CN106399529B - A kind of Phyllosticta musarum molecular detection primer and detection method - Google Patents
A kind of Phyllosticta musarum molecular detection primer and detection method Download PDFInfo
- Publication number
- CN106399529B CN106399529B CN201610900830.9A CN201610900830A CN106399529B CN 106399529 B CN106399529 B CN 106399529B CN 201610900830 A CN201610900830 A CN 201610900830A CN 106399529 B CN106399529 B CN 106399529B
- Authority
- CN
- China
- Prior art keywords
- banana
- primer
- detection
- phyllosticta
- musarum
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 241000770397 Phyllosticta musarum Species 0.000 title claims abstract description 60
- 238000001514 detection method Methods 0.000 title claims abstract description 52
- 235000018290 Musa x paradisiaca Nutrition 0.000 claims abstract description 80
- 235000013399 edible fruits Nutrition 0.000 claims abstract description 21
- 241000234295 Musa Species 0.000 claims abstract description 18
- 238000012360 testing method Methods 0.000 claims abstract description 7
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 5
- 238000006243 chemical reaction Methods 0.000 claims description 22
- 230000003321 amplification Effects 0.000 claims description 18
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 18
- 238000004925 denaturation Methods 0.000 claims description 17
- 230000036425 denaturation Effects 0.000 claims description 17
- 239000000872 buffer Substances 0.000 claims description 12
- 238000001962 electrophoresis Methods 0.000 claims description 11
- 241000196324 Embryophyta Species 0.000 claims description 10
- 238000012408 PCR amplification Methods 0.000 claims description 10
- 238000004519 manufacturing process Methods 0.000 claims description 10
- 108090000790 Enzymes Proteins 0.000 claims description 9
- 102000004190 Enzymes Human genes 0.000 claims description 9
- 238000000137 annealing Methods 0.000 claims description 9
- 239000012154 double-distilled water Substances 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 4
- 238000000605 extraction Methods 0.000 claims description 3
- 239000002773 nucleotide Substances 0.000 claims 1
- 125000003729 nucleotide group Chemical group 0.000 claims 1
- 239000002304 perfume Substances 0.000 claims 1
- 235000013599 spices Nutrition 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 11
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 10
- 201000010099 disease Diseases 0.000 abstract description 9
- 208000003351 Melanosis Diseases 0.000 abstract description 8
- 206010027146 Melanoderma Diseases 0.000 abstract description 2
- 230000002265 prevention Effects 0.000 abstract description 2
- 208000031968 Cadaver Diseases 0.000 abstract 1
- 238000009792 diffusion process Methods 0.000 abstract 1
- 240000008790 Musa x paradisiaca Species 0.000 description 63
- 108020004414 DNA Proteins 0.000 description 39
- 238000003752 polymerase chain reaction Methods 0.000 description 35
- 241000894006 Bacteria Species 0.000 description 19
- 241000233866 Fungi Species 0.000 description 16
- 238000000034 method Methods 0.000 description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 244000052769 pathogen Species 0.000 description 11
- 230000009182 swimming Effects 0.000 description 10
- 210000001519 tissue Anatomy 0.000 description 10
- 230000001717 pathogenic effect Effects 0.000 description 9
- 239000000047 product Substances 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 7
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 6
- 210000003705 ribosome Anatomy 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 241000223600 Alternaria Species 0.000 description 5
- 241000223211 Curvularia lunata Species 0.000 description 5
- 240000000905 Nymphoides indica Species 0.000 description 5
- 235000017590 Nymphoides indica Nutrition 0.000 description 5
- 238000013461 design Methods 0.000 description 5
- 238000003745 diagnosis Methods 0.000 description 5
- 239000013642 negative control Substances 0.000 description 5
- 125000006850 spacer group Chemical group 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 208000024891 symptom Diseases 0.000 description 4
- 235000005340 Asparagus officinalis Nutrition 0.000 description 3
- 108091023242 Internal transcribed spacer Proteins 0.000 description 3
- 206010039509 Scab Diseases 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000007689 inspection Methods 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 244000003416 Asparagus officinalis Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 101100425538 Pseudomonas aeruginosa (strain UCBPP-PA14) tis1 gene Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
- 230000002969 morbid Effects 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000012409 standard PCR amplification Methods 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 241001246233 Aecidium Species 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 241000207199 Citrus Species 0.000 description 1
- 240000007582 Corylus avellana Species 0.000 description 1
- 235000007466 Corylus avellana Nutrition 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 206010014970 Ephelides Diseases 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 244000017020 Ipomoea batatas Species 0.000 description 1
