CN103060436A - Real-time fluorescent PCR (polymerase chain reaction) detection kit for Phomopsis helianthi M.Muntanola-Cvetkovic et al. - Google Patents
Real-time fluorescent PCR (polymerase chain reaction) detection kit for Phomopsis helianthi M.Muntanola-Cvetkovic et al. Download PDFInfo
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- CN103060436A CN103060436A CN2012104367167A CN201210436716A CN103060436A CN 103060436 A CN103060436 A CN 103060436A CN 2012104367167 A CN2012104367167 A CN 2012104367167A CN 201210436716 A CN201210436716 A CN 201210436716A CN 103060436 A CN103060436 A CN 103060436A
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Abstract
The invention provides a real-time fluorescent PCR (polymerase chain reaction) detection kit for Phomopsis helianthi M.Muntanola-Cvetkovic et al. Nucleotide sequences of primers adopted by the kit are shown in a sequence table PHDf&PHDr and a nucleotide sequence of probes is shown in a sequence table PHDp. The kit takes the total DNA of samples as a template, utilizes the primers and the probes to carry out real-time fluorescent PCR amplification, collects data after each cycle ends and judges the results according to the amplification curve after reaction ends. The kit has very good specificity and stability, and the detection method is quick and simple and has high accuracy and flexibility, thus providing guarantees for import and export safety of plant materials possibly carrying Phomopsis helianthi M.Muntanola-Cvetkovic et al.
Description
Technical field
The present invention relates to Measurement for Biotechnique, relate to specifically Sunflower Receptacle stem canker detection test kit.
Background technology
The U.S. in 1975 have reported the hazardness of Sunflower Receptacle stem canker sick (Phomopsis helianthi M.Muntanola-Cvetkovic et al.), the therefore sick underproduction 60% of the Minnesota State at first.1979-1982 Yugoslavia should disease occur greatly, and production loss is more than 50%, and the percentage of germination of susceptible seed, 100-grain weight, oil yield and quality reduce.1981-1986 Hungary, Romania, France in succession reported should disease a situation arises.In recent years, Italian, Bulgarian, former Czechoslovakia, the U.S., the states such as Canada and Argentina all reported should disease large generation.The former Soviet Union was in 1985, Wu Rigeluode district, Carpathians state finds this disease first on hybrid sunflower cringle Dao Er outside Ukraine, 1988-1989 is at the Moldova most areas, the outer Carpathians state of Ukraine, state, Odessa and Fu Gele state, Killough have also been found should disease, this disease is popular in most of sunflower planting country, produces to Sunflower Receptacle and causes very big harm.This disease can cause stem's ulcer, and plant is withered, a little less than the stem stalk, and easily lodging.The diseased plant floral disc is little, and seed is light.1980-1981 Former Yugoslavia's Wal fourth that, diseased plant rate 20%-94%, the diseased plant grain yield not as good as healthy tree half.
Less to the research of this germ both at home and abroad, calendar year 2001, the human aflp analysis such as Veronique the phylogeny of this germ; 2004, the human PCR such as Mariarosaria, RFLP and Southern hybridization technique have been checked the hereditary biotype of this bacterium and the relation between the epidemiology difference; 2007, the people such as Mara studied cause of disease form, biological characteristics and the variety resistance of this germ.Up to the present also not effective especially chemical agent or resistant variety are prevented and treated this disease, owing to the Sunflower Receptacle stem canker is mainly propagated by the Sunflower Receptacle seedling, so the major control method is by quarantine that the Sunflower Receptacle seedling that enters the territory is tested at present.Have no at present the report of the detection kit research of relevant this germ both at home and abroad.Therefore, be necessary to set up a kind of accuracy rate and sensitivity all high quick detection kit detect the Sunflower Receptacle stem canker.Because this disease is the quarantine disease of China, therefore be the means of this disease of major control in China's quarantine, strictly limit or forbid also must testing at the Check and Examination of Port Quarantine Bureau from the Sunflower Receptacle seedling of other national import from the countries and regions import Sunflower Receptacle seedling of this disease occurs.The means that the present Sunflower Receptacle of detection both at home and abroad stem canker is taked mainly are:
1. plant and plant observation: the Sunflower Receptacle seedling that enters the territory is carried out isolation implant according to the requirement of inspection and quarantine mechanism, and routine observation has or not the classical symptom of performance Sunflower Receptacle stem canker between planting season.This method can only be as preliminary judgement, and take a lot of work, accuracy rate time-consuming, that the result judges is low.
2. separation and Culture: the Sunflower Receptacle seedling that enters the territory is carried out separation and purification at substratum, and sense cycle is long, and empirical positive determines that difficulty is large by force, and sensitivity is not high.
