CN1284864C - One-step dual PCR method for detecting fire blight of pear - Google Patents
One-step dual PCR method for detecting fire blight of pear Download PDFInfo
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- CN1284864C CN1284864C CN 200510064520 CN200510064520A CN1284864C CN 1284864 C CN1284864 C CN 1284864C CN 200510064520 CN200510064520 CN 200510064520 CN 200510064520 A CN200510064520 A CN 200510064520A CN 1284864 C CN1284864 C CN 1284864C
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- fire blight
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Abstract
The present invention relates to a one-step double PCR method for detecting pear fire blight pathogenic bacteria, which belongs to the technical field of crop disease control and plant quarantine. The method designs two pairs of specific primers according to the pEA29 plasmid and the genome ams gene order of the pear fire blight pathogenic bacteria so as to simultaneously detect the pear fire blight pathogenic bacteria in the same reaction system; amplification products have the sizes of 1.0 kb and 1.5 kb respectively, and the detection sensibility of the two pairs of primers reaches three bacterial cells. The one-step double PCR method is the supplementary and improvement of the detection method of specific primers for pEA29 plasmid of the pear fire blight pathogenic bacteria, and can also detect the bacterial strain of the pear fire blight pathogenic bacterium strain containing no pEA29 plasmid. Meanwhile, the method adopts the double PCR technology, and the two pairs of primers are simultaneously amplified in the same reaction system; the detection accuracy is enhanced and the detection time is saved; the present invention can be popularized at the ports of China in large scales in a kit mode.
Description
(1) technical field
The present invention " is used to detect an one-step dual PCR method of erwinia amylovora ", is exclusively used in the detection erwinia amylovora, belongs to crop disease control and Plant Quarantine technical field.
(2) background technology
Erwinia amylovora (Erwina amylovora) is a kind of crushing Micobial Disease on pears, apple and other rosaceous plants, find pre-1780 the earliest, existing more than 40 countries such as Middle and North America, Oceania, Europe, West Asia, Egypt, Japan, Korea S that mainly are distributed in.1951-1960 during the decade, the U.S. loses 4,000,000 dollars every year on average.China still finds no the harm of this disease at present, is listed in an external class Quarantine Objects of China.
Fire blight of pear main harm flower, leaf, the tender tip, fruit etc., the general underproduction 25%.
China's cultivation of fruit tree area is very big, and according to statistics in 1998, the cultivated area of apple reached 2,840,000 hm
2, the pear tree area reaches 940,000 hm
2, to compare with countries in the world, apple and pear tree cultivated area and gross output all occupy first place in the world.Along with the continuous expansion of international trade, China begins to introduce from Japanese and Korea S the seedling of good fruit variety, from various fruit of national import such as the U.S., Australia, as not strict quarantine, then brings erwinia amylovora into China probably.In a single day this disease is imported into, will certainly bring serious harm to the cultivation of fruit tree and the production of China, also will influence the foreign exchange earning of China's fancy fruit simultaneously.
Strengthen quarantine and must set up the rapid and precise molecular detecting method of a cover, but existing in the world several different methods: 1) half selective medium separation and Culture is identified; 2) serology detection technique is as ELISA and immunofluorescence; 3) DNA hybridization and round pcr etc.But the present domestic detection method of the detection of fire blight of pear still not being had standard.
Widely used in plant pathogenetic bacteria detects and identifies is round pcr.It is to utilize specific primer that round pcr detects, and under the catalysis of archaeal dna polymerase, target dna is carried out amplification in vitro, judges detected result according to having or not of expection DNA band.Round pcr detect have highly sensitive, high specificity, fast and advantage such as easy.
Abroad, design a pair of special primer and detect erwinia amylovora according to the partial sequence of pEA29 plasmid in the erwinia amylovora.But can't detect for the erwinia amylovora that does not contain the pEA29 plasmid.
