CN104250665A - Rastonia solanacearum 5# microspecies RTQ-PCR (real-time quantitative polymerase chain reaction) detection primer and method - Google Patents
Rastonia solanacearum 5# microspecies RTQ-PCR (real-time quantitative polymerase chain reaction) detection primer and method Download PDFInfo
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Abstract
The invention discloses a ralstonia solanacearum 5# microspecies RTQ-PCR detection primer and a method, and the sequence is RS72F:5'-ATGGATAAAGGGTTCGTGGTG-3'; RS312R:5'-CAGGCTCAGCGAGATTGC-3'. On the basis of the ralstonia solanacearum 5# microspecies different from specific fragments of other ralstonia solanacearum microspecies, a pair of specific primers are designed and screened, at the same time, by use of the primer, a fluorescence quantitative PCR method for detecting pseudomonas solanaeearum is designed, and the method has the characteristics of good specificity and high sensitivity, greatly shortens the desired detection time, and is suitable for early diagnosis of bacterial wilt, and the time from sample processing to obtaining results is only 4-5h.
Description
Technical field
The invention belongs to field of molecular detection, be specifically related to Plant Quarantine technology, particularly relate to a kind of be exclusively used in detect cause primer and the detection method of Ralstonia solanacearum No. 5 microspecies of mulberry tree bacterial wilt.
Background technology
Bacterial wilt (Bacterial wilt) distributes the widest, harm in the world the most seriously and the great bacterial disease of the most difficult control, listed in the list of reply bio-terrorism in 2002 by the U.S..This disease is widely current in the torrid zone, subtropics and Temperate Region in China, to comprising draft, section more than 50 300 various plants of shrub and Qiao Ben works the mischief (Annual review of phytopathology, 1991,29 (1): 65-87).Bacterial wilt can cause the highest underproduction 95% of farm crop, causes direct economic loss up to 50,000,000,000 dollars every year in the world, causes the extensive concern of people.
Bacterial wilt is caused by the withered Raul Salmonella of green grass or young crops (Ralstonia solanacearum), and Ralstonia solanacearum can be divided into 5 physiological strains (physiology No. 1 microspecies-physiology No. 5 microspecies) according to its host range; 5 biochemical types (biochemical type I-biochemical type V) (Acta Horticulturae can be divided into according to the difference of 3 kinds of disaccharide (maltose, lactose and cellobiose) and 3 kinds of hexanols (N.F,USP MANNITOL, sorbyl alcohol and sweet and pure) oxidation acid producing ability, 2005,695:127-136).China Lv Zhi waits by force the result of study of (2007) to show, the withered Raul Salmonella of green grass or young crops of infecting mulberry tree belongs to physiological strain 1 and 5, distinguishes by biochemical type, has biochemical type I, biochemical type III, biochemical type IV and biochemical type V etc.Be otherwise known as mulberry bacterial wilt (Mulberry bacterial wilt) " blight "; bacterialo wilt disease; popular name " pest mulberry ", " subcutaneous ulcer mulberry "; this sick oncoming force is violent, it is fast to spread; that one has destructive soil biography Plant Quarantine venereal disease evil to mulberry tree; novel species mulberry can be caused or Sang Dangnian death of growing into forest, become the important factor of restriction sericulture there Sustainable development.Bacterial wilt is once occur, be difficult to effectively effect a radical cure, and be in preclinical plant tissue from being difficult in appearance separate with healthy tissues, therefore, early diagnosis is fast and effectively for controlling spreading and avoiding the generation of heavy losses significant of bacterial wilt.
Early stage detection of pathogens means are the bases of improving Defect inspection efficiency fast, accurately, are also the prerequisites reducing biological control cost.The generation of mulberry bacterial wilt is that cause pipe obstruction, plant water transport is obstructed because its cause of disease is in the intrafascicular breeding of plant vasular, thus makes plant performance wilting symptom.And other diseases on mulberry tree, the mulberry epidemic disease that the blight caused as mulberry tree enterobacteria (Enterobacter mori), mulberry pseudomonas syringae (Pseudomonas syringae pv.mori) cause, often bring erroneous judgement to diagnosis, thus increase cost accounting.
