CN103031383B - Method for detecting phytophthora hibernalis carne and sphaeropsis tumefaciens hedges - Google Patents
Method for detecting phytophthora hibernalis carne and sphaeropsis tumefaciens hedges Download PDFInfo
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- CN103031383B CN103031383B CN201210583426.5A CN201210583426A CN103031383B CN 103031383 B CN103031383 B CN 103031383B CN 201210583426 A CN201210583426 A CN 201210583426A CN 103031383 B CN103031383 B CN 103031383B
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Abstract
The invention provides a method for detecting phytophthora hibernalis carne and sphaeropsis tumefaciens hedges based on double PCR (Polymerase Chain Reaction) amplification. The method comprises the following steps that an aseptic centrifuge tube is fetched; sterile deionized water, a PCR buffer solution, a dNTPs (Diethyl-nitrophenyl Thiophosphate Solution) mixture, a sense primer PHIB1, an antisense primer PHIB2, a sense primer SPT1, an antisense primer SPT2, a target object DNA, and TaqDNA polymerase are added to the centrifuge tube in sequence, and mixed uniformly for centrifugation; reaction liquid is centrifuged to the bottom of the tube; the centrifuge tube is placed on a PCR instrument for amplification to form a PCR product after the amplification; the PCR product is separated by 2%-3% of agarose gel electrophoresis, and dyed by an ethidium bromide solution for 12-20 min; and a PCR result is observed on an ultraviolet transmissometer. The method is simple in step, and can detect the phytophthora hibernalis carne and the sphaeropsis tumefaciens hedges effectively.
Description
Technical field
The present invention relates to a kind of oranges and tangerines winter raw mould brown rot pathogenic bacteria of epidemic disease and the method for oranges and tangerines branch knurl pathogenic bacteria that detects, relate in particular to a kind of raw mould brown rot pathogenic bacteria of epidemic disease and the method for oranges and tangerines branch knurl pathogenic bacteria of double pcr amplification technology for detection oranges and tangerines winter of application of high sensitivity.
Background technology
It is wide that the mould brown rot pathogenic bacteria of raw epidemic disease of oranges and tangerines winter Phytophthora hibernalis Carne has host range, and the Orange Producing of causing harm is serious, the feature that distributional region is wide, and fruit, tissue or asexual propagation material carry out long-distance communications in spite of illness.China's oranges and tangerines planting area is widely distributed, and these pathogenic bacterias are along with the probability of seed nursery stock successor China of introduction high-quality is increasing in recent years.Once this disease is imported into, will cause very big harm to China's Citrus Industry.The mould brown rot disease of raw epidemic disease of oranges and tangerines winter raw epidemic disease of microbial oranges and tangerines winter of mould brown rot cause of disease is to find first on the citrusfruit of western australia, also finds successively subsequently in other countries and area.This epidemic disease is that what to infect that oranges and tangerines cause is a kind of destructive disease, infects the initial stage, and light brown variable color appears in fruit surface, and infection site cortex is hard, and humidity grows white hypha body while being applicable to.Be injured fruit bitter, tool rotten odor.The oranges and tangerines planting area of China is extensive, and geography and climate is various, and the area that has applicable disease to occur, therefore it is large surely to grow possibility.Closely circulation way has the route of transmission such as wind and rain, instrument, and long-distance communications are mainly propagated with soil, fruit, reproductive material.This disease continues perplexing the Orange Producing on the ground such as Italy, Portugal always.
Oranges and tangerines branch knurl pathogenic bacteria Sphaeropsis tumefaciens Hedges, main harm Mexican lime, comes lemon and most tangerine section plant.Performance branch knurl and clump branch two class symptoms, but taking branch knurl and branch knurl ulcer as main.Plant tissue in spite of illness can pass pathogenic bacteria.Pathogenic bacteria can borrow wind, rain etc. closely to propagate, plant tissue in spite of illness as branch, sick leaf and reproductive material be the approach of long-distance communications pathogenic bacteria.Many citriculture kinds are all sick responsive to this, and impact greatly.On May 22nd, 2008, food safety office of European Union issues the assessment that the Risk Assessment Report about oranges and tangerines harmful organism being completed by French authorities has just comprised this pathogenic bacteria.
For the existing part Study of phylogeny of phytophthora fungi, also there is the report of PCR detection for the molecular detection technology of the mould brown rot pathogenic bacteria of raw epidemic disease of oranges and tangerines winter at present.2008, the comparative analysiss such as Zhang Haifeng the mould and mould ITS sequence of other epidemic disease of raw epidemic disease of winter, design on this basis 1 pair and detected the mould special primer 751F/752R of raw epidemic disease of winter, this to primer can be from raw phytophthora of winter amplification to the DNA band of long 616 bp, and other 19 kinds of epidemic diseases are mould and other fungi all without amplified band.Its detection limit can reach every 25 μ L reaction systems only needs the level of 10 fg genomic dnas, but the amplified band of the oranges and tangerines sample that carries disease germs is unobvious especially.