(3) summary of the invention
Technical problem the purpose of this invention is to provide an one-step dual PCR method that is used to detect erwinia amylovora.This method designs two pairs of Auele Specific Primers according to the pEA29 plasmid and the genome ams gene order of erwinia amylovora, detects erwinia amylovora in same reaction system simultaneously.Be replenishing and improving to erwinia amylovora pEA29 plasmid Auele Specific Primer detection method.Also can detect for the fire blight of pear bacteria strain that does not contain the pEA29 plasmid.Simultaneously, present method adopts multiple PCR technique, and two pairs of primers amplifications simultaneously in same reaction system have improved the accuracy that detects, and have saved detection time, and form that can test kit is in China's port large-scale promotion.
Technical scheme
Be used to detect an one-step dual PCR method of erwinia amylovora, comprise:
1) is used to detect the specimen preparation of erwinia amylovora
A) with bacterial ooze as template
With the erwinia amylovora activation of-80 ℃ of preservations, at first (beef extract 3 grams, yeast soak powder 0.5 gram to picking bacterium liquid, peptone 5 grams at the NA substratum, glucose 10 grams, 1000 milliliters of distilled water, pH 7.0-7.2) on, 28 ℃, cultivated 1-2 days, the picking bacterial ooze directly is added in the PCR reaction system.
B) with the bacterium liquid of fresh culture as template
Picking bacterial ooze from the NA substratum is added in the NA liquid nutrient medium, and 28 ℃, shaking culture 1 day is got the template of 1 μ l bacterium liquid as pcr amplification, directly is added in the PCR reaction system.
2) be used to detect the Auele Specific Primer of erwinia amylovora
PrimerA/B and primier E/F, its primer sequence is:
PrimerA：5’-CGGTTTTTAACGCTGGG-3’
PrimerB：5’-GGGCAAATACTCGGATT-3’
PrimerE：5’-AATAAGGTGCCTGGTAATG-3’
PrimerF：5’-GGTGGAATGAGACTGGGTA-3’
Wherein primer A/B primer sequence is from the distinctive pEA29 plasmid of this bacterium, and specific amplification goes out the product of 1.0kb in erwinia amylovora; Primer E/F primer sequence is from the karyomit(e) that changes bacterium, and specific amplification goes out the product of 1.5kb in erwinia amylovora.
3) reaction system of pcr amplification
Primer A/B primer to primer E/F primer in same reaction system, carrying out double PCR reaction simultaneously, 10 * PCR buffer, 5 μ l wherein, 15mM MgCl
210 μ l, 10mM dNTPs 2 μ l, each 3 μ l of 10mM primerA/B and primer E/F, Taq enzyme (5U/ μ l) 1 μ l, template is the erwinia amylovora 1 μ l or the bacterial ooze of fresh culture, supplies with sterilized water, final volume is 50 μ l.
4) pcr amplification program
95 ℃ of pre-sex change 3min, 93 ℃ of sex change 1min, 52 ℃ of annealing 2min, 72 ℃ are extended 2min, 30 circulations, last 72 ℃ are extended 10min.
4) evaluation of PCR product
Get 10 μ lPCR products and separate with 0.1% (weight/volume) agarose gel electrophoresis, through behind the ethidium bromide staining under ultraviolet lamp according to the big or small result of determination of amplified production.
Beneficial effect
The present invention is used to detect an one-step dual PCR method of erwinia amylovora, can be used for detecting the pears fire vaccine body from the scab separate tissue, and whether the vegetable material that also can be used for detecting no disease symptom has erwinia amylovora.Compare with external existing method, the present invention has following technical superiority:
1) detection accuracy height.Present method detects simultaneously from genome and two aspects of pEA29 plasmid of erwinia amylovora, also can detect for not containing pEA29 plasmid fire blight of pear bacteria strain.
2) simple and efficient to handle.The present invention adopts multiple PCR technique, two pairs of primer amplifications simultaneously in same reaction system, and amplification template can be the bacterium liquid of bacterial ooze or fresh culture, has saved the complicated processes of DNA of bacteria preparation.Whole process is simple, quick, efficient.General whole testing process can be finished in 4 hours.
