer that the present invention is provided, including (Seq ID No:1-4)：
Forward primer Fg18TF：5’-TTGTAAGTGTTTTCCTCTGACCT-3’
Reverse primer Fg18TR：5’-AGAAAGCAGCACCGATTTGG-3’；And
Positive nido outer primer HW18TF：5’-AACATGCGTGAGATTGTAAGTG TT-3’
Reverse nido outer primer HW18TR：5’-TAAACACCATTGCTGTCGAGA-3’
The present invention also provides the kit for detecting F.graminearum schw containing above-mentioned PCR primer.
Also include dNTPs, Taq archaeal dna polymerase, Mg in aforementioned agents box2+, PCR reaction buffers, standard positive template
At least one in.
The present invention also provides the application of above-mentioned PCR primer or kit in F.graminearum schw is detected, comprises the following steps：
1) DNA in sample is extracted；
2) with step 1) in extract DNA be template, using primers F g18TF and Fg18TR carry out single-wheel PCR amplification instead
Should；Or,
When template DNA concentration is too low leads to not amplify band, using nest-type PRC；First with nido outer primer
HW18TF and HW18TR carries out first round pcr amplification reaction, 1~2 μ L amplified productions is then taken as template, using primer
Fg18TF and Fg18TR carries out the second wheel pcr amplification reaction；
3) pcr amplification product is analyzed.
Foregoing PCR reaction systems are：The μ L of 0.25 μ L, 10 × PCR reaction buffer of 1U/ μ L Taq archaeal dna polymerases 1.0,
2.5mM dNTPs 0.5 μ L, 10 μm of ol/L forward directions, each 0.5 μ L of reverse primer, DNA profiling 1.0 μ L, ddH2O 6.25μL。
Carry out the second wheel pcr amplification reaction in single-wheel pcr amplification reaction and nest-type PRC, the response procedures for being used for：
94℃5min；95 DEG C of 50s, 58 DEG C of 50s, 72 DEG C of 1min, 35 circulations；72℃10min.
First round pcr amplification reaction in nest-type PRC, the response procedures for being used for：94℃5min；95 DEG C of 50s, 56 DEG C
50s, 72 DEG C of 1min, 35 circulations；72℃10min.
1~2% agarose gel electrophoresis is carried out to above-mentioned pcr amplification product, if electrophoresis detection result size occurs about
It is the characteristic bands of 291bp, then shows to contain F.graminearum schw in sample.
Sample of the present invention is the mycelia containing F.graminearum schw, conidial sample.
The present invention also provides the specific PCR detection architecture of F.graminearum schw, including：The μ of 1U/ μ L Taq archaeal dna polymerases 0.25
The μ L of 1.0 μ L, 2.5mM dNTPs of L, 10 × PCR reaction buffer 0.5,10 μm of ol/L forward directions, each 0.5 μ L, DNA moulds of reverse primer
Plate 1.0 μ L, ddH2O 6.25μL。
Beta tubulin (β-tubulin) the gene order difference of the present invention based on F.graminearum schw with other fusarium kinds, design
Go out can quick detection F.graminearum schw Specific PCR primers, and establish PCR rapid detection systems on this basis, can be from hair
Complicated pathogen environment quickly and accurately detects F.graminearum schw in sick plant tissue and soil.Built according to the method
Detection kit is easy to operate, specificity it is good, sensitivity is high, can to brood body such as the mycelia of the various forms of F.graminearum schw, divide
Raw spore etc. is detected that the aspect such as cause of disease monitoring of early warning, epidemic-stricken area to F.graminearum schw epidemic situation is significant.
Brief description of the drawings
Fig. 1 is the β-tubulin gene orders part comparison result of F.graminearum schw of the present invention and other fusarium kinds.
Fig. 2 be the embodiment of the present invention 2 in using primers F g18TF/Fg18TR enter performing PCR amplification result；Wherein, 1：Point
Fusarium；2：Layer goes out fusarium；3：Yellow fusarium；4：Eggplant fusarium；5：F.graminearum schw；6：Intend wheel branch fusarium；7：Redden fusarium；8：Teng Cang
Fusarium；9：Pythium spp；10：Penicillium notatum；11：Aspergillus flavus；12：Trichoderma, M is 100bp DNA Ladder.