- 235000002678 Ipomoea batatas Nutrition 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 241000233614 Phytophthora Species 0.000 description 1
- 241000233616 Phytophthora capsici Species 0.000 description 1
- 239000007984 Tris EDTA buffer Substances 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- 244000193174 agave Species 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 210000001367 artery Anatomy 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 235000021015 bananas Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 235000020971 citrus fruits Nutrition 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000012631 diagnostic technique Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 208000002173 dizziness Diseases 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 238000009313 farming Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000007857 nested PCR Methods 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 244000039328 opportunistic pathogen Species 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- 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
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Botany (AREA)
- Mycology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to a kind of Phyllosticta musarum molecular detection primer and its detection methods, belong to corps diseases detection and field of biotechnology.The specific primer includes upstream primer BMF:5 '-GCTACAACGCCGAAATGACC-3 ', downstream primer BMR:5 '-GACGTTGCCCAATACCAAGC-3 '.The detection primer and detection method invented can be used for the Testing and appraisal of Phyllosticta musarum pure culture, can also detect to Leaf of banana and fruit；Detection primer high specificity of the present invention, high sensitivity, detection method practicability is good, simple and efficient to handle；The present invention is able to achieve the early detection of banana freckle, similar anthracnose on banana, the mould leaf spot of curved spore, black spot, checkers spore leaf spot, dark double spore leaf spots can also effectively be distinguished, the diffusion sprawling of early warning and prevention and control to banana freckle, controlling disease is of great significance.
Description
Technical field
The present invention relates to a kind of Phyllosticta musarum molecular detection primer and its detection methods, are exclusively used in Phyllosticta musarum
Rapid molecular detection, while can realize field Phyllosticta musarum early diagnosis and germ monitoring identify, belong to farming
Object Defect inspection and field of biotechnology.
Background technique
Banana be in the world the maximum fruit crop of yield and it is world-renowned the torrid zone, subtropical fruit,.In being also
The main fruit of state's tropical and subtropical zone, banana its with unique taste and higher nutritive value, always can be rated as a kind of superior
Health fruit.China is one of World banana main production country, the banana planting production with long cultivation history and the world
One of big country, the cultivated area and yield of banana occupy the 4th of national fruit.
Banana freckle is also known as tar spot, ephelides, and can cause harm Leaf of banana, carpopodium and fruit, in each banana kind in China
Growing area is generally existing.Blade and middle arteries generate many scattered or all living creatures small blackspot of protrusion, diameter about l mm, periphery when morbidity
Filbert, middle part is slightly sunk, and is gone up and is given birth to small black grain.The intensive blocking spot of scab, it is withered and yellow to finally result in blade, fruit morbidity, symptom
Occur within often 2~4 weeks after cutting off male flower cluster, mostly refer to curved abdominal section in fruit, Quan Guojun has when serious, and initial stage is rufous, peripheral dirty-green
Water is dizzy, and as fruit meat degree increases, scab density increases, serious to extend to full fruit, influences the appearance and storage property of fruit, closely
Banana freckle generally occurs and causes harm to get worse in China banana producing region over year, it has also become the important disease in banana production
One of.
Phyllosticta musarum (Phyllosticta musarum) grown on the common culture mediums such as PDA, PSA and V8 it is very slow
Slowly, when being separated, bacterium colony is usually covered by miscellaneous bacteria, and pycnidia generation time evening.It is difficult in Early Identification.
Pure culture was not obtained also by 1972, in addition to this disease conventional diagnostic techniques based on symptom, need using Ke He Shi
Rule by pathogenicbacteria separation culture, pathogen identification, connect bacterium, symptom analysis, time-consuming, low efficiency, accuracy are poor,
It is difficult to propagation and the plant disease epidemic for detecting and effectively controlling pathogen when disease occurs in time, it is difficult to meet banana freckle
The actual needs of diagnosis, therefore a set of convenient and efficient, result is reliable, quick diagnosis technology of high sensitivity there is an urgent need to establish.
