CN103122375A - Kit for real-time fluorescence PCR (Polymerase Chain Reaction) detection of phoma macdonaldii boerma - Google Patents
Kit for real-time fluorescence PCR (Polymerase Chain Reaction) detection of phoma macdonaldii boerma Download PDFInfo
- Publication number
- CN103122375A CN103122375A CN2012104367190A CN201210436719A CN103122375A CN 103122375 A CN103122375 A CN 103122375A CN 2012104367190 A CN2012104367190 A CN 2012104367190A CN 201210436719 A CN201210436719 A CN 201210436719A CN 103122375 A CN103122375 A CN 103122375A
- Authority
- CN
- China
- Prior art keywords
- kit
- real
- sunflower receptacle
- probe
- detection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a kit for real-time fluorescence PCR (Polymerase Chain Reaction) detection of phoma macdonaldii boerma. A primer nucleotide sequence adopted by the kit is shown in a sequence table LEPf or LEPr; a probe nucleotide sequence is shown in the sequence table Pmp. The total DNA of the sample is adopted as a template, so that the kit can perform real-time PCR amplification by utilizing the primer and the probe; the data after each circulation is collected; and the result is judged according to an amplification curve after the reaction is completed. The kit disclosed by the invention is good in specificity and stability, simple and quick in detection method, and high in accuracy and flexibility, so that guarantee is provided for the import and export safety of a plant material which is probably carried with the phoma macdonaldii boerma.
Description
Technical field
The present invention relates to Measurement for Biotechnique, relate to specifically Sunflower Receptacle black stem bacterium detection test kit.
Background technology
Sunflower Receptacle black stem (Phoma macdonaldii Boerma) is found in Europe in 20 century 70 later stages first, and all there is generation in the states such as France, Romania, Hungary, Serbia, USSR (Union of Soviet Socialist Republics), Former Yugoslavia, Australia, Canada, the U.S., Argentina, Iran, Iraq now.Mainly generate typical large-scale scab on cane, black, glossy, tool is the edge clearly.Betide at first petiole base, to the expansion of stem stalk, form the black scab rapidly, can reach several modest ability, cause that the blade atrophy is withered.When serious, on stem, scab can be around the stem stalk, and whole stem stalk is all blackening also, and the plant dead ripeness is withered in advance.In addition, at blade, the floral disc back side, the black scab also occurs in collar and basal part of stem.The black small grain point occurs in stem surface, i.e. the pycnidium of pathogenic bacteria, and naked eyes do not see Chu, need observe with hand magnifier.2008, the people such as Chen Weimin reported Yili of Xinjiang discovery Sunflower Receptacle black stem.At present this disease in Xinyuan County, Xinjiang of China Ili Prefecture, Tekesi County, Nileke County occur, and area 16200hm occurs
2, financial loss 28～80%.
Less to the research of Sunflower Receptacle black stem bacterium both at home and abroad, more to the mould research of other stem point, external Victor Morales etc. is studied the mould 5.8S of part stem point and ITS regional sequence; Franco Rollod etc. utilizes PCR to carry out rapid detection to lemon top drying pathogenic bacteria (Phoma tracheiphila).The people such as the Xinjiang Yili of China Chen Wei people of Vocationl Technical College in 2008 have reported that Yili of Xinjiang finds the Sunflower Receptacle black stem, and the morphology of this germ, occurrence degree etc. have been made preliminary study.Up to the present also not effective especially chemical agent or resistant variety are prevented and treated this disease, because Sunflower Receptacle black stem bacterium 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 Sunflower Receptacle black stem bacterium.It is the quarantine disease of China due to this disease, 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 black stem bacterium is taked are mainly:
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 black stem bacterium between planting season.This method can only be as preliminary judgement, and take a lot of work, accuracy rate time-consuming, the result judgement is low.
2. separation and Culture: the Sunflower Receptacle seedling that enters the territory is carried out separation and purification on substratum, the method 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 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, introduce a large amount of sunflower seeds in China every year from the U.S., Germany and other places, 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.Mechanism lacks the effective detection kit to Sunflower Receptacle black stem bacterium but each Check and Examination of Port of present China is quarantined.Disease control, prediction and Plant Quarantine need to have detection kit quickly and accurately, especially need China to have the detection kit of independent intellectual property right, thereby it can be put in commercial production and application, also there is no at present the report of Sunflower Receptacle black stem bacterium 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 the problems such as long for the plantation observation post's need cycle that solves Sunflower Receptacle black stem bacterium in prior art, that evaluation is difficult, accuracy rate is low, the quick detection kit of Sunflower Receptacle black stem bacterium is provided.
