CN106636401A - Anoectochilus roxburghii stalk rot pathogen molecular detection primers and detection method thereof - Google Patents
Anoectochilus roxburghii stalk rot pathogen molecular detection primers and detection method thereof Download PDFInfo
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- CN106636401A CN106636401A CN201611196903.7A CN201611196903A CN106636401A CN 106636401 A CN106636401 A CN 106636401A CN 201611196903 A CN201611196903 A CN 201611196903A CN 106636401 A CN106636401 A CN 106636401A
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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
- C12Q1/6895—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
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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
- C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
Abstract
The invention provides anoectochilus roxburghii stalk rot pathogen molecular detection primers and a detection method thereof, specially applicable to specific molecular detection of anoectochilus roxburghii stalk rot pathogens. A pair of specific primers (ITS403f: 5' CCCAACCCCTGTGACATAC 3'; ITS403r: 5' ATTACCAGTAACGAGGGTT 3') for the anoectochilus roxburghii stalk rot pathogens are mainly designed; and based upon PCR amplification and agarose gel electrophoresis, a specific amplification product, which is 403bp in segment length, can be specifically amplified in pure DNA of the anoectochilus roxburghii stalk rot pathogens. The specific molecular detection primers and the using method thereof provided by the invention, in practical production, are applicable to the rapid, sensitive and specific detection of the stalk rot pathogens in anoectochilus roxburghii stalk rot pathogen infected plants; and meanwhile, the specific molecular detection primers and the using method are applicable to early diagnosis of field diseases as well as monitoring and identification of the pathogens; therefore, reliable technology and theoretical basis are provided for preventing and controlling diseases caused by the anoectochilus roxburghii stalk rot pathogens.
Description
Technical field
The present invention relates to a kind of Herba Anoectochili roxburghii Pathogen molecular detection primer and its detection method, are exclusively used in Herba Anoectochili roxburghii stem rot
The detection of pathogenic bacteria high sensitivity rapid molecular, overcomes traditional shortcoming that culture identification method is numerous and diverse and accuracy is low, belongs to agriculture
The field of crop pest detection, identification and Prevention Technique.
Background technology
Also known as Anoectochilus nefiliforme (Nakai) hara, orchid family opens lip orchid etc. to Herba Anoectochili roxburghii, belongs to herbaceos perennial, and rare Chinese herbal medicine among the people have " medicine
The laudatory titles such as king " " gold grass " " god's medicine ", the effects such as annealing, nourish strong, lung moistening liver protection and subduing swelling and detoxicating with heat clearing away, extensively should
The aspects such as the treatment and health care for hypertension, diabetes, nephropathy, tumor etc..Wild gold lotus are harsh to growing environmental requirement,
Low reproduction rate, poor growth, and due to its exclusive nutritive value and medical value, artificial excessively excavation is caused, face and gone out
Exhausted condition.At present Herba Anoectochili roxburghii is mainly by artificial culture, but in the course of cultivation disease problem is also day by day highlighted.
Recently, stem rot disease sample is occurred in that in the multiple Herba Anoectochili roxburghii planting sites in Fujian Province, the disease is to Herba Anoectochili roxburghii different growing stages
All can work the mischief, the water stain shape scab of yellowish-brown first occurs in plant basal part of stem during morbidity, scab is extended to around stem week, sick portion's tissue
Withered contracting of hanging rot in wire.The disease spreads rapidly, and the state of an illness is increasingly severe, and seedling lodges rapidly death, large area occurs sudden
, it is serious to threaten the health of Herba Anoectochili roxburghii to produce.By the way that to the collection of sick sample, pathogenic bacteria isolation identification, Koch's Postulates checking is final true
The pathogen of deposit Anoectochilus roxburghii stem rot is Fusarium oxysporum（Fusarium oxysporum）.
Classical culture protocols are still continued to use in identification to Herba Anoectochili roxburghii stem rot at present mostly.Traditional pathogen identification technology is
On the basis of separation and Culture obtains corresponding pathogen, the kind of pathogen is judged by morphological observation and Koch's Postulates
Class.Whole process usually needs to expend substantial amounts of labour force and time, and generally requiring several days just can complete, and require operator
Possess pathogenicbacteria separation, Morphological Identification knowledge and the rich experience of specialty, therefore be characterized as judging with Pathogens
Traditional diagnosis method on basis, because of it, time-consuming, and efficiency is low, it is difficult to meets the actual need to the diagnosis of Herba Anoectochili roxburghii stem rot
Will, because it easily misses the best period of disease control.