In recent years, Protocols in Molecular Biology is quickly grown, as Protocols in Molecular Biology is constantly sent out in plant pathology subject
Exhibition and application, some molecular marking techniques provide new approach for the diagnosis detection of phytopathogen, wherein PCR
(polymerase chain reaction) technology with high specificity, high sensitivity, it is convenient and efficient the features such as be used for phytopathy
The diagnosis of opportunistic pathogen.Guarantor in fungi ribosomes transcribed spacer (internal transcribed spacer, ITS) sequence kind
The characteristics of keeping property and section belong to inter-species changeability, design specific primer carry out PCR, are used for quickly detecting and identify to pathogen
Technology has been widely applied, and researchers at home and abroad successfully develop soybean phytophthora, Phytophthora capsici, citrus bacterial canker disease
The specific primer and detection method of a variety of pathogens such as bacterium, sweet potato black rot pathogen and corn south aecidium, realize quickly,
Sensitive and accurate identification.But there is not been reported for the current research in relation to Phyllosticta musarum Molecular Detection.
Summary of the invention
For pathogen morpha feature is based primarily upon to the detection and identification of Phyllosticta musarum in the prior art, banana is black
Star pathogen growth is slow, and separating difficulty is big, and separation program is cumbersome, the dead length of consumption, high to identification skill requirement, accuracy is low, it is difficult to
The actual needs problem for meeting banana freckle diagnosis provides a kind of Phyllosticta musarum molecular detection primer and its detection side
Method.
To achieve the above object, this invention takes following technical schemes:
Present invention firstly provides a kind of Phyllosticta musarum molecular detection primer, nucleotide sequences are as follows:
Upstream primer BMF:5 '-GCTACAACGCCGAAATGACC-3 '；
Downstream primer BMR:5 '-GACGTTGCCCAATACCAAGC-3 '.
The primer BMF and BMR goes out the product of 382bp to Phyllosticta musarum specific amplification.
The present invention also provides a kind of rapid detection methods of Phyllosticta musarum, comprising the following steps:
(1) DNA is extracted from Leaf of banana or fruit；
When for detecting pathogen pure culture, strains tested genomic DNA is extracted using CTAB method；For detecting
When Banana Tissue whether there is Phyllosticta musarum, banana plant tissue gene group DNA is extracted using NaOH rapid cleavage method；
(2) to extract the DNA of Leaf of banana or fruit as template progress PCR amplification: 25 μ L of PCR reaction system includes
2.5 μ 10 × PCR of L buffer, 2.0 μ L concentration are the dNTP Mixture of 2.5mmol/L, and 0.15 μ L concentration is 5U/ μ L's
Taq enzyme, each 0.5 μ L of the BMF and BMR of 10 μm of ol/L, 1 μ L of DNA profiling add ddH2O is to total volume up to 25 μ L；PCR reaction condition
Are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 45sec, 56 DEG C of annealing 45sec, 72 DEG C of extension 45sec, totally 35 recycle；72 DEG C are prolonged
Stretch 10min；
(3) resulting PCR product is walked on 1.0% Ago-Gel with the electrophoretic analysis of 0.5 × tbe buffer liquid, voltage is
4-5V/cm；Testing result is determined according to the size of amplified production, if energy specific amplification goes out the product of 382bp, can determine that
There are Phyllosticta musarums in the Leaf of banana or fruit, and otherwise there is no banana is black in the Leaf of banana or fruit
Star germ.
Positive beneficial effect of the invention is:
(1) accuracy is high: the present invention be according to fungi ribosomes transcribed spacer (rDNA-ITS) sequence in fungi kind
The characteristics of the belonging to inter-species changeability design of well-conserved and section there is the PCR of specific amplified effect to draw Phyllosticta musarum
Object.The plant leaf blade to the Phyllosticta musarum of different geographic origins, carrying Phyllosticta musarum, carrying banana freckle
The pericarp of bacterium and the Banana Tissue of health have carried out detection verifying, only Phyllosticta musarum and the Banana Tissue for carrying the germ
The electrophoretic band that 382 bp can specifically be amplified illustrates primer designed by the present invention for detecting the black star of banana
Germ is accurate and reliable；
(2) high specificity: primer pair Phyllosticta musarum designed by the present invention has very strong specificity, can be used in
Distinguish Phyllosticta musarum, banana anthracnose, Curvularia lunata bacterium, banana alternaria, banana checkers spore leaf spot
Bacterium and banana secretly pathogen common on the bananas such as double spore leaf spot fungis, so as to the raw symptom characteristic on banana of effective district distribution
Similar disease；
(3) high sensitivity: the present invention has combined the special primer of design with ITS gene universal primer (ITS1/ITS4)
After carrying out nest-type PRC amplification, 1fg can reach in DNA level to the detection sensitivity of Phyllosticta musarum；
(4) applicability is wide, practicability is good: the detection method of Phyllosticta musarum of the invention, can not only be to germ mycelia
Body is detected, and also can be detected the early detection, it can be achieved that Phyllosticta musarum to susceptible Leaf of banana and pericarp, i.e.,
It is detected before disease shows disease, it is significant to the eruption and prevalence of scab after prevention Banana Growth phase and picking.
