CN103031383A - 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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- CN103031383A CN103031383A CN2012105834265A CN201210583426A CN103031383A CN 103031383 A CN103031383 A CN 103031383A CN 2012105834265 A CN2012105834265 A CN 2012105834265A CN 201210583426 A CN201210583426 A CN 201210583426A CN 103031383 A CN103031383 A CN 103031383A
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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 method that the oranges and tangerines winter gives birth to the mould brown rot pathogenic bacteria of epidemic disease and oranges and tangerines branch knurl pathogenic bacteria that detects, relate in particular to the method that a kind of application two-fold pcr amplification technology for detection oranges and tangerines winter of high sensitivity gives birth to the mould brown rot pathogenic bacteria of epidemic disease and oranges and tangerines branch knurl pathogenic bacteria.
Background technology
It is wide that the mould brown rot pathogenic bacteria of living epidemic disease of oranges and tangerines winter Phytophthora hibernalis Carne has host range, and the Orange Producing of causing harm is serious, the characteristics 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 the seed nursery stock successor China that introduces high-quality is increasing in recent years.In case this disease is imported into, will cause very big harm to China's Citrus Industry.It is to find first on the citrusfruit of western australia that the oranges and tangerines winter is given birth to the mould brown rot disease of living epidemic disease of microbial oranges and tangerines winter of the mould brown rot cause of disease of epidemic disease, also finds successively in other countries and area subsequently.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 the white hypha body when being fit 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 suitable disease to occur is so it is large surely to grow possibility.Closely circulation way has the routes 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, comes lemon and most tangerine section plant at the main harm Mexican lime.Performance branch knurl and clump branch two class symptoms, but take 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, and plant tissue in spite of illness such as branch, sick leaf and reproductive material are 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 issue has just comprised the assessment of this pathogenic bacteria by what French authorities finished about the Risk Assessment Report of oranges and tangerines harmful organism.
For the existing part Study of the phylogeny of phytophthora fungi, the report of PCR detection is arranged also for the molecular detection technology of the mould brown rot pathogenic bacteria of living epidemic disease of oranges and tangerines winter at present.2008, Zhang Haifeng etc. have relatively analyzed the winter and have given birth to the mould and mould ITS sequence of other epidemic disease of epidemic disease, design on this basis 1 pair and detected the mould special primer 751F/752R of living epidemic disease of winter, this can give birth to the phytophthora amplification to the DNA band of long 616 bp from the winter to primer, and other 19 kinds of epidemic diseases are mould and other fungi all without amplified band.Its detection limit can reach the level that per 25 μ L reaction systems only need 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 detection of oranges and tangerines branch knurl pathogenic bacteria is only seen and is used universal primer ITS4 and ITS5 to carry out the report that PCR detects.From the database of NCBI, search for, also only have trivial 2 rrna sequences.Therefore, the PCR design of primers has become a difficult problem.
Therefore, it is two kinds of common two kind germs very large to the harm of oranges and tangerines in the external oranges and tangerines that oranges and tangerines branch knurl cause of disease and oranges and tangerines winter are given birth to the mould brown rot pathogenic bacteria of epidemic disease, detect the detection method of above-mentioned two kinds of germs when therefore providing a kind of more high sensitivity, become the study hotspot in this area.
Summary of the invention
The invention solves the difficult problem in the above-mentioned background technology, provide a kind of and detected the method that oranges and tangerines branch knurl cause of disease and oranges and tangerines winter give birth to the mould brown rot pathogenic bacteria of epidemic disease based on double pcr amplification, the method step is simple, can resultful two kinds of germs be detected.
Realize that the technical scheme that above-mentioned purpose of the present invention adopts is:
A kind of method of giving birth to the mould brown rot pathogenic bacteria of epidemic disease and oranges and tangerines branch knurl pathogenic bacteria based on the double pcr amplification detection oranges and tangerines winter, it is characterized in that may further comprise the steps: (1), get an aseptic centrifuge tube, in centrifuge tube, add successively sterilization 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 it is mixed rear centrifugal, reaction solution is centrifugal to managing the end;
(2), centrifuge tube placed on the PCR instrument increases, obtain the PCR reaction product after the amplification;
(3), the PCR product is with the separation of 2%-3% agarose gel electrophoresis, behind ethidium bromide solution dyeing 12-20min, at ultraviolet transilluminator observation PCR reaction result.
PCR damping fluid described in the step (1) is 10 * PCR damping fluid, the deionized water that adds, the PCR damping fluid, the dNTPs mixture, sense primer PHIB1, antisense primer PHIB2, sense primer SPT1, antisense primer SPT2, volume ratio between target compound DNA and the TaqDNA polysaccharase is 16:2.5:1:1:1:1:1:1: 0.5, sense primer PHIB1 wherein, 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, and the concentration of target compound DNA is 100-200 ng/ μ l.
