阅读说明：本技术 一种检测枸杞根腐病尖孢镰孢菌的环介导等温扩增检测方法及其检测引物和验证方法 (A kind of loop-mediated isothermal amplification detection method and its detection primer and verification method detecting fructus lycii root rot Fusarium oxysporum ) 是由 漆永红 李敏权 李雪萍 曹素芳 李建宏 李继平 申培增 陈爱昌 于 2019-08-22 设计创作，主要内容包括：本发明公开了一种检测枸杞根腐病尖孢镰孢菌的环介导等温扩增检测方法及其检测引物和验证方法；检测引物采用LAMP设计软件primer software PrimerExplorer V4进行设计,包括2条外引物F3和B3,2条内引物FIP、BIP。本发明的有益效果为：采用本发明的检测方法、引物及验证方法进行检测,5个不同地理来源的枸杞根腐病尖孢镰孢菌LAMP检测均呈黄绿色,即阳性,而对照和其他病原菌均呈橘色,即阴性。本专利具有引物特异性强、检测灵敏度高、反应速度快、直接用肉眼观察结果、准确性高、不易污染、对疑似病害样本直接检测等优点。同时通过显色进行比对判断,大幅提升检测准确性。(The invention discloses a kind of loop-mediated isothermal amplification detection method for detecting fructus lycii root rot Fusarium oxysporum and its detection primer and verification methods；Detection primer is designed using LAMP design software primer software PrimerExplorer V4, including 2 outer primer F3 and B3,2 inner primers FIP, BIP.The invention has the benefit that being detected using detection method of the invention, primer and verification method, the fructus lycii root rot Fusarium oxysporum LAMP detection of 5 different geographic origins is in yellow green, it is i.e. positive, and compareing with other pathogens is in orange, i.e., it is negative.This patent have many advantages, such as primer specificity is strong, detection sensitivity is high, reaction speed is fast, the result that directly detects by an unaided eye, accuracy are high, it is not easy to pollute, doubtful disease sample is directly detected.Judgement is compared by colour developing simultaneously, detection accuracy is substantially improved.)

1. a kind of ring mediated isothermal amplification detection primer for detecting fructus lycii root rot Fusarium oxysporum, which is characterized in that described Ring mediated isothermal amplification detection primer are as follows:

F3:5 '-GTCCGAAAATTTTGCGGTGC-3 '；

B3:5 '-TTCCAGTGGTTA GTGACTGC-3 '；

FIP:5 '-AGTGGCGGGGTAAGATACCCC-3 '；

BIP:5 '-GCTTGCCCTGTTCCCACAAAAC-3 '.

2. a kind of loop-mediated isothermal amplification detection method for detecting fructus lycii root rot Fusarium oxysporum, which is characterized in that the right to use Benefit require 1 described in ring mediated isothermal amplification detection primer carry out ring mediated isothermal amplification.

3. a kind of ring mediated isothermal amplification detection side for detecting fructus lycii root rot Fusarium oxysporum according to claim 2 Method, which comprises the following steps:

A, sick sample acquisition:

Fructus lycii root rot diseased plant is acquired, interior separates pathogen in conventional manner, and pathogen saves at 4 DEG C；

B, strain culturing:

Bacteria culturing in PDA culture medium, PDA culture medium formula be potato 200g, glucose 20g, agar 15-20g, from Water 1000mL is cultivated for 25 DEG C of room temperature under illumination condition；

C, strain gene group DNA extracts:

Using E.Z.N.A^TMHP Fungal DNA Kit kit, takes cultured mycelia in step B to grind under protection of liquid nitrogen Powder is worn into, the CPL buffer of 600 μ L is added in powder, adds 10 μ L beta -mercaptoethanols；65 DEG C of water-bath 30min, water bath processing Period taking-up is rocked 2 times, is added 600 μ L chloroforms to be made and is extracted mixed liquor, and extracting mixed liquor and dehydrated alcohol, 24:1 is mixed by volume It closes, centrifugal treating 5min under revolving speed 12000r/min；