(5), easy to operate quick: the present invention can sentence after need to only carrying out DNA extraction, PCR amplification and agarose electrophoresis
It is simple and efficient to handle calmly as a result, general entire detection process can be completed within a few hours.
Detailed description of the invention
Fig. 1 is primer pair Phyllosticta musarum specific amplification electrophoretogram of the present invention: wherein swimming lane M is 2000bp DNA
Marker, swimming lane 2, swimming lane 3 are Phyllosticta musarum, and swimming lane 4-11 is respectively as follows: Glorosprium musarum Cookeet Mass, Curvularia lunata
Bacterium, banana alternaria, banana checkers spore leaf spot fungi, banana secretly double spore leaf spot fungis, banana blight bacteria, Asparagus Stem Blight
Bacterium, negative control.
Fig. 2 is the sensitivity detection amplification electrophoretogram of primer Phyllosticta musarum of the present invention: A: regular-PCR sensitivity inspection
Survey, wherein swimming lane M be 2000bp DNA Marker, swimming lane 2-11 be respectively as follows: 100ng, 10ng, 1ng, 100pg, 10pg, 1pg,
100fg, 10fg, negative control, positive control；B is nest-type PRC sensitivity technique, and wherein swimming lane M is 2000bp DNA
Marker, swimming lane 2-13 are respectively as follows: 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg, 100ag, feminine gender
Control, positive control.
Fig. 3 is that Leaf of banana of the present invention and the detection of Banana peel kind black star germ expand electrophoretogram, and wherein swimming lane M is
2000bp DNA Marker, swimming lane 2-10 are respectively natually morbid Leaf of banana, natural occurrence banana, artificial infection
Early stage banana after the Leaf of banana of morbidity, artificial infection, healthy Leaf of banana, the Banana peel of health, health
Banana carpopodium, negative control, positive control.
Specific embodiment
In order to make content of the present invention easily facilitate understanding, With reference to embodiment to of the present invention
Technical solution is described further.
Test method as used in the following examples is conventional method unless otherwise specified.
Test material as used in the following examples, reagent etc., unless otherwise specified, commercially obtain.
The design of 1 molecular detection primer of embodiment and the foundation of Phyllosticta musarum special molecular detection method
1. the extraction of Phyllosticta musarum genomic DNA:
The genomic DNA of 6 plants of Phyllosticta musarums of this laboratory preservation is extracted using CTAB method, specific steps are such as
Under:
(1) it takes 0.1 g hypha powder in 1.5 mL centrifuge tubes, 900 μ L2%CTAB extracting solutions is added, are shaken using oscillator
Mixing is swung, 60 DEG C of water-bath 60min, under room temperature, 12000r/min are centrifuged 15 min；
(2) 700 μ L of supernatant is taken, adds isometric phenol, chloroform, isoamyl alcohol mixed liquor (each volume ratio is 25:24:1), mildly
It shakes, under room temperature, 8000 r/min are centrifuged 10min；
(3) 500 μ L of supernatant is taken, isometric chloroform is added and extracts again once, under room temperature, 8000 r/min centrifugation
10min；
(4) 350 μ L of supernatant is taken, 1/10 volume, 3 mol/L NaAc and 2 times of volume dehydrated alcohols are added, -20 DEG C heavy
Form sediment 60 min, and under the conditions of 4 DEG C, 8000 r/min are centrifuged 5 min；
(5) liquid is discarded supernatant, 700 μ L volumetric concentrations of addition are 70% ice ethyl alcohol, jog 10sec, under the conditions of 4 DEG C, 8000
R/min is centrifuged 10sec, dries, and 50 μ L TE buffers is added, -20 DEG C save backup.