For the detection of Ralstonia solanacearum, current topmost selective substratum and molecular biological method.Selective medium is TTC/TZC (the Tripheny tetrazolium chloride) substratum worked out in 1954 by Kelman the earliest, and Rica equals the SM-1 substratum that nineteen eighty-three obtains a kind of improvement on TZC basis.Ralstonia solanacearum cultivates 2-5d under 28 DEG C of conditions on substratum, there will be that typical mobility is strong, the tool white halo of irregular cycle pink bacterium colony.Elphinstone obtains SMSA substratum after equaling further improvement in 1996, the separable Ralstonia solanacearum detected in band soil bacteria.The withered isolation medium of green grass or young crops of current use is also based on above several substratum or improvement mostly.Plant indicator detection method is also detect one of method of pathogenic bacteria, but due to its cycle oversize, and by seasonal restriction, therefore this method is not suitable for the former bacterium of rapid detection bacterial wilt.
Molecular biological method mainly comprises making nucleic acid molecular hybridization and PCR method, Chen Yongfang etc. utilize RAPD technology screening to 1 distinctive DNA fragmentation of Ralstonia solanacearum, and according to its base sequence, devise PCR special primer, can infect other pathogenetic bacterias of potato for contrast, increase to sample DNA, result shows, only have Ralstonia solanacearum can amplify the DNA segment of 773bp, other contrast bacteriums all do not amplify these bands of a spectrum.The pathogenic bacterium to African eucalyptus and potato such as Fouche detect, use PCR-RFLP technology, carry out increasing with Hrp gene region thus determine biotype (the Journal of General Plant Pathology of ralstonia solanacearum, 2006,72 (6): 369 – 373).
Various method all can only be separated Ralstonia solanacearum in green grass or young crops withered kind of level above, can not distinguish withered No. 5 microspecies of green grass or young crops as mulberry tree pathogenic bacteria from microspecies level.Patent CN103602727A discloses a kind of detection primer sets of rapid detection Ralstonia solanacearum, detection kit and detection method, patent CN103468794A discloses a kind of tobacco bacterial wilt molecular detection primer, detection method and application, patent CN102465173A discloses the PCR method for detecting specificity of Ralstonia solanacearum No. 2 microspecies, and the another kind that patent CN101724692A discloses mulberry tree causes Auele Specific Primer and the diagnostic method of the bacterium mulberry tree enterobacteria of blight.Z.C.Pan etc. utilize suppress ion subtractive hybridization to establish, and difference that Ralstonia solanacearum No. 5 microspecies are different from standard cyan dry strain GMI1000 subtracts Sequence Library (Eur J Plant Pathol, 2013,137:377 – 391).But above method is all detect based on the regular-PCR of Auele Specific Primer, at present and have no and utilize the single amplified fragments special primer of Ralstonia solanacearum No. 5 microspecies to be combined the report detecting mulberry Ralstonia solanacearum with fluorescent quantitative PCR technique.
The present invention attempts using the method for quantitative fluorescent PCR to spend detection by quantitative absolutely to mulberry Ralstonia solanacearum, improve specificity and the sensitivity of detection, the erroneous judgement to mulberry tree disease on producing can be avoided, reduce the time needed for detection simultaneously, for mulberry bacterial wilt early diagnosis and take prophylactico-therapeutic measures to lay the foundation early.
Summary of the invention
The technical problem solved: the invention provides a kind of Ralstonia solanacearum No. 5 microspecies RTQ-PCR and detect primer and method, sets up a kind of fluorescent quantitative PCR detection method that is sensitive, stable, mulberry tree Ralstonia solanacearum reliably by design Ralstonia solanacearum No. 5 little species-specific primers.
