CN109517916A - Specific primer, kit and the detection method of Rapid identification orchid bacterial brown rot germ - Google Patents
Specific primer, kit and the detection method of Rapid identification orchid bacterial brown rot germ Download PDFInfo
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- CN109517916A CN109517916A CN201910019193.8A CN201910019193A CN109517916A CN 109517916 A CN109517916 A CN 109517916A CN 201910019193 A CN201910019193 A CN 201910019193A CN 109517916 A CN109517916 A CN 109517916A
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- brown rot
- pcr
- bacterial brown
- dna
- rot germ
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- 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
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- 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
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention discloses a kind of specific primers, kit and detection method for Rapid identification orchid bacterial brown rot germ.Primer sequence is as follows: EcUP:5'-TCAACGCCAAGCCGATTTCT-3'；EcNP:5'-ACGAATGAACCGTCGTCGGC-3'.The present invention establishes a kind of quarantine identification method of specific detection orchid bacterial brown rot germ, standard and judgment is established for quarantine orchid bacterial brown rot germ, help to reinforce host plant inspection and quarantine of the port to orchid bacterial brown rot germ, prevent orchid bacterial brown rot germ and China is passed to by port, it is avoided to spread within the border in China.More rapidly than traditional identification method, specificity is stronger, and sensitivity is higher for the method for foundation of the invention, and the DNA sensitivity of primer identification is about 0.01ng/ μ l.
Description
Technical field
The present invention relates to specific primer, kit and the detection sides of a kind of Rapid identification orchid bacterial brown rot germ
Method belongs to microorganism detection identification technology field.
Background technique
Orchid bacterial brown rot germ (Erwinia cypripedii) be enterobacteriaceae Erwinia (Erwinia)
Plant pathogenetic bacteria,ErwiniaCategory is by bacteriologist Erwin F. Smith name in nineteen twenty, most members
It is plant pathogen, some members are an important composition, a small number of bacterium in the ecological masses of the non-plant pathogenic bacteria of plant surface
System is the accidental pathogen of human and animal.Classification Change is larger, is divided into 3 groups by what majority approved:
Amylovora group, carotovora group, herbicola group.1945, Waldee proposed that soft rotten class Erwinia should be one
A individual category ---PectobacteriumBelong to, however, being just recognized since absence of proof is supported until 1980, leads
Want member from carotovora group.Currently,PectobacteriumSubordinate includes 9 kinds/subspecies of carotovora group:P. atroseptica 、P. betavasculorum 、P. carotovora subsp. Carotovora、P. carotovora subsp. Brasiliense、P. carotovora subsp. Odorifera、P. wasabiae、P. parmentieri 、P. cacticida、P. cypripedii .2010, Brady et al. is proposed by the methods of MLSA willP. cypripediiDivide into general Pseudomonas (Pantoea).The bacterium is in latest edition " primary Jie Shi systematic bacteriology handbook "Pectobacterium (Erwinia) cypripedii。
Orchid bacterial brown rot germ is distributed mainly on South Africa, Japan, Australia, the U.S. and China Taiwan, can pass through
Plant carry out long-distance communications, seriously endanger orchid cause brown rot, generally infect orchid meat blade, start for
Then small water soaking mode spot extends simultaneously micro-pits, filbert oil immersion shape, and expands to stem and growing point, be the one kind for endangering orchid
Critically important bacteriosis.The germ is classified as quarantine plant-pest by Malaysia.With the development of trade, in recent years
China introduces cymbidium variety and quantity increases considerably, and the incoming risk of new expression increasingly increases, since the germ can be by posting
Main plant carries out long-distance communications, therefore the incoming domestic risk to cause damages to agricultural production, ecological safety is higher, at present I
The germ is repeatedly intercepted and captured from the plant product that the commodity of immigration and passenger carry in state port, and State General Administration for Quality Supervision repeatedly assigns
Warning notification, and " the defeated Hua Zhi in " bulletin about import Cymbidium hyridus plant inspection quarantine request " [2014 No. 131]
Object quarantine request protocol " is inner to be classified as the germ harmful organism for forbidding immigration.To protect China's agricultural production ecological safety,
It effectively prevent bacteriosis to spread, the research for reinforcing the germ rapid detection method is of great significance.
