CN106480227A - A kind of Citrullus vulgariss Acidovorax avenae subsp nested PCR detection method - Google Patents
A kind of Citrullus vulgariss Acidovorax avenae subsp nested PCR detection method Download PDFInfo
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Abstract
The present invention provides a kind of Citrullus vulgariss Acidovorax avenae subsp nested PCR detection method.This method is passed through to design Citrullus vulgariss Acidovorax avenae subsp VapB specific primer, with sample DNA as template, carry out the amplification of first round PCR using above-mentioned universal primer, again with first round PCR primer as template, carry out the second wheel PCR amplification using two couples of Citrullus vulgariss Acidovorax avenae subsp specific primer VapB 1F/VapB 1R and VapB 1F/VapB 2R, amplified production carries out electrophoresis detection, and the specific band if there is 175bp is it is determined that the pathogen being detected is Citrullus vulgariss Acidovorax avenae subsp.Instant invention overcomes the shortcoming of the method such as Biology identification, Enzyme-multiplied immune technique and Standard PCR, provide quick, sensitive, the special Citrullus vulgariss Acidovorax avenae subsp detection method of specificity, can be used for the early diagnosiss of Citrullus vulgariss Acidovorax avenae subsp, the prevention to this disease and timely preventing and treating tool are of great significance.
Description
Technical field
The present invention relates to specialized plant pathogen PCR detection method is and in particular to a kind of Citrullus vulgariss Acidovorax avenae subsp
（Acidovorax citrulli）Nested PCR detection method, belongs to biological technical field.
Background technology
Melon bacterial fruit blotch (Bacterial fruit blotch, BFB) is weight on the ground family crops such as western Fructus Melo
The bacteriosises wanted.Melon bacterial fruit blotch is a kind of typical seed-borne disease, bites sour bacterium (Acidovorax by Citrullus vulgariss
Citrulli) cause.This condition of disease infects blade, fruit and seed, has a strong impact on the quality and yield of fruit, to melon produce and
Related industry constitutes great threat.China from after the nineties in last century reported first, in Xinjiang, Ningxia, Hebei, interior
The ground such as Mongolia, Beijing, Fujian, Hainan, Shandong, Jilin, Hunan occur in a large number.Melon bacterial fruit blotch cause land for growing field crops Citrullus vulgariss and
The Fructus Melo underproduction is even had no harvest, and produces to western Fructus Melo and brings massive losses, the current generation of this disease is in rising trend.Therefore build
The fast and accurately detection technique that sour bacterium bitten by vertical Citrullus vulgariss is the Important Action preventing this disease from occurring.
Traditional detection of pathogens technology is to separate and further cultivated thus obtaining the base of corresponding pathogen
The species of pathogen on plinth, is judged by morphological observation and section's He Shi rule.But, separate, culture all processes are often
All very elapsed time and need substantial amounts of human resources.Although immunoserology authentication method is sensitive, high specificity,
Preparation process takes time and effort.In recent years, the molecular detecting method with round pcr as representative had obtained rapid development and had applied,
PCR rapid molecular detection method generally has high precision, quickly, effective feature, and round pcr is relatively special, sensitive.And
Sleeve type PCR detection method has than traditional passing PCR molecular detecting method more accurately sensitivity and higher specificity.Cause
This, set up Citrullus vulgariss Acidovorax avenae subsp rapid molecular detection system, in Citrullus vulgariss premorbid or morbidity early stage, disease plant can be entered
Row quick and precisely detects, the timely forecast to Citrullus vulgariss fruit spot disease, and controls the disease of its generation, the melon industry of protection China
It is significant.
Content of the invention
It is an object of the invention to provide a kind of Citrullus vulgariss Acidovorax avenae subsp nested PCR detection method, it has high specificity, spirit
The high feature of sensitivity, can be used for the early diagnosiss of field Citrullus vulgariss fruit blotch.This technology causes disease to show for Citrullus vulgariss Acidovorax avenae subsp
Early monitoring before disease and timely preventing and treating tool are of great significance.
