CN106119387A - A kind of primer detecting potato pseudomonas solanacearum and PCR detection method thereof - Google Patents
A kind of primer detecting potato pseudomonas solanacearum and PCR detection method thereof Download PDFInfo
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Abstract
A kind of primer detecting potato pseudomonas solanacearum and PCR detection method thereof, it relates to a kind of primer and PCR detection method thereof.The invention solves the problems that the existing method detection of pathogens narrow range to potato bacterial wilt, poor specificity, poor sensitivity, the problem that detection efficiency is low.This primer sequence is: QK5 1:5'GCTAATACCGCATACGAC 3'；QK3 1:5'GAGCGTCAGTGTTATCCC 3'.Detection method is: extracts complete genome DNA, carries out PCR detection, verify detection primer, has amplified band in 591bp position, represents that sample is positive.Detection sensitivity of the present invention can reach the template DNA of 1pg.Detection efficiency is high, and detection sample can be potato plant DNA, tuber DNA, it might even be possible to directly carries out PCR detection using bacterium solution as template.
Description
Technical field
The present invention relates to a kind of detection primer and PCR detection method thereof.
Background technology
Potato bacterial wilt (Potato bacterial wilt), has another name called bacterialo wilt disease.From region or harm occur
For property, bacterial wilt is a kind of commonly encountered diseases and the frequently-occurring disease being only second to late blight in Rhizoma Solani tuber osi disease, for worldwide great antibacterial
Sexually transmitted disease (STD) evil.Bacterial wilt distribution is extremely wide, is mainly distributed on the subtropical and tropical zones that warm moist, rainfall are abundant.Due to
The more difficult control of bacterial wilt, both without immunizing antigen, can once occur bacterial wilt through soil infection again, the field significantly underproduction of falling ill,
The production loss of morbidity weight reaches about 80%.
This disease is to be referred to as the bacterial invasion of Ralstonia solanacearum (Ralstonia solanacearum) by one to cause.
This bacterium is the plant pathogenetic bacteria that a kind of vascular bundle is parasitic, and pathogenic bacteria breeds rapidly after invading vascular bundle, and obstruction conduit, hinders water
Partite transport is defeated, finally results in plant and wilts.The host range of this kind of antibacterial is very wide, can infect 33 sections more than 100 and plant plant, except Solanaceae vegetables
Dish such as Fructus Lycopersici esculenti, Fructus Solani melongenae, Fructus Capsici etc. are outer also infects the multiple kinds of crops such as Nicotiana tabacum L., Semen Sesami, Semen arachidis hypogaeae, Semen sojae atricolor, Radix Raphani, and pathogen growth is grown
Optimum temperature is 30-37 degree Celsius, is a kind of antibacterial requiring high temperature, and the requirement to humidity is the highest.This bacterium is depending mainly on invalid
Body is stayed and is survived the winter on field or potato ball, and during without host, pathogenic bacteria can be in Tu Zhongying saprogenesis up to 14 months～6 years
A kind of facultative saprophyte of stubbornness.
Bacterial wilt is the disease that harm is serious.How the general less aobvious disease of Seedling Stage, show acute aobvious disease at Post flowering of buddingging,
Infected plant slightly stunts than healthy tree, and leaf color is shallower, is usually the indivedual lobule in top or compound leaf appearance wilting, evening at the noon of fine day
Between and still can recover early morning, the most gradually increase the weight of, cause Herb blade wither hang down can not recover again, Herb stem and leaf wilt death, but still
Keeping dark green color, blade does not also fall off, subsequently vein gradually browning, and brown stripes also occurs in stem.Diseased plant tied potato block is such as
Catching an illness light, appearance non-evident sympton, the graying brown of umbilical part of weight of catching an illness moistens shape, cuts sick potato, bundle ring browning, slightly extrudes
Also overflowing dirty white antibacterial pus, serious tuber crust be full of cracks, the soft decomposed of marrow is rotten.This disease has latent infection to Rhizoma Solani tuber osi,
Though appearance non-evident sympton, but invade because of antibacterial and hidden in potato block, at storing selling period, if condition properly will continue to
Development and cause cause tuber to rot in a large number.