When studying Testing and appraisal method, the bacterium often withErwiniaOrchid soft rot can equally be caused in category
Chrysanthemum Phyllostachys pubescens (E.chrysanthemi) and carrot soft rot germ (E.carotovora var. carotovora) into
The identification of row physiology, biochemistry and cultural characteristic.At present aboutE.chrysanthemiWithE.carotovora var.carotovoraThe rapid detection method research carried out is relatively more, but the detection of orchid bacterial brown rot germ is reflected
Determine the rare report of method.In port quarantine qualification process, it is generally also confined to traditional Screening of Media, Physiology and biochemistry mirror
It is fixed, and the tediously long step such as Pathogenicity need to be carried out, detecting step is many and diverse, and the time is tediously long or even allows beyond plant survival
Time domain the defects of, the identification technology about orchid bacterial brown rot germ has at present: (1) Determination of Physiological And Biochemical Indices,
This is the identification most traditional reliable method of bacterium, it is desirable that pathogen must be first isolated from host plant, purified by germ,
Measurement comparison one by one is carried out in conjunction with every physiological and biochemical property of genus and species, identifying this method for daily port, there are two
It is insufficient: first is that authentication step is many and diverse, to need the longer pre-treatment time, the time is tediously long, second is that even with quick Physiology and biochemistry
Assessing instrument, also only the primary dcreening operation of physiological and biochemical index compares, and cannot realize ultimate judgement to germ.(2) 16S rDNA gene sequence
Column analysis refers to and carries out Species estimation to bacterium using the method for bacterial 16 S rDNA sequence.It is mentioned including bacterium group DNA
Take, 16S rDNA specific primer PCR amplification, amplified production purifying, DNA sequencing, sequence alignment, be it is a kind of effectively, can
The pathogenic bacteria certification method leaned on, but the strain having due to interspecific difference it is small, rely solely on 16S rDNA identification cannot identify
Kind, it needs in conjunction with other identification methods, often as auxiliary identification of means, is unable to satisfy daily port quick and precisely sensitive mirror
Fixed work requirements.
Currently, the rapid quarantine for orchid bacterial brown rot germ identifies that method is also marked without corresponding country not yet
Quasi-, professional standard.Both at home and abroad for the detection of orchid bacterial brown rot germ without the molecular biology method using specificity
The detection method of (such as PCR- gel electrophoresis, real-time fluorescence PCR) is reported.
Summary of the invention
The technical problems to be solved by the present invention are: providing a kind of specificity of Rapid identification orchid bacterial brown rot germ
Primer, kit and detection method.
In order to solve the above technical problems, the technical solution adopted by the present invention is that:
The present invention devises the specific primer of one group of Rapid identification orchid bacterial brown rot germ, and primer sequence is as follows:
EcUP:5'- TCAACGCCAAGCCGATTTCT -3';
EcNP:5'- ACGAATGAACCGTCGTCGGC -3'.
The present invention also provides a kind of kits of Rapid identification orchid bacterial brown rot germ, including P CR amplified reaction
Liquid, positive control D N A two parts:
P C R amplification reaction solution total volume is 25 μ L, wherein containing: the sample DNA of 2 μ L 10 μ g/mL-100 μ g/mL, on 10 μM
Each 1 μ L, 5U/ μ L Taq archaeal dna polymerase of EcNP 0.5 μ l, 10 μm of 2 μ L, 10 × PCR buffer of ol/L dNTP, 2.5 μ L, water are mended
Enough to 25 μ L of total volume；
The primer sequence for the PCR amplification detection being related to is as follows:
EcUP:5'- TCAACGCCAAGCCGATTTCT -3';
EcUP:5'- ACGAATGAACCGTCGTCGGC -3'；
Positive control dna is the DNA extracting solution containing orchid bacterial brown rot germ.
The present invention also provides a kind of methods of quickly detection orchid bacterial brown rot germ, successively include the following steps:
(1) extraction of measuring samples DNA
DNA extraction is carried out using the CTAB method of improvement or applicable commercial kit；
(2) PCR amplification
A. 25 μ L PCR reaction systems: PCR Premix (2X) 12.5 μ L, H are used28.5 μ L of O, 10 μM of primers each 1
μ L, 2.0 μ L of DNA profiling；
The ingredient for including inside PCR Premix: 0.1U Taq polymerase/ μ L, 500 μM of DNTP each, 20mM
Tris-HCL (PH8.3), 100Mm KCl, 3mMgCl.