Fig. 3 is the pcr amplification reaction system sensitivity technique result of primers F g18TF/Fg18TR in the embodiment of the present invention 2,
Wherein, 1-8 distinguishes representation DNA sample through 101、102、103、104、105、106、107With 108Dilute again, M is 100bp DNA
Ladder。
Fig. 4 be the embodiment of the present invention 2 in entered using nido outer primer HW18TF/HW18TR and primers F g18TF/Fg18TR
Row two-wheeled pcr amplification reaction system sensitivity technique result, wherein, 1-8 distinguishes representation DNA sample through 101、102、103、104、
105、106、107With 108Dilute again, M is 100bp DNA Ladder.
Specific embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.If not specializing, embodiment
According to conventional laboratory conditions, such as Sambrook equimoleculars Cloning: A Laboratory Manual (Sambrook J＆Russell DW,
Molecular Cloning:A Laboratory Manual, 2001), or the condition advised according to manufacturer's specification.
Embodiment 1 is used for the synthesis of the Specific PCR primers for detecting F.graminearum schw
The β micro-pipes of F.graminearum schw are downloaded from Fusarium Center's database at Penn State databases
Albumen (β-tubulin) gene order (KX087134.1), using biological softwares such as DNAMAN to F.graminearum schw and other sickles
β-tubulin the gene orders of spore kind compare, it was found that several high specials and exist only in F.graminearum schw
Site, a series of primer of detection F.graminearum schws is designed using software Primer 5, therefrom filters out specificity, sensitivity most
Strong primers F g18TF/Fg18TR (table 1), nido outer primer HW18TF/HW18TR (table 2) simultaneously establishes detection on this basis
The PCR reaction systems of F.graminearum schw.
The F.graminearum schw specific PCR detection primer Fg18TF/Fg18TR information of table 1
The nest-type PRC outer primer HW18TF/HW18TR information of table 2
Primer synthesis is completed by Shanghai Ying Jun Bioisystech Co., Ltd.
PCR reaction systems are：0.25 μ L, 10 × PCR reaction buffer of 1U/ μ L Taq archaeal dna polymerases 1.0 μ L, 2.5mM
DNTPs 0.5 μ L, 10 μm of ol/L forward directions, each 0.5 μ L of reverse primer, DNA profiling 1.0 μ L, ddH2O 6.25μL。
The response procedures that single-wheel pcr amplification reaction is used for：94℃5min；95 DEG C of 50s, 58 DEG C of 50s, 72 DEG C of 1min,
35 circulations；72℃10min.
When template DNA concentration is too low leads to not amplify band, using nest-type PRC.
The response procedures of nest-type PRC are：
First round pcr amplification reaction：94℃5min；95 DEG C of 50s, 56 DEG C of 50s, 72 DEG C of 1min, 35 circulations；72℃
10min。
Second wheel pcr amplification reaction：94℃5min；95 DEG C of 50s, 58 DEG C of 50s, 72 DEG C of 1min, 35 circulations；72℃
10min。
Embodiment 2 using PCR primer Fg18TF/Fg18TR and HW18TF/HW18TR detection F.graminearum schw specificity and
Sensitivity analysis
1.1 samples sources：
The pathogen used in the present embodiment includes that sharp fusarium, F.graminearum schw, yellow fusarium, eggplant fusarium, layer go out fusarium, intend
Wheel branch fusarium, the fusarium that reddens, rattan storehouse fusarium, pythium spp, Penicillium notatum, Aspergillus flavus, Trichoderma, make by the Chinese Academy of Agricultural Sciences
Thing Science Institute provides.
1.2 Fusariumsp extracting genome DNAs：
To be respectively transferred in different PDA culture mediums through the different Fusariumsp separators of single spore separation, 25 DEG C of culture 7d are scraped
Take after mycelia is dried loaded in 2.0mL centrifuge tubes.DNA is extracted with fungal genomic DNA Rapid extraction kit (Shanghai life work).