2. Phyllosticta musarum ITS sequence measures:
With fungi ribosomes internal gene transcribed spacers (rDNA-ITS) universal primer TS1:5'-
TCCGTAGGGAACCTGCGG-3' and ITS4:5'-TCCTCCGCTTATTGATATGC-3' is that primer pair extracts banana freckle
The DNA of bacterium carries out PCR amplification, amplification reaction system and response procedures are as follows: 25 μ L of PCR reaction system, comprising 2.5 μ L 10 ×
PCR buffer, 2.0 μ L concentration are the dNTP Mixture of 2.5mmol/L, and 0.15 μ L concentration is the Taq enzyme (Takara of 5U/ μ L
Dalian treasured bioengineering Co., Ltd), each 0.5 μ L of the TIS1 and ITS4 of 10 μm of ol/L, 1 μ L of DNA profiling add ddH2O is to totality
It accumulates up to 25 μ L；PCR reaction condition are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of 1 min of denaturation, 55 DEG C of annealing 45sec, 72 DEG C extend 1
Min, totally 35 recycle；72 DEG C of extension 10min；Gained PCR product send Dalian treasured bioengineering Co., Ltd to be sequenced, and submits
GenBank obtains Serial No. KX611161.
3. the design of Phyllosticta musarum special molecular detection primer:
According to Phyllosticta musarum ribosomes the Internal Transcribed Spacer (rDNA-ITS) sequence fungi inter-species height variation and
Kind internal stability will be sequenced in ITS sequence and the GenBank of 6 plants of obtained Phyllosticta musarums with Clustal X softwarePhyllostictaCategory ITS sequence not of the same race, Glorosprium musarum Cookeet Mass ITS sequence, Curvularia lunata bacterium ITS sequence
Column, banana alternaria ITS sequence, banana checkers spore leaf spot fungi ITS sequence, banana secretly double spore leaf spot fungi ITS sequences
Column, banana blight bacteria ITS sequence carry out homology analysis and difference site is compared, and is set with BMRimer BMRimer5 software
Having counted has one couple of PCR primers of specific amplification effect (by Shanghai Sheng Gong bioengineering Co., Ltd Phyllosticta musarum
Synthesis), i.e. the sequence of special molecular detection primer are as follows:
Upstream primer BMF:5 '-GCTACAACGCCGAAATGACC-3 '；
Downstream primer BMR:5 '-GACGTTGCCCAATACCAAGC-3 '
4. the foundation of Phyllosticta musarum rapid molecular detection method:
(1) DNA is extracted from Leaf of banana or fruit:
1. extracting strains tested genomic DNA using CTAB method for when detecting pathogen pure culture;
2. when for detecting banana plant tissue with the presence or absence of Phyllosticta musarum, being extracted using NaOH rapid cleavage method
Banana plant tissue gene group DNA, the specific steps are as follows:
A. 0.1 g of plant tissue to be detected is weighed, 0.5 mol/L NaOH, 30 μ L is added, tissue is sufficiently milled to
Paste；
B. paste tissue is transferred in 1.5 mL centrifuge tubes, 12000r/min is centrifuged 6 min, takes 5 μ l of supernatant；
C. 0.1 Tris-HCl(pH=8.0 mol/L 495 μ L are added in supernatant), it is uniformly mixed, takes 1.0 μ L conducts
PCR template is expanded；
(2) to extract Leaf of banana or banana DNA as template progress PCR amplification: 25 μ L of PCR reaction system includes
2.5 μ 10 × PCR of L buffer, 2.0 μ L concentration are the dNTP Mixture of 2.5mmol/L, and 0.15 μ L concentration is 5U/ μ L's
Taq enzyme, each 0.5 μ L of the BMF and BMR of 10 μm of ol/L, 1 μ L of DNA profiling add ddH2O is to total volume up to 25 μ L；PCR reaction condition
Are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 45sec, 56 DEG C of annealing 45sec, 72 DEG C of extension 45sec, totally 35 recycle；72 DEG C are prolonged
Stretch 10min；
(3) resulting PCR product is walked on 1.0% Ago-Gel with the electrophoretic analysis of 0.5 × tbe buffer liquid, voltage is
4-5V/cm；Testing result is determined according to the size of amplified production, if energy specific amplification goes out the product of 382bp, can determine that
There are Phyllosticta musarums in the Leaf of banana or banana, otherwise do not deposit in the Leaf of banana or banana
In Phyllosticta musarum.
2 Phyllosticta musarum specific amplification of embodiment
1. extracting 2 plants of Phyllosticta musarums, Glorosprium musarum Cookeet Mass, Curvularia lunata bacterium, banana using CTAB method
The base of alternaria, banana checkers spore leaf spot fungi, banana secretly double spore leaf spot fungis, banana blight bacteria and asparagus stem wilt bacteria
Because of a group DNA.
