CN101805796A - Primer for detecting the soybean phytophthora and kit and method thereof - Google Patents
Primer for detecting the soybean phytophthora and kit and method thereof Download PDFInfo
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Abstract
The invention provides a primer for detecting the soybean phytophthora, and a kit and a method thereof. The primer and the detecting method for detecting the soybean phytophthora have high accuracy, high specificity and high sensitivity, and can detect the various propagules of the soybean phytophthora such as hypha, oospore, zoospore and the like. The whole technique is fast, sensitive and accurate, which has important significance in the prevention of foreign soybean phytophthora, the detection of pathogenic bacteria in domestic affected area, the international trade and the like.
Description
Technical field
The present invention relates to a kind of primer, test kit and method that is used to detect soybean phytophthora, belong to biological technical field.
Background technology
By soyabean phytophthora Phytophthora sojae Kaufmann﹠amp; It is one of destructive disease on soybean produces that Gerdemann infects the phytophthora root rot that soybean causes, also is the class Quarantine Objects (Xu Zhigang, 2003) that China externally announces.Soyabean phytophthora can infect soybean in each period of soybeans they grow, the season disease rainy in humidity takes place especially serious, because soybean phytophthora carries out syngenesis in homothallic mode, on disease plant, can produce a large amount of oospore, remaining in oospore in the soil after the plant results just becomes the primary source of infection of soybean phytophthora, it is reported the oospore of soybean phytophthora (Xu Xiuhong etc., 2003) more than 5 years of can in soil, surviving.Therefore (Zhu Zhendong etc., 1999) in case take place will be difficult to eliminate in soybean phytophthora root rot.
The method of traditional detection soybean phytophthora is to carry out Soybean Leaves dish trapping and dull and stereotyped cultivate or the two is combined (Canaday and Schmitthenner, 1982; Oudemans, 1999; Zhu Zhendong etc., 2003; Prince meets etc., and 2005).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 (Tsao, 1990; Dobrowolski and O ' Brien, 1993), so be difficult to promote the use aborning.
Round pcr has fast and the sensitive advantage, and is more and more important in the evaluation and the status in the detection of pathogen.In recent years, the PCR detection method of some phytophthoras is developed.The source of the primer that designs in these methods mainly all is transcribed spacer (ITS) (Cooke et al., 1995a, b; Bonants et al., 1997; Tooley et al., 1997; Troutet al., 1997; Liew et al., 1998; Tooley et al., 1998; Schubert et al., 1999; Bonants et al., 2000; Judelson and Tooley, 2000; Winton﹠amp; Hansen, 2001; Grote et al., 2002; Ippolito et al., 2002) or elicitin gene (Coelho et al., 1997; Lacourt and Duncan, 1997; Kong et al., 2003b).These zones or gene all are high copy in genome, so be easy to be detected.But discover that more and more the ITS sequence difference of the mould kind of part epidemic disease is very little, the primer that designs as target is difficult to these phytophthoras and its sibling species are made a distinction.Wang etc. (2006) find during according to ITS sequences Design soybean phytophthora Auele Specific Primer, the similarity of the ITS sequence of soybean phytophthora and P.melonis is up to 97%, the Auele Specific Primer 3 ' of soybean phytophthora is terminal only to differ 1 base with the corresponding sequence of P.melonis, annealing temperature need be brought up to could be with these two kinds of mould making a distinction of epidemic disease more than 66 ℃, and still too high annealing temperature can reduce the sensitivity of PCR reaction.And improve thoroughly head it off of annealing temperature, when the concentration of P.melonis is higher, still can cause false-positive phenomenon.
Summary of the invention
Technical problem to be solved by this invention provides a kind of be used to the detect primer of soybean phytophthora, the detection kit that comprises this primer and detection method, utilizes this primer and detection method to detect soybean phytophthora accuracy height, high specificity, susceptibility height.
Technical scheme provided by the invention is: a kind of primer that is used to detect soybean phytophthora, that is: TrapF1/TrapR1, its upstream primer nucleotide sequence be as described in the SEQ ID No.1, and the downstream primer nucleotide sequence is as described in the SEQ ID No.2.
A kind of test kit that detects soybean phytophthora comprises above-mentioned primer.
Described test kit also comprises 4 kinds of dNTP, 10 * PCR reaction buffer, 2mM Mg
2+, 1%BSA and Taq enzyme.