Technical scheme: Ralstonia solanacearum No. 5 microspecies RTQ-PCR detect primer, it is characterized in that sequence is: RS72F:5'-ATGGATAAAGGGTTCGTGGTG-3'; RS312R:5'-CAGGCTCAGCGAGATTGC-3'.Utilize described primer to detect the RTQ-PCR method of Ralstonia solanacearum No. 5 microspecies, step comprises: (1) uses TM liquid nutrient medium to cultivate tested bacterium, and the tested bacterium DNA of extracting, preparation bacterium liquid sample and DNA sample; (2) use primer pair step 1) in bacterium liquid sample and DNA sample carry out pcr amplification, obtain amplified production; (3) according to step 2) in the presence or absence of amplified production can judge whether institute's test sample product are Ralstonia solanacearum No. 5 microspecies.
PCR reagent and step comprise: amplification reaction system: in 25 μ L reaction solutions, comprise 2 μ L bacterium liquid template or DNA profilings, 12.5 μ L 2 × Taq MasterMix: containing 2.5U Taq Polymerase/ μ L, 20mM Tris-HCl, 3mM MgCl
2, 500 μMs of dNTP and 100mM KCl, 10pmoles primer RS72F, 10pmoles primer RS312R; Pcr amplification program is 94 DEG C of denaturation 5min, 94 DEG C of sex change 15s, 53 DEG C of annealing 30s, and 72 DEG C extend 30s, 30 circulations, and last 72 DEG C extend 10min; Electrophoresis detection: after reaction terminates, get 10 μ L reaction product and detect on 1.5% agarose gel electrophoresis, and use gel imaging system to take pictures under ultraviolet lamp, according to the length judged result that has that it's too late of product.
Described Ralstonia solanacearum No. 5 microspecies RTQ-PCR detect the application of primer in detection by quantitative soil, tissue in pathogenic bacteria.Step comprises: the genomic dna of (1) extracting mulberry Ralstonia solanacearum, and carries out gradient dilution; (2) mulberry Ralstonia solanacearum is cultivated, and carry out gradient dilution; (3) with above-mentioned steps 1) and 2) in sample be that template carries out quantitative fluorescent PCR, and drawing standard curve; (4) amplification reaction system: in 20 μ L reaction systems, comprise 10 μ L SYBR Premix Ex Taq II (Tli RNaseH Plus) (2 ×), 0.8 μ L RS72F 10 μMs, 0.8 μ L RS312R 10 μMs, 0.4 μ L ROX Reference Dye (50 ×), 2.0 μ L templates; (5) amplification program: two-step approach amplification program: the first step: 95 DEG C of denaturation 10min; Second step: 95 DEG C of sex change 15s, 60 DEG C extend 31s, react 45 circulations.
Concrete grammar is: be different from the diversity sequence of other Ralstonia solanacearum microspecies by Ralstonia solanacearum No. 5 microspecies in search GenBank, Primer Premier 5 is utilized to design primer, by PCR and electrophoresis detection, obtain 1 pair of Ralstonia solanacearum No. 5 little species-specific primer RS72F/RS312R, after PCR reaction, product is detected by agarose gel electrophoresis, directly can judge whether testing sample is Ralstonia solanacearum No. 5 microspecies according to product clip size.Meanwhile, the RTQ-PCR detection method of the mulberry Ralstonia solanacearum utilizing the sensitivity of this design of primers higher.
For detecting the PCR method of Ralstonia solanacearum No. 5 microspecies, comprising:
(1) sample preparation for detecting
Use TM liquid nutrient medium to cultivate the control strain of No. 5 microspecies Ralstonia solanacearums, other microspecies Ralstonia solanacearums and other kind, reach about 10 to its concentration
8cfu/mL; Bioflux Biospin Genomic DNA Extraction Kit is used to extract sample gene group DNA.
(2) Auele Specific Primer for detecting
Primer sequence is:
RS72F：5'-ATGGATAAAGGGTTCGTGGTG-3'；
RS312R：5'-CAGGCTCAGCGAGATTGC-3'；
RS72F/312R primer pair can amplify the product that fragment length is 241bp specifically from Ralstonia solanacearum No. 5 microspecies bacterial strains;
Use the public specificity primer AU759/760 of all types Ralstonia solanacearum in contrast, its sequence is simultaneously:
AU759：5'-GTCGCCGTCAACTCACTTTCC-3'；
AU760：5'-GTCGCCGTCAGCAATGCGGAATCG-3'；
AU759/760 primer pair can amplify the product that fragment length is 282bp specifically from the Ralstonia solanacearum of all little types.
