CN102643925A - Loop-mediated isothermal amplification (LAMP) primer composition for detecting phytophthora rot and application thereof - Google Patents

Abstract

The invention belongs to the field of a genetic engineering, and relates to a loop-mediated isothermal amplification (LAMP) primer composition for detecting phytophthora rot and an application thereof. The primer composition comprises a forward inner primer FIP showed in SEQ ID NO.1, a backward inner primer BIP showed in SEQ ID NO.2, a forward outer primer F3 showed in SEQ ID NO.3, a backward outer primer B3 showed in SEQ ID NO.4, and a reverse annular primer LB showed in SEQ ID NO.5. The LAMP primer composition can be applied to detection or identification of phytophthora rot. The LAMP primer composition has the advantages of good sensibility for specificity, rapid amplification, high efficiency and simple identification in the application of detecting phytophthora rot.

Description

Be used to detect the LAMP primer sets compound and the application thereof of soybean phytophthora

Technical field

The invention belongs to the genetically engineered field, relate to the LAMP primer sets compound and the application thereof that are used to detect soybean phytophthora.

Background technology

It is one of destructive disease on soybean produces that soyabean phytophthora Phytophthora sojae Kaufmann&Gerdemann infects the phytophthora root rot that soybean causes, also is one type of Quarantine Objects that China externally announces.Soybean phytophthora root rot came to light at Indiana, United States early than 1948 ^[3], after this, more than 20 soybean such as Canada, Brazil, Argentina produces the generation that country has reported this disease in succession ^[2,6]Su Yanchun ^[10]Equal reported first in 1993 the soybean main producing region, Heilungkiang of China soybean phytophthora root rot takes place, after this find the harm of soybean phytophthora successively in the soybean main producing region of northern China.The annual at present financial loss that causes to global soybean production is above 1,000,000,000 dollars ^[8]In order to stop the continuous expansion of soybean phytophthora spread scope, make soybean phytophthora root rot controlled, need detect quickly and accurately it.

The method of traditional detection soybean phytophthora is to carry out the trapping of Soybean Leaves dish with dull and stereotyped the cultivation perhaps the two to be combined ^[1,10]Traditional method has been brought into play vital role in soybean phytophthora detects, but wastes time and energy and the mould separation of epidemic disease, the morphology that require the operator to possess specialty is identified knowledge and rich experience.Along with the development of the relevant authentication method of nucleic acid, the method for PCR-based successfully has been used to detect soybean phytophthora ^[7]Though the PCR method is greatly improved on specificity and susceptibility, detection time is still long, general 4～5h, and PCR method relies on accurate temperature cycle device simultaneously.Its detection sensitivity is than higher, but testing process is complicated, can not satisfy the demand of rapid detection.

(Loop-mediated isothermal amplification LAMP) is a kind of new nucleic acid amplification technologies that Japanese Rong Yan strain formula can be invented to loop-mediated isothermal amplification technique ^[4],, become the new nucleic acid amplification technologies that can substitute PCR because it is simple to operate, quick, specificity is high, low cost and other advantages.It is 6 zone design, the 4 species specific primers to target gene; Under the effect of the big fragment polysaccharase of Bst, cause the self-circulation strand replacement reaction; In 60～65 ℃ of scope 80min, being attended by by product when synthesizing target dna in a large number---the magnesium pyrophosphate deposition of white produces.Because the LAMP amplification procedure relies on 6 isolated areas of identification target sequence; So atopic is very strong; And amplification process is under constant temperature, to carry out, and the ortho-water bath perhaps has the equipment of stable heat just can satisfy the reaction requirement, detects cost and reduces greatly.

The selection of target gene is one of important factor of LAMP detection.Detect soyabean phytophthora with round pcr and be based on the ribosomal gene sequence ^[7], but, therefore develop the focus that the detection target that makes new advances becomes detection because the rrna sequence does not have abundant site to distinguish all pathogenic bacterias.

Summary of the invention

The purpose of this invention is to provide the LAMP primer sets compound that is used to detect soybean phytophthora.

Another object of the present invention provides the application of this primer sets compound.

The object of the invention can be realized through following technical scheme:

Being used to detect the LAMP primer sets compound of soybean phytophthora, is that forward inner primer FIP, the sequence shown in the SEQ ID No.1 is that reverse inner primer BIP, the sequence shown in the SEQ ID No.2 is that forward outer primer F3, the sequence shown in the SEQ ID No.3 is that reverse outer primer B3, the sequence shown in the SEQID No.4 is that the reverse ring primer LB shown in the SEQ ID No.5 forms by sequence.

