CN106591452A - Real-time fluorescence PCR method for quantitatively detecting Pseudomonas syringae pv. Actinidiae - Google Patents
Real-time fluorescence PCR method for quantitatively detecting Pseudomonas syringae pv. Actinidiae Download PDFInfo
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Abstract
The invention discloses a real-time fluorescence PCR method for quantitatively detecting Pseudomonas syringae pv. Actinidiae. The method comprises the following steps: extracting an Actinidia chinensis sample genome DNA; electrophoretically detecting the band size and purity of the Actinidia chinensis sample genome DNA; carrying out fluorescent quantitative detection on a PSA pathogen; carrying out specific primer-based fluorescent quantitative amplification, and preliminarily determining whether the PSA pathogen is contained or not according to an amplification curve; purifying a PCR product with an obvious amplification peak, and sequencing the purified PCR product; and comparing a sequence obtained after the sequencing by Internet to further determine the PSA pathogen. The method allows a fluorescent quantitative PCR-based rapid detection method of the pathogen to be established, realizes rapid identification of the Pseudomonas syringae pv. Actinidiae infected sample, has the characteristics of simplicity in operation, high sensitivity, high specificity, and accurate and objective detection result, and is of great significance to improve the detection efficiency and the detection sensitivity of the Pseudomonas syringae pv. Actinidiae infected sample.
Description
Technical field
The invention belongs to pathogenic detection technique field, and in particular to a kind of reality of quantitative determination Prospect on Kiwifruit Bacterial Canker bacterium
When fluorescence PCR method.
Background technology
Kiwi berry (Actinidia chinensis) is a kind of very high fruit of economic worth, there is a large amount of kinds both at home and abroad
Plant.However, being continuously increased recently as Kiwifruit Culture area, bacterial canker quickly spreads, since two thousand five,
Serious harm is caused in world main kiwifruit producing region, becomes most destructive disease, serious prestige during Kiwifruit Culture
The production safety of side of body world's Kiwi berry.Prospect on Kiwifruit Bacterial Canker is a kind of with destructive bacterial disease, most earlier than last century
The early stage eighties is found and has made more detailed report in Japan and the U.S..Subsequently, this disease is in succession in Korea, Iran, meaning
Great Li Deng states occur.2008 start, the disease in European domestic large-scale outbreak, in succession in France, Portugal, Spain, soil
Ear its, the state such as Switzerland occurs.In addition, the disease also happens occasionally in states such as Australia, New Zealand.In China, the disease is most
Early Hunan Dongshan peak farm is found in, just spreads to the province such as Sichuan, Anhui, Fujian rapidly within shorter a period of time afterwards,
Many orchards force orchard worker to chop at a tree to ruin garden, suffer heavy losses because dead tree rate is too high.Canker has become the main of Kiwi berry production
Limiting factor.With the development of China's Kiwi berry foreign trade, Kiwi berry bursts to swing disease and be listed in national forestry quarantine to be had
Evil is biological, and quarantining and control spreading for the disease becomes the most important thing for ensureing that China's Kiwi berry industry develops in a healthy way.
Prospect on Kiwifruit Bacterial Canker main harm plant young sprout, limb and blade, cause branch withered, and blade is wilted；Morbidity is tight
Even whole strain is withered during weight dies.The disease can be produced by pathogen infection kiwi fruit plant interior tissue on its ground histoorgan
The obvious symptom of life：On limb, start morbidity from side shoot, spray, eye, carpopodium etc. more.Killed initial stage scab is in water stain shape,
Gradually expand afterwards, color burn, cause cortex and xylem to separate, in soft shape；Subsequently disease portion cortex is longitudinally cracked, and flows out big
Amount milky thick liquid, after be oxidized to bronzing；Tissue sink at morbidity later stage gummosis, and white or bronzing are oozed out in formation
The ulceration of mucus, finally causes the withered death of morbidity limb.On blade, formed with yellow dizzy irregular crineous scab, it is wet
There is bacterial ooze to overflow when spending big at scab, later stage scab is changed into the big spot of black, cause that blade is shrivelled to come off when serious.The disease once
Generation will have a strong impact on the yield and quality of Kiwi berry, cause serious economic loss.