Recently as the development of Protocols in Molecular Biology, the molecular detecting method with round pcr as representative has obtained fast
The development and application, the especially application of polymerase chain reaction (PCR) technology of speed compensate for a great extent traditional method
Deficiency.This kind of method generally have it is quick, accurate, sensitive and the characteristics of pathogenicbacteria separation culture need not be carried out, in control
The propagation of plant disease, cause disaster in have begun to play a significant role.Therefore, a set of quick, sensitive, accurate Herba Anoectochili roxburghii is set up
Pathogen Examination and diagnosis are not only very necessary, and very urgent.
Ribosome transcribed spacer ITS sequence is made up of alternate conserved region and variable region, in microbial genome
With multicopy appearance, become in the fabulous target of the horizontal pathogen identification planted with the specificity planted, therefore the sequence.For
This, we expand the transcribed spacer of Herba Anoectochili roxburghii Pathogen and carry out cloning and sequencing using funguses universal primer ITS1/ITS4,
By sequence alignment, design a pair of Herba Anoectochili roxburghii Pathogen specific primers, set up Herba Anoectochili roxburghii Pathogen it is quick, easy,
The high monitoring technology system of high specificity, sensitivity.This technology can be applicable to Herba Anoectochili roxburghii Pathogen and cause before the aobvious disease of disease
Early monitoring, for determine disease control best period there is important effect, for control strategy formulation provide science according to
According to.
The content of the invention
The purpose of the present invention is for experiment of the Herba Anoectochili roxburghii Pathogen traditional detection method to operator in prior art
Technical ability and practical experience have very high requirement, and quite time-consuming, it is impossible to reach the problem of quick inspection, there is provided Herba Anoectochili roxburghii stem rot
The high Herba Anoectochili roxburghii Pathogen of the special molecular detection primer of bacterium and reliable results, easily operated, high specificity, sensitivity
Rapid molecular detection system and program.The method can be used for Herba Anoectochili roxburghii Pathogen and cause disease to show the early monitoring before disease,
Pair determine that disease control best period tool is of great significance.
For achieving the above object, the present invention is adopted the following technical scheme that：
1. the spy being made up of alternate conserved region and variable region according to Herba Anoectochili roxburghii Pathogen ribosome transcribed spacer ITS sequence
Point, devises one couple of PCR primers that there is specific amplified to act on to Herba Anoectochili roxburghii Pathogen, and sequence is as follows：
ITS403f：5’-CCCAACCCCTGTGACATAC-3’；
ITS403r：5’-ATTACCAGTAACGAGGGTT-3’.
2. the foundation of Herba Anoectochili roxburghii Pathogen rapid detection system
（1）The foundation of Herba Anoectochili roxburghii Pathogen specific PCR rapid detection system
The μ l of the PCR reaction systems 25.0, including 2 ×TaqPCR Master Mix12.5 μ L, the primer of 10 μm of ol/L
(ITS403f/ITS403r) each 0.5 μ L, DNA profiling 25ng, insufficient section is by dd H2O is supplied.PCR reaction conditions are：
95 DEG C of denaturations 3min；94 DEG C of degeneration 1min, 58 DEG C of annealing 30sec, 72 DEG C of extension 1min, totally 25 circulations；72 DEG C of extensions
10 min。
（2）The foundation of Herba Anoectochili roxburghii Pathogen nest-type PRC rapid detection system
With funguses universal primer（ITS1/ ITS4）Carry out the amplification of first round PCR, the μ L of reaction system 25.0, including 2 ×Taq
PCR Master Mix12.5 μ L, the primer of 10 μm of ol/L（（ITS1/ ITS4）Each 0.2 μ L, DNA profiling 25ng, not foot
Divide by dd H2O is supplied.PCR reaction conditions are:94 DEG C of denaturations 3min, 94 DEG C of degeneration 1min, 55 DEG C of 30 sec of annealing, 72
DEG C extend 1min, totally 30 circulation, 72 DEG C extension 10 min.