The present invention by analyzing the order-checking of this laboratory result and reported the mould ribosomal gene sequence of stem point, design primer and fluorescent probe.Primer pair is comprised of forward and reverse primer, its nucleotide sequence such as sequence table LEPf﹠amp; Shown in LEPr; The nucleotide sequence of probe is as shown in sequence table PMp, 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/dabcy1/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 both distance becomes far away, restraining effect weakens, and report fluorescence dye signal strengthens.In the amplified reaction process, probe is hybridized with the purpose amplified fragments on 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 Sunflower Receptacle black stem bacterium.
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.
The present invention specifically carries out the real-time fluorescence PCR reaction take sample total DNA as template.Namely add Real-time PCR Mix10 μ L in 25 μ L reaction systems, 5 μ M primer (LEPf﹠amp; LEPr) each 2 μ L, the PMp5 μ L of 2 μ M/L, sample DNA template 2ng.
The real-time fluorescence PCR response procedures adopts two-step approach: 95 ℃ of the first steps, and then 3min enters the second step circulation: 95 ℃, 15s; 54 ℃, 25s, 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 black stem bacterium vegetable material provides assurance.
Description of drawings
Fig. 1. the present invention detects Sunflower Receptacle black stem bacterium figure as a result
1. Sunflower Receptacle black stem blade; 2 Sunflower Receptacle black stem canes; 3. Sunflower Receptacle black stem seed; 4-7. other disease plant of healthy Sunflower Receptacle blade and Sunflower Receptacle
Fig. 2. the present invention detects Sunflower Receptacle black stem bacterium sensitivity figure as a result
1、1.953μg；2、390.625ng；3、78.125ng；4、15.625ng；5、3.125ng；6、625pg；7、125pg；8、25pg；9、5pg；10、1pg；11、200fg；12、ofg。
Embodiment
Following embodiment is used for further illustrating of the present invention, but is not used for limiting the scope of the invention.
According to the result of this laboratory order-checking and the mould ribosomal gene sequence of stem point of having reported, adopt primer probe design software DNAMAN design primer and probe, primer sequence is:
LEPf：5’-TCACCTCTTTCTGATTCTACCC-3’
LEPr：5’-CATTGTTACTGACGCTGACTG-3’
Probe sequence is: PMp:5 '-TTGCGTACTACTTGGTTTCCTCGGCG-3 ', and 5 ' end of this probe is labeled as report fluorescence dye FAM, and 3 ' end is labeled as cancellation fluorescence dye TAMRA.
Embodiment 2 Sunflower Receptacle sample detection
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.
Add Real-time PCR Mix10 μ L in 25 μ L reaction systems, each 2 μ L of 5 μ M primers (LEPf/LEPr), the PMp 5 μ L of 2 μ M/L, sample DNA template 2ng.
3. real-time fluorescence PCR reaction conditions
After sample hose is put into Applied Biosystems7300 fluorescent PCR instrument, response procedures is set: 95 ℃ of the first steps, then 3min enters the second step circulation: 95 ℃, 15s; 54 ℃, 25s, 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 black stem bacterium real-time fluorescence PCR assay kits
Take the total DNA of Sunflower Receptacle black stem bacterium plant of reveal any symptoms 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: take the total DNA of Sunflower Receptacle black stem bacterium plant of reveal any symptoms as template, detect by real-time fluorescence PCR and can be observed obvious fluorescence intensity and change, 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 black stem bacterium real-time fluorescence PCR assay kits
With 5 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 1.953 μ g, 390.625ng, 78.125ng, 15.625ng, 3.125ng, 625pg, 125pg, 25pg, 5pg, 1pg, 200fg, detected result is as shown in Figure 2, still fluorescent signal can be detected when nucleic acid concentration during lower than 200fg, the low DNA concentration that explanation can detect is lower than 200fg.
Claims (5)
1. a Sunflower Receptacle black stem bacterium detects and uses primer, and its nucleotides sequence is classified as:
LEPf：5’-TCACCTCTTTCTGATTCTACCC-3’
LEPr：5’-CATTGTTACTGACGCTGACTG-3’。
2. probe that is used in conjunction with the described primer of claim 1, its nucleotides sequence is classified as: an end of PMp:5 '-this probe of TTGCGTACTACTTGGTTTCCTCGGCG-3 ' is labeled as the report fluorescence dye, and the other end is labeled as the cancellation fluorescence dye.
3. a method that detects Sunflower Receptacle black stem bacterium, is characterized in that the annealing temperature in the real-time fluorescence PCR reaction process is 54 ℃.