(3) pcr amplification reaction system
In 25 μ L reaction solutions, comprise 2 μ L bacterium liquid template or DNA profilings, 12.5 μ L 2 × Taq MasterMix are (containing 2.5U Taq Polymerase/ μ L, 20mM Tris-HCl, 3mM MgCl
2, 500 μMs of dNTP, 100mM KCl), 10pmoles upstream primer, 10pmoles downstream primer.
(4) pcr amplification program
94 DEG C of denaturation 5min, 94 DEG C of sex change 15s, 53 DEG C of annealing 30s, 72 DEG C extend 30s, 30 circulations, and last 72 DEG C extend 10min.
(5) qualification of PCR primer
Get 10 μ L reaction product to detect on 1.5% agarose gel electrophoresis, and use gel imaging system to take pictures under ultraviolet lamp, according to the length judged result that has that it's too late of product.
(6) preparation of RTQ-PCR standard model
Use the Elution Buffer in DNA extraction kit to carry out ten times of gradient dilutions the DNA of Ralstonia solanacearum No. 5 microspecies, make its final concentration be respectively 5.0 × 10
2ng/ μ L, 5.0 × 10
1ng/ μ L, 5.0 × 10
0ng/ μ L, 5.0 × 10
-1ng/ μ L, 5.0 × 10
-2ng/ μ L, 5.0 × 10
-3ng/ μ L, 5.0 × 10
-4ng/ μ L.
It is 1 × 10 that Ralstonia solanacearum No. 5 microspecies bacterium liquid of pure culture are mixed with concentration gradient
8cfu/mL, 5 × 10
7cfu/mL, 2 × 10
7cfu/mL, 1 × 10
7cfu/mL, 5 × 10
6cfu/mL, 2 × 10
6cfu/mL, 1 × 10
6cfu/mL, 1 × 10
5cfu/mL, 1 × 10
4cfu/mL, 1 × 10
3cfu/mL, 1 × 10
2cfu/mL cell suspending liquid.
(7) RTQ-PCR reaction system
In 20 μ L reaction systems, comprise 10 μ L SYBR Premix Ex Taq II (Tli RNaseH Plus) (2 ×), 0.8 μ L RS72F (10 μMs), 0.8 μ L RS312R (10 μMs), 0.4 μ L ROX Reference Dye (50 ×), 2.0 μ L templates.
(8) RTQ-PCR amplification program
Two-step approach amplification program: the first step: 95 DEG C of denaturation 10min; Second step: 95 DEG C of sex change 15s, 60 DEG C extend 31s, react 45 circulations.
(9) drafting of typical curve
Respectively with the LOG value of the LOG value of DNA concentration and bacterial concentration for X-coordinate, corresponding CT value (cycle number experienced when the fluorescent signal in each reaction tubes arrives the threshold value of setting) is ordinate zou drawing standard curve.
Beneficial effect
The present invention is based on Ralstonia solanacearum No. 5 microspecies be different from the specific fragment design of other Ralstonia solanacearum microspecies and screened a pair Auele Specific Primer, utilize the fluorescent quantitative PCR detection method of this design of primers mulberry Ralstonia solanacearum simultaneously, have the advantages that specificity is good, highly sensitive, and substantially reducing detection required time, sample preparation only needs 4 ~ 5h to obtaining a result.Be applicable to the early diagnosis of mulberry bacterial wilt.
