CN101178379A - Method for detecting No.4 race of Fusarium oxysporum f sp. Cubense - Google Patents
Method for detecting No.4 race of Fusarium oxysporum f sp. Cubense Download PDFInfo
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- CN101178379A CN101178379A CNA2007100321182A CN200710032118A CN101178379A CN 101178379 A CN101178379 A CN 101178379A CN A2007100321182 A CNA2007100321182 A CN A2007100321182A CN 200710032118 A CN200710032118 A CN 200710032118A CN 101178379 A CN101178379 A CN 101178379A
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Abstract
The invention discloses a rapid detection method of No.4 race of fusarium oxysporum f.sp.cubense, including the steps of extraction of DNA of banana plants, electrophoresis detection of the template DNA, PCR amplification of DNA amplification, PCR circulation and electrophoresis detection, the invention makes use of a primer of the No.4 race of the fusarium oxysporum f.sp.cubense to carry out the PCR amplification and circulation of the DNA of the banana plants, and then the electrophoresis detection is carried out, the whole detection process consumes the time of less than 6 hours, which greatly improves the detection efficiency, the invention does not need to carry out the separation and the purification of pathogens in the sample, the steps are simple, the operation is easy, the complicated and precise instruments are unnecessary, the invention is more accurate and sensitive, so the invention is more conductive to the rapid and convenient development of a plant quarantine work.
Description
Technical field
The present invention relates to plant protection and biological technical field, relate in particular to the method for quick of No. 4 microspecies of banana blight bacteria.
Background technology
The banana fusarium wilt claims banana Panama disease, yellowtop again, is the soil-borne disease that is caused the vascular bundle necrosis by fusarium infection, and pathogen is Fusarium oxysporum Cuba specialized form (Fusarium oxysporum f.sp.cubense).Pathogen is invaded from injured rhizome, develops on stem by host's vascular bundle, and further spreads to leaf portion.After plant was withered, pathogen was retained in the soil with residual body, along with current or the farm implements that carry disease germs carry out natural propagation again in any of several broadleaf plants garden, and its chlamydospore 8-10 of can in soil, surviving.Because this bacterium survival period is long, propagate rapidly, Shang Weiyou prevents and treats this sick specific medicament, and therefore large tracts of land breaks out and causes heavy losses easily.
Fusarium oxysporum Cuba specialized form has 4 biological strains, No. 1 microspecies (FOC1) infect cultigen " big honey is breathed out " [Grosmichel (AAA)] and the fossilia dentis mastodi any of several broadleaf plants (MusaAAB) of banana, [Dwarfcavendish (AAA)] is more disease-resistant for short banana, these microspecies are the world and distribute, the existing record in the China's Mainland; No. 2 microspecies (FOC2) only infect triploid hybrid shuttle banana [Bluggoe (AAB)] in Central America, do not infect " big honey is breathed out "; No. 3 microspecies (FOC3) infect wild castrated ram tail any of several broadleaf plants and belong to (Heliconia); No. 4 microspecies (FOC4) can infect nearly all banana species, refer to any of several broadleaf plants as Cevendish, " big honey is breathed out ", short banana, wild any of several broadleaf plants (BB) and shuttle, and are crushing maximum.At present, do not cultivate the anti-No. 4 microspecies kinds of effective banana as yet.
At present, No. 4 microspecies of banana blight bacteria are still adopted traditional biology and pathogenic inspection technical appraisement, need the long time could distinguish No. 4 microspecies and No. 1 microspecies.Even adopt pathogen to separate and determine microspecies No. 4 in conjunction with the proterties on VCG or the Komada improved culture medium, also need the long period, generally needed 30 days even the more time evaluation is made a definite diagnosis, be unfavorable for the timely prevention and control of banana blight.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, the method for quick of No. 4 microspecies of a kind of banana blight bacteria is provided, the DNA that utilizes the primer of No. 4 microspecies of banana blight bacteria to carry out banana plant carries out carrying out electrophoresis detection after pcr amplification and the circulation.