3) high specificity.But two pairs of primer specific detection erwinia amylovoras of the present invention's design do not have amplified production in its sibling species, genus bacterium.
Therefore present method is practical, can satisfy the needs of Plant Quarantine and disease monitoring.
(4) description of drawings
Fig. 1: two pairs of primers of primer primerA/B and primer E/F are to the double PCR amplification M of erwinia amylovora and sibling species thereof, genus bacterium: molecular weight standard DGL2000; 1,5: erwinia amylovora (Ea1); 2,6: erwinia amylovora (Ea3); 3,7: erwinia amylovora (Ea65); 4,8: erwinia amylovora (Ea67); 9: agrobacterium tumefaciens Agrobacterium tumefaciens; 10: radish soft-rot bacterium Erwinia carotovora var.carotovora; 11: corn bacterial stem rot germ Erwinia chrysanthemi; 12: corn bacterial wilt Erwinia stewartii; 13: grass living Erwinia Erwinia herbicola (non-pathogenic bacteria); 14: pseudomonas syringae Pseudonomas syringae; 15: pears top drying germ Pseudonomas syringae; 16: rice leaf spot bacteria Xanthomonas oryzae.
* 1,2,3,4 bacterial ooze with erwinia amylovora are template; 5,6,7, the 8 bacterium liquid with the erwinia amylovora fresh culture are template.
Fig. 2: primer primerA/B and primer primer E/F detect erwinia amylovora sensitivity and the minimum mensuration Maker that detects the bacterium amount: molecular weight standard DGL2000; 1: erwinia amylovora Ea1 original bacteria liquid; 2: dilution 10
-13: dilution 10
-24: dilution 10
-35: dilution 10
-46: dilution 10
-5CK: negative control (template is a water)
(5) embodiment
Embodiment 1: primer primerA/B and primer primer E/F with the erwinia amylovora bacterial ooze as template.
1) preparation of erwinia amylovora bacterial ooze template
With the erwinia amylovora activation of-80 ℃ of preservations, at first (beef extract 3 grams, yeast soak powder 0.5 gram to picking bacterium liquid, peptone 5 grams at the NA substratum, glucose 10 grams, 1000 milliliters of distilled water, pH 7.0-7.2) on, 28 ℃, cultivated 1-2 days, the picking bacterial ooze directly is added in the PCR reaction system.
2) Auele Specific Primer of detection erwinia amylovora is synthetic
Primer?A：5’-CGGTTTTTAACGCTGGG-3’；
Primer?B：5’-GGGCAAATACTCGGATT-3’
And
Primer?E：5’-AATAAGGTGCCTGGTAATG-3’
Primer F:5 '-GGTGGAATGAGACTGGGTA-3 ' gives birth to worker company by Shanghai and synthesizes.
3) pcr amplification reaction
Primer A/B primer to primer E/F primer in same reaction system, carrying out double PCR reaction simultaneously, 10 * PCR buffer, 5 μ l wherein, 15mM MgCl
210 μ l, 10mM dNTPs 2 μ l, each 3 μ l of 10mM primer A/B and primer E/F, Taq enzyme (5U/ μ l) 1 μ l, template is the bacterial ooze of erwinia amylovora, supplies with sterilized water, final volume is 50 μ l.
4) pcr amplification program
95 ℃ of pre-sex change 3min, 93 ℃ of sex change 1min, 52 ℃ of annealing 2min, 72 ℃ are extended 2min, 30 circulations, last 72 ℃ are extended 10min.
5) evaluation of PCR product
Get 10 μ l PCR products and separate with 0.1% (weight/volume) agarose gel electrophoresis, through behind the ethidium bromide staining under ultraviolet lamp according to the big or small result of determination of amplified production.