300 μ L of Aspirate supernatant adds 150 μ L CXD buffers, then plus the obtained supernatant of 300 μ L dehydrated alcohols into new centrifuge tube Above-mentioned 750 μ L supernatant mixed liquor is added in HiBind DNA column, centrifugal treating 1min under revolving speed 12000r/min by mixed liquor, It leaves and takes sediment and discards filtrate；

650 μ L SPW washing lotions, centrifugal treating 1min under revolving speed 12000r/min are added in sediment；650 μ L SPW are added to wash Liquid, centrifugal treating 1min under revolving speed 12000r/min, leaves and takes sediment and discards filtrate, sediment is put into revolving speed in new centrifuge tube Centrifugal treating 1min under 12000r/min；

Sediment after washing is transferred in new centrifuge tube, the 100 μ L of Elution buffer of 65 DEG C of preheating, revolving speed is added DNA profiling is made in centrifugal treating 1min dissolving DNA under centrifugal treating 2min under 12000r/min, last revolving speed 12000r/min；

D, the amplification of pathogen ITS sequence and the design of LAMP primer:

ITS sequence in the dissolving DNA of fructus lycii pine root fungus is expanded, cloned, is sequenced, its germ Fusarium oxysporum is obtained ITS sequence carries out design of primers, including 2 using LAMP design software primer software PrimerExplorer V4 Outer primer F3 and B3,2 inner primers FIP, BIP；Its sequence is respectively as follows:

F3:5 '-GTCCGAAAATTTTGCGGTGC-3 '；

B3:5 '-TTCCAGTGGTTA GTGACTGC-3 '；

FIP:5 '-AGTGGCGGGGTAAGATACCCC-3 '；

BIP:5 '-GCTTGCCCTGTTCCCACAAAAC-3 '；

E, the foundation of LAMP reaction system:

Within the scope of 60~80 DEG C carry out LAMP reaction, reaction time 20min-1h, LAMP reaction system be 10 × 1~3 μ L of Isothermal Amplification Buffe, 100mmol/L MgSO₄0.5~2 μ L, inner primer FIP0.5~ 1.5 μ L, 0.5~1.5 μ L of inner primer BIP, 0.5~1.5 μ L of outer primer F3,0.5~1.5 μ L of outer primer B3,100mmol/L The Bst 2.0DNA polymerization of 2.0~4.0 μ L of deoxyribonucleoside triphosphate dNTP, DNA profiling 2.0~4.0 μ L, 8 000U/mL 0.5~1.5 μ L of enzyme plus ddH₂8.0~11.0 μ L of O to 25 μ L of total system；

F, amplification judgment method:

3 μ L products are taken after reaction, detect amplified production with 2% agarose gel electrophoresis of mass concentration, successive steps occur Shape band is then determined as the positive, detects fructus lycii root rot Fusarium oxysporum；Do not occur band, be then determined as feminine gender, Chinese holly is not detected Qi root rot Fusarium oxysporum.

4. a kind of ring mediated isothermal amplification detection side for detecting fructus lycii root rot Fusarium oxysporum according to claim 3 Method, it is characterised in that be with 65 DEG C of progress LAMP reactions, reaction time 1h in the step E.

5. a kind of ring mediated isothermal amplification detection side for detecting fructus lycii root rot Fusarium oxysporum according to claim 3 Method, it is characterised in that be with 80 DEG C of progress LAMP reactions, reaction time 20min in the step E.

6. a kind of ring mediated isothermal amplification detection side for detecting fructus lycii root rot Fusarium oxysporum according to claim 3 Method, it is characterised in that in the step F, 0.5 μ L fluorescent dye SYBR Green I can also be added, it is anti-to observe by the naked eye LAMP It answers liquid color change whether occurs and carrys out judging result, positive reaction is yellow green, detects fructus lycii root rot Fusarium oxysporum, negative Reaction is orange, and fructus lycii root rot Fusarium oxysporum is not detected.

7. being examined according to a kind of ring mediated isothermal amplification of any detection fructus lycii root rot Fusarium oxysporum of claim 2-6 The specificity verification method of survey method, which is characterized in that it is to choose the fructus lycii pine root fungus in multiple areas to prepare DNA profiling, with ddH₂The DNA profiling that O substitutes object bacteria is negative control, with close kind of Fusaium solani Fusarium of Fusarium oxysporum The genomic DNA work of sonali and other 2 non-Fusariumsp Phytophthora infestans, Rhizoctonia solani For control group, LAMP reaction, observing response pipe color change are carried out respectively；The multiple regional respectively Gansu Province Jingyuan County, Jingtai County, Wuwei, Yumen City, Zhongweiof Ningxia, northwest China.