For oranges and tangerines branch knurl pathogenic bacteria, at present, the report that uses universal primer ITS4 and ITS5 to carry out PCR detection is only seen in the detection of oranges and tangerines branch knurl pathogenic bacteria.From the database of NCBI, search for, also only have trivial 2 rrna sequences.Therefore, PCR design of primers has become a difficult problem.
Therefore, the mould brown rot pathogenic bacteria of raw epidemic disease of oranges and tangerines branch knurl cause of disease and oranges and tangerines winter is two kinds of common two kind germs very large to the harm of oranges and tangerines in external oranges and tangerines, when therefore a kind of more high sensitivity is provided, detect the detection method of above-mentioned two kinds of germs, become the study hotspot in this area.
Summary of the invention
The invention solves the difficult problem in above-mentioned background technology, a kind of method that detects oranges and tangerines branch knurl cause of disease and the mould brown rot pathogenic bacteria of raw epidemic disease of oranges and tangerines winter based on double pcr amplification is provided, the method step is simple, can resultful two kinds of germs be detected.
Realizing the technical scheme that above-mentioned purpose of the present invention adopts is:
A kind of method based on the double pcr amplification detection oranges and tangerines winter life mould brown rot pathogenic bacteria of epidemic disease and oranges and tangerines branch knurl pathogenic bacteria, it is characterized in that comprising the following steps: (1), get an aseptic centrifuge tube, in centrifuge tube, add successively sterilizing deionized water, PCR damping fluid, dNTPs mixture, sense primer PHIB1, antisense primer PHIB2, sense primer SPT1, antisense primer SPT2, target compound DNA and TaqDNA polysaccharase, then mixed rear centrifugal, by centrifugal reaction solution to managing the end;
(2), centrifuge tube is placed on PCR instrument and is increased, after amplification, obtain PCR reaction product;
(3), PCR product separates with 2%-3% agarose gel electrophoresis, after ethidium bromide solution dyeing 12-20min, observes PCR reaction result on ultraviolet transilluminator.
PCR damping fluid described in step (1) is 10 × PCR damping fluid, the deionized water adding, PCR damping fluid, dNTPs mixture, sense primer PHIB1, antisense primer PHIB2, sense primer SPT1, antisense primer SPT2, volume ratio between target compound DNA and TaqDNA polysaccharase is 16:2.5:1:1:1:1:1:1: 0.5, wherein sense primer PHIB1, antisense primer PHIB2, the concentration of sense primer SPT1 and antisense primer SPT2 is 10 μ M, the concentration of dNTPs mixture is 2.5 mM each, the concentration of TaqDNA polysaccharase is 5U/ μ l, the concentration of target compound DNA is 100-200 ng/ μ l.
In step (2), the program of amplification is: 94 DEG C of 3min, 94 DEG C of 30S, 62 DEG C of 30S, 72 DEG C of 30S, circulation 32-36 time, last 72 DEG C of added time 5min.
Provided by the invention based on double pcr amplification detect the oranges and tangerines winter the raw mould brown rot pathogenic bacteria of epidemic disease and the method for oranges and tangerines branch knurl pathogenic bacteria have following advantage: (1), the method energy Accurate Diagnosis go out oranges and tangerines and whether carry the pathogenic fungi of these two kinds of quarantines; (2) the method adopts the method for double PCR, only needs half a day and just can identify the oranges and tangerines winter raw mould brown rot pathogenic bacteria of epidemic disease and the kind of oranges and tangerines branch knurl pathogenic bacteria, can speed passage through customs; (3) the method is reproducible, highly sensitive, the kind that target compound DNA only needs 100ng/ μ L just can identify oranges and tangerines to carry pathogenic fungi.
Brief description of the drawings
Fig. 1 is the detected through gel electrophoresis result figure in embodiment 1.
Embodiment
Below in conjunction with specific embodiment, the present invention is done to detailed specific description.
In the present embodiment, the raw mould brown rot pathogenic bacteria of epidemic disease of bacterial classification oranges and tangerines winter (Phytophthora hibernalis Carne), purchased from ATCC DSMZ of the U.S., is numbered 32995
tM, and carry out Operation & use in strict accordance with the dangerous bacterial classification of quarantine.The pcr amplification of process DNA, the checking of Cloning and sequencing before using.
The present embodiment bacterial classification oranges and tangerines used branch knurl pathogenic bacteria (Sphaeropsis tumefaciens Hedges), purchased from ATCC DSMZ of the U.S., is numbered 20908
tM, and carry out Operation & use in strict accordance with the dangerous bacterial classification of quarantine.The pcr amplification of process DNA, the checking of Cloning and sequencing before using.
