CN105543382A - Rapid detection method of pathogens of corn southern leaf blight - Google Patents
Rapid detection method of pathogens of corn southern leaf blight Download PDFInfo
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- CN105543382A CN105543382A CN201610058100.9A CN201610058100A CN105543382A CN 105543382 A CN105543382 A CN 105543382A CN 201610058100 A CN201610058100 A CN 201610058100A CN 105543382 A CN105543382 A CN 105543382A
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Abstract
The invention belongs to the technical field of gene engineering, relates to a method for rapidly detecting pathogens by a specific primer, and in particular relates to the specific primer for rapidly detecting corn southern leaf blight and a detection method of the corn southern leaf blight. A rapid detection method of pathogens of the corn southern leaf blight comprises the following steps: (1) collecting fungal hyphae; (2) extracting fungal DNAs; (3) carrying out PCR (Polymerase Chain Reaction) amplification, wherein a PCR system is as follows: 1 microliter of a forward primer X-EF, 1 microliter of a reverse primer X-ER, 1 microliter of a DNA template, 12.5 microliters of Taq PCR Master Mix, and the balance of ddH2O which is added until the volume is 25 microliters; a PCR procedure: pre-denaturing for 3 minutes at 94 DEG C; denaturing for 30 seconds at 94 DEG C, annealing for 30 seconds at 52.9 DEG C, and extending for 30 seconds at 72 DEG C, circulating for 32 times; extending for 10 minutes at 72 DEG C and preserving at 4 DEG C. In order to provide evidences for rapidly and accurately monitoring whether corn leaves are latently infected by bipolaris maydis or not, prevention and control measures can be easily and effectively adopted as soon as possible.
Description
Technical field
The invention belongs to gene engineering technology field, relate to the method for a species-specific primer rapid detection pathogen, be specifically related to a kind of Auele Specific Primer and detection method thereof of rapid detection corn southern leaf blight.
Background technology
Southern corn leaf blight is imperfect fungi, Bipolaris Pseudomonas, Fusarium oxysporum, formal name used at school is: Bipolarismaydis (NisikadoeTMiyake) Shoem, different name is: HelminthosporiummaydisNisikadoeTMiyake, Drechsleramaydis (NisikadoetMiyake) Subram & Jain.Perfect stage is different cochliobolus Cochliobolusheterostrophus (Drechsler) Drechsler, and different name is: OphiobolusHeterostrophusDrechsler.This bacterium ascostroma is black, subsphaeroidal, 357-642 × 276-443 (μm), the blunt circle in ascus top, base portion tool short handle, size 124-183 × 23-29 (μm).Thecaspore number in each ascus not etc., is not generally about three, thecaspore in long linear, about 7.3 × 6.3-8.8 (μm) size.The scattered scab both sides on sick leaf of conidiophore of Invisible element, stretch out from the pore of epidermic cell or leaf tissue, single raw or Shu Sheng, olive brown is to brown, stretch or bend, base cells is large, and top is slightly thin and color is more shallow, and bottom color depth is thicker, spore trace is obvious, raw on summit or break, generally there are 6 to 8 barrier films, size 80 ~ 156 × 5 ~ 10 (μm).Conidium bears from the top of sporophore or side, oblong, bend towards more side tool every, omphalion is truncate.
This pathogenic bacteria can cause corn southern leaf blight, is a kind of comparatively serious leaf diseases extensively betiding corn producing region.In area, Jiangsu and Zhejiang Provinces, the large silk ribbon for holding a jade seal through its nose of twentieth century China's twenties famous phytopathologist Yu finds that this is sick, and carried out detailed deep report in 1933 to this disease.Since the seventies in last century, american corn helminthosporium maydis was very popular, corn southern leaf blight has generation in corn producing region, countries in the world, becomes one of Major Diseases on corn, all can cause certain production loss every year.Therefore, detect this pathogenic bacteria as early as possible fast to have great importance to alleviating the loss that this disease causes.The classification of current Bipolaris class germ and qualification are mainly based on morphological feature, Pathogenicity etc.Traditional germ discrimination method length consuming time, sensitivity are low and by force empirical, are separated and pathogen identification needs time a couple of days in disease plant, are difficult to the timely monitoring accomplishing occur disease and the propagation effectively controlling pathogenic bacteria and plant disease epidemic.Along with molecular biological development, differing molecular technology is used for the detection of plant compacted spore class disease.The partial sequence of other EF-1 α gene of several kinds in Bipolaris in the Fusarium oxysporum checked order by comparison and GenBank, utilize DNAMAN have devised to can be used for the Auele Specific Primer detecting Fusarium oxysporum, for whether quick from maize leaf, accurate measurements Fusarium oxysporum latent infection lay a good foundation, be conducive to effectively taking prophylactico-therapeutic measures early.