8. being examined according to a kind of ring mediated isothermal amplification of any detection fructus lycii root rot Fusarium oxysporum of claim 2-6 The sensitivity verification method of survey method, which is characterized in that be to use 10 times of concentration series dilution methods by the fructus lycii root rot of extraction The DNA profiling of Fusarium oxysporum carries out gradient dilution, its DNA profiling mass concentration gradient is made to be followed successively by 100ng/ μ L, 10ng/ μ L, 1ng/ μ L, 100pg/ μ L and 10pg/ μ L take 2 μ L to carry out LAMP reaction, observing response pipe color change as template respectively.

9. being examined according to a kind of ring mediated isothermal amplification of any detection fructus lycii root rot Fusarium oxysporum of claim 2-6 The field verification method of survey method, which is characterized in that be to randomly select sample 200mg diseased tissues preparation DNA in field to be made LAMP amplification, observing response pipe color change are carried out for template.

10. according to a kind of ring mediated isothermal amplification of any detection fructus lycii root rot Fusarium oxysporum of claim 2-6 The soil germ verification method of detection method, which is characterized in that be the spore system for taking cause of disease bacteria strain Fusarium oxysporum in step B Standby spore suspension；Every part of 0.25g of soil sample without Fusarium oxysporum is taken, is put into the EP pipe of 2mL, is added respectively not in EP pipe With 10 000,1 000,100,10,1, spore of quantity, according to E.Z.N.A^TM.HP Fungal DNA Kit extracts kit Specification extracts pedotheque total DNA, carries out LAMP amplification as template, and using Fusarium oxysporum pure dna as the positive Control, aqua sterilisa is as negative control, observing response pipe color change.

Technical field

The present invention relates to plant disease detection of pathogens technical fields, and in particular to a kind of detection fructus lycii root rot point spore sickle The loop-mediated isothermal amplification detection method and its detection primer and verification method of spore bacterium.

Background technique

Fructus lycii root rot disease symptom: early stage, diseased plant blade is grayish green, and noon wilts, and can restore sooner or later.Thereafter no longer Restore, blade becomes withered and yellow, slowly dead.Rhizome portion slightly swelling, bast redden brown, feel like jelly, there is white hypha in outside, wooden There is bronzing stria in portion, penetrates into deep layer sometimes.The bast softening browning of main root and lateral root is dead.Epidermis lobe, bast In have many white silty dots, easily peeled off from xylem.

Now studies have found that the main pathogenic fungi of fructus lycii root rot is Fusarium oxysporum (Fusarium oxysporum), In Bacteria colony white, pink colour to purple in PDA culture medium.Colony diameter 90mm after 7d, protuberance are in ulotrichy, and bacterium back generates pink colour or purple Color pigment.Hyphae colorless, have every.Falx single bottle stalk is produced, size is 1.8 μm of 8.2~16.5 μ m (average 11.8 μ m, 1.8 μ m).Macroconidium fusarium shape, intermediate thick, both ends are tapering, have 3~5 diaphragms, mostly 3 every, size be 23.5~ 32.9 2.4~3.5 μm of μ ms (average 2.7 μm of 27.5 μ m), foot is obvious.Microconidia ellipse, oblong, it is single Born of the same parents, colourless, false head, size are 1.8~2.9 μm of 3.5~14.1 μ m (average 2.5 μm of 6.8 μ m).There are many chlamydospore, Dan Sheng, to life, raw or basidixed, spherical, size is 5.5~10.0 μm.Although above-mentioned cultural method can carry out fusarium oxysporum Bacterial examination is surveyed, but needs staff to observe change in shape situation in real time and carry out record just to can be carried out judgement.Above method training The feeding period is long, criterion is different.Field can not be especially applicable in quickly to test, field after the too long testing result of cultivation cycle determines Occurrence of large-area disaster can not shift to an earlier date prevention and control.