In the present embodiment, the extracting method of target compound DNA is as follows: be 10 by 100 μ l concentration
7spore suspension is coated on the PDA and V8 flat board that is covered with sterilizing glassine paper, cultivates 7-10 days at 18 DEG C, and scraping mycelia is freezing for subsequent use.Get 0.5g mycelia, be placed in centrifuge tube, add in the CTAB Extraction buffer of the own preheating of 2ml (65 DEG C), centrifuge tube is put upside down and mixed, 65 DEG C of water-bath 5min, mix gently; Add isopyknic phenol: chloroform: primary isoamyl alcohol (25:24:l), mix, the centrifugal 15min of 12000r/min, gets supernatant liquor in centrifuge tube, then adds equal-volume chloroform: primary isoamyl alcohol (24:l) mixes, the centrifugal 15min of 12000r/min, get supernatant liquor in centrifuge tube, add 100% ethanol of 2 times of volumes to put upside down and mix, the centrifugal 10min of 12000r/min, the supernatant liquor that inclines, with 70% ethanol by twice of washing of precipitate.After washing finishes, the centrifuge tube that contains DNA is placed in to 37 DEG C of incubators dry.After dry, add 30 μ l TE dissolution precipitations, save backup in-20 DEG C.
In following examples, the nucleotide sequence of sense primer PHIB1 is as shown in SEQ ID NO:1: 5 '-TCGGGTCTGAGCTAGTAGTCTT-3 '; The nucleotide sequence of antisense primer PHIB2 is as shown in SEQ ID NO:2: 5 '-CTTCCACAACCAATTCCATTATGC-3.The nucleotide sequence of sense primer SPT1 is as shown in SEQ ID NO:3: 5 '-GAACGCAGCGAAATGCGATAAG-3 '; The nucleotide sequence of antisense primer SPT2 is as shown in SEQ ID NO:4: 5 '-ATGCTNAAGTTCAGCGGGTAT-3 '.
In the present embodiment, the cumulative volume of double PCR reaction system is 25 μ l, concrete detection method is as follows: (1) gets an aseptic centrifuge tube, in centrifuge tube, add successively sterilizing deionized water, PCR damping fluid, concentration is the dNTPs mixture of 2.5mM each, concentration is the sense primer PHIB1 of 10 μ M, concentration is the antisense primer PHIB2 of 10 μ M, concentration is the sense primer SPT1 of 10 μ M, concentration is the antisense primer SPT2 of 10 μ M, concentration is that target compound DNA and the concentration of 100ng/ μ l is the TaqDNA polysaccharase of 5U/ μ l, the volume of each material is respectively: 16 μ l, 2.5 μ l, 1 μ l, 1 μ l, 1 μ l, 1 μ l, 1 μ l, 1 μ l, 0.5 μ l.Then mixed rear centrifugal, by centrifugal reaction solution to managing the end.
(2), centrifuge tube is placed on PCR instrument and is increased, amplification program is 3min at 94 DEG C, 30S at 94 DEG C, and 30S at 62 DEG C, 30S at 72 DEG C, circulates 36 times, and added time 5min at last 72 DEG C, obtains PCR reaction product after amplification.
(3), PCR product separates with 3% agarose gel electrophoresis, after ethidium bromide solution dyeing 20min, observes PCR reaction result on ultraviolet transilluminator.
Result display application primer pair PHIB1/PHIB2 and SPT1/SPT2 carry out double pcr amplification; detect the oranges and tangerines winter simultaneously and give birth to the mould brown rot pathogenic bacteria of epidemic disease and oranges and tangerines branch knurl pathogenic bacteria; on sepharose, can observe only to have and on swimming lane 5, have 2 specific DNA fragmentations (307bp and 407 bp) clearly; as shown in Figure 1; swimming lane M in Fig. 1, DL2000; Swimming lane 1, raw phytophthora+water of winter; Swimming lane 2, branch knurl pathogenic bacteria+water; Swimming lane 3, raw phytophthora+PHIB of winter, swimming lane 4, branch knurl pathogenic bacteria+SPT; Swimming lane 5, winter raw phytophthora+branch knurl pathogenic bacteria+PHIB+SPT; Swimming lane 6, raw phytophthora+SPT of winter; Swimming lane 7, branch knurl pathogenic bacteria+PHIB.
In the present embodiment, the cumulative volume of double PCR reaction system is 25 μ l, concrete detection method is as follows: (1) gets an aseptic centrifuge tube, in centrifuge tube, add successively sterilizing deionized water, PCR damping fluid, concentration is the dNTPs mixture of 2.5mM each, concentration is the sense primer PHIB1 of 10 μ M, concentration is the antisense primer PHIB2 of 10 μ M, concentration is the sense primer SPT1 of 10 μ M, concentration is the antisense primer SPT2 of 10 μ M, concentration is that target compound DNA and the concentration of 200ng/ μ l is the TaqDNA polysaccharase of 5U/ μ l, the volume of each material is respectively: 14 μ l, 2.5 μ l, 1 μ l, 1 μ l, 1 μ l, 1 μ l, 1 μ l, 1 μ l, 0.5 μ l.Then mixed rear centrifugal, by centrifugal reaction solution to managing the end.