Technical scheme of the present invention provides the method for quick of No. 4 microspecies of a kind of banana blight bacteria, may further comprise the steps:
(1) DNA of extracting banana plant;
(2) template DNA of electrophoresis detection (1);
(3) pcr amplified dna amplification;
(4) PCR circulation;
(5) electrophoresis detection.
The described electrophoresis detection template DNA of step (2) is that the banana plant DNA sample of getting the extraction of 5 μ L (1) steps mixes with the load sample damping fluid, the agarose gel electrophoresis 40min with 1%, and 5v/cm, observation has or not the DNA band under gel imaging system.6 * loading buffer that described load sample damping fluid is produced for TAKARA company.
The amplification reaction system of the described pcr amplified dna of step (3) is as follows:
10×PCR?Buffer 2.5μL；
Mg
2+(25mmoL/L) 2.5μL；
dNTPs(25mmoL/L) 2μL；
Primer RF4-1 (10 μ moL/L) 1 μ L;
Primer RF4-2 (10 μ moL/L) 1 μ L;
Taq enzyme (5U/ μ L) 0.2 μ L;
Sterilization ddH
2O is to cumulative volume 25 μ L.
Described primer is:
RF4-1：5’-TGCGGGTCCTATTAGTAC-3’；
RF4-2：5’-GGTCCTCACACTCCAATC-3’。
The described PCR circulation of step (4) is 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s, 52 ℃ of annealing 30s, 72 ℃ are extended 45s, 35 circulations; 72 ℃ are extended 10min.
The described electrophoresis detection of step (5) is to take out step (4) reaction mixture 5 μ L to mix with the load sample damping fluid, and 1% agarose gel electrophoresis detects amplified production 903bp specific band.Running gel is: 1% Ago-Gel, prescription is: the 1g agarose, 100mL0.5 * TBE electrophoretic buffer, 5 μ L ethidium bromides (EB), wherein, 0.5 * TBE electrophoretic buffer is by 5 * TBE (54g Tris, 27.5g boric acid, 20ml 0.5mol/L EDTA (pH8.0) is settled to 1000ml) dilution ten times re-use.
The present invention compared with prior art, its beneficial effect shows:
(1) the invention provides a detection method fast, use this method and can within 6 hours, carry out fast, accurately detect No. 4 microspecies of banana blight bacteria.
(2) primer provided by the invention and method need not the pathogen in the sample is separated and purifying, and step is simple, and operation need not complexity, exact instrument easily, helps quick, the convenient plant quarantine work of carrying out.
(3) monitoring method of the present invention has specificity and the accuracy at No. 4 microspecies of banana blight bacteria.
(4) the present invention can Sensitive Detection arrive No. 4 microspecies of banana blight bacteria that low bacterium is measured.
Description of drawings
The specificity electrophoretogram that Fig. 1 adopts the inventive method to carry out the PCR detection in 23 bacterial strains detects result's (903bp specific band) of FOC4
Fig. 2 the inventive method is carried out the sensitivity electrophoretogram testing result (903bp specific band) that PCR detects variable concentrations pathogen FOC4 DNA
Fig. 3 separate sources banana is intended the result's (903bp specific band) like droop pattern detection FOC4
Specific embodiment
Below in conjunction with the drawings and specific embodiments the present invention is done explanation in further detail.
Use the inventive method and in 23 bacterial strains, carry out the specificity electrophoretogram detection that PCR detects.
1. the DNA of extracting plant: the CTAB method is carried out routinely.
2. electrophoresis detection template DNA: get the DNA sample that 5 μ L are extracted and mix the agarose gel electrophoresis 40min with 1%, 5v/cm with the load sample damping fluid, observe down in gel imaging system, if the DNA band is arranged, then DNA extracting success, can continue to use, if no DNA band then needs extracting again.
