CN103882108B - Method for detecting temperature sensitive microspecies gx2 of F.moniliforme - Google Patents

Method for detecting temperature sensitive microspecies gx2 of F.moniliforme Download PDF

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CN103882108B
CN103882108B CN201310677403.5A CN201310677403A CN103882108B CN 103882108 B CN103882108 B CN 103882108B CN 201310677403 A CN201310677403 A CN 201310677403A CN 103882108 B CN103882108 B CN 103882108B
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张木清
林镇越
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Guangxi University
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Abstract

The invention relates to a method for detecting temperature sensitive microspecies gx2 of F.moniliforme. The method comprises the steps of: designing a primer and probe combination; extracting pathogenic fungus DNA; establishing a reaction system and a reaction process of qPCR detection; obtaining a qPCR detection result. As a real-time quantitative PCR detection method of a Taqman probe is adopted, the method has the characteristics of high sensitivity, good specificity and small amount of required DNA, can realize rapid quantitative detection and accurate identification of the temperature sensitive microspecies gx2, can be used for diagnosing and detecting the temperature sensitive microspecies gx2 of F.moniliforme in a field and is high in detection efficiency.

Description

Detect the method for sugarcane toppers maize ear rot responsive to temperature type microspecies gx2
Technical field
The present invention relates to a kind of detection method of Plant diseases, be specifically related to a kind of method detecting sugarcane toppers maize ear rot (F.moniliforme) responsive to temperature type microspecies gx2, belong to biological technical field.
Background technology
Sugarcane toppers maize ear rot is fungal disease, and this disease pathogen bacterium is sickle-like bacteria (F.moniliforme), mostly occurs in 6 ~ September of high temperature and humidity.Seedling, ill plant and the upper invalid withered strain of soil table are the primary infection sources of disease in spite of illness.The conidium of germ is propagated with wind and rain, air-flow, drops on tip of a branch lobus cardiacus, relies on lobus cardiacus moisture, sprouts grow the tender base portion of germ tube intrusion lobus cardiacus children when condition is suitable for, then the sugarcane stem near intussusception point, and sick portion produces conidium row superinfection more subsequently.The yellow of their early stage spire base portion chlorisis, rear blade narrows, significantly gauffer, tussle or cripetura, on blade, sorrel longitudinal stripe splits becomes damaged leaf, and severe patient is in deformity; Infect leaf sheath and form red necrotic plaque or trapezoidal scab; Vegetative point of causing harm causes top rot and the necrosis of young axle; Sick stem is the step of the parallel lobe of sorrel, and have many horizontal breaches as the shape that lancinates, blackening brown, internode bends.
Nearest more than 20 years, along with the establishing in large scale of new platform sugar series of products (ROC10, ROC16, ROC22), kind simplification caused increasing the weight of year by year of top rot, and current sickness rate reaches more than 10%, is up to 60%.Particularly in the top rot of the rainy generation in season of summer high temperature, also happen occasionally in autumn and winter at present.From 2012, seminar is by investigating the biogeography research of sampling and pathogenic bacteria to the top rot in each producing region of China's Mainland sugarcane, the pathogenicbacteria separation of all collected specimens is cultivated, through Morphological Identification and molecular biology identification, with ITS sequence cluster analysis, preliminary evaluation China's Mainland sugarcane toppers maize ear rot cause of disease mainly single kind sickle-like bacteria (F.moniliforme), but have two microspecies gx1 and gx2.Not homophylic culture studies shows, gx1 and gx2 is completely different to the susceptibility of temperature, and gx2 under the high temperature conditions (higher than 28 DEG C) stops growing substantially, and lower than at 20 DEG C, its growth is obviously faster than gx1.Illustrate that gx2 is the microspecies of responsive to temperature type, the generation of top rot generation at home, particularly autumn and winter season is closely related with recent years.For this reason, how Testing and appraisal is fast carried out to the former microspecies gx2 of Chinese sensitive type sugarcane toppers maize ear rot, the pathogenic bacteria differentiation of research top rot and disease control tool are of great significance.