(2), centrifuge tube is placed on PCR instrument and is increased, amplification program is 3min at 94 DEG C, 30S at 94 DEG C, and 30S at 62 DEG C, 30S at 72 DEG C, circulates 33 times, and added time 5min at last 72 DEG C, obtains PCR reaction product after amplification.
(3), PCR product separates with 2% agarose gel electrophoresis, after ethidium bromide solution dyeing 13min, observes PCR reaction result on ultraviolet transilluminator.
Result display application primer pair PHIB1/PHIB2 and SPT1/SPT2 carry out double pcr amplification, detect the oranges and tangerines winter simultaneously and give birth to the mould brown rot pathogenic bacteria of epidemic disease and oranges and tangerines branch knurl pathogenic bacteria, on sepharose, can observe 2 specific DNA fragmentations (307bp and 407 bp) clearly.
Claims (3)
1. the method based on the double pcr amplification detection oranges and tangerines winter life mould brown rot pathogenic bacteria of epidemic disease and oranges and tangerines branch knurl pathogenic bacteria, it is characterized in that comprising the following steps: (1), get an aseptic centrifuge tube, in centrifuge tube, add successively sterilizing deionized water, PCR damping fluid, dNTPs mixture, sense primer PHIB1, antisense primer PHIB2, sense primer SPT1, antisense primer SPT2, target compound DNA and TaqDNA polysaccharase, then mixed rear centrifugal, by centrifugal reaction solution to managing the end;
The nucleotide sequence of described sense primer PHIB1 is as shown in SEQ ID NO:1: 5 '-TCGGGTCTGAGCTAGTAGTCTT-3 ', the nucleotide sequence of antisense primer PHIB2 is as shown in SEQ ID NO:2: 5 '-CTTCCACAACCAATTCCATTATGC-3, and the nucleotide sequence of sense primer SPT1 is as shown in SEQ ID NO:3: 5 '-GAACGCAGCGAAATGCGATAAG-3 '; The nucleotide sequence of antisense primer SPT2 is as shown in SEQ ID NO:4: 5 '-ATGCTNAAGTTCAGCGGGTAT-3 ';
(2), centrifuge tube is placed on PCR instrument and is increased, after amplification, obtain PCR reaction product;
(3), PCR product separates with 2%-3% agarose gel electrophoresis, after ethidium bromide solution dyeing 12-20min, observes PCR reaction result on ultraviolet transilluminator.
2. the method based on the double pcr amplification detection oranges and tangerines winter life mould brown rot pathogenic bacteria of epidemic disease and oranges and tangerines branch knurl pathogenic bacteria according to claim 1, it is characterized in that: the PCR damping fluid described in step (1) is 10 × PCR damping fluid, the deionized water adding, PCR damping fluid, dNTPs mixture, sense primer PHIB1, antisense primer PHIB2, sense primer SPT1, antisense primer SPT2, volume ratio between target compound DNA and TaqDNA polysaccharase is 16:2.5:1:1:1:1:1:1:0.5, wherein sense primer PHIB1, antisense primer PHIB2, the concentration of sense primer SPT1 and antisense primer SPT2 is 10 μ M, the concentration of dNTPs mixture is 2.5mM each, the concentration of TaqDNA polysaccharase is 5U/ μ l, the concentration of target compound DNA is 100-200ng/ μ l.
3. the method based on the double pcr amplification detection oranges and tangerines winter life mould brown rot pathogenic bacteria of epidemic disease and oranges and tangerines branch knurl pathogenic bacteria according to claim 1, it is characterized in that: in step (2), the program of amplification is: 94 DEG C of 3min, 94 DEG C of 30S, 62 DEG C of 30S, 72 DEG C of 30S, circulation 32-36 time, last 72 DEG C of added time 5min.
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Non-Patent Citations (3)
| Title |
|---|
| Avijit Roy,et al.A multiplex polymerase chain reaction method for reliable, sensitive and simultaneous detection of multiple viruses in citrus trees.《Journal of Virological Methods》.2005,47-55页. * |
| AvijitRoy，etal.Amultiplexpolymerasechainreactionmethodforreliable sensitive and simultaneous detection of multiple viruses in citrus trees.《Journal of Virological Methods》.2005 |
| D. Montenegro，et al.A selective PCR-based method for the identification of Phytophthora hibernalis Carne.《Spanish Journal of Agricultural Research》.2008,78-84页. * |
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