3.PCR amplification: the dna amplification reaction system is as follows:
10×PCR?Buffer 2.5μL
Mg
2+(25mmoL/L) 2.5μL
dNTPs(25mmoL/L) 2μL
Primer RF4-1 (10 μ moL/L) 1 μ L
Primer RF4-1 (10 μ moL/L) 1 μ L
Taq enzyme (5U/ μ L) 0.2 μ L
Sterilization ddH
2O is to cumulative volume 25 μ L
4.PCR circulation:
94 ℃ of pre-sex change 5min
72 ℃ are extended 10min
5. electrophoresis detection: take out reaction mixture 5 μ L and mixes with the load sample damping fluid, 1% agarose gel electrophoresis detects amplified production, and observation has or not the appearance of 903bp specific band, and then positive amplification is arranged.
Using the inventive method detects following germ: No. 4 microspecies F.oxysporum of banana blight bacteria f.sp.cubense race4 (FOC4) and No. 1 microspecies F.oxysporum of banana blight bacteria f.sp.cubense race1 (FOC1), tomato wilt bacterium F.oxysporum f.sp.lyscopersici (FOL), withered germ of water-melon F.oxysporumf.sp.niveum (FON), balsam pear wilt F.oxysporum f.sp.momodicae (FOM), Kidney bean wilt F.oxysporum f.sp.phaseoli (FOP), cotton-wilt fusarium F.oxysporum f.sp.vasinfectum (FOV), fusarium graminearum F.graminearum (FG), colletotrichum gloeosporioides Penz Colletotrichum gloeosporioides (CG), the result who detects as shown in Figure 1, only No. 4 microspecies of banana blight bacteria are at 903bp place generation specific band.In the accompanying drawing 1, M:DL2000 Marker; 1～15:FOC4; 16:FOC1; 17: the tomato wilt bacterium; 18: withered germ of water-melon; 19: the balsam pear wilt; 20: the Kidney bean wilt; 21: cotton-wilt fusarium; 22: fusarium graminearum; 23: colletotrichum gloeosporioides Penz; 24: the clear water contrast.
Experimental procedure detects variable concentrations pathogen DNA with embodiment 1 with PCR, and its sensitivity electrophoresis result is seen accompanying drawing 2, wherein, and M:DL2000 Marker; The total DNA dilution of 1～7:FOC4 gradient (10
0-10
-6); 8: the clear water contrast.Accompanying drawing 2 shows: the DNA extract is in dilution 10
4Still can amplify the specific band of 903bp doubly, promptly can detect the fresh mycelia that is equivalent to 2ng, the sensitivity of the inventive method is described.
(1) extracting banana plant DNA
The CTAB method is carried out routinely, with banana tissue 100～200mg liquid nitrogen grinding powdering to be checked, add 600 μ L reagent C TAB and 12 μ L2-mercaptoethanols, lapping liquid changes in the 1.5mL centrifuge tube, 65 ℃ of water-baths 30 minutes, add 600 μ L phenol the chloroform mixed liquor, under room temperature, 12000rpm condition centrifugal 5 minutes, get supernatant and place new centrifuge tube.NaAc and 2 times of cold absolute ethyl alcohols of volume of adding 1/10 volume 3mol/L, pH5.2 were put-20 ℃ of precipitations 20 minutes; Under 4 ℃, 12000rpm condition centrifugal 10 minutes, with 70% ethanol drip washing DNA precipitation, air-dry under the room temperature respectively; Be dissolved in the 50 μ L sterilization distilled water and obtain dna solution.
(2) electrophoresis detection template DNA
Get the DNA sample that 5 μ L are extracted and mix with the load sample damping fluid, the agarose gel electrophoresis 40min with 1%, 5v/cm observes down in gel imaging system, if the DNA band is arranged, then DNA extracting success can continue to use, if no DNA band, then need extracting again.6 * loading buffer that described load sample damping fluid is produced for TAKARA company.