Along with China's opening development, agricultural-food are imported and exported day by day frequent, and the probability that passes with the agricultural plants harmful organism further strengthens, and the hazardness of harmful organism further strengthens.Sunflower Receptacle developed rapidly since last century, and more than the domestic investigation mission outside the city or town kind batch, quantity is large, and the source is complicated, and the quarantine problem is very outstanding.Since two thousand, China introduces a large amount of sunflower seeds from the U.S., Germany and other places every year, and the number/weight of import all occupies the prostatitis of the plant propagation material that enters the territory, and Check and Examination of Port Quarantine Bureau every year, the sunflower seeds from import detected multiple harmful organism.But each Check and Examination of Port quarantine mechanism of China lacks the effective detection kit to the Sunflower Receptacle stem canker at present.Disease control, prediction and Plant Quarantine need to have quickly and accurately detection kit, especially need China to have the detection kit of independent intellectual property right, thereby it can be put in commercial production and the application, also not have at present the report of Sunflower Receptacle stem canker real-time fluorescence PCR assay kit both at home and abroad in this field.The present invention has satisfied these demands.
Summary of the invention
The objective of the invention is provides the quick detection kit of Sunflower Receptacle stem canker for the plantation observation post that solves Sunflower Receptacle stem canker in the prior art needs cycle length, identifies the problems such as difficult, that accuracy rate is low.
The present invention is by the result who analyzes this laboratory order-checking and other Phomopsis germ ribosomal gene sequences of having reported, design primer and fluorescent probe.Primer is to being made of its nucleotide sequence such as sequence table PHDf﹠amp forward and reverse primer; Shown in the PHDr; The nucleotide sequence of probe is shown in sequence table PHDp, and this probe one end is marked with the report fluorescence dye, and the other end is marked with the cancellation fluorescence dye.Available report fluorescence dye has FAM/hex/tet/joe/vic/fitc/cy3/cy5, and available cancellation fluorescence dye has TAMRA/rox/dabcyl/bhq1/bhq2.
According to the TaqMan technology, the nucleotide probe of two fluorescence dye groups that added a mark on the basis of conventional PCR, for example will report that fluorochrome label is at 5 of probe ' end, the cancellation fluorochrome label is at 3 of probe ' end, both consist of the energy transfer organization, report that namely the fluorescence that fluorescence dye is launched can be absorbed by the cancellation fluorescence dye, when the two is far away apart from change, restraining effect weakens, and report fluorescence dye signal strengthens.In the amplified reaction process, probe is hybridized with the purpose amplified fragments on the template, because the Taq archaeal dna polymerase has 5 ' end to the 5 prime excision enzyme activity of 3 ' end, in the amplification extension stage probe is cut off, the restraining effect of cancellation fluorescence dye disappears, report fluorescence dye signal strengthens, thereby realizes the detection to the Sunflower Receptacle stem canker.
The present invention uses above-mentioned primer and probe carries out the real-time fluorescence PCR detection, and it carries out the real-time fluorescence PCR reaction take sample total DNA as template, each loop ends image data, and reaction finishes rear according to the amplification curve result of determination.
Specifically the present invention carries out the real-time fluorescence PCR reaction take sample total DNA as template.Namely in 25 μ L reaction systems, add Real-time PCR Mix10 μ L, 5 μ M primer (PHDf﹠amp; PHDr) each 2 μ L, the PHDp5 μ L of 2 μ M/L, sample DNA template 2ng.
The real-time fluorescence PCR response procedures adopts three-step approach: 50 ℃ of the first steps, 2min; 95 ℃ of second steps, 10min; Then enter the circulation of the 3rd step, 95 ℃, 10s, 60 ℃, 1min is totally 40 circulations.
The present invention has extraordinary specificity and stability, and detection method is simple fast, and accuracy and susceptibility are high, for the imports and exports safety that may carry Sunflower Receptacle stem canker vegetable material provides assurance.
Description of drawings
Fig. 1. the present invention detects as a result figure of Sunflower Receptacle stem canker
1. Sunflower Receptacle stem canker bacterial strain one; 2. Sunflower Receptacle stem canker bacterial strain two; 3-11. other disease plant of healthy Sunflower Receptacle blade and Sunflower Receptacle
Fig. 2. the present invention detects as a result figure of Sunflower Receptacle stem canker sensitivity
1、5.8μg，2、580ng，3、58ng，4、5.8ng，5、580pg，6、58pg，7、5.8pg，8、580fg，9、ofg
Embodiment
Following embodiment is used for further specifying of the present invention, but is not used for limiting the scope of the invention.
Embodiment 1 primer and probe design
Put mould ribosomal gene sequence according to the result of this laboratory order-checking and the stem of having reported, adopt primer probe design software DNAMAN design primer and probe, primer sequence is:
PHDf：5’-CGTTCAAAGATTCGATGA-3’
PHDr：5’-CAGTGGATCTCTGAGTAA-3’
Probe sequence is: PHDp:5 '-ACTTTCAACAACGGATCTCTTGGTT-3 ', 5 of this probe, end are labeled as report fluorescence dye FAM, and 3 ' end is labeled as cancellation fluorescence dye TAMRA.