Summary of the invention
The technical problem to be solved in the present invention is: southern corn leaf blight can cause corn southern leaf blight, is a kind of comparatively serious leaf diseases extensively betiding corn producing region, all can causes certain production loss every year.Therefore, detect this pathogenic bacteria as early as possible fast and have great importance to alleviating the loss that this disease causes, the invention provides a kind of Auele Specific Primer and using method of rapid detection corn southern leaf blight.
Technical scheme of the present invention is:
A method for quick for corn southern leaf blight pathogen, described detection method comprises the following steps:
(1) collection of hypha,hyphae;
(2) extraction of fungal DNA;
(3) pcr amplification: PCR reaction system is, each 1 μ L of forward primer X-EF and reverse primer X-ER, DNA profiling 1 μ L, TaqPCRMasterMix12.5 μ L, ddH
2o mends to 25 μ L; PCR response procedures is 94 DEG C of denaturation 3min; 94 DEG C of sex change 30s, 52.9 DEG C of annealing 30s, 72 DEG C of extensions 30s, totally 32 circulations; 72 DEG C extend 10min, 4 DEG C of preservations.
Described forward primer X-EF sequence is as shown in SEQIDNO:1, and reverse primer X-ER sequence is as shown in SEQIDNO:2.
The concentration of described forward primer and reverse primer is 5 μm of ol/ μ L, DNA profiling concentration is 25ng/ μ L, TaqPCRMasterMix is purchased from Lai Feng bio tech ltd, Shanghai, described TaqPCRMasterMix contains 0.1 μ/μ LTaqDNApolymerase, 0.4mMeachdNTP, 2 × TaqBuffer, PCR toughener and protein stabilizing agent.
A kind of method for quick of corn southern leaf blight is as the application of helminthosporium maydis pathogenic bacteria early monitoring in maize leaf.
The invention has the beneficial effects as follows: for whether quick from maize leaf, accurate measurements Fusarium oxysporum latent infection provide foundation, be conducive to effectively taking prophylactico-therapeutic measures early.
Accompanying drawing explanation
Fig. 1 is corn southern leaf blight substance PCR primer specific detection figure, wherein 1:(ZM09358), 2:(ZM10094-2), 3:(ZM10233-3), 4:(09316-5), 5:(ZM09313-2-1), 6:(ZM10321), 7:(ZM10599), 8:(ZMLC025-2), 9:(ZM10322-3), 10:(ZM10587-2), 11:(ZTY030032), 12:(ZM10239-1), 13:(ZM09362-2), 14:(DH020637), 15:(ZM10601), 16:(ZM10602), 17:(ZM10603), 18:(ZM10604), 19:(ZM10605), CK: blank, M:DL2000Marker,
Fig. 2 is the detection figure of corn southern leaf blight primer sensitivity, and wherein M is molecule scalar DL1000; CK is negative control; Swimming lane 1-12 is the amplification containing 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg, 100ag, 10ag, 1ag, 0.1agDNA amount in the system of 25 μ L respectively;
Fig. 3 is the detection of corn incidence of leaf, and M is the marker of molecule scalar DL2000; CK is negative control; Swimming lane 1 is that healthy maize leaf extracts DNA cloning result; Swimming lane 2 is morbidity maize leaf extraction DNA cloning result; Swimming lane 3 is positive control.