The purpose of the present invention can be achieved through the following technical solutions：
Citrullus vulgariss Acidovorax avenae subsp nested PCR detection method, comprises the following steps：
Step A, extracting genome DNA：Citrullus vulgariss Acidovorax avenae subsp genomic DNA or morbidity plant tissue DNA are extracted using CTAB method；
Step B, first round PCR amplification：The DNA sample being obtained with step A as template, using Citrullus vulgariss Acidovorax avenae subsp VapB-like
Toxin-resistant gene primer VapB -1F/ VapB -1R obtains first round PCR primer through PCR amplification.
Primer sequence：VapB-1F：5’-GGGCAATAGCTTGGCGGTTCG-3’；
VapB -1R：5’-CATTGGCCTCGTCCCGGTTGA-3’；
PCR reaction system 25 μ l：Genomic DNA 1 μ l, 10 × PCR reaction buffer is (containing Mg2+) 2.5 μ l, each 2.5 mmol/
L dNTPs mixed liquor 2 μ l, DNA Taq polymerase 0.5 μ l (5U/ μ l), VapB-1F primer 1 the μ l, 10 μ of 10 μm of ol/L
The VapB-1R primer 1 μ l of mol/L, sterilizing ultra-pure water complements to 25 μ l；PCR amplification program：94 DEG C of denaturations 4min, 94 DEG C of changes
Property 30sec, 55 DEG C annealing 30 sec, 72 DEG C extension 45sec, totally 30 circulation；72 DEG C of extension 10min；4 DEG C of preservations；
Step C, the second wheel PCR amplification：With the diluent of first round pcr amplification product or first round pcr amplification product as mould
Plate, obtains the second wheel pcr amplification product using specific primer VapB-2F/VapB-2R through PCR amplification.
Primer sequence：VapB-2F：5’-GCGAACGTAGTCGAAGCGCTCG-3’；
VapB-2R：5’-TGGCCTCGTCCCGGTTGAACT-3’；
PCR reaction system 25 μ l：Diluent 1 the μ l, 10 × PCR of first round pcr amplification product or first round pcr amplification product
Reaction buffer is (containing Mg2+) 2.5 μ l, each 2.5 mmol/L dNTPs mixed liquor 2 μ l, DNA Taq polymerase 0.5 μ l (5U/ μ
L), the VapB-2F primer 1 μ l of 10 μm of ol/L, the VapB-2R primer 1 μ l of 10 μm of ol/L, sterilizing ultra-pure water complements to 25 μ l；
PCR amplification program：94 DEG C of denaturations 4min, 94 DEG C of degeneration 30sec, 55 DEG C of annealing 30 sec, 72 DEG C of extension 40sec,
Totally 30 circulations；72 DEG C of extension 8min；4 DEG C of preservations；
Step D, pcr amplification product detection：8 μ l second are taken to take turns pcr amplification product, using 1% agarose gel, in 80 V electricity
Pressure electrophoresis 30min, is placed in afterwards in EB and takes out after dyeing 20min, observe and take pictures under gel imaging system, if there is
The specific band of 175bp is it is determined that the pathogenic bacteria being detected is Citrullus vulgariss Acidovorax avenae subsp.
The present invention has following technical advantage and good effect
1st, sensitivity is high：Detection sensitivity of the present invention is 0.5 fg Citrullus vulgariss Acidovorax avenae subsp genomic DNA, more conventional PCR sensitivity
Improve 1000 times.
2nd, high specificity：The present invention targetedly detects Citrullus vulgariss Acidovorax avenae subsp, can be from Citrullus vulgariss Acidovorax avenae subsp genome
Amplify the single goal band of 175bp, and oat acidovorax avenae Herba bromi japonici subspecies, xanthomonas campestrises Radix Malvae sylvestriss pvs oryzae and oryzicola, fourth
Fragrant pseudomonass, rhubarb horsetails Erwinia sp, Fang Shi clavibacter, solution Erzvinia amylovora etc. 30 participate in the experiment strain gene group DNA and
Equal no amplified production in watermelon plant tissue DNA, is disturbed little, accuracy rate is high by other mushrooms.