The present invention also provides a kind of method that detects soybean phytophthora: the DNA that extracts testing sample is as template, utilize described primer to carry out the PCR reaction, get PCR reaction amplified production and carry out gel electrophoresis, voltage is 50-100V, after 30 minutes under UV-light detected result, if exist molecular weight to be about the DNA band of 267bp, then prove in institute's sample product and contain soybean phytophthora.
For improving the sensitivity that detects, the present invention also provides the another kind of primer that is used to detect soybean phytophthora, it comprises two pairs of primers, first pair of primer be promptly: TrapF1/TrapR1, its upstream primer nucleotide sequence be as described in the SEQ ID No.1, and the downstream primer nucleotide sequence is as described in the SEQ ID No.2, second pair of primer, that is: TrapF2/TrapR2, its upstream primer nucleotide sequence be as described in the SEQ ID No.3, and the downstream primer nucleotide sequence is as described in the SEQ ID No.4.
The present invention also provides the test kit of another kind of detection soybean phytophthora, and it comprises above-mentioned first pair of primer and second pair of primer.
Above-mentioned test kit also comprises 4 kinds of dNTP, 10 * PCR reaction buffer, 2mM Mg
2+, 1%BSA, tween 20 and Taq enzyme.
The present invention also provides the another kind of method that detects soybean phytophthora, its detailed process is: the DNA that extracts testing sample, carry out the sleeve type PCR reaction, the template of first round PCR reaction is for extracting the DNA of testing sample, primer is described first pair of primer (TrapF1/TrapR1), second template of taking turns the PCR reaction is a first round PCR reaction product, primer is described second pair of primer (TrapF2/TrapR2), getting second takes turns PCR reaction amplified production and carries out gel electrophoresis, voltage is 50-100V, after 30 minutes under UV-light detected result, if exist molecular weight to be about the DNA band of 267bp, then prove in institute's sample product and contain soybean phytophthora.
Simultaneously, the present invention also provides a kind of PCR reaction system of utilizing described primer to set up.The mixed solution cumulative volume of PCR reaction is 25 μ l, comprising: the template DNA of respective concentration, 0.5 μ M primer, 4 kinds of each 50 μ M of dNTP, 2.5 μ l10 * PCR reaction buffer, 2mM Mg
2+, 2.5 μ l 1%BSA, 1.25 Taq of unit enzymes (TaKaRa) carry out amplified reaction on PTC200 (MJ company) PCR instrument.Response procedures is: 94 ℃ of pre-sex change 5min; Enter circulation then, 94 ℃ of sex change 30sec, 60 ℃ of annealing 30sec, 72 ℃ are extended 30sec, totally 35 circulations; Last 72 ℃ are extended 7min.Getting 7 μ l amplified productions electrophoresis 30min (100V) in 1.0% sepharose after reaction finishes, detect on gel imaging system and take pictures, is the DNA band of 267bp if there is molecular weight, then proves in institute's sample product and contains soybean phytophthora.
In order to improve detection sensitivity, the present invention has further set up the sleeve type PCR reaction system.Make up respectively as first round reaction primer and primer TrapF2/TrapR2 with TrapF1/TrapR1 and to carry out sleeve type PCR.The mixed solution cumulative volume of first round PCR reaction is 25 μ l, comprising: 0.5 μ M primer, 4 kinds of each 50 μ M of dNTP, 2.5 μ l, 10 * PCR reaction buffer, 2mM Mg
2+, 2.5 μ l 1%BSA, 0.5 μ l tween 20,1.25 Taq of unit enzymes (TaKaRa), template DNA is some, carries out amplified reaction on PTC200 (MJ company) PCR instrument.Response procedures is: 94 ℃ of pre-sex change 5min; Enter circulation then, 94 ℃ of sex change 30sec, 60 ℃ of annealing 30sec, 72 ℃ are extended 30sec, totally 35 circulations; Last 72 ℃ are extended 7min.Get 1 μ l first round PCR reaction product and carry out second as template and take turns reaction, reaction system and response procedures are with described in the 1.5.2.Getting 7 μ l amplified productions electrophoresis 30min (100V) in 1.0% sepharose after reaction finishes, detect on gel imaging system and take pictures, is the DNA band of 267bp if there is molecular weight, then proves in institute's sample product and contains soybean phytophthora.