Accompanying drawing explanation
Fig. 1 a primer RS72F/312R is to the amplification figure of Ralstonia solanacearum:
M:DNA molecular weight standard Marker (600bp, 500bp, 400bp, 300bp, 200bp, 100bp); 1: Ralstonia solanacearum No. 5 microspecies (GIM1.73); 2: Ralstonia solanacearum No. 1 microspecies (GIM1.74); 3: Ralstonia solanacearum No. 2 microspecies (GIM1.76); 4: Ralstonia solanacearum No. 3 microspecies (RS-3); 5: blue or green withered reference culture GMI1000 (No. 3 microspecies); 6: blue or green withered No. 4 microspecies (GIM1.71); CK: blank TM nutrient solution.It is primer AU759/760 amplification on the left of dotted line; It is primer RS72F/312R amplification on the right side of dotted line.
Fig. 1 b primer RS72F/312R is to the amplification figure of other kind control strains:
M:DNA molecular weight standard Marker (600bp, 500bp, 400bp, 300bp, 200bp, 100bp); 1: Ralstonia solanacearum No. 5 microspecies (GIM1.73); 7: mulberry pseudomonas syringae (M4-13); 8: mulberry enterobacteria (JX-6); 9: intestinal bacteria (DCG); 10: Pseudomonas fluorescens (A-S1); 11: pseudomonas putida (ECJ); 12: subtilis (KCY); 13: bacillus cereus (LYY); 14: bacillus thuringiensis (SYJ); 15: streptococcus aureus (PTQ); 16: tetrads (SLQ); 17: tangerine green trichoderma (JLM); 18: yellow streptomycete (HLM).
Fig. 2 a is with CT value for ordinate zou, and the LOG value of DNA concentration is X-coordinate drafting fluorescent quantitation absolute quantitation canonical plotting:
DNA concentration is 10
-4~ 10
2within the scope of ng/ μ L, its LOG value and CT value are good linear relationship, Y=-2.747X+21.834, R
2=0.993.
Fig. 2 b with CT value for ordinate zou, the fluorescent quantitation absolute quantitation canonical plotting that the LOG value of bacterial concentration is drawn for X-coordinate:
Bacterial concentration is 10
3~ 10
7within the scope of cfu/mL, its LOG value and CT value are good linear relationship, Y=-3.418X+45.447, R
2=0.998.
Embodiment
Embodiment 1
(1) to participate in the experiment the cultivation of bacterial strain and DNA extraction
The 18 strain bacterial strains of participating in the experiment comprise a strain mulberry Stalk Rot GIM1.73 (blue or green withered No. 5 microspecies, its pathogenic on mulberry tree through the conspicuous rule checking of section), Ralstonia solanacearum (the GIM1.74 of 5 other microspecies of strain, GIM1.76, GIM1.71, RS-3, GMI1000), wherein GIM1.73, GIM1.74 (blue or green withered No. 1 microspecies), GIM1.76 (blue or green withered No. 2 microspecies), GIM1.71 (blue or green withered No. 4 microspecies) are purchased from Guangdong Province's Culture Collection (GIMCC); RS-3 (blue or green withered No. 3 microspecies) is provided by plant protection institute of Agricultural University Of Nanjing; GMI1000 (blue or green withered No. 3 microspecies) purchased from American Type culture collecting center (ATCC).Control strain (M4-13, JX-6, the DCG of other 12 kinds of withered genus of non-green grass or young crops, A-S1, ECJ, KCY, LYY, SYJ, PTQ, SLQ, JLM, HLM) preserved by this laboratory, wherein M4-13 is mulberry epidemic disease pathogenic bacteria (Pseudomonas syringae pv.mori), JX-6 is mulberry bacterial wilt disease pathogenic bacteria (Enterobacter mori), and all the other are then some Pseudomonas common in soil.By inoculation to (glucose 5g, peptone 10g, caseinhydrolysate 1g, distilled water 1000mL) in TM liquid nutrient medium, 28 DEG C, cultivate 2 days.Biospin Genomic DNA Extraction Kit is used to extract the DNA of bacterial strain of participating in the experiment.
(2) synthesis of primer
RS72F:5'-ATGGATAAAGGGTTCGTGGTG-3'；
RS312R:5'-CAGGCTCAGCGAGATTGC-3'；
AU759:5'-GTCGCCGTCAACTCACTTTCC-3'；
AU760:5'-GTCGCCGTCAGCAATGCGGAATCG-3'；
Primer is synthesized by Shanghai Sheng Gong company.