3rd, practicality is good：The present invention can be used for Defect inspection when Citrullus vulgariss fruit blotch incubation period or susceptible initial stage.To disease
The early diagnosiss of evil and timely preventing and treating report tool are of great significance.
4th, easy and simple to handle quick：Application the inventive method, carries out pathogenic bacteria DNA to the plant tissue with Citrullus vulgariss Acidovorax avenae subsp and carries
Take, PCR amplification and routine sepharose electrophoresis after get final product result of determination, restricted enzyme enzyme need not be carried out to amplified production
Cut.Typically whole detection process can complete in 8 hours.
Brief description
Fig. 1 is the specificity verification result of Citrullus vulgariss Acidovorax avenae subsp VapB specific primer.Wherein, M：DL2000 DNA
Marker, 1 is positive control, and 2-3 is the DNA of the Citrullus vulgariss Acidovorax avenae subsp of different sources ground, 6：Oat acidovorax avenae Herba bromi japonici subspecies, 7：
Xanthomonas campestrises Radix Malvae sylvestriss pvs oryzae and oryzicola, 8：Pseudomonas syringae, 9：Rhubarb horsetails Erwinia sp, 10：Fang Shi clavibacter, 11：
Solution Erzvinia amylovora, 12：Negative control.
Fig. 2 is Citrullus vulgariss Acidovorax avenae subsp VapB susceptiveness the result.Wherein, M is DL2000 DNA Marker, 1-9：5.0
Ng/ 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, 5.0 × 10-
The variable concentrations Citrullus vulgariss Acidovorax avenae subsp DNA of 5ng/ μ L, 5.0 × 10-6ng/ μ L, 5.0 × 10-7ng/ μ L, 5.0 × 10-8ng/ μ L.
Tu3Shi incidence tissue VapB specific detection result.Wherein, M is 2000 DNA marker, 1,8：Health plant
Tissue, 2：Positive control, 3-4：Invasioning delitescence plant tissue, 5-7,9：It is respectively morbidity plant tissue.
Specific embodiment
It is specifically described the present invention with reference to embodiments.
The nest-type PRC detection of embodiment 1 Citrullus vulgariss Acidovorax avenae subsp
1. sample DNA extracts
1.1 extraction thallus DNAs
Citrullus vulgariss Acidovorax avenae subsp is inoculated in LB fluid medium, 28 DEG C, 200rpm incubated overnight.Extract bacterial gene with CTAB method
Group DNA, concrete operations are as follows：
1) take 1.5ml culture in 2.0ml centrifuge tube, 12000rpm is centrifuged 2min；
2) abandon supernatant, add 567 μ l TE buffer Eddy diffusion thalline, add 30 μ l 10%SDS solution and 3 μ l 20mg/
The E.C. 3.4.21.64 of ml, gently mixes, and incubates 1h in 37 DEG C；
3) add 100 μ l 5mol/L NaCl solution, fully mix, add 80ul CTAB/NaCl solution, 65 after mixing
DEG C incubate 10min；
4) add the phenol/chloroform/isoamyl alcohol of 800 μ l（Three's volume ratio is 25:24:1）, 12000rpm centrifugation after mixing
5min；
5) take supernatant to proceed in new 1.5ml centrifuge tube, add 0.6-0.8 times of volume isopropanol, be gently mixed until
DNA precipitates, and 12000rpm is centrifuged 15 min；
6) abandon supernatant, add 1000 μ l dehydrated alcohol, mix, 12000 rpm are centrifuged 5 min；
7) abandon supernatant, superclean bench dries naturally, add 60 μ l ddH2O dissolving DNA, standby in -20 DEG C of preservations
With.