The present invention has following advantage:
In carrying out soybean phytophthora Study on Genome process, the inventor has found the class transposon sequence of one section soybean phytophthora, in the analytic process of this sequence being carried out information biology, find this sequence height repetition and only be present in the soybean phytophthora therefore very suitable target as the soybean phytophthora Molecular Detection.The specificity test-results confirms that this primer possesses the specificity of planting.The inventor according to the class transposon sequences Design of soybean phytophthora the special primer TrapF1/TrapR1 of soybean phytophthora, utilize that this is mould to 26 kinds of epidemic diseases to primer, 29 kinds of other fungies totally 232 bacterial strains carry out the specificity checking.Test-results proves that this primer can only increase and obtain the band of a 267bp from the soybean phytophthora bacterial strain.Other bacterial strain, the very proximate phytophthora P.melonis of ITS sequence and soybean phytophthora and also can infect the Fusarium solani f.sp.glycines that soybean is caused the root-rot symptom particularly, Rhizoctonia solani, no amplified band such as Pythium aphanidermatum and Colletotrichum glycines occurs.The inventor utilizes this primer that strain soybean phytophthora bacterial strain surplus the soybean phytophthora 100 of various regions and nations has been carried out pcr amplification, the result can both amplify the band of a 267bp from the different soybean phytophthora bacterial strain in these sources, show that such transposon sequence is suitable as the target of soybean phytophthora Molecular Detection, can diagnose exactly and detect the root rot that soybean phytophthora causes based on the special primer of this sequences Design.
The checking result of primer sensitivity confirms, only passes through single-wheel PCR, this primer can detect 10pg the soybean phytophthora genomic dna.In order to improve the sensitivity of detection, the inventor designs another again to primer TrapF2/TrapR2 in the fragment of TrapF1/TrapR1 amplification, take turns amplimer as second of sleeve type PCR.The sleeve type PCR reaction that primer TrapF1/TrapR1 and TrapF2/TrapR2 combination are carried out can make detection sensitivity improve 1000 times, can detect the soybean phytophthora genomic dna of 10fg.In the testing process of zoospore and oospore,, just can detect 1 oospore and 1 zoospore respectively only by single-wheel PCR.。
Soybean blight is a kind of destructive disease on soybean produces, and also is simultaneously the important Quarantine Objects in China's port quarantine, is not still having at present under the prerequisite of effectively preventing and treating medicine, and controlling its propagation is one of major measure that reduces harm.The inventor has found a new soybean phytophthora Molecular Detection target, and detects special primer according to this sequence for target has designed soybean phytophthora, sets up soybean phytophthora regular-PCR Molecular Detection system.This system can to the propagulum of the various forms of soybean phytophthora for example mycelia, oospore and zoospore etc. detect, a whole set of technology fast, sensitive, accurately.Therefore, no matter preventing external soybean phytophthora invasion, still to equal tool significances in aspect such as monitoring of domestic epidemic-stricken area pathogenic bacteria and international trade contacts.
Description of drawings
Fig. 1 is a soybean phytophthora class transposon sequence.
Fig. 2 is the pcr amplification electrophorogram of primer TrapF1/TrapR1, and wherein, M is 2000bp DNA marker; 1 is standard soybean phytophthora bacterial strain 6497; 2-18 is mould kind of bacterial strains of other epidemic diseases; 19 negative controls.
Fig. 3 be primer TrapF1/TrapR1 the pcr amplification electrophorogram wherein, M is 2000bp DNA marker; 1 is standard soybean phytophthora bacterial strain 6497; 2-18 is other fungal bacterial strains; 19 negative contrasts.
Fig. 4 is the pcr amplification electrophorogram of primer TrapF1/TrapR1, and wherein, M is 2000bp DNA marker; 1 is standard soybean phytophthora bacterial strain 6497; 2-22 is from country variant and geographic soybean phytophthora bacterial strain; 23 negative contrasts.
Fig. 5 is the detected result of the sensitivity of primer TrapF1/TrapR1 amplification soybean phytophthora different concns genomic dna, and wherein, M is 2000bp DNA marker; 1-9 is the amplification that contains 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg, 100ag, 10ag DNA in the reaction system of 25 μ l respectively; 10 negative contrasts.
Fig. 6 is the detected result of the sensitivity of primer TrapF1/TrapR1 and primer TrapF2/TrapR2 combination sleeve type PCR amplification soybean phytophthora different concns genomic dna, and wherein, M is 2000bp DNA marker; 1-8 is the amplification that contains 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg, 100ag DNA in the reaction system of 25 μ l respectively; 9 negative contrasts.