(3) pcr amplification reaction
1. in 25 μ L reaction systems, comprise 2 μ L DNA profilings, 12.5 μ L 2 × Taq MasterMix are (containing 2.5U Taq Polymerase/ μ L, 20mM Tris-HCl, 3mM MgCl
2, 500 μMs of dNTP, 100mM KCl), 10pmoles primer RS72F, 10pmoles primer RS312R.
2. in 25 μ L reaction systems, comprise 2 μ L DNA profilings, 12.5 μ L 2 × Taq MasterMix are (containing 2.5U Taq Polymerase/ μ L, 20mM Tris-HCl, 3mM MgCl
2, 500 μMs of dNTP, 100mM KCl), 10pmoles primer AU759,10pmoles primer AU760.
(4) pcr amplification program
94 DEG C of denaturation 5min, 94 DEG C of sex change 15s, 53 DEG C of annealing 30s, 72 DEG C extend 30s, 30 circulations, and last 72 DEG C extend 10min.
(5) qualification of PCR primer
Get 10 μ L reaction product to detect on 2% agarose gel electrophoresis, and use gel imaging system to take pictures under ultraviolet lamp, according to the length judged result that has that it's too late of product.
(6) preparation of quantitative fluorescent PCR template
1. use the Elution Buffer in DNA extraction kit to carry out ten times of gradient dilutions the DNA of Ralstonia solanacearum No. 5 microspecies, make its final concentration be respectively 5.0 × 10
2ng/ μ L, 5.0 × 10
1ng/ μ L, 5.0 × 10
0ng/ μ L, 5.0 × 10
-1ng/ μ L, 5.0 × 10
-2ng/ μ L, 5.0 × 10
-3ng/ μ L, 5.0 × 10
-4ng/ μ L.
2. use nutrient solution to carry out gradient dilution Ralstonia solanacearum No. 5 microspecies bacterium liquid, make its final concentration be respectively 5.0 × 10
7cfu/mL; 2.0 × 10
7cfu/mL; 1.0 × 10
7cfu/mL; 5.0 × 10
6cfu/mL; 2.0 × 10
6cfu/mL; 1.0 × 10
6cfu/mL; 1.0 × 10
5cfu/mL; 1.0 × 10
4cfu/mL; 1.0 × 10
3cfu/mL.
(7) fluorescent quantitative PCR system
In 20 μ L reaction systems, comprise 10 μ L SYBR Premix Ex Taq II (Tli RNaseH Plus) (2 ×), 0.8 μ L RS72F (10 μMs), 0.8 μ L RS312R (10 μMs), 0.4 μ L ROX Reference Dye (50 ×), 2.0 μ L templates.Each sample carries out 5 times to be repeated.
(8) fluorescent quantitative PCR program
Two-step approach amplification program:
The first step: 95 DEG C of denaturation 10min; Second step: 95 DEG C of sex change 15s, 60 DEG C extend 31s, react 45 circulations.
(9) DNA is the absolute quantitation typical curve of template
With CT value for ordinate zou, the LOG value of DNA concentration is X-coordinate drawing standard curve.
(10) bacterium liquid is the absolute quantitation typical curve of template
With CT value for ordinate zou, the LOG value of bacterial concentration is X-coordinate drawing standard curve.
Result of implementation
The primer RS72F/312R utilizing the present invention to design, with the Ralstonia solanacearum of Ralstonia solanacearum No. 5 microspecies and other microspecies for template, carry out pcr amplification, result as shown in Figure 1a, the special primer AU759/760 in Ralstonia solanacearum kind level is used on the left of dotted line, often kind of Ralstonia solanacearum all can amplify the fragment of 282bp, kind of level proves bacterial strain of participating in the experiment is Ralstonia solanacearum; Use the primer RS72F/312R that the present invention designs on the right side of dotted line, only have blue or green withered No. 5 microspecies can be amplified out the specific fragment of 241bp, prove that primer of the present invention has specificity in Ralstonia solanacearum microspecies level with this.