1.2 extraction Plant tissue samples DNA to be checked
1) weigh Fructus Litchi incidence tissue 100mg, add liquid nitrogen grinding to become powder, proceed in 2.0ml centrifuge tube；
2) add 1000 μ l CTAB/NaCl solution, in 65 DEG C of water-bath 30min, often each 10min mixes once, 12000rpm from
Heart 10min；
3) take supernatant 800 μ l in 2.0ml centrifuge tube, add isopyknic phenol/chloroform/isoamyl alcohol（Three's volume ratio
For 25:24:1）, 12000rpm centrifugation 5min after mixing；
4) take supernatant 750 μ l in 2.0ml centrifuge tube, add isopyknic chloroform/aqueous isopropanol（Volume ratio is 24:1）,
Mix, 12000rpm is centrifuged 5min；
5) take supernatant 500 μ l in 2.0ml centrifuge tube, add 3mol/L NaAC solution (PH=5.2) of 50 μ l pre-coolings, mix
Even, add the isopropanol of 50 μ l pre-coolings, mix, place 30min in -20 DEG C；
6) 4 DEG C of 10000rpm are centrifuged 5min, abandon supernatant, add 75% absolute ethanol washing of 1000 μ l pre-coolings to precipitate 2 times,
With absolute ethanol washing 1 time, superclean bench dries naturally, add 60 μ l ddH2O dissolving DNA, preserves in -20 DEG C
Standby.
2. first round PCR amplification
With the DNA sample of extraction as template, using Citrullus vulgariss Acidovorax avenae subsp VapB-like toxin-resistant gene primer VapB -1F/
VapB -1R obtains first round PCR primer through PCR amplification.
Primer sequence：VapB-1F：5’-GGGCAATAGCTTGGCGGTTCG-3’
VapB -1R：5’-CATTGGCCTCGTCCCGGTTGA-3’
PCR reaction system 25 μ l：Genomic DNA 1 μ l, 10 × PCR reaction buffer is (containing Mg2+) 2.5 μ l, each 2.5 mmol/
L dNTPs mixed liquor 2 μ l, DNA Taq polymerase 0.5 μ l (5U/ μ l), VapB-1F primer 1 the μ l, 10 μ of 10 μm of ol/L
The VapB-1R primer 1 μ l of mol/L, sterilizing ultra-pure water complements to 25 μ l；PCR amplification program：94 DEG C of denaturations 4min, 94 DEG C of changes
Property 30sec, 55 DEG C annealing 30 sec, 72 DEG C extension 45sec, totally 30 circulation；72 DEG C of extension 10min；4 DEG C of preservations；
2. the second wheel PCR amplification：With the diluent of first round pcr amplification product or first round pcr amplification product as template,
Second wheel pcr amplification product is obtained through PCR amplification using specific primer VapB-2F/VapB-2R.
Primer sequence：VapB-1F：5’-GCGAACGTAGTCGAAGCGCTCG-3’
VapB-2R：5’-TGGCCTCGTCCCGGTTGAACT-3’
PCR reaction system 25 μ l：Diluent 1 the μ l, 10 × PCR of first round pcr amplification product or first round pcr amplification product
Reaction buffer is (containing Mg2+) 2.5 μ l, each 2.5 mmol/L dNTPs mixed liquor 2 μ l, DNA Taq polymerase 0.5 μ l (5U/ μ
L), the VapB-2F primer 1 μ l of 10 μm of ol/L, the VapB-2R primer 1 μ l of 10 μm of ol/L, sterilizing ultra-pure water complements to 25 μ l；
PCR amplification program：94 DEG C of denaturations 4min, 94 DEG C of degeneration 30sec, 55 DEG C of annealing 30 sec, 72 DEG C of extension 40sec,
Totally 30 circulations；72 DEG C of extension 8min；4 DEG C of preservations；
4. pcr amplification product detection
Take 8 μ l second to take turns pcr amplification product, using 1% agarose gel, electrophoresis 30min under 80 V voltages, be placed in afterwards
Take out after dyeing 20min in EB, observe under gel imaging system and take pictures, the specific band if there is 175bp is it is determined that institute
The pathogenic bacteria of detection is Citrullus vulgariss Acidovorax avenae subsp.