Through agarose gel electrophoresis and UV-VIS spectrophotometer Q5000 ultraviolet-visible spectrophotometers (Quawell,
USA) its content of double check and purity, save backup in -20 DEG C.
The gradient dilution of 1.3 sample DNAs：
Detected with UV-VIS spectrophotometer Q5000 ultraviolet-visible spectrophotometers (Quawell, USA)
The DNA concentration of the F.graminearum schw sample of said extracted, is 1089 μ g/mL.DNA sample is carried out into 10 times of gradient dilutions, 8 are diluted altogether
Secondary, highest is diluted to 108Times, saved backup in -20 DEG C.
1.4PCR amplified reactions：
Amplified reaction is carried out in Biometra T3000 thermal cyclers.PCR reaction systems and response procedures are with embodiment 1
It is described.
1.5 results：
After reaction terminates, by 2 μ L sample-loading buffers (the 10mmol/L EDTA of pH8.0,98% deionized formamide,
0.025% dimethylbenzene green grass or young crops FF) it is sufficiently mixed with pcr amplification product, take 5 μ L on 1% Ago-Gel added with nucleic acid dye
Carry out electrophoresis detection, voltage is 50-100V, after 30min under gel imaging system observed and recorded, according to 100bp DNA
Ladder estimates stripe size.Electrophoresis detection result as shown in Fig. 2 swimming lane 5 be F.graminearum schw sample, about 291bp can be amplified
Characteristic bands, and there is not amplified band in other fusarium kinds.The above results show the specificity of primers F g18TF/Fg18TR
By force, F.graminearum schw can substantially be made a distinction with other fungies using the primer.
Respectively single-wheel pcr amplification reaction is carried out by template of the DNA sample of above-mentioned 8 concentration gradients dilution.PCR expands journey
Sequence and amplified production detection method are ibid.Result is not as shown in figure 3, when being diluted to 100 times, band is obvious.Therefore, the primer
To sensitivity up to 100 μ g/mL, detection sensitivity is relatively low.
The DNA sample with the dilution of above-mentioned 8 concentration gradients utilizes nest-type PRC primer HW18TF/HW18TR as template respectively
Wheel PCR amplifications are first carried out, then take 1 μ L product to carry out the second wheel PCR amplifications, PCR amplification programs and amplified production are detected
Method is ibid.Result is diluted to 10 as shown in figure 4, working as8Times when, band is not obvious.Therefore, can be by primer spirit using nest-type PRC
Sensitivity is promoted to 100fg/mL, and detection sensitivity is very high.
Using PCR reaction systems of the invention, the sample that can be caught an illness to different F.graminearum schws carries out qualitative Molecular Detection, application
The method can Accurate Prediction crop field F.graminearum schw growth and decline state and epidemic situation development trend, when being easy to determine in time the optimal prevention and control of disease
Phase, block the breeding of source germ and spread, reach the purpose for efficiently administering F.graminearum schw disease.With the popularization of round pcr,
Using round pcr detection disease, compared with traditional Morphological Identification method, its operation is more simple, quickly, low cost.And this
Invention make use of nest-type PRC, and its sensitivity has reached 100fg/mL, be examined based on LAMP method relative to ZL201410456539.8
The lower limit of survey is 100pg, and the sensitivity of this method detection improves 1000 times, can be widely used for detecting the cause of disease of lower concentration
Bacterium.