Embodiment
A method for quick for corn southern leaf blight pathogen, described detection method comprises the following steps:
(1) collection of hypha,hyphae;
(2) extraction of fungal DNA;
(3) pcr amplification reaction.
Described forward primer X-EF sequence is as shown in SEQIDNO:1, and reverse primer X-ER sequence is as shown in SEQIDNO:2.
The method for quick of corn southern leaf blight is as an application for helminthosporium maydis pathogenic bacteria early monitoring in maize leaf, and concrete steps are as follows:
One, the collection of hypha,hyphae
The bacterial strain (seeing attached list one) deposited of going bail for is inoculated on PDA flat board, grows 3 days, choose fresh mycelia, be inoculated in the 250mL triangular flask that 1/3 volume PDB is housed at 25 DEG C.25 DEG C of 120r/min, cultivate 3-4 days, to generating a large amount of hypha body.Double gauze filters, and distilled water continuous flushing, filter paper suck dry moisture, is placed in 1.5mL centrifuge tube-20 DEG C and saves backup.
The source of table one strains tested and numbering
Two, the extraction of fungal DNA
Adopt the CTAB method improved to extract hypha,hyphae DNA, concrete steps are as follows:
(1) get appropriate mycelia (about 0.5g) in mortar, add liquid nitrogen and clay into power fast (at least adding liquid nitrogen grinding 3 times);
(2) spoon of sample powder sterilizing is transferred in the centrifuge tube of 1.5mL, add the CTAB Extraction buffer of 700 μ L preheating in 65 DEG C of water-baths, then centrifuge tube is placed in the water bath with thermostatic control of 65 DEG C and keeps 45min, every 10min gently put upside down several times, powder and damping fluid are mixed;
(3) 12000rpm, 4 DEG C of centrifugal 10min, Aspirate supernatant;
(4) add 700 μ L extracts I (phenol: chloroform: primary isoamyl alcohol=25:24:1), put upside down several to solution gently and mix;
(5) 12000rpm, 4 DEG C of centrifugal 10min, Aspirate supernatant;
(6) add 700 μ L extracts I, put upside down several to solution gently and mix;
(7) 12000rpm, 4 DEG C of centrifugal 10min, Aspirate supernatant;
(8) add 600 μ L extracts II (chloroform: primary isoamyl alcohol=24:1), repeatedly mix;
(9) 12000rpm, 4 DEG C of centrifugal 10min, Aspirate supernatant;
(10) add the Virahol of 600 μ L precoolings, shake up gently, be placed on 30min on ice;
(11) abandon supernatant, and the washing with alcohol adding 600 μ L70% precipitates twice;
(12) be placed on aseptic operating platform, dry up (about 20min);
(13) 20-200 μ LddH is added
2o, dissolving DNA;
(14) use spectrophotometer to detect DNA concentration, and concentration is adjusted to about 100ng/ μ L-20 DEG C and saves backup.
Three, pcr amplification reaction
(1) substance PCR reaction system: TaqPCRMasterMix is (containing 0.1 μ/μ LTaqDNApolymerase, 0.4mMeachdNTP, 2 × TaqBuffer, PCR toughener and protein stabilizing agent) 12.5 μ L, forward primer X-EF (5 μm of ol) and reverse primer X-ER (5 μm of ol) each 1 μ L, DNA profiling (25ng/ μ L) 1 μ L, ddH
2o mends to 25 μ L.
(2) PCR reaction conditions: 94 DEG C of denaturation 3min; 94 DEG C of sex change 30s, 52.9 DEG C of annealing 30s, 72 DEG C of extensions 30s, totally 32 circulations; 72 DEG C extend 10min; Amplified production detects and UVI gel imaging system analytical results (see Fig. 1) in 1.2% agarose gel electrophoresis.By the known band of 205bp that only had the pathogen of corn southern leaf blight to run out of of figure bis-, other bacterial strain does not all run out of band.