(3) pcr amplification: synthesize with full-automatic dna synthesizer.The dna amplification reaction system is as follows:
10×PCR?Buffer 2.5μL
Mg
2+25mmoL/L 2.5μL
dNTPs 25mmol/L 2μL
Primer RF4-1 10 μ mol/L 1 μ L
Primer RF4-2 10 μ mol/L 1 μ L
Taq enzyme 5U/ μ L 0.2 μ L
Sterilization ddH
2O is added to reaction system in right amount
(4) PCR circulation
94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s, 52 ℃ of annealing 30s, 72 ℃ are extended 45s, 35 circulations; 72 ℃ are extended 10min.Tabulation is expressed as follows:
94 ℃ of pre-sex change 5min
72 ℃ are extended 10min
(5) electrophoresis detection: take out reaction mixture 5 μ L and mix with the load sample damping fluid, 1% agarose gel electrophoresis detects amplified production, and testing result is seen accompanying drawing 3, wherein, and M:DL2000Marker; 1: Lianjiang City's banana sample; 2～10: Gaozhou City's banana sample; 11～16: Xuwen County banana sample; 17～21: Shantou City's banana sample; 22～23: Zhongshan city's banana sample; 24～29: Jieyang City's banana sample; 30: Zhuhai City's banana sample; 31: the clear water contrast; 32: the healthy plant contrast, testing result is consistent with germ isolation and identification method result.If the 903bp specific band is arranged, then positive amplification has No. 4 microspecies of banana blight bacteria; If no 903bp specific band is then negative, there are not No. 4 microspecies of banana blight bacteria.
Claims (5)
1. the method for quick of No. 4 microspecies of a banana blight bacteria is characterized in that may further comprise the steps:
(1) DNA of extracting banana plant;
(2) template DNA of electrophoresis detection (1);
(3) pcr amplified dna amplification;
(4) PCR circulation;
(5) electrophoresis detection.
2. according to the method for quick of No. 4 microspecies of the described banana blight bacteria of claim 1, it is characterized in that the amplification reaction system of the described pcr amplified dna of step (3) is as follows:
Template DNA 1 μ L;
10×PCR?Buffer 2.5μL；
Mg
2+ 25mmoL/L，2.5μL；
dNTPs 25mmoL/L，2μL；
Primer RF4-1 10 μ moL/L, 1 μ L;
Primer RF4-2 10 μ moL/L, 1 μ L;
Taq enzyme 5U/ μ L, 0.2 μ L;
Sterilization ddH
2O is added to reaction system cumulative volume 25 μ L in right amount.
3. according to the method for quick of No. 4 microspecies of the described banana blight bacteria of claim 2, it is characterized in that described primer is respectively:
Primer RF4-1:5 '-TGCGGGTCCTATTAGTAC-3 ';
Primer RF4-2:5 '-GGTCCTCACACTCCAATC-3 '.
4. according to the method for quick of No. 4 microspecies of the described banana blight bacteria of claim 1, it is characterized in that the described PCR circulation of step (4) is 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s, 52 ℃ of annealing 30s, 72 ℃ are extended 45s, 35 circulations; 72 ℃ are extended 10min.
5. according to the method for quick of No. 4 microspecies of the described banana blight bacteria of claim 1, it is characterized in that the described electrophoresis detection of step (5) is to take out step (4) reaction mixture 5 μ L to mix with the load sample damping fluid, 1% agarose gel electrophoresis detects amplified production 903bp specific band.