Nest-type PRC is a kind of polymerase chain reaction of variation (PCR), and complete fragment is expanded using two pairs of PCR primers, by
It is located inside first round PCR primer in second set of primer, rather than purpose segment includes two sets of possibility poles of primer binding site
It is small, therefore second set of primer can not possibly expand non-purpose segment.This nested PCR amplification effectively prevent the second wheel PCR primer
Because primer pairing specificity is not strong and caused by non-specific amplification phenomenon, therefore nest-type PRC specificity is very strong.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be modified or improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Institute of Crop Science, Chinese Academy of Agricultural Science
<120>The specific PCR amplimer and detection architecture of F.graminearum schw and application
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Claims (10)
1. the specific PCR amplimer of F.graminearum schw (Fusarium graminearum), it is characterised in that including：
Forward primer Fg18TF：5’-TTGTAAGTGTTTTCCTCTGACCT-3’
Reverse primer Fg18TR：5’-AGAAAGCAGCACCGATTTGG-3’；And
Positive nido outer primer HW18TF：5’-AACATGCGTGAGATTGTAAGTG TT-3’
Reverse nido outer primer HW18TR：5’-TAAACACCATTGCTGTCGAGA-3’.
2. the kit for detecting F.graminearum schw containing primer described in claim 1.
3. kit according to claim 2, it is characterised in that the kit is also polymerized including dNTPs, Taq DNA
Enzyme, Mg2+, PCR reaction buffers, at least one in standard positive template.
4. the application of primer described in claim 1 or kit described in Claims 2 or 3 in F.graminearum schw is detected.
5. application according to claim 4, it is characterised in that comprise the following steps：
1) DNA in sample is extracted；
2) with step 1) in extract DNA be template, carry out single-wheel pcr amplification reaction using primers F g18TF and Fg18TR；Or
Person,
When template DNA concentration is too low leads to not amplify band, using nest-type PRC；First with nido outer primer HW18TF
Carry out first round pcr amplification reaction with HW18TR, then take 1~2 μ L amplified productions as template, using primers F g18TF and
Fg18TR carries out the second wheel pcr amplification reaction；
3) pcr amplification product is analyzed.
6. application according to claim 5, it is characterised in that PCR reaction systems are：1U/ μ L Taq archaeal dna polymerases
0.25 μ L, 10 × PCR reaction buffer, 1.0 μ L, 2.5mM dNTPs 0.5 μ L, 10 μm of ol/L forward directions, each 0.5 μ L of reverse primer,
DNA profiling 1.0 μ L, ddH2O 6.25μL。
7. application according to claim 5, it is characterised in that carry out second in single-wheel pcr amplification reaction and nest-type PRC
Wheel pcr amplification reaction, the response procedures for being used for：94℃5min；95 DEG C of 50s, 58 DEG C of 50s, 72 DEG C of 1min, 35 circulations；
72℃10min；
First round pcr amplification reaction in nest-type PRC, the response procedures for being used for：94℃5min；95 DEG C of 50s, 56 DEG C of 50s, 72
DEG C 1min, 35 circulations；72℃10min.
8. the application according to claim any one of 5-7, it is characterised in that 1~2% agar is carried out to pcr amplification product
Sugared gel electrophoresis, if the characteristic bands that size is 291bp occurs in electrophoresis detection result, shows to contain cereal sickle in sample
Spore.
9. application according to claim 8, it is characterised in that the sample is the mycelia containing F.graminearum schw, mitogenetic spore
The sample of son.
10. the specific PCR detection architecture of F.graminearum schw, it is characterised in that including：The μ L of 1U/ μ L Taq archaeal dna polymerases 0.25,
10 × PCR reaction buffers, 1.0 μ L, 2.5mM dNTPs 0.5 μ L, 10 μm of ol/L forward directions, each 0.5 μ L of reverse primer, DNA profiling
1.0 μ L, ddH2O 6.25μL；
Wherein, the positive, definition of reverse primer is with described in claim 1.
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| CN110373412A (en) * | 2019-07-25 | 2019-10-25 | 中国农业大学 | Primer for detecting F.graminearum schw combines and detection method |
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| WO2006005770A2 (en) * | 2004-07-15 | 2006-01-19 | Technische Universität Wien | Method for the detection of fusarium graminearum |
| WO2016203740A1 (en) * | 2015-06-17 | 2016-12-22 | 東洋製罐グループホールディングス株式会社 | Method for examining microorganism, kit for examining microorganism, microarray for examining microorganism, carrier for detecting mold, method for detecting mold and kit for detecting mold |
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