The application of described LAMP primer sets compound in the detection reagent of preparation detection soybean phytophthora.

A kind of LAMP test kit that detects soybean phytophthora contains described LAMP primer sets compound.The LAMP test kit of described detection soybean phytophthora preferably comprises: the Tris-HCl, 10mM KCl, the 10mM (NH4) that are oppositely encircled primer LB, 1.4mM dNTPs, 20mM pH 8.8 by 1.6 μ M forward inner primer FIP, the reverse inner primer BIP of 1.6 μ M, 0.2 μ M forward outer primer F3, the reverse outer primer B3 of 0.2 μ M, 0.2 μ M ₂SO4,6mM MgSO ₄, 0.1%Triton X-100,8U Bst DNA polymerase 320 units, 180mM hydroxynaphthol blue, add ultrapure water and be prepared into detection solution.

A kind of method that detects soybean phytophthora comprises the DNA that extracts mikrobe to be checked, is template with the DNA that extracts, and utilizes the described LAMP primer sets of claim 1 compound to carry out LAMP; Amplified production carries out agarose gel electrophoresis, and detected result under UV-light (hydroxynaphthol blue can not influence electrophoresis result) if there is characteristic scalariform band, then proves in institute's test sample to have soybean phytophthora; (hydroxyl naphthol blue HNB) belongs to a kind of of metals ion indicator to hydroxynaphthol blue.HNB is Mg ²⁺Titrating solution, its color changes with pH value of solution and changes, therefore can be through Mg in the monitoring LAMP reaction system ²⁺The variation of concentration and pH value of solution and play the effect of color indicator.Before the reaction HNB is joined in the reaction solution, reaction system is purple, Mg in the reaction process ²⁺By product P with the LAMP reaction ₂O ₇ ^4-In conjunction with producing a large amount of depositions, Mg in the solution ²⁺Concentration reduces, and pH changes, thereby makes the color of HNB become sky blue by purple.Therefore, reaction finishes the colour-change of back through reaction system, judges having or not of soybean phytophthora: there is soybean phytophthora in sky blue expression test positive; Purple representes that detected result is negative, does not have soybean phytophthora.

The method of described detection soybean phytophthora, preferred: as to extract the DNA of mikrobe to be checked, get 1 μ l dna solution; Add described detection solution of 23 μ l claims 4 and 1 μ l sterilization deionized water and carry out LAMP, the LAMP response procedures is: 60 ℃～65 ° C, 55～90min; Preferred 64 ℃, 80min;

Amplified production carries out agarose gel electrophoresis, and detected result under UV-light (hydroxynaphthol blue can not influence electrophoresis result) if there is characteristic scalariform band, proves that then the cause of disease that is detected is a soybean phytophthora; Perhaps with the colour-change of hydroxynaphthol blue as criterion as a result, there is soybean phytophthora in sky blue expression test positive; Purple representes that detected result is negative, does not have soybean phytophthora.

Beneficial effect:

The present invention compared with prior art, its advantage and positively effect show:

(1) practicality is good.Common PCR reaction is carried out gel electrophoresis to product and is easy to cause the product diffusion, and this is a main source of laboratory pollution; And ethidium bromide (EB) has huge poison, can accumulate carcinogenic; The long-term observation uv lamp also can cause injury to a certain degree to the experimenter.And LAMP reaction only needs in thermostat water bath, carry out, and the colour-change through HNB after reaction finishes is direct judged result just, thereby has increased its using value in the field.

(2) realized constant-temperature amplification; Must thermal cycling unlike the PCR method, so just broken away from dependence, as long as there is stable thermal source LAMP reaction just can take place to the thermal cycling instrument; Expanded the scope that LAMP uses greatly; Why LAMP can be because of in the LAMP reaction solution, having added trimethyl-glycine, double-stranded DNA is in the running balance of unwinding, under the effect of Bst archaeal dna polymerase, having realized amplification reacting under the constant thermal source.