1. the extraction of Sunflower Receptacle material DNA
Adopt test kit method (TaKaRa Universal Genomic DNA Extraction Kit Ver3.0 or DNA Extraction Kit for GMO Detection Ver.2.2) to extract Sunflower Receptacle material nucleic acid.
2. real-time fluorescence PCR reaction system
Take the total DNA of Sunflower Receptacle material as template, carry out the real-time fluorescence PCR reaction.
In 25 μ L reaction systems, add Real-time PCR Mix10 μ L, each 2 μ L of 5 μ M primers (PHDf/PHDr), the PHDp5 μ L of 2 μ M/L, sample DNA template 2ng.
3. real-time fluorescence PCR reaction conditions
After sample hose put into Applied Biosystems7300 fluorescent PCR instrument, response procedures is set: 50 ℃ of the first steps, 2min; 95 ℃ of second steps, 10min; Then enter the circulation of the 3rd step, 95 ℃, 10s, 60 ℃, 1min, totally 40 circulations.
Each loop ends image data, reaction finish rear according to the amplification curve result of determination.
The specificity experiment of embodiment 3 Sunflower Receptacle stem canker real-time fluorescence PCR assay kits
Take the total DNA of Sunflower Receptacle stem canker bacterial strain as template, take healthy Sunflower Receptacle blade and other disease plant of Sunflower Receptacle as contrast, change by the real-time fluorescence PCR fluorescence intensity.
Experimental result: the total DNA of Sunflower Receptacle stem canker bacterial strain is template, detect by real-time fluorescence PCR and can be observed obvious fluorescence intensity variation, and the fluorescence intensity of other disease plant of normal healthy controls and Sunflower Receptacle does not change, and the results are shown in Figure 1.
The sensitivity experiment of embodiment 4 Sunflower Receptacle stem canker real-time fluorescence PCR assay kits
With 10 times of total DNA of dilution of tri-distilled water, carry out relative sensitivity and detect.In 25 μ L reaction systems, total DNA is respectively 5.8 μ g, 580ng, 58ng, 5.8ng, 580pg, 58pg, 5.8pg, 580fg, detected result still can detect fluorescent signal as shown in Figure 2 when nucleic acid concentration is lower than 580fg, and the low DNA concentration that explanation can detect is lower than 580fg.
Claims (5)
1. a Sunflower Receptacle stem canker detects and uses primer, and its nucleotides sequence is classified as:
PHDf：5’-CGTTCAAAGATTCGATGA-3’
PHDr：5’-CAGTGGATCTCTGAGTAA-3’。
2. probe that is used with the described primer of claim 1, its nucleotides sequence is classified as: an end of PHDp:5 '-this probe of ACTTTCAACAACGGATCTCTTGGTT-3 ' is labeled as the report fluorescence dye, and the other end is labeled as the cancellation fluorescence dye.
3. a method that detects the Sunflower Receptacle stem canker is characterized in that the annealing temperature in the real-time fluorescence PCR reaction process is 60 ℃.
4. the test kit that contains the described primer of claim 1.
5. test kit as claimed in claim 4, it also comprises probe claimed in claim 2.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106048010A (en) * | 2016-06-03 | 2016-10-26 | 伊犁出入境检验检疫局综合技术服务中心 | RPA (recombinase polymerase amplification) technology based method for detecting phomopsis helianthi, RPA primers and kit |
| CN106520999A (en) * | 2016-12-20 | 2017-03-22 | 宁波出入境检验检疫局检验检疫技术中心 | Detection method for diaporthe helianthi munt.-cvetk.,mihaljc.et m.petrov on single sunflower seed, and reagent |
-
2012
- 2012-11-06 CN CN2012104367167A patent/CN103060436A/en active Pending
Non-Patent Citations (4)
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| 《中国优秀硕士学位论文全文数据库》 20111215 刘彬等 "新疆向日葵上两种检疫性病原菌生物学特性及快速检测技术研究" , 第12期 * |
| 《新疆农业科学》 20120330 陈燕等 "应用PCR法检测向日葵茎溃疡病菌的研究" 第49卷, 第3期 * |
| 刘彬等: ""新疆向日葵上两种检疫性病原菌生物学特性及快速检测技术研究"", 《中国优秀硕士学位论文全文数据库》 * |
| 陈燕等: ""应用PCR法检测向日葵茎溃疡病菌的研究"", 《新疆农业科学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106048010A (en) * | 2016-06-03 | 2016-10-26 | 伊犁出入境检验检疫局综合技术服务中心 | RPA (recombinase polymerase amplification) technology based method for detecting phomopsis helianthi, RPA primers and kit |
| CN106520999A (en) * | 2016-12-20 | 2017-03-22 | 宁波出入境检验检疫局检验检疫技术中心 | Detection method for diaporthe helianthi munt.-cvetk.,mihaljc.et m.petrov on single sunflower seed, and reagent |
| CN106520999B (en) * | 2016-12-20 | 2020-06-16 | 宁波出入境检验检疫局检验检疫技术中心 | Detection method and reagent for stem canker bacteria of sunflower on single sunflower seed |
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Application publication date: 20130424 |