Four, primer sensitivity technique
The genomic dna of original corn southern leaf blight is diluted from 10ng/ μ L 10 times and obtain 12 process (1,10
-1, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, 10
-8, 10
-9, 10
-10), adopt the amplification system and program optimized, increase respectively, detect, result is as shown in Figure 3.As shown in Figure 2,10ng-1ng (diluting 10 times) all can amplify PCR band (swimming lane 1,2) clearly, and dilutes 100 times, the 1000 times bands increased afterwards and also may be seen indistinctly (swimming lane 3,4); Illustrate that the sensitivity that this method detects corn southern leaf blight is very high, be about 10pg/ μ L, visible the method is reliable.
Five, the rapid detection of germ in disease plant tissue
Can in order to verify the pathogenic bacteria that corn southern leaf blight be detected from corn disease tissue, with the maize leaf that Bipolaris maydis inoculation is healthy, the natural infection of simulation corn southern leaf blight pathogenic bacteria, carries out detection of pathogens after inoculation morbidity.Organize STb gene for template with the maize leaf inoculating morbidity, utilize primer X-EF/X-ER to carry out pcr amplification, the specific fragment of 205bp can be amplified equally, and healthy maize leaf tissue DNA fails to amplify specific band (Fig. 3).
Claims (4)
1. a method for quick for corn southern leaf blight pathogen, is characterized in that, described detection method comprises the following steps:
(1) collection of hypha,hyphae;
(2) extraction of fungal DNA;
(3) pcr amplification: PCR reaction system is, each 1 μ L of forward primer X-EF and reverse primer X-ER, DNA profiling 1 μ L, TaqPCRMasterMix12.5 μ L, ddH
2o mends to 25 μ L; PCR response procedures is 94 DEG C of denaturation 3min; 94 DEG C of sex change 30s, 52.9 DEG C of annealing 30s, 72 DEG C of extensions 30s, totally 32 circulations; 72 DEG C extend 10min, 4 DEG C of preservations.
2. the method for quick of corn southern leaf blight pathogen as claimed in claim 1, it is characterized in that, described forward primer X-EF sequence is as shown in SEQIDNO:1, and reverse primer X-ER sequence is as shown in SEQIDNO:2.
3. the method for quick of corn southern leaf blight pathogen as claimed in claim 1, it is characterized in that, the concentration of described forward primer and reverse primer is 5 μm of ol/ μ L, and DNA profiling concentration is 25ng/ μ L.
4. the method for quick of a corn southern leaf blight is as the application of helminthosporium maydis pathogenic bacteria early monitoring in maize leaf.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108384882A (en) * | 2018-05-18 | 2018-08-10 | 福建省农业科学院植物保护研究所 | Southern corn leaf blight LAMP detection primer and its visible detection method and application |
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| US5800997A (en) * | 1996-11-01 | 1998-09-01 | Novartis Finance Corporation | Detection of maize fungal pathogens using the polymerase chain reaction |
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2016
- 2016-01-28 CN CN201610058100.9A patent/CN105543382A/en active Pending
Patent Citations (3)
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|---|---|---|---|---|
| US5800997A (en) * | 1996-11-01 | 1998-09-01 | Novartis Finance Corporation | Detection of maize fungal pathogens using the polymerase chain reaction |
| CN102399888A (en) * | 2011-11-28 | 2012-04-04 | 江苏省农业科学院 | Molecular identification method for cochliobolus miyabeanus pathogens |
| CN102586470A (en) * | 2012-04-14 | 2012-07-18 | 黑龙江八一农垦大学 | Polymerase chain reaction (PCR) detection kit for maize curvularia leaf spot and detection method for PCR detection kit |
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| Title |
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| IGNAZIO CARBONE 等: "A method for designing primer sets for speciation studies in filamentous ascomycetes", 《MYCOLOGIA》 * |
| P.NICHOLSON 等: "Use of Random Amplified Polymorphic DNA (RAPD) Analysis and Genetic Fingerprinting to Differentiate Isolates of Race O, C and T of Bipolaris maydis", 《J.PHYTOPATHOLOGY》 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108384882A (en) * | 2018-05-18 | 2018-08-10 | 福建省农业科学院植物保护研究所 | Southern corn leaf blight LAMP detection primer and its visible detection method and application |
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Application publication date: 20160504 |