Nearly 5 years, according to biological characteristics, 16S and 16S-23S (ITS) rDNA sequence alignment analysis, New Zealand, France,
The states such as Spain are Pseudomonas syringae pv.actinidiae (Pseudomonas all by Prospect on Kiwifruit Bacterial Canker pathogen identification
Syringae pv.actinidae, PSA).The traditional quarantine method of the disease is more by the classical symptom after morbidity and dependence
Separate cause of disease to differentiate disease.These methods not only take and have some limitations, because the disease is present hiding
Infection, killed plant can infect there is a period of time after just show classical symptom, now entered by chemical method again
Row preventing and treating, much falls flat.Therefore, being highly desirable to set up one kind can be in pathogen infection Rapid＆Early diagnosis disease
Harmful method, prevents disease from further occurring targetedly to take measures.
Real-time fluorescence quantitative PCR (Real-time PCR), because of its high sensitivity and specificity, is applied to more and more widely
The detection of plant disease.Prospect on Kiwifruit Bacterial Canker opportunistic pathogen real-time fluorescence quantitative PCR detection method is set up, and verifies its actual effect,
This is for carrying out the germ precise Identification, early diagnosis, and then effectively preventing and treating Prospect on Kiwifruit Bacterial Canker has extremely important meaning.
At present, real time fluorescence quantifying PCR method is used to detect that the pathogen not yet has at home patent report.
The content of the invention
For above-mentioned technical problem, the present invention provides a kind of real-time fluorescence PCR side of quantitative determination Prospect on Kiwifruit Bacterial Canker bacterium
Method, realizes the Rapid identification of the susceptible sample of Prospect on Kiwifruit Bacterial Canker.
Technical scheme is as follows：
A kind of real time fluorescent PCR method of quantitative determination Prospect on Kiwifruit Bacterial Canker bacterium, comprises the following steps：
Step a, extraction Kiwi berry sample gene group DNA；The band of Kiwi berry sample gene group DNA is big described in electrophoresis detection
Little and purity；
Step b, fluorogenic quantitative detection PSA pathogen；Fluorescent quantitative PCR is carried out based on specific primer, according to expansion
Increase curve whether to primarily determine that containing PSA pathogens；
Step c, sequencing；PCR primer to there is obvious amplified peak is sequenced after purification；Gained sequence carries out comparison by Internet
After further determine that the PSA pathogens.
Preferably, in step a using Plant Genome extracts kit extract the doubtful Kiwi berry branch caught an illness or
Kiwi berry sample gene group DNA of blade, the Kiwi berry sample gene group DNA band of extraction adopts trace dna analyzer
Detection purity.
Preferably, the specific primer in step b includes upstream primer P3F：5’-GGTTTCGGACACCGCAGGTT
CTACCGAG-3 ', and downstream primer P5R：5’-CTTCCTGATCCCCGTTACCCATCGAC-3’；It is glimmering in step b
Fluorescent Quantitative PCR amplification reaction system be：Upstream primer and each 0.5 μ L of downstream primer, SYBR Premix Ex TaqTM 6.75
μ L, template DNA 0.5 μ L, ddH2O 4.75μL；The reaction condition of the fluorescent quantitative PCR in step b is：94℃
3min；94 DEG C of 10s, 68 DEG C of 15s, 72 DEG C of 30s, often circulation detects fluorescence after extending, totally 45 circulations；72 DEG C of extension 10s；Each
Reaction arranges three repetitions；In 55～95 DEG C, 0.5 DEG C of detection fluorescence signal is often raised.
Preferably, the positive sequencing primer of the sequencing in step c is P3F, and reverse sequencing primer is P5R.