LAMP detection has the characteristics that easy to operate, high specificity, high sensitivity, product easily detect, and is widely used in true The detection of the pathogenic microorganisms such as bacterium, bacterium, virus, nematode.The principle of LAMP reaction, using 4 species-specific primers by a kind of high Active strand displacement archaeal dna polymerase.So that strand displacement DNA synthesis ceaselessly self is recycling, amplification is in two stages.1st stage For initial phase, when any one primer carries out base pairing extension to the complementary portions of double-stranded DNA, another chain will be solved From becoming single-stranded.The F2 sequence of upstream internal primers F IP is first in conjunction with template F2c, in the work of strand displacement type archaeal dna polymerase Extend starting strand displacement synthesis forward under.External primers F3 is in conjunction with template F3c and extends, and displaces complete FIP connection Complementary single strand.F1c on FIP and this it is single-stranded on Fl be complementary structure.Self base pairing forms cyclic structure.With this chain For template.Downstream primer BIP and B3 successively starts the synthesis similar to FIP and F3, forms the single-stranded of dumbbell structure.Rapidly with The Fl section of 3 ' ends is starting point using itself as template, carries out DNA synthesis and extends to form stem loop structure.The structure is LAMP The initial structure of gene magnification circulation.2nd stage was the amplification cycles stage.Using stem loop structure as template, FIP and stem ring The area F2c combines.Start strand displacement synthesis, also will form cyclic structure on the single-chain nucleic acid dissociateed.Rapidly with the B1 of 3 ' ends Section is starting point, using itself as template.DNA synthesis extension and strand displacement are carried out, 2 new stem loop structures different in size are formed DNA, the B2 on BIP primer is hybrid with it.Start new round amplification.And product DNA length doubles.In the reaction system 2 Loop primers LF and LB are added, they also start strand displacement respectively in conjunction with stem loop structure and synthesize, in cycles.Amplification End product be the mixture with different number loop-stem structures, different length DNA.And product DNA is amplification target sequence Alternately inverted repetitive sequence.

LAMP react the characteristics of be (1) high sensitivity: the detection sensitivity of LAMP usually than 10~1000 times of PCR high, with Real-time PCR sensitivity is close.(2) high specific: 4 species-specific primers designed for 6 regions of target sequence, 6 Any region and primer mismatch not can be carried out nucleic acid amplification in region, therefore its specificity is high.(3) rapidly and efficiently: being not required to Want preparatory double-stranded DNA thermal denaturation avoid temperature cycles and caused by loss of time nucleic acid amplification it is achievable in l h, It is faster completed than PCR reaction.Amplified reaction can be accelerated by increasing by 1 ring primer in LAMP amplification system, shortened the reaction time, made Amplified reaction can be completed in 30min, improve the efficiency of LAMP detection.(4) product detection is intuitive: LAMP amplification generates a large amount of Double-stranded DNA, into reaction tube be added burn photoinitiator dye SYBR Green I, according to color change judge expand whether occur.

Summary of the invention

The purpose of the present invention is to above-mentioned defect in the prior art, provide a kind of easy to operate, high specificity, The loop-mediated isothermal amplification detection method for the detection fructus lycii root rot Fusarium oxysporum that high sensitivity, product easily detect and its detection Primer and verification method.

The gene order of fructus lycii root rot Fusarium oxysporum:

TTAAGTTCAGCGGGTATTCCTACCTGATCCGAGGTCAACATTCAGAAGTTGGGGTTTAACGGCGTGGCC GCGACGATTACCAGTAACGAGGGTTTTACTACTACGCTATGGAAGCTCGACGTGACCGCCAATCAATTTGAGGAACG CGAATTAACGCGAGTCCCAACACCAAGCTGTGCTTGAGGGTTGAAATGACGCTCGAACAGGCATGCCCGCCAGAATA CTGGCGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTTGCT GCGTTCTTCATCGATGCCAGAACCAAGAGATCCGTTGTTGAAAGTTTTGATTTATTTATGGTTTTACTCAGAAGTTA CATATAGAAACAGAGTTTAGGGGTCCTCTGGCGGGCCGTCCCGTTTTACCGGGAGCGGGCTGATCCGCCGAGGCAAC AAGTGGTATGTTCACAGGGGTTTGGGAGTTGTAAACTCGGTAATG

Specifically as shown in sequence 1 in sequence table.