Conventional potato pseudomonas solanacearum identification and detection method such as Gram staining method and serological method.Gram’s staining
Method needs, plus observing morphological features, to be measured biochemical reactions characteristic, could finally judge, this
Detection method needs to separate pathogen and purification, and the result of test becomes because of cultivation temperature, incubation time length, culture medium
Divide different with determining negative positive standard and change, poor stability, and waste time and energy, be not easy to the application on producing.Serum
Although method sensitivity increases, but still there is shortcoming, in the cross reaction of pathogenic bacteria close with other or resisting of preparation
Serum can not be with all strain hybrids of all of a certain pathogenic bacteria to be detected.Along with Protocols in Molecular Biology is in many fields
Development and application, especially with genome base sequence specific to various biologies, and the specificity polymerization grown up
The application of enzyme chain reaction (PCR) technology, but this technology needs strict requirements to detection process, and primer aspect is also to close
Key, different primers is relatively big on testing result impact, and to detection range, the sensitivity of detection, specificity etc. all has the biggest shadow
Ring.
Summary of the invention
The invention aims to the detection of pathogens narrow range solving existing method to potato bacterial wilt, specificity
Difference, poor sensitivity, the problem that detection efficiency is low.And provide a kind of primer detecting potato pseudomonas solanacearum and PCR detection thereof
Method.
A kind of primer detecting potato pseudomonas solanacearum of the present invention, this primer is to as follows:
QK5-1:5'-GCTAATACCGCATACGAC-3'
QK3-1:5'-GAGCGTCAGTGTTATCCC-3'.
A kind of PCR method detecting potato pseudomonas solanacearum of the present invention, it follows the steps below:
One, the genomic DNA of sample to be detected is extracted；
Two, as template, PCR amplification is carried out as detection primer, the DNA that step one is extracted using the primer of claim 1
Reaction；
Three, PCR primer is through 1% agarose gel electrophoresis；
Four, amplification sheet is analyzed: have amplified band in 591bp position, represents that sample is positive, in detected sample
Containing bell potato ralstonia solanacearum.
The present invention comprises following beneficial effect:
The potato pseudomonas solanacearum PCR detection primer of the present invention, this primer can be to the cause of disease causing potato bacterial wilt
The bacterial strain of tetra-kinds of biochemical types of bacterium Ralstonia solanacearum (BV II～BV V) carries out augmentation detection, and detection range is wide
General.Detection specificity is good, can avoid the non-specific amplification that other Rhizoma Solani tuber osi bacteria pathogeny produce are raw.Detection sensitivity is permissible
Reach the template DNA of 1pg.Detection efficiency is high, and detection sample can be potato plant DNA, tuber DNA, it might even be possible to directly
PCR detection is carried out as template using bacterium solution.
The detection primer of the present invention, detects high specificity, and qualitative detection is effective.Rotten at common Rhizoma Solani tuber osi disease ring
All occurring without amplified band in the pathogen such as disease, balck shank, soft rot and late blight, only ralstonia solanacearum is obtained in that purpose sheet
Section.(avoid ring rot and balck shank in the PCR detection method that Chen Yongfang etc. utilizes RAPD method to set up for 2005 and have amplified band,
The problem that simply banding pattern is different).