The invention has the beneficial effects as follows：
1st, by methods such as genome extraction, fluorescent quantitation amplification and molecule sequencings, Prospect on Kiwifruit Bacterial Canker is realized susceptible
The Rapid identification of sample, establishes method for quick of the pathogen based on quantitative fluorescent PCR；
2nd, with simple to operate, sensitivity is high and the features such as high specificity, and testing result is accurate, objective, to improving Mi
The detection efficiency of the susceptible sample of monkey peach canker and sensitivity are significant；
3rd, suitable for the accurate early diagnosis for detecting PSA germs the tissue such as Kiwi berry branch, blade, disaster area is prevented
Expand, reduce the economic loss for therefore causing；Apply to the method in quarantine is imported and exported, be also greatly improved the speed of quarantine
And the degree of accuracy, to control Prospect on Kiwifruit Bacterial Canker further spreading in China.
Description of the drawings
Fig. 1 is the flow chart of the real time fluorescent PCR method of quantitative determination Prospect on Kiwifruit Bacterial Canker bacterium of the present invention；
Fig. 2 is primer pair P3F/P5R of the real time fluorescent PCR method of quantitative determination Prospect on Kiwifruit Bacterial Canker bacterium of the present invention
Quantitative fluorescent PCR specific detection result figure；
Fig. 3 is primer pair P3F/P5R of the real time fluorescent PCR method of quantitative determination Prospect on Kiwifruit Bacterial Canker bacterium of the present invention
Real-time PCR sensitivity technique result figures；
Fig. 4 is the PSA pathogenic bacteria genes of the real time fluorescent PCR method of quantitative determination Prospect on Kiwifruit Bacterial Canker bacterium of the present invention
The quantitative fluorescent PCR canonical plotting of group DNA；
In Fig. 2：
1st, catch an illness branch；2nd, PSA pathogenic bacteria；3rd, healthy branch；4-13,10 kinds of other bacterium；14、ddH2O；
In Fig. 3：
1st, 1ng/ μ L PSA pathogenic bacteria genomic DNAs；2nd, 100pg/ μ L PSA pathogenic bacteria genomic DNAs；3、10pg/μL
PSA pathogenic bacteria genomic DNAs；4th, 1pg/ μ L PSA pathogenic bacteria genomic DNAs；5th, 100fg/ μ L PSA pathogenic bacteria genomic DNAs；
6th, 10fg/ μ L PSA pathogenic bacteria genomic DNAs；7、ddH2O。
Specific embodiment
Below in conjunction with the accompanying drawings the present invention is described in further detail, to make those skilled in the art with reference to specification text
Word can be implemented according to this.
As illustrated, the present invention a kind of real time fluorescent PCR method of quantitative determination Prospect on Kiwifruit Bacterial Canker bacterium is disclosed, including with
Lower step：
Step a, extraction Kiwi berry sample gene group DNA；The band of Kiwi berry sample gene group DNA is big described in electrophoresis detection
Little and purity；The doubtful Kiwi berry branch caught an illness or blade are extracted using Plant Genome extracts kit in step a
Kiwi berry sample gene group DNA, the Kiwi berry sample gene group DNA band of extraction detects pure using trace dna analyzer
Degree, for the fluorescent quantitative PCR template in step b.
Step b, fluorogenic quantitative detection PSA pathogen；Fluorescent quantitative PCR is carried out based on specific primer, according to expansion
Increase curve whether to primarily determine that containing PSA pathogens；Specific primer in step b includes upstream primer P3F：5’-
GGTTTCGGACACCGCAGGTTCTACCGAG-3 ', and downstream primer P5R：5’-CTTCCTGATCCCCGTTACCCATCGAC
-3’；The reaction system of the fluorescent quantitative PCR in step b is：Upstream primer and downstream primer each O.5 μ L, SYBR
The μ L of Premix Ex TaqTM 6.75, the μ L of template DNA 0.5, the μ L of ddH2O 4.75；Quantitative fluorescent PCR in step b expands
The reaction condition of increasing is：94℃3min；94 DEG C of 10s, 68 DEG C of 15s, 72 DEG C of 30s, often circulation detects fluorescence after extending, and totally 45 are followed
Ring；72 DEG C of extension 10s；Each reaction arranges three repetitions；In 55～95 DEG C, O.5 DEG C detection fluorescence signal is often raised.