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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012104367190A CN103122375A (en) | 2012-11-06 | 2012-11-06 | Kit for real-time fluorescence PCR (Polymerase Chain Reaction) detection of phoma macdonaldii boerma |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012104367190A CN103122375A (en) | 2012-11-06 | 2012-11-06 | Kit for real-time fluorescence PCR (Polymerase Chain Reaction) detection of phoma macdonaldii boerma |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103122375A true CN103122375A (en) | 2013-05-29 |
Family
ID=48453516
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012104367190A Pending CN103122375A (en) | 2012-11-06 | 2012-11-06 | Kit for real-time fluorescence PCR (Polymerase Chain Reaction) detection of phoma macdonaldii boerma |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103122375A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998042866A1 (en) * | 1997-03-26 | 1998-10-01 | Institut National De La Recherche Agronomique | MARKER OF RESISTANCE TO $i(SCLEROTINIA SCLEROTIORUM) |
| RS52018B (en) * | 2008-11-21 | 2012-04-30 | Snežana DUKIĆ | Method for determining the purity of cytoplasmic sterile sunflower line (helianthus annuus) |
| CN102747137A (en) * | 2011-12-05 | 2012-10-24 | 内蒙古农业大学 | Identifying method for sunflower phoma black stem bacteria |
-
2012
- 2012-11-06 CN CN2012104367190A patent/CN103122375A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998042866A1 (en) * | 1997-03-26 | 1998-10-01 | Institut National De La Recherche Agronomique | MARKER OF RESISTANCE TO $i(SCLEROTINIA SCLEROTIORUM) |
| RS52018B (en) * | 2008-11-21 | 2012-04-30 | Snežana DUKIĆ | Method for determining the purity of cytoplasmic sterile sunflower line (helianthus annuus) |
| CN102747137A (en) * | 2011-12-05 | 2012-10-24 | 内蒙古农业大学 | Identifying method for sunflower phoma black stem bacteria |
Non-Patent Citations (2)
| Title |
|---|
| 《菌物学报》 20120715 宋娜等 "向日葵黑茎病菌的快速分子检测" 第630-638页 第31卷, 第4期 * |
| 宋娜等: ""向日葵黑茎病菌的快速分子检测"", 《菌物学报》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101608235B (en) | Padlock probe for detecting Xanthomonas oryzae pv. oryzae and anthomonas oryzae pv.oryzicola and multiple detection method | |
| Engelbrecht et al. | Development of a nested quantitative real-time PCR for detecting Phytophthora cinnamomi in Persea americana rootstocks | |
| Li et al. | Simultaneous detection and quantification of Phytophthora nicotianae and P. cactorum, and distribution analyses in strawberry greenhouses by duplex real-time PCR | |
| CN108060257A (en) | It is a kind of that strong male rotten mould Primer composition and its detection method are detected based on loop-mediated isothermal amplification technique | |
| Monti et al. | EvaGreen real-time PCR protocol for specific ‘Candidatus Phytoplasma mali’detection and quantification in insects | |
| CN108893557A (en) | A kind of method of three kinds of wheat rhizome portion diseases of quick detection | |
| Newman et al. | Molecular diagnosis of mango malformation disease and phylogeny of Fusarium mangiferae | |
| US20090246767A1 (en) | Compositions and methods for detecting Cryptococcus neoformans | |
| JP5522820B2 (en) | Method for detecting pathogens of strawberry important diseases and primers for detection | |
| CN104232782A (en) | PCR (polymerase chain reaction) primer for detecting tobacco soil-borne fungal pathogens as well as application and method of PCR primer | |
| CN104830984B (en) | The fluorescence PCR detecting method and the primer and probe of melon anthrax bacteria | |
| CN101608236A (en) | The padlock probe and the multiple detection method of erwinia amylovora and Asia erwinia amylovora | |
| CN104263841A (en) | Real-time fluorescence LAMP (loop-mediated isothermal amplification) detection method for pectobacterium atrosepticum in potatoes as well as kit | |
| CN103205485B (en) | Method of detecting nucleotides of Bacillus cereus, primers for detecting and probes | |
| CN107828905A (en) | Tobacco smoke pollution LAMP detection primer and detection method | |
| CN103397108A (en) | HCLV (hog cholera lapinized virus) fluorescent quantitation RT-PCR (reverse transcription-polymerase chain reaction) detection kit | |
| WO2011068218A1 (en) | Kit and method for identification of causative bacterium of nail tinea | |
| CN103060436A (en) | Real-time fluorescent PCR (polymerase chain reaction) detection kit for Phomopsis helianthi M.Muntanola-Cvetkovic et al. | |
| CN103122375A (en) | Kit for real-time fluorescence PCR (Polymerase Chain Reaction) detection of phoma macdonaldii boerma | |
| EP2875155A1 (en) | Endpoint zygosity assay to detect rf4 gene in maize | |
| KR102212379B1 (en) | Primer set for simultaneous detection of three viruses in Grapevine and method for detecting three viruses in Grapevine using the same | |
| CN104962659A (en) | Pseudomonas syringae pv.cucumber cucumber strain Padlock probe and application thereof | |
| CN104830999A (en) | LAMP method for detecting bovine ephemeral fever virus | |
| CN106244702B (en) | Real-time fluorescence isothermal amplification primer group, reaction system and detection method for quantitatively detecting lotus rot germs | |
| CN104164486B (en) | The LAMP detection kit of the shrivelled pathogen of a kind of Eucalyptus and using method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20130529 |