Accompanying drawing explanation
Fig. 1 is DNA agarose gel electrophoresis figure in embodiment 1；Wherein, 1-6 swimming lane is respectively as follows: the bacterial wilt of RS1～6
Bacterium；
Fig. 2 is the electrophoretogram that in embodiment 1, annealing temperature optimizes；
Fig. 3 is that in embodiment 1, potato pseudomonas solanacearum PCR expands electrophoretogram；Wherein, M is DL2000；1: negative control；2
～the DNA electrophoretogram of 13:RS1～12；
Fig. 4 is the specificity identification electrophoretogram of PCR detection in embodiment 1；Wherein, M:DL2000；1: negative control；2～
The DNA electrophoretogram of 4:RS1～4；6～8: blakleg of potato bacterium DNA electrophoretogram；9～11: Potato Ring Rot DNA electrophoresis
Figure；12～14: phytophthora infestans DNA electrophoretogram；
Fig. 5 is that in embodiment 1, the susceptiveness of PCR detection identifies electrophoretogram；Wherein, M is DL2000；1: negative control；2～
7 represent: the sample electrophoresis figure of 1ng, 100pg, 10pg, lpg, 100fg, 10fg；
Fig. 6 be the Guangxi province of embodiment 2, Guangdong Province, Fujian Province sample amplification electrophoretogram first；Wherein, by a left side extremely
Right order is followed successively by Marker DL2000, water comparison, negative control, 1-1～6,2-2～4,3-1～12；
Fig. 7 is the sample amplification electrophoretogram second batch in the Guangxi province of embodiment 2, Guangdong Province, Fujian Province；Wherein, by a left side extremely
Right order is followed successively by Marker DL2000,3-13～16,5-1～4；
Fig. 8 is the sample amplification electrophoresis (primer RS32 and RS37) first in the Guangxi province of embodiment 2, Guangdong Province, Fujian Province
Batch；Wherein, from left to right order be followed successively by Marker DL2000, water comparison, negative control, 1-1～6,2-2～4,3-1～
12；
Fig. 9 is the sample amplification electrophoresis (primer RS32 and RS37) second in the Guangxi province of embodiment 2, Guangdong Province, Fujian Province
Batch；Wherein, order is followed successively by Marker DL2000,3-13～16,5-1～4 from left to right；
Figure 10 is the amplification electrophoresis knot that four samples in the Inner Mongol of embodiment 2 and reference culture carry out two pairs of primers simultaneously
Fruit figure；Wherein A is the primer amplification rear electrophoresis figure of the present invention, and B is that quarantine standard recommends primer amplification rear electrophoresis figure.
Detailed description of the invention
Detailed description of the invention one: a kind of primer detecting potato pseudomonas solanacearum of present embodiment, this primer is to as follows:
QK5-1:5'-GCTAATACCGCATACGAC-3'
QK3-1:5'-GAGCGTCAGTGTTATCCC-3'.
Detailed description of the invention two: a kind of PCR method detecting potato pseudomonas solanacearum of present embodiment, it be according to
Lower step is carried out:
One, the genomic DNA of sample to be detected is extracted；
Two, as template, PCR amplification is carried out as detection primer, the DNA that step one is extracted using the primer of claim 1
Reaction；
Three, PCR primer is through 1% agarose gel electrophoresis；
Four, amplification sheet is analyzed: have amplified band in 591bp position, represents that sample is positive, in detected sample
Containing bell potato ralstonia solanacearum.
The PCR system of detailed description of the invention three: present embodiment is unlike detailed description of the invention two: PCR amplification is such as
Under:
PCR amplification condition: 94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 50～63 DEG C of annealing 40s, 72 DEG C extend 100s,
35 circulations, 72 DEG C extend 10min, 4 DEG C of preservations.Other is identical with detailed description of the invention two.
Detailed description of the invention four: present embodiment is unlike detailed description of the invention two: extract genome in step one
DNA, is to extract from potato plant or potato tubers.Other is identical with detailed description of the invention two.
Detailed description of the invention five: present embodiment is unlike detailed description of the invention two: extracting genome DNA step is such as
Under:
One, take potato tubers or potato plant 0.1g puts in homogenate tube, add 2mL homogenate buffer, by material
Liquid is extracted out after crushing immediately by material, after being placed in 5mL centrifuge tube, adds 20 μ L mercaptoethanols, immediately mistake in 65 DEG C of water-baths
Night；
Two, after the centrifuge tube of previous step being taken out from water-bath, add the saturated phenol of equal-volume, cover tightly centrifuge tube, back and forth
Reverse centrifuge tube mixes in 20min；
Three, centrifugal 10min under 10000rpm rotating speed, carefully takes out supernatant, puts in another centrifuge tube, abandon with pipet
Lower floor's organic facies；
Four, centrifuge tube is overturned back and forth after adding isopyknic saturated phenol/chloroform/isoamyl alcohol in the supernatant that step 3 takes,
After mixing 10min, under 10000rpm rotating speed, centrifugal 10min, repeats this step 1-2 time, until the aqueous phase taken out adds organic
Without white cloud thing during solvent；
Five, take the supernatant after step 4 is centrifuged, add isopyknic chloroform/isoamyl alcohol, mix 10min,
Centrifugal 10min under 10000rpm rotating speed；
Six, take previous step centrifugal after supernatant, add 2 times of volumes frozen after dehydrated alcohol, horizontally rotate 50～100
After Zhuaning ,-20 DEG C of refrigerators are placed more than 30min, precipitates DNA；
The centrifuge tube that seven, previous step is positioned over-20 DEG C takes out, and under 10000rpm rotating speed after centrifugal 10min, it is heavy to collect
Form sediment, obtain DNA；
Eight, adding 1mL volumn concentration in the DNA obtained one step up is the ethanol of 75%, back and forth reverse for several times from
Heart pipe, is centrifuged 4～5min under 8000rpm rotating speed, outwells ethanol, be upside down in by pipe in clean absorbent paper, and dry DNA sinks
Form sediment；
Nine, the most dried DNA adds the deionized water of 100-200 μ L sterilizing, dissolves in 55 DEG C of water-baths
DNA；
Ten, extracting solution one step up adds the RNase of 1 μ L, places 30min at normal temperatures, then with 0.1% solidifying
Glue carries out electrophoresis detection, preserves in-20 DEG C of refrigerators.Other is identical with detailed description of the invention two.