Step c, sequencing；PCR primer to there is obvious amplified peak is sequenced after purification；Sequencing gained sequence is carried out on the net
The PSA pathogens are further determined that after comparison.The positive sequencing primer of the sequencing in step c be P3F, reverse sequencing primer
For P5R, sequencing result carries out sequence alignment, and such as comparison result reaches the above 99% with PSA bacterial strain similarities, in determining the sample
Containing Prospect on Kiwifruit Bacterial Canker pathogen.
Embodiment：
A kind of real time fluorescent PCR method of quantitative determination Prospect on Kiwifruit Bacterial Canker bacterium, comprises the following steps：
Step a, extraction Kiwi berry sample gene group DNA；The band of Kiwi berry sample gene group DNA is big described in electrophoresis detection
Little and purity；The doubtful Kiwi berry branch caught an illness or blade are extracted using Plant Genome extracts kit in step a
Kiwi berry sample gene group DNA, the Kiwi berry sample gene group DNA band of extraction detects pure using trace dna analyzer
Degree, for the fluorescent quantitative PCR template in step b.
Step b, fluorogenic quantitative detection PSA pathogen；Fluorescent quantitative PCR is carried out based on specific primer, according to expansion
Increase curve whether to primarily determine that containing PSA pathogens；Specific primer in step b includes upstream primer P3F：5’-
GGTTTCGGACACCGCAGGTTCTACCGAG-3 ', and downstream primer P5R：5’-CTTCCTGATCCCCGTTACCCATCGAC
-3’；The reaction system of the fluorescent quantitative PCR in step b is：Upstream primer and each 0.5 μ L of downstream primer,
The μ L of SYBRPremix Ex TaqTM 6.75, the μ L of template DNA 0.5, the μ L of ddH2O 4.75；Fluorescent quantitation in step b
PCR amplification reaction condition be：94℃3min；94 DEG C of 10s, 68 DEG C of 15s, 72 DEG C of 30s, often circulation detects fluorescence after extending, altogether
45 circulations；72 DEG C of extension 10s；Each reaction arranges three repetitions；In 55～95 DEG C, 0.5 DEG C of detection fluorescence letter is often raised
Number.
Step c, sequencing；PCR primer to there is obvious amplified peak is sequenced after purification；Sequencing gained sequence is carried out on the net
The PSA pathogens are further determined that after comparison.The positive sequencing primer of the sequencing in step c be P3F, reverse sequencing primer
For P5R, sequencing result carries out sequence alignment, and such as comparison result reaches the above 99% with PSA bacterial strain similarities, in determining the sample
Containing Prospect on Kiwifruit Bacterial Canker pathogen.
Experiment 1, the specificity of checking real-time fluorescence reaction system
To verify the specificity of primer of the present invention and real-time fluorescence method, the present embodiment is caught an illness branch respectively with Kiwi berry
Bar, PSA pathogens and other 10 kinds of bacteriums (bacterial canker of tomato Pseudomonas.syringae pv.tomato, Japanese plums
Ulcer bacteria Pseudomonas syringae pv.morsprunorum, cloves ulcer bacteria Pseudomonas syringae
Pv.syringe, muskmelon ulcer bacteria Pseudomonas syringae pv.lachryma, Peach canker disease bacterium
Pseudomonas syringae pv.persicae, citrus processing Xanthomonas axonopdis pv.citri,
Kidney bean ulcer bacteria Pseudomonas syringae pv.phaseolicola, Pseudomonas viridiflava Pseudomonas
Viridiflava, olive pseudomonad Pseudomonas savastanoi, pseudomonas aeruginosa Pseudomonas
Aeruginosa) genomic DNA be template, Chinese goosebeery healthy branch DNA be negative control, carry out fluorescent quantitative PCR and
Analysis.
As shown in Fig. 2 in fluorescence quantitative PCR detection figure, only catch an illness branch and PSA to cause strain gene group DNA to occur molten
Solution curve is unimodal, and the miscellaneous peak without notable non-specific amplification and primer dimer, and healthy branch and 10 kinds of pseudomonads and
Mutation bacterium is not detected by fluorescence signal, shows the specificity height of the primer and reaction condition is more excellent.