Summary of the invention
The object of the invention is to provide a kind of method detecting sugarcane toppers maize ear rot (F.moniliforme) responsive to temperature type microspecies gx2.Described sugarcane toppers maize ear rot responsive to temperature type microspecies gx2, refer to and cause China's sugarcane toppers maize ear rot, particularly at the cause of disease microspecies gx2 that autumn and winter occurs, have the cause of disease microspecies to sensitive that can not grow at temperature is higher than 28 DEG C, its Testing and appraisal characteristic sequence is:
GAGACCGCCACTAGATTTCGGGGCCGGCTTGCCGCAAGGGCTCGCCGATCCCCAACACCAAACCCGGGGGCTTGAGGGTTGAAATGACGCTCGAACAG。
To achieve these goals, the molecular detecting method of sugarcane toppers maize ear rot responsive to temperature type microspecies gx2 of the present invention, is first the characteristic sequence according to gx2 microspecies, is designed for some primers and the Taqman probe combinations FAM-gx2 of real-time quantitative PCR.
Utilize molecular detecting method to detect primer and a probe combinations FAM-gx2 of sugarcane toppers maize ear rot responsive to temperature type microspecies gx2, it is characterized in that described primer and probe combinations are 1 combination in following 6 combinations:
FAM-gx2 first group:
Primer: FAM-gx2-F1:5 '-CTGATCCGAGGTCAACATTCAG-3 '
FAM-gx2-R1：5’-GCAGCTTCCATTGCGTAGTA-3’
Probe: FAM-gx2-P1:5 '-CGCGTACCAGTTGCGAGGGTTT-3 ';
FAM-gx2 second group:
Primer: FAM-gx2-F2:5 '-ACTACTACGCAATGGAAGCTG-3 '
FAM-gx2-R2：5’-AGCGTCATTTCAACCCTCAA-3’
Probe: FAM-gx2-P2:5 '-CAGCGAGACCGCCACTAGATTTCG-3 ';
FAM-gx2 the 3rd group:
Primer: FAM-gx2-F3:5 '-CGCAATGTGCGTTCAAAGAT-3 '
FAM-gx2-R3：5’-ACAACGGATCTCTTGGTTCTG-3’
Probe: FAM-gx2-P3:5 '-TTTGCTGCGTTCTTCATCGATGC-3 ';
FAM-gx2 the 4th group:
Primer: FAM-gx2-F4:5 '-GTACCAGTTGCGAGGGTTT-3 '
FAM-gx2-R4：5’-CCTGTTCGAGCGTCATTTCA-3’
Probe: FAM-gx2-P4:5 '-CAGCGAGACCGCCACTAGATTTCG-3 ';
FAM-gx2 the 5th group:
Primer: FAM-gx2-F5:5 '-GGGCTTGAGGGTTGAAATGA-3 '
FAM-gx2-R5：5’-TCGATGAAGAACGCAGCAA-3’
Probe: FAM-gx2-P5:5 '-AATCTTTGAACGCACATTGCGCCC-3 ';
FAM-gx2 the 6th group:
Primer: FAM-gx2-F6:5 '-TCATTTCAACCCTCAAGCCC-3 '
FAM-gx2-R6：5’-GAGACCGCCACTAGATTTCG-3’
Probe: FAM-gx2-P6:5 '-CGCCGATCCCCAACACCAAAC-3 ';
5 ' end, the 3 ' end of above-mentioned Taqman probe report fluorescence dye and BHQ-1 quenching group mark with FAM respectively.
In described 6 primed probe combination, FAM ?gx2 the 6th group be the best.
Utilize the method for primed probe combine detection sugarcane toppers maize ear rot responsive to temperature type microspecies gx2 of the present invention, comprise extraction pathogenic fungi DNA, the reaction system setting up qPCR detection, the response procedures of qPCR detection, quantitatively qPCR detection, it is characterized in that:
(1) pathogenic fungi DNA is extracted: gather sugarcane toppers maize ear rot sample, through the sample extraction pathogenic bacteria DNA of isolation and purification culture pathogenic bacteria; Or extracting directly disease builds the leaf DNA of intersection;
(2) reaction system that qPCR detects is set up: the reaction system that qPCR detects is 10 times of PCR reaction buffers of 1/10 volume; Each 0.1 ~ the 0.5mmol/L of dNTP; Taq archaeal dna polymerase 0.1 ~ 5 international unit; Taqman probe 0.1 ~ 0.5mmol/L; DNA profiling liquid 0.1 ~ 5 μ l of separation and purification; The primer of a combination and probe, each 0.1 ~ 2nmol/L; Adding aseptic deionized water makes cumulative volume reach 10 ~ 100 μ l;
(3) response procedures of qPCR detection: the response procedures that qPCR detects is: 90-100 DEG C of 30-60s; 90-100 DEG C of 5-30s, 50-70 DEG C of 20-90s, 20 ~ 50 circulations, at the end of the extension of each circulation, carry out fluorescent signal detection, fluorescence mode is set to FAM;
(4) quantitative qPCR detected result: qPCR detected result Ct value is 0, represents that sample does not detect sugarcane toppers maize ear rot responsive to temperature type microspecies gx2.