Present invention is not limited only to the content of the respective embodiments described above, one of them or the group of several detailed description of the invention
Contract sample can also realize the purpose of invention.
Be the method have the benefit that by following example checking
Embodiment 1
1, test material
The potato pseudomonas solanacearum bacterial strain that the present embodiment is used is as shown in table 1, and bacterial strain RS1～4 is by international Rhizoma Solani tuber osi
The heart (being called for short CIP) provides for scientific research, and it is withered to Rhizoma Solani tuber osi green grass or young crops by TZC isolation medium that remaining bacterial strain is this seminar
Sick disease plant carry out isolated and purified after, making aqueous solution, to be placed in 4 DEG C of Refrigerator stores standby.
Table 1 strains tested title and source
2, test method
2.1, the extracting genome DNA of test antibacterial used
DNA extraction method sees the description of commercial kit.
2.2, the DNA extraction of potato tubers
1, take potato tubers 0.1g and put in homogenate tube, add 2mL homogenate buffer, will after material is crushed rapidly
Liquid is extracted out, after being placed in 5mL centrifuge tube, adds 20 μ L mercaptoethanols, rapidly in 65 DEG C of water-baths overnight；
2, after centrifuge tube being taken out from water-bath, add the saturated phenol of equal-volume, cover tightly centrifuge tube.Slowly overturn back and forth from
Heart pipe at least 20min is with mixing.The most violent, in case DNA is interrupted；
3,10000rpm is centrifuged 10min.Aqueous phase containing DNA on upper strata, the organic facies containing protein and cell debris under
Layer；
4, carefully take out supernatant with pipet, put in another centrifuge tube, abandon lower floor's organic facies；
5, centrifuge tube is slowly overturned after adding isopyknic saturated phenol/chloroform/isoamyl alcohol back and forth, after mixing 10min
10000rpm is centrifuged 10min.Repeat this step 1-2 time, until can't see white cloud and mist when the aqueous phase taken out adds organic solvent
Shape thing；
6, taking supernatant, add isopyknic chloroform/isoamyl alcohol, mixing 10min, 10000rpm are centrifuged 10min.
7, take supernatant, add dehydrated alcohol 2 times of volumes, ice-cold, after horizontally rotating 50～100 turns, be placed on-20 DEG C of ice
More than 30min in case, precipitates DNA；
8, centrifuge tube is taken out, after 10000rpm is centrifuged 10min, it is seen that there is precipitation the lower section of pipe, i.e. DNA；
9, outwell liquid carefully, add the ethanol of 1mL 75%, back and forth reverse centrifuge tube for several times, to remove in precipitation block
Presumable foreign body, 8000rpm is centrifuged 4-5min, carefully outwells ethanol, is upside down in clean absorbent paper by pipe, allows ethanol stream
To the greatest extent, dry DNA precipitates；
10, the deionized water of 100-200 μ L sterilizing, dissolving DNA in 55 DEG C of water-baths is added according to the size of precipitation block；
11, in order to remove the RNA extracted, extracting solution adds 1 μ L RNase, places 30min at normal temperatures, then
Gel with 0.1% carries out electrophoresis detection.-20 DEG C of refrigerators preserve.
Above-mentioned steps extracts the mode of DNA, it is also possible to extract DNA from plant body.