Embodiment 2 nest-type PRC sensitivity test
The DNA being 50 ng/ μ L by the Citrullus vulgariss Acidovorax avenae subsp initial concentration of extraction using the method for 10 times of dilutions, is diluted to respectively
5.0 ng/ 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, 5.0 × 10- 5Ng/ μ L, 5.0 × 10-6Ng/ μ L, 5.0 × 10-7Ng/ μ L, 5.0 × 10-8Ng/ μ L, 5.0 × 10-9Ng/ μ L variable concentrations are covered
Formula PCR.The DNA 1 μ l taking each concentration respectively, as template, by the step of embodiment 1, carries out the first round and the second wheel two successively
Wheel PCR amplification.See Fig. 2 through the electrophoresis result that two-wheeled PCR expands products therefrom.Nest-type PRC detection sensitivity of the present invention is 0.5
Fg Citrullus vulgariss Acidovorax avenae subsp genomic DNA, more conventional PCR sensitivity improves 100 times.
Embodiment 3：The detection of incidence tissue's Chinese and Western melon Acidovorax avenae subsp
1. sample collecting：Plant tissue samples pick up from Fujian Foochow different regions supermarket and the market of farm produce.
2.DNA extracts and detects
Morbidity plant tissue adopts NaOH rapid cleavage method to extract Citrullus vulgariss Acidovorax avenae subsp DNA.Detailed process is as follows：(1) by Citrullus vulgariss
Tissue is cleaned, dries, clip site of pathological change；(2) press 1mg disease leaf and add 10 μ l（0.5mol/L NaOH, 0.5%PVP）Metering, will
Tissue is fully milled to paste, and is centrifuged 5min in 12,000g centrifuge；(3) take supernatant 20 μ l and isopyknic 0.1 mol/L's
Tris-HCl（pH8.0）Mixing；(4) dilute 10 times, 100 times, 1000 times of liquid, take respectively 1 μ l stock solution, 10 times, 100 times, 1000
Times liquid is expanded as pcr template.
Carry out the first round and the second wheel two-wheeled PCR amplification by the step 2 and step 3 of embodiment 1 successively.After two-wheeled amplification
Product electrophoresis result is shown in Fig. 3.Wherein, M is 2000 DNA marker, 1：Health plant is organized, and 2：Positive control, 3-7：Respectively
For plant tissue of falling ill, 8-11：It is respectively invasioning delitescence plant tissue.As seen from Figure 3, using nest-type PRC to 35 samples
The purpose band of 175bp is all amplified, health tissues and negative control all no amplified productions when being detected.This shows, this
The bright Defect inspection that can be used for when Citrullus vulgariss fruit blotch incubation period or susceptible initial stage.
The foregoing is only presently preferred embodiments of the present invention, all impartial changes done according to scope of the present invention patent with
Modify, all should belong to the covering scope of the present invention.
SEQUENCE LISTING
<110>Inst. of Plant Protection, fujian Academy of Agricultural Science
<120>A kind of Citrullus vulgariss Acidovorax avenae subsp nested PCR detection method
<130> 4
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence
<400> 1
gggcaatagc ttggcggttc g 21
<210> 2
<211> 21
<212> DNA
<213>Artificial sequence
<400> 2
cattggcctc gtcccggttg a 21
<210> 3
<211> 22
<212> DNA
<213>Artificial sequence
<400> 3
gcgaacgtag tcgaagcgct cg 22
<210> 4
<211> 21
<212> DNA
<213>Artificial sequence
<400> 4
tggcctcgtc ccggttgaac t 21
Claims (2)
1. a kind of Citrullus vulgariss Acidovorax avenae subsp nest-type PRC detection primer it is characterised in that：Described detection primer includes as follows：VapB-
1F：5’-GGGCAATAGCTTGGCGGTTCG-3’；
VapB -1R：5’-CATTGGCCTCGTCCCGGTTGA-3’；
VapB-2F：5’-GCGAACGTAGTCGAAGCGCTCG-3’；
VapB-2R：5’-TGGCCTCGTCCCGGTTGAACT-3’.