The experiment 2, sensitivity of real-time fluorescence reaction system
Primer according to designed by embodiment, establishes a kind of real time fluorescent quantitative detection of detection Prospect on Kiwifruit Bacterial Canker bacterium
Method.Reaction system is：The each 0.5 μ L of primer, SYBR Premix Ex TaqTM 6.75 μ L, template DNA 0.5 μ L, ddH2O
4.75μL.Reaction condition：94℃3min；94 DEG C of 10s, 68 DEG C of 15s, 72 DEG C of 30s, often circulation detects fluorescence after extending, totally 45
Circulation；72 DEG C of extension 10s.Each reaction sets 3 repetitions.In 55～95 DEG C, 0.5 DEG C of detection fluorescence signal is often raised.
To verify the sensitivity of primer of the present invention and real-time fluorescence method, extracted with Plant Genome extracts kit
PSA pathogenic bacteria genomic DNAs, after putting forward DNA sample trace dna analyzer detection purity, are diluted to 1ng/ μ L, 100pg/ μ
L, 10pg/ μ L, 1pg/ μ L, 100fg/ μ L, 10fg/ μ L, carry out fluorescent quantitative PCR, detection primer sensitivity.
As shown in figure 3, concentration is 100pg/ μ L, 10pg/ μ L, 1pg/ μ L, 100fg/ μ L PSA pathogenic bacteria genomic DNAs going out
Existing smooth stable S-shaped solubility curve, Exponential growth stage and plateau, substantially, and increase with dilution factor, and Exponential growth stage occurs
Required period is reduced, and 10fg/ μ LPSA pathogenic bacteria genomic DNAs and control show quantitative fluorescent PCR without amplification curve
Method detection sensitivity is 100fg/ μ L.
The experiment 3, calibration curve of real-time fluorescence reaction system
As shown in figure 4,10ng PSA pathogenic bacteria genomic DNAs are carried out into fluorescence using 10 times of gradient dilutions as standard items
Quantitative pcr amplification.Calibration curve is repeated 3 times altogether, is as a result displayed between 100fg/ μ L-10ng/ μ L and good linear pass is presented
System, coefficient correlation is 0.997, and amplification efficiency is 115.6%.
Experiment 4, Kiwi berry sample detection
5 parts are gathered on the spot without illness and 5 parts in Jiangshan of Zhejiang Province, Wenzhou District of Zhejiang Province, Yangling Shaanxi, Sichuan Pujiang, Yichuan
The Kiwi berry branch of typical illness, the pathogenic bacteria concentration of real-time PCR systems detection set up with this research.
5 parts without in illness Kiwi berry branch, real-time PCR methods detect pathogen containing PSA in 3 parts of samples, most
Low concentration is 8.45 × 10-5ng/μL；Real-time PCR methods detect PSA in the Kiwi berry branch of 5 parts of typical illnesss
Pathogen, maximum concentration is 2.18ng/ μ L, and illness to manifest the PSA concentration for more substantially detecting higher.10 parts gather on the spot
The Detection results of sample demonstrate this research foundation real-time PCR methods specificity, sensitivity degree it is all higher, the detection
System is relatively reliable stable.
The Real-time PCR quantitative determinations of SPA pathogen DNA in the Kiwi berry sample of table 1
The present invention realizes Prospect on Kiwifruit Bacterial Canker by methods such as genome extraction, fluorescent quantitation amplification and molecule sequencings
The Rapid identification of susceptible sample, establishes method for quick of the pathogen based on quantitative fluorescent PCR；With it is simple to operate,
The features such as sensitivity height and high specificity, and testing result is accurate, objective, the detection to improving the susceptible sample of Prospect on Kiwifruit Bacterial Canker
Efficiency and sensitivity are significant；The early stage of PSA germs examines suitable for the accurately tissue such as detection Kiwi berry branch, blade
Disconnected, prevention disaster area expands, and reduces the economic loss for therefore causing；Apply to the method in quarantine is imported and exported, also can be big
The big speed for improving quarantine and the degree of accuracy, to control Prospect on Kiwifruit Bacterial Canker further spreading in China.