(3) accuracy is high: because the mould detection technique of traditional soybean epidemic disease is just confirmed Quarantine Objects according to morphological specificity, can't get rid of the interference of human factor, be difficult to distinguish the close kind of form, detection accuracy has only 60-80%; And the present invention is according to YPT1 gene (the Genbank accession number: sequence DQ162958.1) of soybean phytophthora; Genome evolution district and the conserved regions of this sequence in soybean phytophthora taken turns at interval; Utilize Bioedit software that the YPT1 gene order of soybean phytophthora and the sequence of the mould kind of other epidemic diseases are compared, choose the specific LAMP primer of the distinctive one section sequences Design of soybean phytophthora.The LAMP reaction is through 6 isolated areas on 4 primers (FIP, BIP, F3, B3) specific recognition target sequence, and its specificity and sensitivity are all than higher.In addition, oppositely encircle primer LB and can improve speed of reaction and other four primers together, react under the situation of accuracy guaranteeing, make the present invention can carry out soybean phytophthora fast and detect.

The LAMP method of detection soybean phytophthora provided by the invention overcome in the prior art the required cycle of the biological detection method of soybean phytophthora grow, waste time and energy, problem and PCR detection technique loaded down with trivial details, poor specificity need the thermal cycling instrument, problem that can't the rapid detection soyabean phytophthora.Detection method of the present invention is under 64 ° of C isothermal conditions; Ability is quick, convenient, efficient, height is special, detect soyabean phytophthora with sensitivity; Do not need complex instrument; Can better satisfy the scene of soyabean phytophthora is detected; For the detection of quarantine venereal disease evil soyabean phytophthora provides new technology platform, can be used for customs and pass in and out soybean institute with the highly sensitive rapid detection of soybean phytophthora (Phytophthora sojae), the while also can be used for the early diagnosis of field soybean eqpidemic disease and the monitoring of germ.

Description of drawings

Fig. 1 LAMP detects the specificity of soybean phytophthora

(a) LAMP detects the specific gelose gel electrophoresis figure of soyabean phytophthora.

Wherein, M is 100bp DNA marke.1: standard soybean phytophthora bacterial strain 6497; 2-7: be respectively soybean phytophthora physiological strain R3, R6, R8, R12, R14, R17; 8: negative control; 9-12: be respectively soybean phytophthora physiological strain R19, R20, R28, R31; 13: negative control.

(b) color judges that LAMP detects the specificity colour developing figure of soyabean phytophthora.Show among the figure that the 1st～7 pipe and the 9th～12 pipe show sky blue, are positive; The 8th pipe and the 13rd pipe show purple, are negative.

Wherein, 1: standard soybean phytophthora bacterial strain 6497; 2-7: be respectively soybean phytophthora physiological strain R3, R6, R8, R12, R14, R17; 8: negative control; 9-12: be respectively soybean phytophthora physiological strain R19, R20, R28, R31; 13: negative control.

Fig. 2 carries out the specificity that LAMP detects soybean phytophthora through the bacterial strain to soybean phytophthora kind and other kind

(a) LAMP detects the specific gelose gel electrophoresis figure of soyabean phytophthora.

Wherein, 1: standard soybean phytophthora bacterial strain 6497; 2: ramie mould; 3: the Oak Tree epidemic disease is mould; 4: the Jue Shi epidemic disease is mould; 5: Phytophthora cactorum; 6: melon epidemic disease is mould; 7: phytophthora infestans; 8: negative control; 9: standard soybean phytophthora bacterial strain 6497; 10: ultimate corruption is mould; 11: Fusarium equiseti; 12: the tack anthrax-bacilus; 13: Pyricularia oryzae; 14: dry thread Pyrenomycetes; 15: big beautiful Verticillium; 16: negative control.

(b) color judges that LAMP detects the specificity colour developing figure of soyabean phytophthora.Show among the figure that the 1st pipe and the 9th pipe show sky blue, are positive; All the other each pipes show purple, are negative.

Wherein 1: standard soybean phytophthora bacterial strain 6497; 2: ramie mould; 3: the Oak Tree epidemic disease is mould; 4: the Jue Shi epidemic disease is mould; 5: Phytophthora cactorum; 6: melon epidemic disease is mould; 7: phytophthora infestans; 8: negative control; 9: standard soybean phytophthora bacterial strain 6497; 10: ultimate corruption is mould; 11: Fusarium equiseti; 12: the tack anthrax-bacilus; 13: Pyricularia oryzae; 14: dry thread Pyrenomycetes; 15: big beautiful Verticillium; 16: negative control.

Fig. 3 LAMP detects the sensitivity of soybean phytophthora

LAMP amplification different concns genomic dna; From left to right be respectively the amplification that contains 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg DNA in the reaction system of 25 μ L respectively.