Be sugarcane toppers maize ear rot responsive to temperature type microspecies gx2 in order to ensure bacterial strain of participating in the experiment, the present invention by ex-vivo purified cultivation and ITS ?PCR method pathogenic bacteria microspecies gx2 carried out to gathered sample identified.
Advantage of the present invention: the present invention, with the ITS sequence of gx2 microspecies easy region of variability design special primer and TanMan probe combinations (FAM-gx2), can realize the precise Identification of Quantitative detection to responsive to temperature type microspecies gx2 and microspecies by qPCR technology.
Accompanying drawing illustrates:
The qPCR of the fungal DNA of the different weaker concn of Fig. 1 reacts amplification curve, and X-coordinate represents cycle number Ct value, and ordinate zou represents fluorescent absorption value.
The relation of Fig. 2 fungal DNA concentration and Ct value.X-coordinate (x) represents cycle number Ct value, ordinate zou (Y) representation DNA concentration.Y gx2=-0.2997x+7.6674(R 2=0.992)
Embodiment
Below by way of specific embodiment, technical scheme of the present invention is further detailed and is described.
Embodiment 1: the separation of strains tested and cultivation
Gather the typical scab on the disease plant of sugarcane toppers maize ear rot responsive to temperature type microspecies gx2 field, carry out the separation and Culture of pathogenic bacteria, specific procedure is as follows: the plant tissue of getting the strong intersection of disease (scab and healthy intersection partly), be cut into the square that the length of side is 0.5cm size, soaked for 10 seconds in the spirituous solution of 70%, then sterilizing 40 seconds in 0.1% mercuric chloride, last aseptic water washing three times, be placed on sterilizing filter paper and naturally dry, be connected on the PDA substratum containing penbritin 100 μ g/ml, be inverted for 16-22 DEG C and cultivate 3-4 days.Carry out line purifying after mycelia grows to cultivate, be inverted and cultivate after 2-3 days, single bacterium colony mycelium that picking is sprouted purifying of again ruling is cultivated, purifying three generations.Adopt sterilized water dilution to cultivate with water agar the method combined and be separated monospore, it is spread upon uniformly on the water agar containing penbritin 100 μ g/ml with bending sterile glass rod, be inverted and cultivate after 2-3 days, the single spore of picking is containing in the Cha Shi liquid nutrient medium containing Streptomycin sulphate 100 μ g/ml, shaking culture is carried out at 16-22 DEG C, hunting speed is 250r/min, and the method is for the preparation of the mycelium of sugarcane toppers maize ear rot pathogenic bacteria.
Embodiment 2: the extraction of pathogenic bacteria DNA
Utilize centrifugal method to collect the thalline of embodiment 1, be placed in the centrifuge tube of 10ml by bacterium liquid, 4 DEG C, 13,000rpm, centrifugal 15min, after removing supernatant liquor, the thalline of precipitation be placed in aseptic Bechtop and dry up.Then, mycelial genomic dna is extracted to employing SDS method; First, the mycelium obtained by collected by centrifugation is placed in mortar more, adds liquid nitrogen and grind into powder, transfers to 1.5ml eppendorf and manages; Secondly, DNA extraction liquid and isopyknic phenol, chloroform, primary isoamyl alcohol (25:24:1) mixed solution is added, violent vortex 5min, collected by centrifugation supernatant liquor.Finally, add isopyknic chloroform/primary isoamyl alcohol (24:1), switch for several times fully mixing, 4 DEG C, 11,000rpm, centrifugal 5min, get supernatant liquor in new centrifuge tube.Again (to remove impurity, improve purification efficiency) after extracting once, Aspirate supernatant, in new centrifuge tube, adds the dehydrated alcohol (-20 DEG C) of two volumes precooling, and about 30min placed by-20 DEG C of refrigerators.12,000rpm, centrifugal 12min after taking out, dry on concentrated frozen centrifugal drying instrument, then add the Rnase aqueous solution, 37 DEG C of water-baths digest 1 hour, (removing RNA impurity).After agarose gel electrophoresis and pcr amplification judge that DNA quality is put forward by institute ,-20 DEG C save backup.The final concentration of the sample DNA of the representative strain YN41 of adjustment sugarcane toppers maize ear rot sick temperature sensitive pathogenic bacteria gx2 microspecies is 1,000 μ g/ml.