2. for the DNA for trying bacterium be that template carries out PCR amplification: 25 μ L of PCR reaction system to extract, include 2.5 μ L 10 ×
PCR buffer, 2.0 μ L concentration are the dNTP Mixture of 2.5mmol/L, and 0.15 μ L concentration is the Taq enzyme of 5U/ μ L, 10 μ
Each 0.5 μ L of the BMF and BMR of mol/L, 1 μ L of DNA profiling, adds ddH2O is to total volume up to 25 μ L；PCR reaction condition are as follows: 94 DEG C pre-
It is denaturalized 5min；94 DEG C of denaturation 45sec, 56 DEG C of annealing 45sec, 72 DEG C of extension 45sec, totally 35 recycle；72 DEG C of extension 10min；
Electrophoresis detection amplified production.
3. specific amplification result
As shown in Figure 1,2 plants of Phyllosticta musarums can go out the band of 382bp with specific amplification, and Glorosprium musarum Cookeet Mass,
Secretly double spore leaf spot fungis, banana are withered for Curvularia lunata bacterium, banana alternaria, banana checkers spore leaf spot fungi, banana
Germ, asparagus stem wilt bacteria and negative control wither without amplified band, shows that molecular detection primer of the invention can be by banana
Other pathogens of black star germ and banana of causing harm distinguish, and have very strong specificity, and detection method of the invention is available
In the specific amplification of Phyllosticta musarum.
The sensitivity of the primer pair Phyllosticta musarum of the present invention of embodiment 3 detects
1. extracting the genomic DNA of Phyllosticta musarum using CTAB method；
2. by the genomic DNA of the Phyllosticta musarum of extraction, after spectrophotometric determination concentration, with sterile ultrapure water
Dilution, is configured to series of concentrations, spare；
It include 2.5 3. carrying out standard PCR amplification: 25 μ L of PCR reaction system as template at series of concentrations DNA using preparation
μ 10 × PCR of L buffer, 2.0 μ L concentration are the dNTP Mixture of 2.5mmol/L, and 0.15 μ L concentration is the Taq of 5U/ μ L
Enzyme, each 0.5 μ L of the BMF and BMR of 10 μm of ol/L, 1 μ L of DNA profiling add ddH2O is to total volume up to 25 μ L；PCR reaction condition are as follows:
94 DEG C of initial denaturation 5min；94 DEG C of denaturation 45sec, 56 DEG C of annealing 45sec, 72 DEG C of extension 45sec, totally 35 recycle；72 DEG C of extensions
10min；Electrophoresis detection amplified production.
4. carrying out nested PCR amplification as template at series of concentrations DNA using preparation:
(1) first round PCR is expanded: with fungi ribosomes internal gene transcribed spacers (rDNA-ITS) universal primer TS1:
5'-TCCGTAGGGAACCTGCGG-3' and ITS4:5'-TCCTCCGCTTATTG ATATGC-3' be outer primer to preparation at
Series of concentrations DNA carries out first round PCR amplification, amplification reaction system and response procedures are as follows: 25 μ L of PCR reaction system includes
2.5 μ 10 × PCR of L buffer, 2.0 μ L concentration are the dNTP Mixture of 2.5mmol/L, and 0.15 μ L concentration is 5U/ μ L's
Taq enzyme (Dalian Takara treasured bioengineering Co., Ltd), each 0.5 μ L of the TIS1 and ITS4 of 10 μm of ol/L, 1 μ L of DNA profiling,
Add ddH2O is to total volume up to 25 μ L；PCR reaction condition are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of 1 min of denaturation, 55 DEG C of annealing
45sec, 72 DEG C of 1 min of extension, totally 35 recycle；72 DEG C of extension 10min；
(2) second wheel PCR amplifications: using the product of first round PCR amplification as template, second is carried out by primer of BMF/BMR
PCR amplification is taken turns, 25 μ L of PCR reaction system includes 2.5 μ 10 × PCR of L buffer, and 2.0 μ L concentration are the dNTP of 2.5mmol/L
Mixture, 0.15 μ L concentration are the Taq enzyme of 5U/ μ L, and each 0.5 μ L of the BMF and BMR of 10 μm of ol/L, 1 μ L of DNA profiling add ddH2O
To total volume up to 25 μ L；PCR reaction condition are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 45sec, 56 DEG C of annealing 45sec, 72 DEG C
Extend 45sec, totally 30 circulations；72 DEG C of extension 10min；Electrophoresis detection amplified production.