To achieve the goals above, a kind of technical solution provided by the invention are as follows: detection fructus lycii root rot Fusarium oxysporum Ring mediated isothermal amplification detection use primer, the ring mediated isothermal amplification detection primer are as follows:

F3:5 '-GTCCGAAAATTTTGCGGTGC-3 '；

B3:5 '-TTCCAGTGGTTA GTGACTGC-3 '；

FIP:5 '-AGTGGCGGGGTAAGATACCCC-3 '；

BIP:5 '-GCTTGCCCTGTTCCCACAAAAC-3 '.

A second object of the present invention is to provide a kind of ring mediated isothermal expansions for detecting fructus lycii root rot Fusarium oxysporum Increase detection method, carries out ring mediated isothermal amplification using above-mentioned ring mediated isothermal amplification detection primer.

Further, the loop-mediated isothermal amplification detection method of above-mentioned a kind of detection fructus lycii root rot Fusarium oxysporum, The following steps are included:

A, sick sample acquisition:

Fructus lycii root rot diseased plant is acquired, interior separates pathogen in conventional manner, and pathogen saves at 4 DEG C；

B, strain culturing:

For Bacteria culturing in PDA culture medium, PDA culture medium formula is potato 200g, glucose 20g, agar 15- 20g, tap water 1000mL are cultivated for 25 DEG C of room temperature under illumination condition；

C, strain gene group DNA extracts:

Using E.Z.N.A^TMHP Fungal DNA Kit kit takes in step B cultured mycelia in protection of liquid nitrogen Under be ground into powder, in powder plus the CPL buffer of 600 μ L, add 10 μ L beta -mercaptoethanols；65 DEG C of water-bath 30min, water-bath It takes out and rocks 2 times during processing, add 600 μ L chloroforms to be made and extract mixed liquor, extract mixed liquor and dehydrated alcohol by volume 24: 1 mixes, centrifugal treating 5min under revolving speed 12000r/min；

300 μ L of Aspirate supernatant adds 150 μ L CXD buffers into new centrifuge tube, then plus 300 μ L dehydrated alcohols be made Above-mentioned 750 μ L supernatant mixed liquor is added in HiBind DNA column, centrifugal treating under revolving speed 12000r/min by supernatant mixed liquor 1min leaves and takes sediment and discards filtrate；

650 μ L SPW washing lotions, centrifugal treating 1min under revolving speed 12000r/min are added in sediment；Add 650 μ L SPW washing lotion, centrifugal treating 1min under revolving speed 12000r/min, leaves and takes sediment and discards filtrate, sediment is put into new centrifuge tube Centrifugal treating 1min under middle revolving speed 12000r/min；

Sediment after washing is transferred in new centrifuge tube, the 100 μ L of Elution buffer of 65 DEG C of preheating is added, turned DNA mould is made in centrifugal treating 1min dissolving DNA under centrifugal treating 2min under fast 12000r/min, last revolving speed 12000r/min Plate；

D, the amplification of pathogen ITS sequence and the design of LAMP primer:

ITS sequence in the dissolving DNA of fructus lycii pine root fungus is expanded, cloned, is sequenced, its germ fusarium oxysporum is obtained The ITS sequence of bacterium carries out design of primers, packet using LAMP design software primer software PrimerExplorer V4 Include 2 outer primer F3 and B3,2 inner primers FIP, BIP；Its sequence is respectively as follows:

F3:5 '-GTCCGAAAATTTTGCGGTGC-3 '；

B3:5 '-TTCCAGTGGTTA GTGACTGC-3 '；

FIP:5 '-AGTGGCGGGGTAAGATACCCC-3 '；

BIP:5 '-GCTTGCCCTGTTCCCACAAAAC-3 '；

E, the foundation of LAMP reaction system:

Within the scope of 60~80 DEG C carry out LAMP reaction, reaction time 20min-1h, LAMP reaction system be 10 × 1~3 μ L of IsothermalAmplification Buffe, 100mmol/L MgSO₄0.5~2 μ L, inner primer FIP0.5~ 1.5 μ L, 0.5~1.5 μ L of inner primer BIP, 0.5~1.5 μ L of outer primer F3,0.5~1.5 μ L of outer primer B3,100mmol/L The Bst 2.0DNA polymerization of 2.0~4.0 μ L of deoxyribonucleoside triphosphate dNTP, DNA profiling 2.0~4.0 μ L, 8 000U/mL 0.5~1.5 μ L of enzyme plus ddH₂8.0~11.0 μ L of O to 25 μ L of total system；

F, amplification judgment method:

3 μ L products are taken after reaction, detect amplified production with 2% agarose gel electrophoresis of mass concentration, are occurred continuous Ladder-like band is then determined as the positive, detects fructus lycii root rot Fusarium oxysporum；Do not occur band, is then determined as feminine gender, does not examine Fructus lycii root rot Fusarium oxysporum out.

Further, the loop-mediated isothermal amplification detection method of above-mentioned a kind of detection fructus lycii root rot Fusarium oxysporum, It is with 65 DEG C of progress LAMP reactions, reaction time 1h in the step E.

Further, the loop-mediated isothermal amplification detection method of above-mentioned a kind of detection fructus lycii root rot Fusarium oxysporum, It is with 80 DEG C of progress LAMP reactions, reaction time 20min in the step E.

Further, the loop-mediated isothermal amplification detection method of above-mentioned a kind of detection fructus lycii root rot Fusarium oxysporum, In the step F, 0.5 μ L fluorescent dye SYBR Green I can also be added, observe by the naked eye whether LAMP reaction solution occurs Color change carrys out judging result, and positive reaction is yellow green, detects fructus lycii root rot Fusarium oxysporum, and negative reaction is orange, Fructus lycii root rot Fusarium oxysporum is not detected.

Third object of the present invention is to provide a kind of above-mentioned ring mediations etc. for detecting fructus lycii root rot Fusarium oxysporum The specificity verification method of warm amplification detection method is to choose the fructus lycii pine root fungus in multiple areas to prepare DNA profiling, with ddH₂The DNA profiling that O substitutes object bacteria is negative control, with close kind of Fusaium solani Fusarium of Fusarium oxysporum The genomic DNA work of sonali and other 2 non-Fusariumsp Phytophthora infestans, Rhizoctonia solani For control group, LAMP reaction, observing response pipe color change are carried out respectively；The multiple regional respectively Gansu Province Jingyuan County, Jingtai County, Wuwei, Yumen City, Zhongweiof Ningxia, northwest China.

There is provided a kind of above-mentioned ring mediations etc. for detecting fructus lycii root rot Fusarium oxysporum for fourth object of the present invention The sensitivity verification method of warm amplification detection method is to use 10 times of concentration series dilution methods by the fructus lycii root rot point spore of extraction The DNA profiling of Fusariumsp carries out gradient dilution, make its DNA profiling mass concentration gradient be followed successively by 100ng/ μ L, 10ng/ μ L, 1ng/ μ L, 100pg/ μ L and 10pg/ μ L take 2 μ L to carry out LAMP reaction, observing response pipe color change as template respectively.

Fifth object of the present invention is to provide a kind of above-mentioned ring mediations etc. for detecting fructus lycii root rot Fusarium oxysporum The field verification method of warm amplification detection method is to randomly select sample 200mg diseased tissues preparation DNA as mould in field Plate carries out LAMP amplification, observing response pipe color change.

Sixth object of the present invention is to provide a kind of above-mentioned ring mediations etc. for detecting fructus lycii root rot Fusarium oxysporum The soil germ verification method of warm amplification detection method is that the spore of cause of disease bacteria strain Fusarium oxysporum in step B is taken to prepare spore Sub- suspension；Every part of 0.25g of soil sample without Fusarium oxysporum is taken, is put into the EP pipe of 2mL, adds different numbers in EP pipe respectively 10 000,1 000,100,10,1, the spore of amount, according to E.Z.N.A^TM.HP Fungal DNA Kit extracts kit explanation Book extracts pedotheque total DNA, carries out LAMP amplification as template, and using Fusarium oxysporum pure dna as positive control, Aqua sterilisa is as negative control, observing response pipe color change.