2. a kind of Citrullus vulgariss Acidovorax avenae subsp nested PCR detection method it is characterised in that：Comprise the following steps：
Step A, extracting genome DNA：Citrullus vulgariss Acidovorax avenae subsp genomic DNA or morbidity plant tissue DNA are extracted using CTAB method；
Step B, first round PCR amplification：The DNA sample being obtained with step A as template, using the primer described in claim 1
VapB -1F/ VapB -1R obtains first round PCR primer through PCR amplification；
PCR reaction system 25 μ l：Genomic DNA 1 μ l, 10 × PCR reaction buffer is (containing Mg2+) 2.5 μ l, each 2.5 mmol/L
DNTPs mixed liquor 2 μ l, 5U/ μ l DNA Taq polymerase 0.5 μ l, the VapB-1F primer 1 μ l of 10 μm of ol/L, 10 μm of ol/L's
VapB-1R primer 1 μ l, sterilizing ultra-pure water complements to 25 μ l；PCR amplification program：94 DEG C of denaturations 4min, 94 DEG C of degeneration
30sec, 55 DEG C of annealing 30 sec, 72 DEG C of extension 45sec, totally 30 circulations；72 DEG C of extension 10min；4 DEG C of preservations；
Step C, the second wheel PCR amplification：With the diluent of first round pcr amplification product or first round pcr amplification product as mould
Plate, obtains the second wheel pcr amplification product using the primer VapB-2F/VapB-2R described in claim 1 through PCR amplification；
PCR reaction system 25 μ l：Diluent 1 the μ l, 10 × PCR of first round pcr amplification product or first round pcr amplification product
Reaction buffer is (containing Mg2+) 2.5 μ l, each 2.5 mmol/L dNTPs mixed liquor 2 μ l, 5U/ μ l DNA Taq polymerase 0.5 μ l,
The VapB-2R primer 1 μ l of the VapB-2F primer 1 μ l of 10 μm of ol/L, 10 μm of ol/L, sterilizing ultra-pure water complements to 25 μ l；
PCR amplification program：94 DEG C of denaturations 4min, 94 DEG C of degeneration 30sec, 55 DEG C of annealing 30 sec, 72 DEG C of extension 40sec,
Totally 30 circulations；72 DEG C of extension 8min；4 DEG C of preservations；
Step D, pcr amplification product detection：8 μ l second are taken to take turns pcr amplification product, using 1% agarose gel, in 80 V electricity
Pressure electrophoresis 30min, is placed in afterwards in EB and takes out after dyeing 20min, observe and take pictures under gel imaging system, if there is
The specific band of 175bp is it is determined that the pathogenic bacteria being detected is Citrullus vulgariss Acidovorax avenae subsp.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106987654A (en) * | 2017-06-12 | 2017-07-28 | 福建省农业科学院植物保护研究所 | One kind detection watermelon Acidovorax avenae subsp LAMP primer and its quick determination method |
| CN107043825A (en) * | 2017-06-12 | 2017-08-15 | 四川农业大学 | The nest type PCR detection reagent and its detection method of a kind of walnut branch-rot opportunistic pathogen |
| CN107287307A (en) * | 2017-07-04 | 2017-10-24 | 福建省农业科学院植物保护研究所 | A kind of PCR detection method of quick differentiation southern corn leaf blight mating type |
| CN115044687A (en) * | 2022-06-17 | 2022-09-13 | 中国热带农业科学院三亚研究院 | Nested PCR (polymerase chain reaction) detection method for watermelon fruit blotch germs |
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| CN107287307B (en) * | 2017-07-04 | 2020-09-22 | 福建省农业科学院植物保护研究所 | PCR detection method for rapidly distinguishing mating types of corn microsporum |
| CN115044687A (en) * | 2022-06-17 | 2022-09-13 | 中国热带农业科学院三亚研究院 | Nested PCR (polymerase chain reaction) detection method for watermelon fruit blotch germs |
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