5. testing result
As shown in Fig. 2, when carrying out Standard PCR as primer using primer BMF/BMR of the present invention, in 25 μ L reaction systems,
The Phyllosticta musarum DNA of 10pg can obtain view strip band, and detection sensitivity can reach 10pg(Fig. 2-A)；And it is further
With the universal primer TS1:5'-TCCGTAGGGAACCTGCGG-3' of fungi ribosomes internal gene transcribed spacers (rDNA-ITS) and
ITS4:5'-TCCTCCGCTTATTGATATGC-3' is that the product of external primer amplification is template, with primer BMF/BMR of the present invention
When carrying out the second wheel amplification for primer, in 25 μ L reaction systems, the Phyllosticta musarum DNA of 1fg can obtain view strip band,
Its detection sensitivity can reach 1fg (Fig. 2-B).
The detection of Phyllosticta musarum in 4 Leaf of banana of embodiment and fruit
1. extracting banana plant tissue gene group DNA using NaOH rapid cleavage method.
It include 2.5 2. carrying out standard PCR amplification: 25 μ L of PCR reaction system as template at series of concentrations DNA using preparation
μ 10 × PCR of L buffer, 2.0 μ L concentration are the dNTP Mixture of 2.5mmol/L, and 0.15 μ L concentration is the Taq of 5U/ μ L
Enzyme, each 0.5 μ L of the BMF and BMR of 10 μm of ol/L, 1 μ L of DNA profiling add ddH2O is to total volume up to 25 μ L；PCR reaction condition are as follows:
94 DEG C of initial denaturation 5min；94 DEG C of denaturation 45sec, 56 DEG C of annealing 45sec, 72 DEG C of extension 45sec, totally 35 recycle；72 DEG C of extensions
10min；Electrophoresis detection amplified production.
3. testing result
As shown in figure 3, natually morbid Leaf of banana, natural occurrence banana, artificial infection morbidity Leaf of banana,
It can produce the view strip band of 382bp or so in artificial infection early stage banana and positive control, and healthy Banana Leaf
Piece, the Banana peel of health, the banana carpopodium of health, negative control occur without any band, show primer of the present invention and inspection
Survey method can also be used in the detection of Phyllosticta musarum in the banana plant of field.
SEQUENCE LISTING
<110>Inst. of Plant Protection, fujian Academy of Agricultural Science
<120>a kind of Phyllosticta musarum molecular detection primer and detection method
<130> 4
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>artificial sequence
<400> 1
gctacaacgc cgaaatgacc 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<400> 2
gacgttgccc aataccaagc 20
<210> 3
<211> 18
<212> DNA
<213>artificial sequence
<400> 3
tccgtaggga acctgcgg 18
<210> 4
<211> 20
<212> DNA
<213>artificial sequence
<400> 4
tcctccgctt attgatatgc 20
Claims (4)
1. a kind of Phyllosticta musarum molecular detection primer, it is characterised in that: the nucleotide sequence of primer are as follows:
Upstream primer BMF:5 '-GCTACAACGCCGAAATGACC-3 '
Downstream primer BMR:5 '-GACGTTGCCCAATACCAAGC-3 '.
2. Phyllosticta musarum molecular detection primer according to claim 1, it is characterised in that: the upstream primer BMF and
Downstream primer BMR goes out the product of 382bp to Phyllosticta musarum specific amplification.
3. a kind of rapid detection method of Phyllosticta musarum, it is characterised in that: the following steps are included:
(1) DNA of Leaf of banana or fruit is extracted；
(2) PCR amplification: 25 μ L of PCR reaction system is carried out by template of the DNA of the Leaf of banana of extraction or fruit, includes 2.5 μ
10 × PCR of L buffer, 2.0 μ L concentration are the dNTP Mixture of 2.5mmol/L, and 0.15 μ L concentration is the Taq enzyme of 5U/ μ L,
Each 0.5 μ L of BMF and BMR described in claim 1 of 10 μm of ol/L, 1 μ L of DNA profiling add ddH2O is to total volume up to 25 μ L；PCR
Reaction condition are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 45sec, 56 DEG C of annealing 45sec, 72 DEG C of extension 45sec, totally 35 are followed
Ring；72 DEG C of extension 10min；
(3) resulting PCR product is walked on 1.0% Ago-Gel with the electrophoretic analysis of 0.5 × tbe buffer liquid, voltage 4-
5V/cm；Testing result is determined according to the size of amplified production, if energy specific amplification goes out the product of 382bp, can determine that perfume (or spice)
There are Phyllosticta musarums for any of several broadleaf plants blade or fruit.