The invention has the benefit that

1, primer specificity is strong:

In LAMP system, 6 regions of 4 primer specificity identification target genes, and there was only 2 in PCR system Primer identifies 2 regions.The invention patent application LAMP design software designs 4 primers, including 2 outer primers F3, B3 and 2 Inner primer FIP, BIP, the fructus lycii root rot Fusarium oxysporum LAMP detection of 5 different geographic origins is in yellow green (positive), And compareing with other pathogens is in orange (feminine gender), electrophoresis detection does not have band.4 primers are designed using LAMP, in realization State effect.Judgement is directly compared by coloration method simultaneously, detection accuracy is substantially improved.

2, detection sensitivity is high:

LAMP technology detection sensitivity is 10~1000 times higher than regular-PCR technology.The invention patent sensitivity verification result Show that LAMP reaction solution detection sensitivity reaches 10pg in DNA level.

3, reaction speed is fast:

PCR generally requires 3~6h, and the LAMP reaction system optimum response program of the invention patent optimization is 65 DEG C of 1h, and 80 ℃20min。

4, directly detect by an unaided eye result:

Fluorescent dye SYBR Green I is added in the invention patent LAMP technology after completion of the reaction, observes by the naked eye Whether LAMP reaction solution, which occurs color change, is carried out judging result, and positive reaction is yellow green, and negative reaction is orange.

5, accuracy is high:

In the invention patent, the yin and yang attribute of amplified production is straight by the way that fluorescent dye SYBR Green I color change is added Connect judgement.

6, not easy to pollute:

Fluorescent dye SYBR Green I can be added in reaction solution before LAMP reaction, it is possible to prevente effectively from after reaction It uncaps pollution problem caused by fluorescent dye is added.

7, doubtful disease sample is directly detected:

The invention patent LAMP technology detects the DNA of doubtful disease sample extraction, it is only necessary to extract fructus lycii morbidity The total DNA at position can detect the presence or absence of Fusarium oxysporum, which is capable of detecting when the sharp spore in fructus lycii incidence tissue Fusariumsp, eliminates and contains multiple-microorganism on disease sample, is not readily separated the human factor influence for being purified to object bacteria by hand.

8, the high sensitivity detected in soil:

The sensitivity that the invention patent LAMP technology detects in the soil is 10 Fusarium oxysporum spore/0.25g soil, Soil K+adsorption sensitivity is very high.

In conclusion the invention patent establishes fusarium oxysporum germ on the basis of Fusarium oxysporum ITS distinguished sequence LAMP is easy, sensitive, method, LAMP reaction solution detection sensitivity reach 10pg/ μ L, soil in DNA level fast and accurately The sensitivity of middle detection is 10 Fusarium oxysporum spore/0.25g soil.The patent of invention is to produce upper fructus lycii root rot point spore The diagnosis and prevention and treatment of Fusariumsp provide important practical value.

Detailed description of the invention

What Fig. 1 was shown as Fusarium oxysporum LAMP reaction system establishes schematic diagram.

Wherein, 1 is Fusarium oxysporum LAMP reaction solution, and 2 be control product.

Fig. 2 is shown as Fusarium oxysporum LAMP specific detection schematic diagram.

Wherein, 1, Jingyuan County；2, Jingtai County；3, Wuwei；4,Rhizoctonia solani；5, Yumen City；6, Phytophthora infestans；7,Fusarium sonali；8, Zhongweiof Ningxia, northwest China；9, negative control.

Fig. 3 is shown as Fusarium oxysporum LAMP sensitivity technique schematic diagram.

Wherein, 1~5: concentration is followed successively by 100ng/ μ L, 10ng/ μ L, 1ng/ μ L, 100pg/ μ L, 10pg/ μ L；6: negative right According to.

Fig. 4 is shown as fructus lycii root rot diseased tissues LAMP detection schematic diagram.

Wherein, 1~7: morbidity sample；8: negative control；9: positive control.

Fig. 5 is shown as the detection schematic diagram of Fusarium oxysporum in soil.

Wherein, No. 1: positive control；2~No. 6: 10 000,1 000,100,10,1 spore suspensions；No. 7: negative right According to.

Specific embodiment