Result of implementation
Utilizing primer primerA/B and primer primerE/F is template with the bacterial ooze of erwinia amylovora, serves as that the double PCR amplification is carried out in contrast with erwinia amylovora sibling species, genus.In the erwinia amylovora of 1-4 swimming lane, amplified the band (seeing the 1-4 swimming lane of accompanying drawing 1) of 1.5kb and 1.0kb two clauses and subclauses.In other 8 sibling specieses, genus bacterium, all there is not amplified production.The high specificity that illustrates that we screen from two pairs of specific PCR amplimers of fire blight of pear bacteria plasmid and chromosomal DNA.
Embodiment 2: primer primerA/B and primer primer E/F with the bacterium liquid of erwinia amylovora fresh culture as template.
1) with the bacterium liquid of fresh culture as template
Picking bacterial ooze from the NA substratum is added in the NA liquid nutrient medium, and 28 ℃, shaking culture 1 day is got the template of 1 μ l bacterium liquid as pcr amplification, directly is added in the PCR reaction system.
2) Auele Specific Primer of detection erwinia amylovora is synthetic
Primer?A：5’-CGGTTTTTAACGCTGGG-3’；
Primer?B：5’-GGGCAAATACTCGGATT-3’
And
Primer?E：5’-AATAAGGTGCCTGGTAATG-3’
Primer F:5 '-GGTGGAATGAGACTGGGTA-3 ' gives birth to worker company by Shanghai and synthesizes.
3) pcr amplification reaction
Primer A/B primer to primer E/F primer in same reaction system, carrying out double PCR reaction simultaneously, 10 * PCR buffer, 5 μ l wherein, 15mM MgCl
210 μ l, 10mM dNTPs 2 μ l, each 3 μ l of 10mM primerA/B and primer E/F, Taq enzyme (5U/ μ l) 1 μ l, template is the erwinia amylovora bacterium liquid 1 μ l of fresh culture, supplies with sterilized water, final volume is 50 μ l.
4) pcr amplification program
95 ℃ of pre-sex change 3min, 93 ℃ of sex change 1min, 52 ℃ of annealing 2min, 72 ℃ are extended 2min, 30 circulations, last 72 ℃ are extended 10min.
5) evaluation of PCR product
Get 10 μ l PCR products and separate with 0.1% (weight/volume) agarose gel electrophoresis, through behind the ethidium bromide staining under ultraviolet lamp according to the big or small result of determination of amplified production.
Result of implementation
Utilizing primer primerA/B and primer primer E/F is template with the bacterial ooze of erwinia amylovora, serves as that the double PCR amplification is carried out in contrast with erwinia amylovora sibling species, genus.In the erwinia amylovora of 5-8 swimming lane, amplified the band (seeing the 5-8 swimming lane of accompanying drawing 1) of 1.5kb and 1.0kb two clauses and subclauses.In other 8 sibling specieses, genus bacterium, all there is not amplified production.The high specificity that illustrates that we screen from two pairs of specific PCR amplimers of fire blight of pear bacteria plasmid and chromosomal DNA.
Embodiment 3: primer primerA/B and primer primer E/F detect erwinia amylovora sensitivity and the minimum mensuration that detects the bacterium amount
1) with the bacterium liquid of fresh culture as template picking bacterial ooze from the NA substratum, be added in the NA liquid nutrient medium, 28 ℃, shaking culture 1 day is got original bacteria liquid respectively and is diluted 10
1-10
5 Bacterium liquid 1 μ l doubly is the template of pcr amplification, directly is added in the PCR reaction system.
2) Auele Specific Primer of detection erwinia amylovora is synthetic
Primer?A：5’-CGGTTTTTAACGCTGGG-3’；
Primer?B：5’-GGGCAAATACTCGGATT-3’
And
Primer?E：5’-AATAAGGTGCCTGGTAATG-3’
Primer F:5 '-GGTGGAATGAGACTGGGTA-3 ' gives birth to worker company by Shanghai and synthesizes.
3) pcr amplification reaction
Primer A/B primer to primer E/F primer in same reaction system, carrying out double PCR reaction simultaneously, 10 * PCR buffer, 5 μ l wherein, 15mM MgCl
210 μ l, 10mM dNTPs 2 μ l, each 3 μ l of 10mM primerA/B and primer E/F, Taq enzyme (5U/ μ l) 1 μ l, template is the erwinia amylovora bacterium liquid 1 μ l of fresh culture, supplies with sterilized water, final volume is 50 μ l.