Fig. 7 is the sensitivity detected result of primer TrapF1/TrapR1 amplification soybean phytophthora different concns oospore, and wherein, M is 2000bp DNA marker; 1 positive contrast; 2～11 is the amplification that contains 10,9,8,7,6,5,4,3,2,1 oospore DNA in the reaction system of 25 μ l respectively; 12 negative contrasts.
Fig. 8 is the sensitivity of primer TrapF1/TrapR1 amplification soybean phytophthora different concns zoospore, and wherein, M is 2000bp DNA marker; 1 positive contrast; 2-11 is the amplification that contains 10,9,8,7,6,5,4,3,2,1 zoospore DNA in the reaction system of 25 μ l respectively; 12 negative contrasts.
Fig. 9 is the PCR detected result of soybean phytophthora in the inoculation soybean plant strain, and wherein, M is 2000bp DNA marker; 1 negative contrast; 2-11 is the amplification of soybean phytophthora in the inoculation soybean; 12 for extracting the amplification of the healthy DNA of soyabean tissue; 13 positive contrasts.
Figure 10 is the sleeve type PCR detected result of pathogen in the soil of the sick field of soybean, and wherein, M is 2000bp DNA marker; 1-5 is a soil sample in the piece of susceptible soybean field; 6 positive contrasts; 7 is healthy field piece soil sample; 8 negative contrasts.
Embodiment
Detailed description below by embodiment is further illustrated the present invention, but is not limitation of the present invention, only does the example explanation.
Embodiment 1: design, synthetic primer are also set up the PCR reaction system of soybean phytophthora detection kit
One, design of primers and synthetic
According to a pair of special primer TrapF1/TrapR1 (specifically seeing nucleotides sequence tabulation SEQ ID NO.1 and SEQ ID NO.2) that is used for the soybean phytophthora Molecular Detection of soybean phytophthora class transposon sequence (see figure 1) design, in order to improve the sensitivity of detection, the present invention is based on this sequences Design another to primer TrapF2/TrapR2 (specifically seeing nucleotides sequence tabulation SEQ IDNO.3 and SEQ ID NO.4), take turns amplimer as second of sleeve type PCR.All primers all entrust Shanghai living worker company synthetic.
Two, set up conventional PCR reaction system
The mixed solution cumulative volume of PCR reaction is 25 μ l, comprising: the template DNA of respective concentration, 0.5 μ MTrapF1/TrapR1 primer, 4 kinds of each 50 μ M of dNTP, 2.5 μ l, 10 * PCR reaction buffer, 2mM Mg
2+, 2.5 μ l 1%BSA, 1.25 Taq of unit enzymes (TaKaRa) carry out amplified reaction on PTC200 (MJ company) PCR instrument.Response procedures is: 94 ℃ of pre-sex change 5min; Enter circulation then, 94 ℃ of sex change 30sec, 60 ℃ of annealing 30sec, 72 ℃ are extended 30sec, totally 35 circulations; Last 72 ℃ are extended 7min.Get 7 μ l amplified productions electrophoresis 30min (100V) in 1.0% sepharose after reaction finishes, on gel imaging system, detect and take pictures,, then prove in institute's sample product and contain soybean phytophthora if exist molecular weight to be about the DNA band of 267bp.
Three, set up the sleeve type PCR reaction system
In order to improve detection sensitivity, further set up the sleeve type PCR reaction system.Make up respectively as first round reaction primer and primer TrapF2/TrapR2 with TrapF1/TrapR1 and to carry out sleeve type PCR.The mixed solution cumulative volume of first round PCR reaction is 25 μ l, comprising: 0.5 μ M primer, 4 kinds of each 50 μ M of dNTP, 2.5 μ l, 10 * PCR reaction buffer, 2mM Mg
2+, 2.5 μ l 1%BSA, 0.5 μ l tween 20,1.25 Taq of unit enzymes (TaKaRa), template DNA is some, carries out amplified reaction on PTC200 (MJ company) PCR instrument.Response procedures is: 94 ℃ of pre-sex change 5min; Enter circulation then, 94 ℃ of sex change 30sec, 60 ℃ of annealing 30sec, 72 ℃ are extended 30sec, totally 35 circulations; Last 72 ℃ are extended 7min.Get 1 μ l first round PCR reaction product and carry out second as template and take turns reaction, reaction system and response procedures are with described popular response system and program.Get 7 μ l amplified productions electrophoresis 30min (100V) in 1.0% sepharose after reaction finishes, on gel imaging system, detect and take pictures,, then prove in institute's sample product and contain soybean phytophthora if exist molecular weight to be about the DNA band of 267bp.