1 μ L first round PCR primer is taken respectively as DNA profiling, and with primer (ITS403f/ITS403r) second is carried out
Wheel PCR amplifications.The μ l of reaction system 25.0, including 2 ×TaqPCR Master Mix12.5 μ L, the primer of 10 μm of ol/L
(ITS403f/ITS403r) each 0.5 μ L, the μ L of 25ng DNA profilings 1, insufficient section is by dd H2O is supplied.PCR reaction conditions
For：95 DEG C of denaturations 3min；94 DEG C of degeneration 1min, 58 DEG C of annealing 30sec, 72 DEG C of extension 1min, totally 25 circulations；72 DEG C are prolonged
Stretch 10 min.
The guardian technique of the present invention is the primer sequence of the efficient specific amplified of Herba Anoectochili roxburghii Pathogen and its amplification side
Method.In order to detect Herba Anoectochili roxburghii Pathogen special primer accuracy, the present invention is with provinces such as China Fujian, Zhejiang, Jiangxi, Taiwan
18 plants of Herba Anoectochili roxburghii Pathogens and other 25 kinds of funguses are material to be tested, and using CTAB methods strains tested genomic DNA, tool are extracted
Body method is as follows：
The mycelium powder after 50mg lyophilizations is taken in 1.5ml centrifuge tubes, the CTAB (cetyl trimethyls of 900 l 2% are added
Ammonium bromide) extracting solution（2% CTAB；100 m mol/L Tris-HCl, PH 8.0；20mmol/L EDTA,pH8.0；1.4
mol/L NaCl）With the SDS of 90 μ l 10%（Dodecylbenzene sodium sulfonate）Vibration afterwards is mixed, in 55～60 DEG C of h of water-bath 1.5, often
10 min are reverse to be mixed once, is centrifuged after water-bath 1.5h（12,000rpm）8min, take supernatant add isopyknic phenol/chloroform/
Isoamyl alcohol（Volume ratio 25:24:1）, it is reverse to make fully mixing, 12,000rpm 12 min of centrifugation take supernatant（Water phase）, add
Equal-volume chloroform/isoamyl alcohol (volume ratio 24:1), overturning makes fully mixing, 12,000rpm centrifugation 5min, sucts clearly, plus 0.1 body
Long-pending 3mol/L NaAC solution and the ice dehydrated alcohol of 2 times of volumes, put and precipitate in -20 DEG C of refrigerators 30 more than min, and 12,
000rpm is centrifuged 5 min, lightly removes supernatant, adds the ethanol of 700 l ice 70% to be washed（Slightly it is centrifuged, inclines and fall supernatant）,
With 1 × TE after alcohol-free taste being dried naturally on superclean bench（10mmol/LTris-HCL,0.1mmol/LEDTA,pH8.0）
Solution is dissolved, and obtains DNA solution, with UV spectrophotometer measuring DNA concentration and to be diluted to 25ng/ μ L stand-by.
Jing enters performing PCR checking to the specificity for 25 kinds of funguses of examination and 18 plants of Herba Anoectochili roxburghii Pathogens.PCR reaction systems
25.0 μ L, including 2 ×TaqThe primer (ITS403f/ITS403r) of PCR Master Mix12.5 μ L, 10 μm of ol/L is each
0.5 μ L, DNA profiling 25ng, insufficient section is by dd H2O is supplied.PCR reaction conditions are：95 DEG C of denaturations 3min；94 DEG C of changes
Property 1min, 58 DEG C annealing 30sec, 72 DEG C extension 1min, totally 25 circulation；72 DEG C of 10 min of extension.This special primer is in gold
The product of 403bp is specifically amplified in Anoectochilus roxburghii Pathogen.This illustrates that the primer can be used for Herba Anoectochili roxburghii in disease plant
The detection and identification of Pathogen fast and reliable.
Beneficial effect of the present invention：The inventive method suitable for plant the detection of the fast and reliable of Herba Anoectochili roxburghii Pathogen and
Identification, for the microbial disease control of Herba Anoectochili roxburghii stem rot has important practical value in agricultural production.The present invention with it is existing
There is technology to compare, with following technical advantage and good effect：
1st, high specificity：Detection method is the characteristics of being made up of alternate conserved region and variable region according to ITS sequence, if
Count a pair of Herba Anoectochili roxburghii Pathogen special primers to be detected.To saving from China Fujian, Zhejiang, Jiangxi, Taiwan etc.