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|---|---|---|---|
| CN2007100321182A CN101178379B (en) | 2007-12-05 | 2007-12-05 | Method for detecting No.4 race of Fusarium oxysporum f sp. Cubense |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899506A (en) * | 2010-05-18 | 2010-12-01 | 华南农业大学 | Detection primer for No.1 and No.4 physiological strains of fusarium oxysporum f. sp cubense and rapid detection method |
| CN101974650A (en) * | 2010-11-30 | 2011-02-16 | 中国农业科学院植物保护研究所 | Polymerase chain reaction (PCR) method for detecting fusarium oxysporum and kit |
| CN103789418A (en) * | 2014-01-09 | 2014-05-14 | 中华人民共和国中山出入境检验检疫局 | TaqMan probe real-time fluorescent primer for detecting fusarium oxysporum cubeba specialized type No. 1 physiological race and application thereof |
| CN104611452A (en) * | 2015-02-16 | 2015-05-13 | 中国热带农业科学院环境与植物保护研究所 | Complete set primers for identification or assisted identification of fusarium oxysporum f.sp.cubense |
| CN104611451A (en) * | 2015-02-16 | 2015-05-13 | 中国热带农业科学院环境与植物保护研究所 | Complete set primers for identification or assisted identification of fusarium oxysporum f.sp.cubense and application of complete set primers |
| CN110669859A (en) * | 2019-10-11 | 2020-01-10 | 华南农业大学 | SRAP molecular marker related to banana wilt resistance and detection method and application thereof |
| CN110714093A (en) * | 2019-10-11 | 2020-01-21 | 华南农业大学 | SCAR molecular marker related to banana wilt resistance and detection method and application thereof |
| CN114480700A (en) * | 2021-12-27 | 2022-05-13 | 仲恺农业工程学院 | PCR primer, method and application for detecting and identifying physiological race 1 and 4 of banana fusarium oxysporum |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1307310C (en) * | 2005-02-28 | 2007-03-28 | 华南农业大学 | Banana wilt bacteria pathogenic polygalactunonic acid enzyme gene and its clone |
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2007
- 2007-12-05 CN CN2007100321182A patent/CN101178379B/en not_active Expired - Fee Related
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899506A (en) * | 2010-05-18 | 2010-12-01 | 华南农业大学 | Detection primer for No.1 and No.4 physiological strains of fusarium oxysporum f. sp cubense and rapid detection method |
| CN101974650A (en) * | 2010-11-30 | 2011-02-16 | 中国农业科学院植物保护研究所 | Polymerase chain reaction (PCR) method for detecting fusarium oxysporum and kit |
| CN101974650B (en) * | 2010-11-30 | 2012-06-27 | 中国农业科学院植物保护研究所 | Polymerase chain reaction (PCR) method for detecting fusarium oxysporum and kit |
| CN103789418B (en) * | 2014-01-09 | 2016-02-24 | 中华人民共和国中山出入境检验检疫局 | TaqMan probe real-time fluorescent primer for detecting fusarium oxysporum cubeba specialized type No. 1 physiological race and application thereof |
| CN103789418A (en) * | 2014-01-09 | 2014-05-14 | 中华人民共和国中山出入境检验检疫局 | TaqMan probe real-time fluorescent primer for detecting fusarium oxysporum cubeba specialized type No. 1 physiological race and application thereof |
| CN104611452A (en) * | 2015-02-16 | 2015-05-13 | 中国热带农业科学院环境与植物保护研究所 | Complete set primers for identification or assisted identification of fusarium oxysporum f.sp.cubense |
| CN104611451A (en) * | 2015-02-16 | 2015-05-13 | 中国热带农业科学院环境与植物保护研究所 | Complete set primers for identification or assisted identification of fusarium oxysporum f.sp.cubense and application of complete set primers |
| CN110669859A (en) * | 2019-10-11 | 2020-01-10 | 华南农业大学 | SRAP molecular marker related to banana wilt resistance and detection method and application thereof |
| CN110714093A (en) * | 2019-10-11 | 2020-01-21 | 华南农业大学 | SCAR molecular marker related to banana wilt resistance and detection method and application thereof |
| CN110669859B (en) * | 2019-10-11 | 2021-01-26 | 华南农业大学 | SRAP molecular marker related to banana wilt resistance and detection method and application thereof |
| CN110714093B (en) * | 2019-10-11 | 2021-03-12 | 华南农业大学 | SCAR molecular marker related to banana wilt resistance and detection method and application thereof |
| CN114480700A (en) * | 2021-12-27 | 2022-05-13 | 仲恺农业工程学院 | PCR primer, method and application for detecting and identifying physiological race 1 and 4 of banana fusarium oxysporum |
| CN114480700B (en) * | 2021-12-27 | 2024-03-19 | 仲恺农业工程学院 | PCR primer, method and application for detecting and identifying banana fusarium wilt bacteria No.1 and No. 4 physiological race |
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