Embodiment 2: the preparation dna profiling
Extract the template of the DNA of all kinds of samples as the PCR reaction, detailed process is as follows:
One, the extracting of hypha powder DNA
Improve a little with reference to (1989) methods such as Sambrook.The hypha powder that takes a morsel adds 900 μ l 2%CTAB extracting solutions and 90 μ l 10%SDS, the whirlpool mixing, and in 55 ℃ of water-bath 1h, middle every 10min turns upside down several times.The centrifugal 10min of 12000rpm gets and resets and add equal-volume phenol/chloroform/primary isoamyl alcohol (25: 24: 1), puts upside down mixing, the centrifugal 10min of 12000rpm; Supernatant is transferred to new pipe, adds the equal-volume chloroform, put upside down mixing gently, the centrifugal 5min of 12000rpm.Supernatant is transferred in the new pipe, adds the dehydrated alcohol of 2 times of volumes and the 3M NaAc of 1/10 volume (pH 5.2) ,-20 ℃ of precipitations (＞1h).The centrifugal 10min of 12000rpm, the supernatant that inclines precipitates with 70% washing with alcohol twice, and room temperature is dried.Add an amount of sterilization ultrapure water or TE (pH 8.0) dissolution precipitation (containing 20 μ g/ml RNase), behind 37 ℃ of processing 1h ,-20 ℃ of preservations are standby.
Two, the extraction of the preparation of oospore suspension and DNA
Slightly change referring to Zheng Xiaobo (1997).The media transfer that will contain oospore adds about water 100ml (decide on the container size) in sterilising vessel, 5000 rev/mins of homogenate 2 minutes is with broken substratum and make oospore and mycelia and agar disengaging.Homogenate filters through 200,300,600 eye mesh screens successively, and water washes each eye mesh screen repeatedly, washes the oospore of collecting on 600 eye mesh screens with a small amount of aqua sterilisa at last, is prepared into oospore suspension.Microscopically to suspension in the number of oospore count, the concentration of oospore is adjusted into 100/μ l, use
(Q-Biogene Ltd USA) extracts DNA to test kit, and extraction step is referring to the test kit specification sheets.
Three, the extraction of the preparation of zoospore suspension and DNA
With reference to the mould release zoospore of Zheng Xiaobo (1997) method inducing soybean epidemic disease, elimination mycelia piece obtains zoospore suspension.Microscopically to suspension in the number of oospore count, the concentration of zoospore is adjusted into 1000/ μ l.Zoospore suspension is joined in eppendorf (EP) pipe of 1.5ml, the centrifugal 10min of 12000g abandons supernatant liquor, and taking precipitate extracts DNA by method 1.4.1.The DNA that extracts is dissolved with the sterilization ultrapure water, and-20 ℃ of preservations are standby.
Four, the extraction of pathogenic bacteria DNA in the biological tissue
With reference to (1993) NaOH methods such as Wang and improvement a little.Get the plant tissue of one section morbidity, every milligram of tissue adds 10 μ l 0.5M NaOH, is transferred in the EP pipe of 1.5ml the centrifugal 5min of 12000rpm after fully grinding in mortar, get 5 μ l supernatant liquors and add 495 μ l 0.1mM Tris (pH 8.0), get 1 μ l behind the mixing and be directly used in the PCR reaction.Each reacts triplicate at least, simultaneously for determining that no PCR inhibition exists in the plant, with present method ITS fragment of extracting the healthy tree tissue DNA after universal primer ITS1/ITS4 increases as blank.
Five, the extraction of pathogenic bacteria DNA in the soil
All pedotheques adopt
(Q-Biogene Ltd USA) carries out the extraction of DNA to the SPIN test kit.The soil DNA extraction step is referring to the test kit specification sheets.Pick up from the not healthy field piece of morbidity with the soil sample that compares.
Carry out the detection of soybean phytophthora with the template of the foregoing description 1 detection primer, test kit and method and embodiment 2 preparations below.