B. PCR reaction tube is put into PCR instrument, completes PCR amplification by following reaction conditions:
PCR reaction condition: initial denaturation: 98 DEG C, 2min；(denaturation: 98 DEG C, 10s；Annealing: 59.4 DEG C, 10s；Extend: 72 DEG C,
10s) 35 circulation；Extend: 72 DEG C, 3min.
Beneficial effects of the present invention:
A kind of quarantine identification method of quickly detection orchid bacterial brown rot germ is established, for orchid bacterial brown rot of quarantining
Bacterium establishes standard and judgment, helps to reinforce host plant inspection and quarantine of the port to orchid bacterial brown rot germ, prevents orchid
Flower Bacterial brown rot bacterium is passed to China by port, it is avoided to spread within the border in China.
The method of foundation of the invention is more stronger than traditional method specificity, and sensitivity is higher, and the DNA of primer identification is sensitive
Degree is about 0.01ng/μ l.
Detailed description of the invention
Specific embodiments of the present invention will be described in further detail with reference to the accompanying drawing.
Fig. 1 is orchid bacterial brown rot germ specific detection result.M:DL5000 DNA Marker；0: ddH2O;
1: orchid bacterial brown rot germ;2: chrysanthemum Phyllostachys pubescens;3: carrot soft rot germ;4: erwinia amylovora;5: agglomerating
General bacterium;6: the general bacterium of pineapple.
Fig. 2 is orchid bacterial brown rot germ primer sensitivity verifying testing result.Note: M:DL5000 DNA Marker；
1:DNA stoste；2:10-1；3:10-2；4:10-3；5:10-4；6:10-5；7:10-6；8:10-7.
Specific embodiment
Embodiment 1
The specific primer of one group of Rapid identification orchid bacterial brown rot germ is present embodiments provided, primer sequence is as follows:
EcUP:5'- TCAACGCCAAGCCGATTTCT -3';
EcNP:5'- ACGAATGAACCGTCGTCGGC -3'.
A kind of kit of Rapid identification orchid bacterial brown rot germ, including P CR amplification reaction solution, positive control D N
A two parts:
P C R amplification reaction solution total volume is 25 μ L, wherein containing: the sample DNA of 2 μ L 10 μ g/mL-100 μ g/mL, on 10 μM
Each 1 μ L, 5U/ μ L Taq archaeal dna polymerase of EcNP 0.5 μ l, 10 μm of 2 μ L, 10 × PCR buffer of ol/L dNTP, 2.5 μ L, water are mended
Enough to 25 μ L of total volume；
The primer sequence for the PCR amplification detection being related to is as follows:
EcUP:5'- TCAACGCCAAGCCGATTTCT -3';
EcNP:5'- ACGAATGAACCGTCGTCGGC -3'；
Positive control dna is the DNA extracting solution containing orchid bacterial brown rot germ.
The present embodiment also provides a kind of method of quickly detection orchid bacterial brown rot germ, successively includes the following steps:
(1) extraction of measuring samples DNA
DNA extraction is carried out using the CTAB method of improvement or applicable commercial kit；
(2) PCR amplification
A. 25 μ L PCR reaction systems: PCR Premix (2X) 12.5 μ L, H are used28.5 μ L of O, 10 μM of primers each 1
μ L, 2.0 μ L of DNA profiling；
The ingredient for including inside PCR Premix: 0.1U Taq polymerase/ μ L, 500 μM of DNTP each, 20mM
Tris-HCL (PH8.3), 100Mm KCl, 3mMgCl.
B. PCR reaction tube is put into PCR instrument, completes PCR amplification by following reaction conditions:
PCR reaction condition: initial denaturation: 98 DEG C, 2min；(denaturation: 98 DEG C, 10s；Annealing: 59.4 DEG C, 10s；Extend: 72 DEG C,
10s) 35 circulation；Extend: 72 DEG C, 3min.
Embodiment 2
The verifying of 2.1 primer specificities
With orchid bacterial brown rot germ (E. cypripedii), chrysanthemum Phyllostachys pubescens (E.chrysanthemi) and carrot soft
Maize ear rot bacterium (E.carotovora var. carotovora), erwinia amylovora (E. amylovory), pantoea agglomerans
(Pantoea agglomerans), the general bacterium of pineapple (Pantoea ananatis) genomic DNA and ddH2O be template, respectively
Routine PCR reaction is carried out with specific primer EcUP, EcNP.