(a) LAMP detects soyabean phytophthora sensitive gelose gel electrophoresis figure.The LAMP reaction can amplify the band of trapezoidal shape specifically from the P.sojoe bacterial strain.Electrophorogram shows that the sensitivity of LAMP reaction reaches 10pg.M is 100bp DNA marker.

(b) color judges that LAMP detects the sensitivity colour developing figure of soyabean phytophthora.The reaction tubes that contains 100ng, 10ng, 1ng, 100pg, 10pg soybean phytophthora DNA in the reaction system of 25 μ L respectively shows sky blue; Be positive; The reaction tubes that contains 1pg, 100fg, 10fg soybean phytophthora DNA in the reaction system of 25 μ L respectively shows purple, and reaction is negative.The colour developing result shows that the sensitivity of LAMP reaction reaches 10pg.

Embodiment

Embodiment 1

A kind of LAMP detection kit that is used to detect soybean phytophthora is oppositely encircled Tris-HCl, 10mM KCl, the 10mM (NH4) of primer LB, 1.4mM dNTPs, 20mM pH 8.8 by 1.6 μ M forward inner primer FIP, the reverse inner primer BIP of 1.6 μ M, 0.2 μ M forward outer primer F3, the reverse outer primer B3 of 0.2 μ M, 0.2 μ M ₂SO4,6mM MgSO ₄, 0.1%Triton X-100,8UBst DNA polymerase 320 units, 180mM hydroxynaphthol blue, add ultrapure water and be prepared into detection solution.

The specificity test one of embodiment 2 soyabean phytophthora LAMP reaction

In order to verify the specificity of LAMP method; Choice criteria soybean phytophthora bacterial strain 6497 is (available from ATCC; Be numbered ATCC 16705, down with) and with soybean phytophthora with the DNA of a kind of different physiological strain R3, R6, R8, R12, R14, R17, R19, R20, R28, R31 as template, get 1 μ l dna solution; The detection solution and the 2 μ l sterilization deionized water that add 23 μ l embodiment, 1 preparation carry out the LAMP reaction, and response procedures is: 64 ° of C 80min.The result shows when removing to increase the dna profiling of physiological strain of soybean phytophthora with the LAMP primer, amplifies band; And negative control does not have to amplify the purpose band.While as criterion as a result, during the dna profiling of amplification soybean phytophthora standard soybean phytophthora bacterial strain 6497 and physiological strain, presents sky blue based on the reaction system color reaction; Negative control presents purple.This LAMP detection method that shows that the present invention sets up has excellent specificity (Fig. 1).

The specificity test two of embodiment 3 soyabean phytophthora LAMP reaction

In order to verify the specificity of LAMP method, select and soybean phytophthora (ramie mould not of the same race; The Oak Tree epidemic disease is mould; The Jue Shi epidemic disease is mould; Phytophthora cactorum; Melon epidemic disease is mould; Phytophthora infestans) and not the generic bacterium (ultimate corruption is mould; Fusarium equiseti; The tack anthrax-bacilus; Pyricularia oryzae; Dry thread Pyrenomycetes; Big beautiful Verticillium) DNA gets 1 μ l dna solution as template, and the detection solution and the 1 μ l sterilization deionized water that add 23 μ l embodiment, 1 preparation carry out the LAMP reaction, and response procedures is: 64 ° of C 80min.The result shows when removing to increase the dna profiling of soybean phytophthora with the LAMP primer, amplifies band; And not of the same race with soybean phytophthora, generic bacterium and negative control all do not have to amplify the purpose band; While as criterion as a result, during the dna profiling of amplification soybean phytophthora, presents sky blue based on the reaction system color reaction; Amplification all presents purple (Fig. 2) with soybean phytophthora dna profiling not of the same race, not generic bacterium and negative control.

The sensitivity test of embodiment 4 soyabean phytophthora LAMP reaction

In order to confirm the sensitivity of LAMP detection method, the DNA of the standard soybean phytophthora bacterial strain 6497 that extracts is then carried out 10 doubling dilutions with DEPC water with spectrophotometric determination concentration (1 μ g/ μ l) ,-70 ℃ of preservations are as template.Get each concentration DNA diluent 1 μ L behind 10 doubling dilutions respectively as template, the detection solution and the 1 μ l sterilization deionized water that add 23 μ l embodiment, 1 preparation carry out the LAMP reaction, and response procedures is: 64 ° of C 80min.Get appearance on the 2 μ L amplified productions, electrophoresis on 2% sepharose, the result shows that the LAMP method can detect the DNA that concentration is the soybean phytophthora of 100pg; The HNB coupling reaction shows that the sensitivity of LAMP reaction also reaches 100pg (Fig. 3).