2.3, the design of PCR specific primer
According to the Ralstonia solanacearum strain TW56 that the accession number in Genebank is DQ924957
16S rRNA sequence, uses the specificity amplification primer of Primers5.0 software design potato pseudomonas solanacearum, and primer is by Shanghai
Sheng Gong biotech firm synthesizes, and primer sequence is as follows:
QK5-1:5'-GCTAATACCGCATACGAC-3'
QK3-1:5'GAGCGTCAGTGTTATCCC-3'；
Purpose fragment length is 591bp.
2.4, PCR amplification
The reaction system of PCR is 25mL.PCR reaction condition determines mainly the suitableeest annealing temperature to amplification program.
2.4.1.PCR reaction system
2.4.2.PCR the optimization of reaction condition
Annealing temperature in PCR reaction condition is optimized:
2.4.5.PCR the specificity verification of detection system
With the specific primer of design synthesis to being supplied examination pathogenic bacteria DNA to carry out PCR amplification, respectively with potato pseudomonas solanacearum
(RS1～4), blakleg of potato bacterium (BYH2, GSH1, NMH1) DNA, Potato Ring Rot (by06-6, hc-42, p06-4)
DNA, phytophthora infestans (ch10, ch11, ncppb) DNA are template, the specificity of detection PCR detection system.
2.4.6.PCR the sensitivity checking of detection system
For identifying the DNA Cmin that designed primer and PCR reaction system can measure, by the dilutest for the DNA extracted
It is interpreted into 1ng, 6 variable concentrations of 100Pg, 10Pg, 1Pg, 100fg, 10fg, carry out PCR amplification with the primer of design, according to solidifying
Gel electrophoresis result measures the sensitivity of primer.Amplified production detects with the agarose gel electrophoresis of 1.0%.
3, result of the test
3.1 ralstonia solanacearum extracting genome DNA
The DNA extracted is carried out agarose gel electrophoresis result as shown in Figure 1.It is seen that the DNA obtained is complete
Whole, quality is preferable, not degraded, it is possible to meets the requirement of PCR isolated genes, may be used for further testing.
4, the amplification of PCR
4.1 the screening of annealing temperature
Being optimized potato pseudomonas solanacearum Standard PCR reaction condition, the condition of optimization is annealing temperature.The selection result
As shown in Figure 2.Result shows: 59 DEG C is the suitableeest annealing temperature.
4.2PCR amplification
Carrying out PCR amplification with potato pseudomonas solanacearum specific primer to for 12 ralstonia solanacearum bacterial strains of examination, result is shown in figure
3。
The specificity identification of 4.3PCR detection
With potato pseudomonas solanacearum specific primer, the DNA of strains tested is carried out PCR amplification, result such as Fig. 4.Can by figure
To find out, ralstonia solanacearum used can amplify the band that size is 591bp, and other all do not expand with reference to pathogen and comparison
Increase and band, illustrate that the primer of design has the specificity of height to ralstonia solanacearum.
The susceptiveness of 4.4PCR detection is identified
Using potato pseudomonas solanacearum specific primer to being diluted to 1ng, 100pg, 10pg, lpg, 100fg, 10fg are total to
The ralstonia solanacearum genomic DNA of 6 variable concentrations carries out PCR amplification, and result is as shown in Figure 5.Be can be seen that by result and detect
Least concentration is the template DNA of 1pg.
The sequencing fragment of 4.5 mesh
Positive bacterium solution through identifying being delivered to Shanghai biotechnology Services Co., Ltd check order, sequence is as follows
(underscore part is primer).