A. 25 μ L PCR reaction systems: PCR Premix (2X) 12.5 μ L, H are used28.5 μ L of O, 10 μM of primers
Each 1 μ L, 2.0 μ L of DNA profiling；
The ingredient for including inside PCR Premix: 0.1U Taq polymerase/ μ L, 500 μM of DNTP each, 20mM
Tris-HCL (PH8.3), 100Mm KCl, 3mMgCl.
B. PCR reaction tube is put into PCR instrument, completes PCR amplification by following reaction conditions:
PCR reaction condition: initial denaturation: 98 DEG C, 2min；(denaturation: 98 DEG C, 10s；Annealing: 59.4 DEG C, 10s；Extend: 72 DEG C,
10s) 35 circulation；Extend: 72 DEG C, 3min.
The result is shown in Figure 1.Specificity verification the result shows that, the orchid bacterial brown rot germ PCR method of foundation has height
Specificity, only occur in the reaction of orchid bacterial brown rot germ DNA profiling clip size be 420 bp specific item
Band, and its allied species, other germs and negative control do not occur specific band.
2.2 primer sensitivity verifying
10 times of concentration gradient dilutions are done with orchid bacterial brown rot germ DNA profiling (60ng/ul), dilute 1000X- respectively
After 7000X, using DNA stoste and dilution as template, orchid is detected according to the reaction system and reaction condition of method design
The sensitivity of flower Bacterial brown rot bacterium PCR reaction.
Sensitivity verification result shows template DNA stoste and 10-1、10-2、10-3、10-4There is specific item in dilution
Band, and 10-5、10-6、10-7Dilution does not occur specific band.It can be seen that 10-4Dilution is the detection of this test
Lower limit.It is found that the sensitivity of orchid bacterial brown rot germ PCR test is up to 0.01ng/μ L after conversion.
Claims (3)
1. the specific primer of Rapid identification orchid bacterial brown rot germ, which is characterized in that primer sequence is as follows:
EcUP:5'- TCAACGCCAAGCCGATTTCT -3';
EcNP:5'- ACGAATGAACCGTCGTCGGC -3'.
2. the kit of Rapid identification orchid bacterial brown rot germ, which is characterized in that right including P CR amplification reaction solution, the positive
According to D N A two parts:
P C R amplification reaction solution total volume is 25 μ L, wherein containing: the sample DNA of 2 μ L 10 μ g/mL-100 μ g/mL, on 10 μM
Each 1 μ L, 5U/ μ L Taq archaeal dna polymerase of EcNP 0.5 μ l, 10 μm of 2 μ L, 10 × PCR buffer of ol/L dNTP, 2.5 μ L, water are mended
Enough to 25 μ L of total volume；
The primer sequence for the PCR amplification detection being related to is as follows:
EcUP:5'- TCAACGCCAAGCCGATTTCT -3';
EcNP:5'- ACGAATGAACCGTCGTCGGC -3'；
Positive control dna is the DNA extracting solution containing orchid bacterial brown rot germ.
3. the quickly method of detection orchid bacterial brown rot germ, which is characterized in that successively include the following steps:
(1) extraction of measuring samples DNA
DNA extraction is carried out using the CTAB method of improvement or applicable commercial kit；
(2) PCR amplification
A. 25 μ L PCR reaction systems: PCR Premix (2X) 12.5 μ L, H are used2O 8.5 μ L, each 1 μ of 10 μM of primers
L, 2.0 μ L of DNA profiling；
The ingredient for including inside PCR Premix: 0.1U Taq polymerase/ μ L, 500 μM of DNTP each, 20mM
Tris-HCL (PH8.3), 100Mm KCl, 3mMgCl；
B. PCR reaction tube is put into PCR instrument, completes PCR amplification by following reaction conditions:
PCR reaction condition: 98 DEG C, 2min；(denaturation: 98 DEG C, 10s；Annealing: 59.4 DEG C, 10s；Extend: 72 DEG C, 10s) it 35 follows
Ring；Extend: 72 DEG C, 3min.
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| CN103789443A (en) * | 2014-02-25 | 2014-05-14 | 广东省农业科学院植物保护研究所 | Multiple intermolecular rapid detection method for banana wilt bacteria and bacterial Erwinia carotovora |
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