The program of amplification is in the step (2): 94 ℃ of 3min, 94 ℃ of 30S, 62 ℃ of 30S, 72 ℃ of 30S, circulation 32-36 time, last 72 ℃ of added time 5min.
Method of giving birth to the mould brown rot pathogenic bacteria of epidemic disease and oranges and tangerines branch knurl pathogenic bacteria based on the double pcr amplification detection oranges and tangerines winter provided by the invention has following advantage: (1), the method can go out the pathogenic fungi whether oranges and tangerines carry these two kinds of quarantines by Accurate Diagnosis; (2) the method adopts the method for double PCR, only needs half a day and just can identify the oranges and tangerines winter to give birth to the kind of the mould brown rot pathogenic bacteria of epidemic disease and oranges and tangerines branch knurl pathogenic bacteria, can speed passage through customs; (3) the method good reproducibility is highly sensitive, and target compound DNA only needs 100ng/ μ L just can identify the kind that oranges and tangerines carry pathogenic fungi.
Description of drawings
Fig. 1 is the detected through gel electrophoresis figure as a result among the embodiment 1.
Embodiment
Below in conjunction with specific embodiment the present invention is done detailed specific description.
The bacterial classification oranges and tangerines winter gives birth to the mould brown rot pathogenic bacteria of epidemic disease (Phytophthora hibernalis Carne) and available from U.S. ATCC DSMZ, is numbered 32995 in the present embodiment
TM, and in strict accordance with the quarantine dangerous bacterial classification carry out Operation ﹠ use.The pcr amplification of process DNA, the checking of Cloning and sequencing before using.
The used bacterial classification oranges and tangerines of present embodiment branch knurl pathogenic bacteria (Sphaeropsis tumefaciens Hedges) available from U.S. ATCC DSMZ, is numbered 20908
TM, and in strict accordance with the quarantine dangerous bacterial classification carry out Operation ﹠ use.The pcr amplification of process DNA, the checking of Cloning and sequencing before using.
The extracting method of target compound DNA is as follows in the present embodiment: be 10 with 100 μ l concentration
7Spore suspension is coated on the PDA and V8 flat board that is covered with the sterilization glassine paper, 18 ℃ of lower cultivations 7-10 days, and the scraping mycelia is freezing for subsequent use.Get the 0.5g mycelia, place centrifuge tube, add in the CTAB Extraction buffer of the own preheating of 2ml (65 ℃), centrifuge tube is put upside down mixing, 65 ℃ of water-bath 5min, gently mixings; Add isopyknic phenol: chloroform: primary isoamyl alcohol (25:24:l), mixing, the centrifugal 15min of 12000r/min gets supernatant liquor in centrifuge tube, adds the equal-volume chloroform again: primary isoamyl alcohol (24:l) mixing, the centrifugal 15min of 12000r/min, get supernatant liquor in centrifuge tube, 100% ethanol that adds 2 times of volumes is put upside down mixing, the centrifugal 10min of 12000r/min, the supernatant liquor that inclines, with 70% ethanol with twice of washing of precipitate.The centrifuge tube that will contain DNA after washing finishes places 37 ℃ of incubators dry.Add 30 μ l TE dissolution precipitations after the drying, save backup in-20 ℃.
The nucleotide sequence of sense primer PHIB1 is shown in SEQ ID NO:1 in following examples: 5 '-TCGGGTCTGAGCTAGTAGTCTT-3 '; The nucleotide sequence of antisense primer PHIB2 is shown in SEQ ID NO:2: 5 '-CTTCCACAACCAATTCCATTATGC-3.The nucleotide sequence of sense primer SPT1 is shown in SEQ ID NO:3: 5 '-GAACGCAGCGAAATGCGATAAG-3 '; The nucleotide sequence of antisense primer SPT2 is shown in SEQ ID NO:4: 5 '-ATGCTNAAGTTCAGCGGGTAT-3 '.
The cumulative volume of double PCR reaction system is 25 μ l in the present embodiment, concrete detection method is as follows: (1) gets an aseptic centrifuge tube, in centrifuge tube, add successively the sterilization deionized water, the 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, and 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 it is mixed rear centrifugal, reaction solution is centrifugal to managing the end.
(2), centrifuge tube placed on the PCR instrument increases, amplification program is 94 ℃ of lower 3min, 94 ℃ of lower 30S, 62 ℃ of lower 30S, 72 ℃ of lower 30S circulate 36 times, last 72 ℃ of 5min of lower added time obtain the PCR reaction product after the amplification.
(3), the PCR product separates with 3% agarose gel electrophoresis, behind ethidium bromide solution dyeing 20min, observes the PCR reaction result at ultraviolet transilluminator.