25 kinds of different funguses of Herba Anoectochili roxburghii Pathogen and other verified, as a result with very strong specificity.
2nd, sensitivity is high：Nest-type PRC can reach 10fg/ to the detection sensitivity of Herba Anoectochili roxburghii Pathogen on DNA level
25 μ L, with very high susceptiveness.
3rd, practicality is good：Designed a pair of specific primers of the invention, can be used for the high sensitivity of Herba Anoectochili roxburghii Pathogen
Quick detection, therefore this method is practical, can meet Herba Anoectochili roxburghii Pathogen carries out the detection and identification of fast and reliable
Need.
4th, it is easy to operate quick：Using the inventive method, pathogenic bacteria DNA is carried out to the plant with Herba Anoectochili roxburghii Pathogen and is carried
Take, PCR amplification and routine agarose gel electrophoresiies after by result of determination, without the need for carrying out restricted enzyme to amplified production
Enzyme action, whole detection process can be completed within a few hours.
Description of the drawings
Fig. 1 is the specific PCR amplification figure of present invention Herba Anoectochili roxburghii Pathogen to be detected
In figure：M is DL2000 DNA Marker；1 is negative control；2-5 is Fusarium oxysporum；6：Fusarium culmorum；7：Herba bromi japonici
Fusarium spp.；8：Fusarium nivale；9：Fusarium graminearum；10：Anthrax bacteria.
Fig. 2 is the susceptiveness detection amplification figure of Herba Anoectochili roxburghii Pathogen of the present invention
In figure：M is DL2000 DNA Marker；1 is negative control；2-9 is respectively 1 ng/ L,
100pg/ μ L, 10 pg/ μ L, 1 pg/ μ L, 100 fg/ μ L, 10 fg/ μ L, 1 fg/ μ L, the variable concentrations of 100 ag/ μ L
Herba Anoectochili roxburghii Pathogen DNA.
Specific embodiment
The specific detection primer of the technology contents including Herba Anoectochili roxburghii Pathogen of the present invention, designed primer and its sequence
For（ITS403f：5 '-CCCAACCCCTGTGACATAC-3 ' and ITS403r：5’-ATTACCAGTAACGAGGGTT-3’）.Utilize
The primer can go out the product of 403bp from specific amplified in Herba Anoectochili roxburghii Pathogen.
Embodiment 1：The specific amplification of primer pair Herba Anoectochili roxburghii Pathogen
1. the specific detection of Herba Anoectochili roxburghii Pathogen
The μ l of PCR reaction systems 25.0, including 2 ×TaqPCR Master Mix12.5 μ L, the primer of 10 μm of ol/L
(ITS403f/ITS403r) each 0.5 μ L, DNA profiling 25ng, insufficient section is by dd H2O is supplied.PCR reaction conditions are：
95 DEG C of denaturations 3min；94 DEG C of degeneration 1min, 58 DEG C of annealing 30sec, 72 DEG C of extension 1min, totally 25 circulations；72 DEG C of extensions
10 min。
2. testing result
The specificity of detection：Can except the Herba Anoectochili roxburghii Pathogen DNA from provinces such as the provinces such as China Fujian, Zhejiang, Jiangxi, Taiwan
Specifically amplify outside the product of 403bp, have detected 25 kinds of other fungal DNAs and fail to amplify spawn, with very strong
Specificity.
Embodiment 2：The sensitivity technique of primer pair Herba Anoectochili roxburghii Pathogen
1.DNA concentration dilutions：The Herba Anoectochili roxburghii Pathogen genomic DNA of extraction, Jing after spectrophotometric determination concentration, using system
Row concentration dilution.
2. the sensitivity technique of Herba Anoectochili roxburghii Pathogen
The Herba Anoectochili roxburghii Pathogen DNA of extraction is diluted to successively using 10 times of concentration series dilution methods, 1 ng/ L, 100 pg/
μ L, 10 pg/ μ L, 1 pg/ μ L, 100 fg/ μ L, 10 fg/ μ L, 1 fg/ μ L, 100 ag/ μ L totally 8 variable concentrations gradients, adopt
It is measured with sensitivity of the nest-type PRC to primer.