Embodiment 3: the primer of pathogenic bacteria and the design of probe
According to the distinguished sequence of sugarcane toppers maize ear rot responsive to temperature type microspecies gx2 design real-time quantitative PCR primer and Taqman probe combinations (FAM-gx2), wherein FAM ?gx2 comprise in following primer probe groups one group:
FAM-gx2 first group:
Primer: FAM-gx2-F1:5 '-CTGATCCGAGGTCAACATTCAG-3 '
FAM-gx2-R1：5’-GCAGCTTCCATTGCGTAGTA-3’
Probe: FAM-gx2-P1:5 '-CGCGTACCAGTTGCGAGGGTTT-3 '
FAM-gx2 second group:
Primer: FAM-gx2-F2:5 '-ACTACTACGCAATGGAAGCTG-3 '
FAM-gx2-R2：5’-AGCGTCATTTCAACCCTCAA-3’
Probe: FAM-gx2-P2:5 '-CAGCGAGACCGCCACTAGATTTCG-3 '
FAM-gx2 the 3rd group:
Primer: FAM-gx2-F3:5 '-CGCAATGTGCGTTCAAAGAT-3 '
FAM-gx2-R3：5’-ACAACGGATCTCTTGGTTCTG-3’
Probe: FAM-gx2-P3:5 '-TTTGCTGCGTTCTTCATCGATGC-3 '
FAM-gx2 the 4th group:
Primer: FAM-gx2-F4:5 '-GTACCAGTTGCGAGGGTTT-3 '
FAM-gx2-R4：5’-CCTGTTCGAGCGTCATTTCA-3’
Probe: FAM-gx2-P4:5 '-CAGCGAGACCGCCACTAGATTTCG-3 '
FAM-gx2 the 5th group:
Primer: FAM-gx2-F5:5 '-GGGCTTGAGGGTTGAAATGA-3 '
FAM-gx2-R5：5’-TCGATGAAGAACGCAGCAA-3’
Probe: FAM-gx2-P5:5 '-AATCTTTGAACGCACATTGCGCCC-3 '
FAM-gx2 the 6th group:
Primer: FAM-gx2-F6:5 '-TCATTTCAACCCTCAAGCCC-3 '
FAM-gx2-R6：5’-GAGACCGCCACTAGATTTCG-3’
Probe: FAM-gx2-P6:5 '-CGCCGATCCCCAACACCAAAC-3 '
5 ' end, the 3 ' end of above-mentioned Taqman probe report fluorescence dye and BHQ-1 quenching group mark with FAM respectively.
Embodiment 4:Taqman fluorescence quantitative PCR detection
Utilize the 6th group of the primer of embodiment 3 and Taqman probe groups FAM-gx2 fluorescence quantitative PCR detection of carrying out responsive to temperature type microspecies gx2:
Amplified reaction cumulative volume is 20 μ L, wherein upstream and downstream primer (FAM-gx2-F6/FAM-gx2-R6) 500nM, probe (FAM-gx2-P6) 250nM, 10 μ l Premix LA Taq (TaKaRa, Dalian), template 1 μ L, remainder distilled water is supplied, put the analysis of Roche480 quantitative fluorescent PCR system cloud gray model, at the end of the extension of each circulation, carry out fluorescent signal detection, fluorescence mode is set to FAM.Response procedures is: 95 DEG C of 30s; 95 DEG C of 5s, 60 DEG C of 30s, 40 circulations.