The display application primer carries out double pcr amplification to PHIB1/PHIB2 and SPT1/SPT2 as a result, detect simultaneously the oranges and tangerines winter and give birth to the mould brown rot pathogenic bacteria of epidemic disease and oranges and tangerines branch knurl pathogenic bacteria, can observe on sepharose only has swimming lane 5 that 2 clearly specific dna fragmentations (307bp and 407 bp) are arranged, as shown in Figure 1, swimming lane M among Fig. 1, DL2000; Swimming lane 1, the winter is given birth to phytophthora+water; Swimming lane 2, branch knurl pathogenic bacteria+water; Swimming lane 3, the winter is given birth to phytophthora+PHIB, swimming lane 4, branch knurl pathogenic bacteria+SPT; Swimming lane 5, the winter is given birth to phytophthora+branch knurl pathogenic bacteria+PHIB+SPT; Swimming lane 6, the winter is given birth to phytophthora+SPT; Swimming lane 7, branch knurl pathogenic bacteria+PHIB.
The cumulative volume of double PCR reaction system is 25 μ l in the present embodiment, concrete detection method is as follows: (1) gets an aseptic centrifuge tube, in centrifuge tube, add successively the sterilization deionized water, the 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, and 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 it is mixed rear centrifugal, reaction solution is centrifugal to managing the end.
(2), centrifuge tube placed on the PCR instrument increases, amplification program is 94 ℃ of lower 3min, 94 ℃ of lower 30S, 62 ℃ of lower 30S, 72 ℃ of lower 30S circulate 33 times, last 72 ℃ of 5min of lower added time obtain the PCR reaction product after the amplification.
(3), the PCR product separates with 2% agarose gel electrophoresis, behind ethidium bromide solution dyeing 13min, observes the PCR reaction result at ultraviolet transilluminator.
The display application primer carries out double pcr amplification to PHIB1/PHIB2 and SPT1/SPT2 as a result, detect simultaneously the oranges and tangerines winter 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 clearly specific dna fragmentations (307bp and 407 bp).
Claims (3)
1. one kind is detected the method that the oranges and tangerines winter gives birth to the mould brown rot pathogenic bacteria of epidemic disease and oranges and tangerines branch knurl pathogenic bacteria based on double pcr amplification, it is characterized in that may further comprise the steps: (1), get an aseptic centrifuge tube, in centrifuge tube, add successively sterilization 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 it is mixed rear centrifugal, reaction solution is centrifugal to managing the end;
(2), centrifuge tube placed on the PCR instrument increases, obtain the PCR reaction product after the amplification;
(3), the PCR product is with the separation of 2%-3% agarose gel electrophoresis, behind ethidium bromide solution dyeing 12-20min, at ultraviolet transilluminator observation PCR reaction result.
2. according to claim 1ly detect the method that the oranges and tangerines winter gives birth to the mould brown rot pathogenic bacteria of epidemic disease and oranges and tangerines branch knurl pathogenic bacteria based on double pcr amplification, it is characterized in that: the PCR damping fluid described in the step (1) is 10 * PCR damping fluid, the deionized water that adds, the PCR damping fluid, the dNTPs mixture, sense primer PHIB1, antisense primer PHIB2, sense primer SPT1, antisense primer SPT2, volume ratio between target compound DNA and the TaqDNA polysaccharase is 16:2.5:1:1:1:1:1:1: 0.5, sense primer PHIB1 wherein, 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, and the concentration of target compound DNA is 100-200 ng/ μ l.
3. according to claim 1ly detect the method that the oranges and tangerines winter gives birth to the mould brown rot pathogenic bacteria of epidemic disease and oranges and tangerines branch knurl pathogenic bacteria based on double pcr amplification, it is characterized in that: the program of amplification is in the step (2): 94 ℃ of 3min, 94 ℃ of 30S, 62 ℃ of 30S, 72 ℃ of 30S, circulation 32-36 time, last 72 ℃ of added time 5min.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109486985A (en) * | 2018-08-07 | 2019-03-19 | 天津出入境检验检疫局动植物与食品检测中心 | The LAMP primer pair and its amplification method and purposes of detection phytophthora hibernalis bacterium |
| CN111004859A (en) * | 2019-12-30 | 2020-04-14 | 南京林业大学 | Phytophthora hibernalis specific detection target Phibe _ s00001g00026.1 and application thereof |
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Non-Patent Citations (2)
| 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》 * |
| D. MONTENEGRO，ET AL: "A selective PCR-based method for the identification of Phytophthora hibernalis Carne", 《SPANISH JOURNAL OF AGRICULTURAL RESEARCH》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109486985A (en) * | 2018-08-07 | 2019-03-19 | 天津出入境检验检疫局动植物与食品检测中心 | The LAMP primer pair and its amplification method and purposes of detection phytophthora hibernalis bacterium |
| CN111004859A (en) * | 2019-12-30 | 2020-04-14 | 南京林业大学 | Phytophthora hibernalis specific detection target Phibe _ s00001g00026.1 and application thereof |
| CN111004859B (en) * | 2019-12-30 | 2020-10-02 | 南京林业大学 | Phytophthora hibernalis specific detection target Phibe _ s00001g00026.1 and application thereof |
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