The Herba Anoectochili roxburghii Pathogen genomic DNA for taking 1 μ L variable concentrations respectively is template, with funguses universal primer（ITS1/
ITS4）Carry out the amplification of first round PCR, the μ L of reaction system 25.0, including 2 ×TaqPCR Master Mix12.5 μ L, 10 μ
The primer of mol/L（（ITS1/ ITS4）Each 0.2 μ L, DNA profiling 25ng, insufficient section is by dd H2O is supplied.
PCR reaction conditions are:94 DEG C of denaturations 3min, 94 DEG C of degeneration 1min, 55 DEG C of 30 sec of annealing, 72 DEG C of extensions
1min, totally 30 circulations, 72 DEG C of 10 min of extension.1 μ L first round PCR primers are taken respectively as DNA profiling, use primer
(ITS403f/ITS403r) carries out the second wheel PCR amplifications.The μ l of reaction system 25.0, including 2 ×Taq PCR Master
The each 0.5 μ L of primer (ITS403f/ITS403r) of Mix12.5 μ L, 10 μm of ol/L, DNA profiling 25ng, insufficient section by
dd H2O is supplied.PCR reaction conditions are：95 DEG C of denaturations 3min；94 DEG C of degeneration 1min, 58 DEG C of annealing 30sec, 72 DEG C of extensions
1min, totally 25 circulations；72 DEG C of 10 min of extension.
3. testing result：
In 25.0 μ L reaction systems, Herba Anoectochili roxburghii Pathogen genomic DNA can obtain obvious amplified band, and detection sensitivity can
Up to 10 fg/25 μ L.
SEQUENCE LISTING
<110>Inst. of Plant Protection, fujian Academy of Agricultural Science
<120>A kind of Herba Anoectochili roxburghii Pathogen molecular detection primer and its detection method
<130> 2
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence
<400> 1
cccaacccct gtgacatac 19
<210> 2
<211> 19
<212> DNA
<213>Artificial sequence
<400> 2
attaccagta acgagggtt 19
Claims (3)
1. a kind of PCR primer of Herba Anoectochili roxburghii Pathogen Molecular Detection, it is characterised in that primer sequence is：
ITS403f：5’-CCCAACCCCTGTGACATAC-3’；
ITS403r：5’-ATTACCAGTAACGAGGGTT-3’；
Above-mentioned primer I TS403f/ ITS403r can go out the product of 403bp from Herba Anoectochili roxburghii Pathogen specific amplification.
2. the method for utilizing PCR primer described in claim 1 for detecting Herba Anoectochili roxburghii Pathogen, it is characterised in that：Including such as
Lower step：
（1）DNA is extracted in the Herba Anoectochili roxburghii plant infected from described Herba Anoectochili roxburghii Pathogen or by Pathogen；
（2）Primer I TS403f/ ITS403r with the DNA as template claim 1 described in are expanded by polymerase chain reaction
Increase：The μ l of PCR reaction systems 25.0, including 2 ×TaqPCR Master Mix12.5 μ L, the primer I TS403f of 10 μm of ol/L
/ ITS403r each 0.5 μ L, DNA profiling 25ng, insufficient section is by dd H2O is supplied；PCR reaction conditions are：95 DEG C of denaturations
3min；94 DEG C of degeneration 1min, 58 DEG C of annealing 30sec, 72 DEG C of extension 1min, totally 25 circulations；72 DEG C of 10 min of extension；
（3）Then step is taken（2）Pcr amplification product with agarose gel electrophoresiies separate, Jing ethidium bromide stainings are after ultraviolet
Observe under lamp, according to the size result of determination of amplified production, if the product of 403bp can be amplified specifically, you can judge
There is Herba Anoectochili roxburghii Pathogen in described Plant samples.
3. application of the PCR primer as claimed in claim 1 in detection Herba Anoectochili roxburghii Pathogen.
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| CN201611196903.7A CN106636401A (en) | 2016-12-22 | 2016-12-22 | Anoectochilus roxburghii stalk rot pathogen molecular detection primers and detection method thereof |
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