AAATCGGTCAGTGCAGCTTGCATGCCTGCAGGTCGACGATTGAGCGTCAGTGTTATCCCAGGGGGCTGCCTTCGCCA
TCGGTATTCCTCCACATCTCTACGCATTTCACTGCTACACGTGGAATTCTACCCCCCTCTGACACACTCTAGCCGTG
CAGTCACCAATGCAATTCCCAAGTTAAGCTCGGGGATTTCACATCGGTCTTGCACAACCGCCTGCGCACGCTTTACG
CCCAGTAATTCCGATTAACGCTTGGACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGTCCTTATTCTT
CCGGTACCGTCATCGACCCCAGGTATTAACCAGAGCCATTTCTTTCCGGACAAAAGTGCTTTACAACCCGAAGGCCT
TCTTCACACACGCGGCATTGCTGGATCAGGCTTTCGCCCATTGTCCAAAATTCCCCACTGCTGCCTCCCGTAGGAGT
CTGGGCCGTGTCTCAGTCCCAGTGTGGCTGATCGTCCTCTCAGACCAGCTACTGATCGTCGCCTTGGTGAGCCTTTA
CCTCACCAACTAGCTAATCAGACATCGGCCGCTCCTATAGCATGAGGCCTTGCGGTCCCCCACTTTCACCCTCAGGT CGTATGCGGTATTAGCAATCTCTAGAGGATCCCCGGGTACCGAGCTCGAATTCGTAATCATGGTCATAGCTGTTTCC
TGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCT
AATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTG
CATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTC
GCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAGGCGGTAATACGGTATCCACAGATCAGGGG
ATACGCAGAAAGACATGTGAGCAAAAGGCCAGCAAAGGCCAGGACCGTAAAAGGCGCGTTGCTGGCGTTTTCCATAG
G。
Embodiment 2
The PCR primer of the present embodiment application present invention and quarantine standard recommend PCR detection primer, to picking up from China Guangxi
The sample of 35 parts of bacterial infection diseases in the provinces such as province, Guangdong Province, Fujian Province, the Inner Mongol compares test.
1 materials and methods
1.1 sample
Healthy potato plant (negative control) is provided by Plant De-toxin Nursery Stock Inst., Heilongjiang Prov. Academy of Agriculture Sc.
Potato bacterial wilt reference culture Ralstonia solanacearum buys from Chinese Academy of Sciences's microbial preservation
Center.
Testing sample: Rhizoma Solani tuber osi producing region, area gathers from China Guangxi province, Guangdong Province, Fujian Province, the Inner Mongol etc., is shown in Table 2.
Table 2 sample message
1.2 reagent
Taq DNA polymerase, dNTP, purchased from TaKaRa (Dalian) company, primer synthesis student on commission's work biological engineering (Shanghai)
Limited company completes.
1.3 plant genome DNAs extract
Method is recommended to extract tuber DNA according to sky root plant genes group DNA extraction kit description.
1.4 PCR amplifications
It is shown in Table 3 for examination primer sequence.The annealing temperature of primer QK5-1 and QK3-1 of the present invention is 59 DEG C, and quarantine standard
The annealing temperature of primer RS32 and RS37 recommended is 60 DEG C.Amplified production is observed through gel imaging system.
Table 3 primer sequence
2 interpretations of result
The amplification of 2.1 primers of the present invention
Test sample result after primer QK5-1 and QK3-1 expands is shown in Fig. 6 and Fig. 7, it can be seen that Guangxi province detection 4
Part bacterial wilt sample；16 parts of Guangdong Province sample all detects ralstonia solanacearum；The ralstonia solanacearum detection of the 4 parts of samples in Fujian Province presents weak sun
Property.
2.2 quarantine standards recommend the amplification of primer
The primer amplification result that test sample is recommended in known quarantine standard is shown in Fig. 8 and Fig. 9, it can be seen that Guangxi
The sample (1-1～6,2-2～4) saved is feminine gender；16 parts of Guangdong Province sample all detects ralstonia solanacearum；The 4 parts of samples in Fujian Province
Ralstonia solanacearum detection presents the weak positive.
The amplification efficiency of 2.3 two pairs of primers compares
Carry out the amplification of two pairs of primers with four samples in the Inner Mongol and reference culture, electrophoresis result is shown in Figure 10 simultaneously.From
Understanding in figure, the DNA concentration of reference culture is 20ng/ μ L, and primer QK5-1 and the QK3-1 amplification of designed, designed obtains purpose and produces
Thing, RS32 and RS37 does not then obtain purpose product.The amplification efficiency aspect of testing sample, the primer QK5-1 of designed, designed and
QK3-1 is better than recommending primer RS32 and RS37.
The above results shows the PCR detection system that the present invention is set up, and accuracy rate is higher than the detection method recommended, amplification
Efficiency is the highest.