4. application of the Phyllosticta musarum molecular detection primer as described in claim 1 in detection Phyllosticta musarum.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610900830.9A CN106399529B (en) | 2016-10-17 | 2016-10-17 | A kind of Phyllosticta musarum molecular detection primer and detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610900830.9A CN106399529B (en) | 2016-10-17 | 2016-10-17 | A kind of Phyllosticta musarum molecular detection primer and detection method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106399529A CN106399529A (en) | 2017-02-15 |
| CN106399529B true CN106399529B (en) | 2019-09-24 |
Family
ID=58011692
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610900830.9A Expired - Fee Related CN106399529B (en) | 2016-10-17 | 2016-10-17 | A kind of Phyllosticta musarum molecular detection primer and detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106399529B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106868147B (en) * | 2017-03-07 | 2020-01-21 | 福建省农业科学院植物保护研究所 | Molecular detection primer for sigatoka bacteria and rapid detection method thereof |
| CN107674924A (en) * | 2017-11-30 | 2018-02-09 | 福建省农业科学院植物保护研究所 | A kind of Phyllosticta musarum LAMP detection primer and its application |
-
2016
- 2016-10-17 CN CN201610900830.9A patent/CN106399529B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| Identification and differentiation of Phyllosticta species causing freckle disease of banana using high resolution melting (HRM) analysis;M.-H. Wong等;《Plant Pathology》;20131231;第62卷;1285-1293 * |
| 瓜黑星病菌、枯萎病菌和蔓枯病菌的三重PCR检测;高永洋 等;《植物病理学报》;20101231;第40卷(第4期);343-350 * |
| 苹果黑星病菌的分子检测;商蓓 等;《西北农林科技大学学报（自然科学版）》;20100831;第38卷(第8期);104-110 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106399529A (en) | 2017-02-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101974650B (en) | Polymerase chain reaction (PCR) method for detecting fusarium oxysporum and kit | |
| CN105063219B (en) | Guava anthrax bacteria specific PCR detection primer and its detection method | |
| CN105648106B (en) | A kind of Exserohilum turcicum molecular detection primer and rapid detection method | |
| CN105256060B (en) | A kind of roxburgh anoectochilus terminal bud anthrax bacteria PCR detection primer and its detection method | |
| CN107699634A (en) | A kind of asparagus stem wilt bacteria LAMP detection primer group and its detection method | |
| CN103290131B (en) | Primer pair and kit for distinguishing Channa argus and Channa maculata, and PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) detection method | |
| CN107815505A (en) | A kind of Glorosprium musarum Cookeet Mass LAMP detection primer group and its detection method | |
| Dung et al. | Morphological and genetic characteristics of Oyster mushrooms and conditions effecting on its spawn growing. | |
| CN106399529B (en) | A kind of Phyllosticta musarum molecular detection primer and detection method | |
| CN104830698A (en) | Pokkah boeng disease pathogen separating and identifying method | |
| CN105734132B (en) | A kind of agaricus bisporus brown rot germ molecular detection primer and rapid detection method | |
| CN104232748B (en) | Whether a kind of red bayberry nursery stock carries the rapid molecular detection method of wilting germ | |
| CN106282386A (en) | A kind of Glorosprium musarum Cookeet Mass molecular detection primer and detection method | |
| CN108546772A (en) | Exserohilum turcicum LAMP detection primer and its rapid detection method and application | |
| CN105755122A (en) | Sweet potato fusarium wilt germ molecule detecting primer and rapid detection method | |
| CN105648107B (en) | A kind of southern corn leaf blight molecular detection primer and rapid detection method | |
| CN107723381A (en) | Banana crown rot bacterium LAMP detection primer and its detection method | |
| CN106244720B (en) | A kind of anthracnose of peach bacterium molecule detection primer and detection method | |
| CN105586431B (en) | A kind of asparagus stem wilt bacteria molecular detection primer and rapid detection method | |
| CN106244721B (en) | A kind of capsicum stain anthrax bacteria molecular detection primer and detection method | |
| CN105063040B (en) | Phytophthora infestans germ PCR detection primers, kit and detection method | |
| CN105219868A (en) | Herba Anoectochili roxburghii anthrax LAMP detection primer and visible detection method thereof | |
| CN106435005A (en) | Roxburgh anoectochilus terminal bud stalk rot pathogen LAMP (loop-mediated isothermal amplification) detection primer and detection method thereof | |
| CN107815504A (en) | A kind of anthracnose of peach collarium mediated isothermality amplification detection primer and its detection method | |
| CN106498043A (en) | A kind of banana crown rot bacterium molecule detection primer and detection method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190924 |