Embodiment 5 detects soybean phytophthora from the inward soybean of customs carries disease germs soil sample:

A kind of LAMP test kit that detects soybean phytophthora comprises: the Tris-HCl, 10mM KCl, the 10mM (NH4) that are oppositely encircled primer LB, 1.4mM dNTPs, 20mM pH 8.8 by 1.6 μ M forward inner primer FIP, the reverse inner primer BIP of 1.6 μ M, 0.2 μ M forward outer primer F3, the reverse outer primer B3 of 0.2 μ M, 0.2 μ M ₂SO4,6mM MgSO ₄, 0.1%Triton X-100,8UBst DNA polymerase 320 units, 180mM hydroxynaphthol blue, add ultrapure water and be prepared into detection solution.

Be used to detect the method for soybean phytophthora with above-mentioned soybean phytophthora detection kit, comprise:

1) enrichment of oospore in the soil:

Get pedotheque 20～100 grams to be checked, grind, successively adopt the big grogs in 200 eye mesh screen places to go, filter through 400,500,800 eye mesh screens then, wash repeatedly with 3～10 premium on currency simultaneously,, use the 1ml aqueous suspension from 800 mesh sieve online collection oospore.Because oospore can not see through 800 eye mesh screens, processing can reach the effect that makes the oospore enrichment like this.

2) from micro-oospore, extract DNA:

To transfer in the centrifuge tube of 1.5mL with the oospore that sterilized water suspends, under the 12000r.min-1 rotating speed centrifugal 5 minutes, pour out liquid;

Add 50 μ L CTAB buffer, grind, add 500 μ L CTAB buffer again, water-bath 30 minutes;

Add the extracting of equal-volume chloroform, under the 12000r.min-1 rotating speed centrifugal 10 minutes, draw supernatant;

The 3M NaAc that adds 1/10 volume, the no water-ice ethanol of 2 times of volumes, precipitation at room temperature 30 minutes under the 12000r.min-1 rotating speed centrifugal 10 minutes, falls dry liquids;

Add 1mL 70% (V/V) washing with alcohol, under the 12000r.min-1 rotating speed centrifugal 10 minutes, fall dry liquids, dry to alcohol-free flavor;

Add the dissolving of 10 μ L aseptic double-distilled waters, be used for the template of LAMP amplification.

3) soybean phytophthora LAMP detects, and comprising:

The LAMP of soybean phytophthora detects: get 1 μ l dna solution, add described detection solution of 23 μ l claims 6 and 1 μ l sterilization deionized water, TV is 25 μ l; Response procedures is: 64 ° of C 80min; As reaction indicator, the color that amplification finishes back LAMP reaction system presents sky blue with HNB (hydroxynaphthol blue), judges that with this from the inward soybean of customs carries disease germs soil sample, can produce positive reaction contains soyabean phytophthora.

The LAMP of embodiment 6 susceptible soybean plantss detects

Adopt the DNA of the soybean plant strain of NaOH alkaline lysis method of extracting inoculation soybean phytophthora, it is used for the LAMP amplification as template.Get 1uLDNA solution, press the method for embodiment 5, carry out the LAMP reaction.The result shows in the soybean plant strain tissue of inoculating soybean phytophthora and carries out LAMP that its color reaction also appears positive sky blue; And healthy plant and negative control present purple.

Reference

1.Canaday，C.，and?Schmitthenner，A.1982.Isolating?Phytophthora?megasperma?f.sp.glycinea?from?soil?with?a?baiting?method?that?minimizes?Pythium?contamination.Soil?Biology?and?Biochemistry?14：67-68.

2.Erwin，D.，Ribeiro，O.，and?Shattock，R.1996.Phytophthora?diseases?worldwide.APS?press?St?Paul，MN，USA.

3.Kaufmann，M.J.，and?Gerdemann，J.1957.Root?and?stem?rot?of?soybean?caused?by?Phytophthora?sojae?n.sp.University?of?Illinois?at?Urbana-Champaign.