4) pcr amplification program
95 ℃ of pre-sex change 3min, 93 ℃ of sex change 1min, 52 ℃ of annealing 2min, 72 ℃ are extended 2min, 30 circulations, last 72 ℃ are extended 10min.
5) evaluation of PCR product
Get 10 μ l PCR products and separate with 0.1% (weight/volume) agarose gel electrophoresis, through behind the ethidium bromide staining under ultraviolet lamp according to the big or small result of determination of amplified production.
6) pave plate calculating minimum and detect the bacterium amount
Get original bacteria liquid respectively and dilute 10
1-10
5Bacterium liquid doubly is coated with flat board on the NA substratum, calculate each dull and stereotyped colony number of going up bacterium.Draw the minimum bacterium amount that pcr amplification detects according to extension rate again.
Result of implementation
Do the mensuration that minimum detects the bacterium amount with primer PrimerE+F and primer PrimerA+B respectively, can detected minimum bacterium amount be original bacteria liquid dilution 10
5Times (see figure 2) is coated with the plate count result and is: Ea1 original bacteria liquid dilution 10
-1There are many bacterium colonies to grow on the plate, dilution 10
-327 bacterium colonies are arranged, dilution 10
-4There are 3 bacterium colonies to grow, dilution 10
-5Do not have bacterium colony to grow, calculate minimum and detect bacterium amount and can reach 3 cells.
Claims (1)
1, a kind of one-step dual PCR detection method that can detect erwinia amylovora 1.0kb plasmid sequence and 1.5kb chromosome sequence simultaneously, it is characterized in that: in same reaction system, only need a pcr amplification just can detect 2 characteristic bands, wherein A and the B primer sequence according to the design of erwinia amylovora plasmid dna sequence is: Primer A:5 '-CGGTTTTTAACGCTGGG-3 ', Primer B:5 '-GGGCAAATACTCGGATT-3 '; E and F primer sequence according to the design of erwinia amylovora chromosome sequence are: Primer E:5 '-AATAAGGTGCCTGGTAATG-3 ', Primer F:5 '-GGTGGAATGAGACTGGGTA-3 '; Pcr amplification adopts 50 μ l reaction systems, 10 * PCR buffer, 5 μ l wherein, 15mM MgCl
210 μ l, 10mM dNTPs 2 μ l, each 3 μ l of 10 μ M primer A/B and primer E/F, 5U/ μ l Taq enzyme 1 μ l, template is the erwinia amylovora 1 μ l or the bacterial ooze of fresh culture, supplies with sterilized water, final volume is 50 μ l; The pcr amplification program is: 95 ℃ of pre-sex change 3min, and 93 ℃ of sex change 1min, 52 ℃ of annealing 2min, 72 ℃ are extended 2min, 30 circulations, last 72 ℃ are extended 10min.
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101509036B (en) * | 2009-03-27 | 2012-05-09 | 南京农业大学 | Detection kit and method for Asia erwinia amylovora |
| CN103114137A (en) * | 2013-01-25 | 2013-05-22 | 郑媛 | Fluorescent PCR (polymerase chain reaction) quick detection primer and kit for pear fire blight pathogenic bacteria |
| CN109988808A (en) * | 2019-03-20 | 2019-07-09 | 新疆农业大学 | A kind of method of the in vitro water culture measurement erwinia amylovora pathogenicity of bergamot pear branch |
| CN113308523A (en) * | 2021-06-08 | 2021-08-27 | 塔里木大学 | Bacterial line for detecting pear pollen branch blight and construction method thereof |
| CN113337628A (en) * | 2021-06-08 | 2021-09-03 | 塔里木大学 | Nested PCR (polymerase chain reaction) reagent and detection system for detecting pear pollen and pear fire blight bacteria |
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