Although embodiment of the present invention is disclosed as above, it is not restricted to listed in specification and embodiment
With, it can be applied to completely various suitable the field of the invention, for those skilled in the art, can be easily
Other modification is realized, therefore under the universal limited without departing substantially from claim and equivalency range, the present invention is not limited
In specific details and shown here as the legend with description.
Claims (4)
1. a kind of real time fluorescent PCR method of quantitative determination Prospect on Kiwifruit Bacterial Canker bacterium, it is characterised in that comprise the following steps：
Step a, extraction Kiwi berry sample gene group DNA；The stripe size of Kiwi berry sample gene group DNA described in electrophoresis detection and
Purity；
Step b, fluorogenic quantitative detection PSA pathogen；Fluorescent quantitative PCR is carried out based on specific primer, it is bent according to amplification
Whether line is primarily determined that containing PSA pathogens；
Step c, sequencing；PCR primer to there is obvious amplified peak is sequenced after purification；Sequencing gained sequence carries out comparison by Internet
After further determine that the PSA pathogens.
2. the real time fluorescent PCR method of quantitative determination Prospect on Kiwifruit Bacterial Canker bacterium according to claim 1, it is characterised in that：
The Kiwi berry sample of the doubtful Kiwi berry branch caught an illness or blade is extracted in step a using Plant Genome extracts kit
Genomic DNA, the Kiwi berry sample gene group DNA band of extraction detects purity using trace dna analyzer.
3. the real time fluorescent PCR method of quantitative determination Prospect on Kiwifruit Bacterial Canker bacterium according to claim 1, it is characterised in that：
Specific primer in step b includes upstream primer P3F：5 '-GGTTTCGGACACCGCAGGTTCTACCGAG-3 ', with
And downstream primer P5R：5’-CTTCCTGATCCCCGTTACCCATCGAC-3’；Fluorescent quantitative PCR in step b
Reaction system is：Upstream primer and each 0.5 μ L of downstream primer, SYBR Premix Ex TaqTM 6.75 μ L, the μ of template DNA 0.5
L, ddH2O4.75μL；The reaction condition of the fluorescent quantitative PCR in step b is：94℃3min；94 DEG C of 10s, 68 DEG C
15s, 72 DEG C of 30s, often circulation detects fluorescence after extending, totally 45 circulations；72 DEG C of extension 10s；Each reaction arranges three repetitions；
In 55～95 DEG C, 0.5 DEG C of detection fluorescence signal is often raised.
4. the real time fluorescent PCR method of quantitative determination Prospect on Kiwifruit Bacterial Canker bacterium according to claim 1, it is characterised in that：
The positive sequencing primer of the sequencing in step c is P3F, and reverse sequencing primer is P5R.
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| CN107586863A (en) * | 2017-10-31 | 2018-01-16 | 青岛农业大学 | The nucleic acid, kit and method of yellows phytoplasma, Pathogenic Fungus of Canker and Leaf blotch pathogeny are detected simultaneously |
| CN108179206A (en) * | 2018-02-10 | 2018-06-19 | 四川省农业科学院植物保护研究所 | RT-PCR detects the primer and detection method of Prospect on Kiwifruit Bacterial Canker opportunistic pathogen |
| CN108192987A (en) * | 2018-02-10 | 2018-06-22 | 绵阳市农业科学研究院 | The primer and its methods and applications of quantitative PCR detection Prospect on Kiwifruit Bacterial Canker opportunistic pathogen |
| CN108315448A (en) * | 2018-02-10 | 2018-07-24 | 绵阳市农业科学研究院 | The primer and its detection method of detection Prospect on Kiwifruit Bacterial Canker opportunistic pathogen and application |
| CN115948407A (en) * | 2022-10-10 | 2023-04-11 | 四川省农业科学院植物保护研究所 | Pseudomonas syringae kiwi fruit pathogenic variety aptamer, screening method and application |
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