4.Notomi，T.，Okayama，H.，Masubuchi，H.，Yonekawa，T.，Watanabe，K.，Amino，N.，and?Hase，T.2000.Loop-mediated?isothermal?amplification?of?DNA.Nucleic?Acids?Research?28：e63-e63.

5.Qutob，D.，Tedman-Jones，J.，Dong，S.，Kuflu，K.，Pham，H.，Wang，Y.，Dou，D.，Kale，S.，Arredondo，F.，and?Tylet，B.2009.Copy?number?variation?and?transcriptional?polymorphisms?of?Phytophthora?sojae?RXLR?effector?genes?Avr1a?and?Avr3a.PLoS?One?4：5066.

6.Schmitthenner，A.1985.Problems?and?progress?in?control?of?Phytophthora?root?rot?of?soybean.Plant?disease69：362.

7.Wang，Y.，Zhang，W.，and?Zheng，X.2006.Rapid?and?sensitive?detection?of?Phytophthora?sojae?in?soil?and?infected?soybeans?by?species-specific?polymerase?chain?reaction?assays.Phytopathology?96：1315-1321.

8.Wrather，J.，Stienstra，W.，and?Koenning，S.2001.Soybean?disease?loss?estimates?for?the?United?States?from1996?to?1998.Canadian?Journal?of?Plant?Pathology?23：122-131.

9. Zhu Zhen is eastern, Wang Huabo, and Wang Xiaoming, normal Ru Zhen, the military little luxuriant and rich with fragrance .2003. Chinese soybean phytophthora of and distributes and the virulence Study on Diversity. Scientia Agricultura Sinica 36:793-799.

10. Su Yan is pure, and and Shen Chong Yao .1993. phytophthora sojae kaufmann&gerdemann is in the discovery and the biological Characteristics Study thereof of China. Plant Pathology.

Claims (6)

1. be used to detect the LAMP primer sets compound of soybean phytophthora, it is characterized in that by sequence being that forward inner primer FIP, the sequence shown in the SEQ ID No.1 is that reverse inner primer BIP, the sequence shown in the SEQ ID No.2 is that forward outer primer F3, the sequence shown in the SEQ ID No.3 is that reverse outer primer B3, the sequence shown in the SEQ ID No.4 is that the reverse ring primer LB shown in the SEQ ID No.5 forms.

2. the application of the described LAMP primer sets of claim 1 compound in the detection reagent of preparation detection soybean phytophthora.

3. a LAMP test kit that detects soybean phytophthora is characterized in that containing the described LAMP primer sets of claim 1 compound.

4. the LAMP test kit of detection soybean phytophthora according to claim 3 is characterized in that described test kit comprises: the Tris-HCl, 10mM KCl, the 10mM (NH4) that are oppositely encircled primer LB, 1.4mM dNTPs, 20mM pH 8.8 by 1.6 μ M forward inner primer FIP, the reverse inner primer BIP of 1.6 μ M, 0.2 μ M forward outer primer F3, the reverse outer primer B3 of 0.2 μ M, 0.2 μ M ₂SO4,6mM MgSO ₄, 0.1%Triton X-100,8U Bst DNA polymerase 320 units, 180mM hydroxynaphthol blue, add ultrapure water and be prepared into detection solution.

5. a method that detects soybean phytophthora is characterized in that extracting the DNA of mikrobe to be checked, is template with the DNA that extracts, and utilizes the described LAMP primer sets of claim 1 compound to carry out LAMP;

Amplified production carries out agarose gel electrophoresis, and detected result under UV-light if there is characteristic scalariform band, proves that then the cause of disease that is detected is a soybean phytophthora;

Perhaps with the colour-change of LAMP reaction system as criterion as a result, there is soybean phytophthora in sky blue expression test positive; Purple representes that detected result is negative, does not have soybean phytophthora.

6. the method for detection soybean phytophthora according to claim 5; It is characterized in that extracting the DNA of mikrobe to be checked, get 1 μ l dna solution, add described detection solution of 23 μ l claims 4 and 1 μ l sterilization deionized water and carry out LAMP; The LAMP response procedures is: 60 ℃～65 ° C; 55～90min, preferred 64 ℃, 80min;

Amplified production carries out agarose gel electrophoresis, detected result under UV-light, if there is characteristic scalariform band, then there is soybean phytophthora in proof;

Perhaps with the colour-change of LAMP reaction system as criterion as a result, there is soybean phytophthora in sky blue expression test positive; Purple representes that detected result is negative, does not have soybean phytophthora.

Images