The present invention is utilized to design, with the contrast bacterium of Ralstonia solanacearum No. 5 microspecies and other kinds for template, carry out pcr amplification, result as shown in Figure 1 b, only have Ralstonia solanacearum No. 5 microspecies can be amplified out the specific fragment of 241bp, illustrate that primer of the present invention has Idiotype in kind of level.
The little species specific DNA sequence dna of No. 5, the Ralstonia solanacearum amplified by primer RS72F/312R:
ATGGATAAAGGGTTCGTGGTGCGGCACATCAAATTTGATATGTGCCGGATGTGCCGTAGTTGGCCGGCATTGGCTACCATAATGCTGAGTGCTTGTTGCGGCACATTCTGAATTGCGAGGCGAGGGGGGCGATTGAGCTATTCCACTGAGAACATTCTCCCGAGTGGCGTTACGGTTGCGCAGGTCGTCGCATTCGCCAAGCTGCTCGGCTACACGCCCGGCGGCAATCTCGCTGAGCCTG
Utilize primer RS72F/312R to design fluorescence quantifying PCR method and carry out absolute quantitation detection to Ralstonia solanacearum No. 5 microspecies DNA, as shown in Figure 2 a, DNA concentration is 10
-4~ 10
2within the scope of ng/ μ L, its LOG value and CT value are good linear relationship, Y=-2.747X+21.834, R
2=0.993.
Utilize primer RS72F/312R to design fluorescence quantifying PCR method and carry out absolute quantitation detection to Ralstonia solanacearum No. 5 microspecies bacterium liquid, as shown in Figure 2 b, bacterial concentration is 10
3~ 10
7within the scope of cfu/mL, its LOG value and CT value are good linear relationship, Y=-3.418X+45.447, R
2=0.998.
The solubility curve display of quantitative fluorescent PCR reaction, there is single peak at 87.9 DEG C in product, the interference without nonspecific amplification or primer dimer in reaction is described.
Above-mentioned embodiment is only and patent of the present invention is described, but can not limit protection scope of the present invention with this.Change and the replacement of any unsubstantiality that those skilled in the art does on basis of the present invention all belong to the present invention's scope required for protection.
SEQUENCE LISTING
<110> Jiangsu University of Science and Technology
<120> Ralstonia solanacearum No. 5 microspecies RTQ-PCR detect primer and method
<130>
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213> artificial sequence
<400> 1
atggataaag ggttcgtggt g 21
<210> 2
<211> 18
<212> DNA
<213> artificial sequence
<400> 2
caggctcagc gagattgc 18
<210> 3
<211> 21
<212> DNA
<213> artificial sequence
<400> 3
gtcgccgtca actcactttc c 21
<210> 4
<211> 24
<212> DNA
<213> artificial sequence
<400> 4
gtcgccgtca gcaatgcgga atcg 24
<210> 5
<211> 241
<212> DNA
<213> Ralstonia solanacearum
<400> 5
atggataaag ggttcgtggt gcggcacatc aaatttgata tgtgccggat gtgccgtagt 60
tggccggcat tggctaccat aatgctgagt gcttgttgcg gcacattctg aattgcgagg 120
cgaggggggc gattgagcta ttccactgag aacattctcc cgagtggcgt tacggttgcg 180
caggtcgtcg cattcgccaa gctgctcggc tacacgcccg gcggcaatct cgctgagcct 240
g 241
Claims (5)
1. Ralstonia solanacearum No. 5 microspecies RTQ-PCR detect primer, it is characterized in that sequence is:
RS72F：5'-ATGGATAAAGGGTTCGTGGTG-3'
RS312R：5'-CAGGCTCAGCGAGATTGC-3'。
2. utilize primer described in claim 1 to detect the RTQ-PCR method of Ralstonia solanacearum No. 5 microspecies, it is characterized in that step comprises:
1) TM liquid nutrient medium is used to cultivate tested bacterium, and the tested bacterium DNA of extracting, preparation bacterium liquid sample and DNA sample;
2) use primer pair step 1) in bacterium liquid sample and DNA sample carry out pcr amplification, obtain amplified production;
3) according to step 2) in the presence or absence of amplified production can judge whether institute's test sample product are Ralstonia solanacearum No. 5 microspecies.