Table 1. detects field sample according to the real-time quantitative PCR special primer of F.moniliforme responsive to temperature type microspecies gx2 sequences Design and probe combinations (FAM-gx2)
Table 1 result shows, the sample of sense gx2 microspecies can detect that Ct value is 16 ~ 25, but the Ct value not infecting the sample of gx2 is 0, the method for Taqman probe FAM-gx2 in conjunction with qPCR is described, has good specificity.
Embodiment 5: the sensitivity analysis of real-time quantitative PCR
(1) sensitivity analysis of Taqman probe qPCR:
The final concentration of typical strain YN41 sample (gx2) DNA of adjustment Pokkah boeng (F.moniliforme) responsive to temperature type is 1,000 μ g/ml.Carry out the dilution of 10 times of series systems, carry out qPCR detection with unique identification Taqman probe FAM-gx1 respectively, obtain kinetic curve as shown in Figure 1.
Conclusion: probe FAM-gx2 to the DNA sample of YN41 at 6 extent of dilution (10 -1~ 10 -5) the S-type amplification curve of scope quantification PCR (Fig. 1); And the Ct value between different extent of dilution differs evenly, meets linear relationship (Fig. 2) strict between the Ct value of quantitative PCR and starting copy number, therefore, has good dependency between sample copy number and Ct value.Because the concentration of fungal DNA template is higher, interference and impact may be produced to the amplification of specific probe, in order to ensure reliability and the accuracy of detected result, in conjunction with repeatedly repeating experimental analysis, determine that Taqman probe FAM-gx2 identifies that the gx2 microspecies limit of detection Ct value of F.moniliforme is 32, namely the threshold concentration of the detection DNA of fungi is 10pg/ μ l.

Claims (2)

1. utilize molecular detecting method to detect primer and a probe combinations FAM-gx2 of sugarcane toppers maize ear rot (F.moniliforme) responsive to temperature type microspecies gx2, it is characterized in that described primer and probe combinations are:
Primer: FAM-gx2-F6:5 '-TCATTTCAACCCTCAAGCCC-3 '
FAM-gx2-R6：5’-GAGACCGCCACTAGATTTCG-3’
Probe: FAM-gx2-P6:5 '-CGCCGATCCCCAACACCAAAC-3 ';
5 ' end, the 3 ' end of above-mentioned Taqman probe report fluorescence dye and BHQ-1 quenching group mark with FAM respectively.
2. a primer as claimed in claim 1 and probe combinations detect the method for sugarcane toppers maize ear rot responsive to temperature type microspecies gx2, comprise extraction pathogenic fungi DNA, the reaction system setting up qPCR detection, the response procedures of qPCR detection, quantitatively qPCR detection, it is characterized in that:
(1) pathogenic fungi DNA is extracted: gather sugarcane toppers maize ear rot sample, through the sample extraction pathogenic bacteria DNA of isolation and purification culture pathogenic bacteria; Or the leaf DNA of the strong intersection of extracting directly disease;
(2) reaction system that qPCR detects is set up: the reaction system that qPCR detects is 10 times of PCR reaction buffers of 1/10 volume; Each 0.1 ~ the 0.5mmol/L of dNTP; Taq archaeal dna polymerase 0.1 ~ 5 international unit; DNA profiling liquid 0.1 ~ 5 μ l of separation and purification; The primer of a combination and probe, each 0.1 ~ 2nmol/L; Adding aseptic deionized water makes cumulative volume reach 10 ~ 100 μ l;
(3) response procedures of qPCR detection: the response procedures that qPCR detects is: 90-100 DEG C of 30-60s; 90-100 DEG C of 5-30s, 50-70 DEG C of 20-90s, 20 ~ 50 circulations, at the end of the extension of each circulation, carry out fluorescent signal detection, fluorescence mode is set to FAM;
(4) quantitative qPCR detected result: qPCR detected result Ct value is 0, represents that sample does not detect sugarcane toppers maize ear rot responsive to temperature type microspecies gx2.
CN201310677403.5A 2013-12-12 2013-12-12 Method for detecting temperature sensitive microspecies gx2 of F.moniliforme Expired - Fee Related CN103882108B (en)

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甘蔗梢腐病病原分子检测及甘蔗组合品种的抗病性评价;张玉娟等;《中国优秀硕士论文全文数据库 农业科技辑》;20091231(第12期);全文 *
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