Claims (5)
1. the primer detecting potato pseudomonas solanacearum, it is characterised in that this primer is to as follows:
QK5-1:5'-GCTAATACCGCATACGAC-3'
QK3-1:5'-GAGCGTCAGTGTTATCCC-3'.
2. the PCR method detecting potato pseudomonas solanacearum, it is characterised in that it follows the steps below:
One, the genomic DNA of sample to be detected is extracted；
Two, as template, pcr amplification reaction is carried out as detection primer, the DNA that step one is extracted using the primer of claim 1；
Three, PCR primer is through 1% agarose gel electrophoresis；
Four, amplification sheet is analyzed: have amplified band in 591bp position, represents that sample is positive, detected sample contains
Bell potato ralstonia solanacearum.
A kind of PCR method detecting potato pseudomonas solanacearum the most according to claim 2, it is characterised in that PCR amplification
PCR system is as follows:
PCR amplification condition: 94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 50～63 DEG C of annealing 40s, 72 DEG C of extension 100s, 35
Circulation, 72 DEG C extend 10min, 4 DEG C of preservations.
A kind of PCR method detecting potato pseudomonas solanacearum the most according to claim 2, it is characterised in that carry in step one
Take genomic DNA, be to extract from potato plant or potato tubers.
A kind of PCR method detecting potato pseudomonas solanacearum the most according to claim 2, it is characterised in that genomic DNA
Extraction step is as follows:
One, take potato tubers or potato plant 0.1g puts in homogenate tube, add 2mL homogenate buffer, material is stood
After i.e. broken, liquid is extracted out, after being placed in 5mL centrifuge tube, add 20 μ L mercaptoethanols, immediately in 65 DEG C of water-baths overnight；
Two, after the centrifuge tube of previous step being taken out from water-bath, add the saturated phenol of equal-volume, cover tightly centrifuge tube, overturn back and forth
Centrifuge tube mixes in 20min；
Three, centrifugal 10min under 10000rpm rotating speed, carefully takes out supernatant, puts in another centrifuge tube, abandon lower floor with pipet
Organic facies；
Four, centrifuge tube is overturned back and forth after adding isopyknic saturated phenol/chloroform/isoamyl alcohol in the supernatant that step 3 takes, mixing
After 10min, under 10000rpm rotating speed, centrifugal 10min, repeats this step 1-2 time, until the aqueous phase taken out adds organic solvent
Time without white cloud thing；
Five, take the supernatant after step 4 is centrifuged, add isopyknic chloroform/isoamyl alcohol, mix 10min, turn at 10000rpm
The lower centrifugal 10min of speed；
Six, take previous step centrifugal after supernatant, add 2 times of volumes frozen after dehydrated alcohol, horizontally rotate 50～100 turns
After ,-20 DEG C of refrigerators are placed more than 30min, precipitates DNA；
The centrifuge tube that seven, previous step is positioned over-20 DEG C takes out, and under 10000rpm rotating speed after centrifugal 10min, collects precipitation,
Obtain DNA；
Eight, adding 1mL volumn concentration in the DNA obtained one step up is the ethanol of 75%, back and forth reverse centrifuge tube for several times,
Being centrifuged 4～5min under 8000rpm rotating speed, outwell ethanol, be upside down in by pipe in clean absorbent paper, dry DNA precipitates；
Nine, the most dried DNA adds the deionized water of 100-200 μ L sterilizing, dissolving DNA in 55 DEG C of water-baths；
Ten, extracting solution one step up adds the RNase of 1 μ L, places 30min at normal temperatures, then enter with the gel of 0.1%
Row electrophoresis detection, preserves in-20 DEG C of refrigerators.
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| CN111154897A (en) * | 2019-12-16 | 2020-05-15 | 福建省农业科学院作物研究所 | RPA primer, kit, method and system for rapidly detecting potato black shank |
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Non-Patent Citations (3)
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| 陈永芳: "我国植物青枯菌的遗传多样性和马铃薯青枯菌的PCR检测技术研究", 《中国博士学位论文全文数据库 农业科技辑》 * |
| 魏兰芳等: "云南马铃薯青枯病菌的PCR检测", 《西南农业大学学报》 * |
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| CN111154897A (en) * | 2019-12-16 | 2020-05-15 | 福建省农业科学院作物研究所 | RPA primer, kit, method and system for rapidly detecting potato black shank |
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