3. RTQ-PCR method according to claim 2, is characterized in that PCR reagent and step comprise:
1) amplification reaction system: comprise 2 μ L bacterium liquid template or DNA profilings in 25 μ L reaction solutions, 12.5 μ L 2 × Taq MasterMix: containing 2.5U Taq Polymerase/ μ L, 20mM Tris-HCl, 3mM MgCl
2, 500 μMs of dNTP and 100mM KCl, 10pmoles primer RS72F, 10pmoles primer RS312R;
2) pcr amplification program is 94 DEG C of denaturation 5min, 94 DEG C of sex change 15s, 53 DEG C of annealing 30s, and 72 DEG C extend 30s, 30 circulations, and last 72 DEG C extend 10min;
3) electrophoresis detection: after reaction terminates, get 10 μ L reaction product and detect on 1.5% agarose gel electrophoresis, and use gel imaging system to take pictures under ultraviolet lamp, according to the length judged result that has that it's too late of product.
4. No. 5 microspecies RTQ-PCR of Ralstonia solanacearum described in claim 1 detect the application of primer in detection by quantitative soil, tissue in pathogenic bacteria.
5. application according to claim 4, is characterized in that step comprises:
1) genomic dna of extracting mulberry Ralstonia solanacearum, and carry out gradient dilution;
2) mulberry Ralstonia solanacearum is cultivated, and carry out gradient dilution;
3) with above-mentioned steps 1) and 2) in sample be that template carries out quantitative fluorescent PCR, and drawing standard curve;
4) amplification reaction system:
In 20 μ L reaction systems, comprise 10 μ L SYBR Premix Ex Taq II (Tli RNaseH Plus) (2 ×), 0.8 μ L RS72F 10 μMs, 0.8 μ L RS312R 10 μMs, 0.4 μ L ROX Reference Dye (50 ×), 2.0 μ L templates;
5) amplification program
Two-step approach amplification program:
The first step: 95 DEG C of denaturation 10min; Second step: 95 DEG C of sex change 15s, 60 DEG C extend 31s, react 45 circulations.
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| CN114686609A (en) * | 2022-03-30 | 2022-07-01 | 中国农业科学院农业资源与农业区划研究所 | Specific primer for identifying ralstonia solanacearum and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101250580A (en) * | 2008-04-11 | 2008-08-27 | 重庆大学 | Primer, probe and real-time fluorescent PCR reagent case for detecting tobacco ralstonia solanacearum |
| CN102465173A (en) * | 2010-11-08 | 2012-05-23 | 中国农业科学院植物保护研究所 | Specific PCR detection method of Ralstonia solanacearum race 2 |
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| CN101250580A (en) * | 2008-04-11 | 2008-08-27 | 重庆大学 | Primer, probe and real-time fluorescent PCR reagent case for detecting tobacco ralstonia solanacearum |
| CN102465173A (en) * | 2010-11-08 | 2012-05-23 | 中国农业科学院植物保护研究所 | Specific PCR detection method of Ralstonia solanacearum race 2 |
Non-Patent Citations (1)
| Title |
|---|
| 潘哲超: ""植物青枯菌遗传多样性及致病力分化研究"", 《中国博士学位论文全文数据库 农业科技辑》, 15 October 2010 (2010-10-15) * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114686609A (en) * | 2022-03-30 | 2022-07-01 | 中国农业科学院农业资源与农业区划研究所 | Specific primer for identifying ralstonia solanacearum and application thereof |
| CN114686609B (en) * | 2022-03-30 | 2024-03-01 | 中国农业科学院农业资源与农业区划研究所 | Specific primer for identifying tomato bacterial wilt and application thereof |
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