Embodiment 3: the specificity that detects primer
With eukaryote universal primer ITS1/ITS4 and special primer TrapF1/TrapR1 the bacterial strain of 26 mould kinds of epidemic disease, 29 other kinds is amounted to 232 strains respectively and carried out pcr amplification, test used soybean phytophthora bacterial strain host, source and quantity and see Table 1, all bacterial strains are single-ascospore strain in the test, are stored in department of plant pathology of Agricultural University Of Nanjing.The result shows that eukaryote universal primer ITS1/ITS4 can amplify the band (seeing Table 1) of an about 700bp from all strains testeds.Prove that the DNA that this institute is carried can be used for pcr amplification, got rid of the influence that the DNA quality causes amplification.Auele Specific Primer TrapF1/TrapR1 can only amplify the band (asking for an interview table 1, Fig. 2-4) of a 267bp specifically from 120 P.sojae bacterial strains for examination.Show that the primer according to the design of soybean phytophthora transposon sequence (see figure 1) has the specificity of planting, P.sojae can be distinguished with mould kind of other epidemic disease and fungi.
Table 1 is used to detect specific fungi of primer TrapF1/TrapR1 and oomycetes bacterial strain
In the last table :+expression has the specific amplification band of primer I TS1/ITS4 or TrapF1/TrapR1; The no amplified production of-expression.
Embodiment 4: detect the sensitivity of primer
1, the sensitivity of soybean phytophthora genomic dna detects
The genomic dna of soybean phytophthora bacterial strain 6497 is begun 10 times from 1ng/ μ l be diluted to 10ag/ μ l downwards, it is that template is carried out pcr amplification with special primer TrapF1/TrapR1 that every concentration gradient is got 1 μ l.This specific band (asking for an interview Fig. 5) that still can increase and obtain 267bp when the result shows the genomic dna that contains 10pg in the reaction system of 25 μ l to primer.
Be template to the product that the genomic dna of above-mentioned different concns carries out pcr amplification earlier, carry out the sleeve type PCR amplification with soybean phytophthora special primer TrapF2/TrapR2 again with primer TrapF1/TrapR1.The result shows, sleeve type PCR product amount is compared with the PCR product amount of carrying out primer amplified separately and is significantly improved, can make original band brightness sample very faint or invisible amplified band produce tangible band (asking for an interview figure-6), show that the sleeve type PCR reaction of carrying out with primer TrapF1/TrapR1 and designed TrapF2/TrapR2 combination of primers can make detection sensitivity improve 1000 times, can detect the genomic dna of 10fg.
2, the sensitivity of soybean phytophthora oospore DNA detects
With concentration is 100 oospore/μ l by gradient dilution to 1 oospore/μ l of 10 times, and the DNA with 1 each concentration of μ l is the pcr amplification that template is carried out primer TrapF1/TrapR1 respectively.The result shows, can detect specific amplified band (asking for an interview Fig. 7) when containing the DNA amount of 1 oospore in 25 μ l reaction systems.
3, the sensitivity of soybean phytophthora zoospore DNA detects
With concentration is 1000 zoospores/μ l by gradient dilution to 1 zoospore/μ l of 10 times, and the DNA with 1 each concentration of μ l is that template is carried out the single-wheel pcr amplification respectively.The result shows, can detect specific amplified band (asking for an interview Fig. 8) when containing the DNA amount of 1 zoospore in 25 μ l reaction systems.
Embodiment 5: the PCR of susceptible soybean plant strain detects
Extract the DNA of the soybean plant strain of inoculation soybean phytophthora with the NaOH cracking process, it is used for pcr amplification as template.The result is that near the tissue of soybean plant strain inoculation point of inoculation soybean phytophthora all can amplify the 267bp band, and healthy plant can only amplify the band of a 700bp with eukaryote ITS district universal primer ITS1/ITS4, and (asking for an interview Fig. 9) occur with the no band of TrapF1/TrapR1 amplification.Show that NaOH method and special primer TrapF1/TrapR1 can be used for the fast PCR detection of susceptible soybean plant strain.
Embodiment 6: the detection of pathogen in the soil
The employing sleeve type PCR detects the pathogen in the sick soil that picks up from the field, different areas, all soil samples of picking up from the sick field of soybean band with the identical size of positive control that all increased, soil sample as negative control does not then amplify any band (asking for an interview Figure 10), illustrates that the soil sample of picking up from these sick fields contains P.sojae.The result of employing soybean leaves mass trapping has also confirmed the inventor's pcr amplification result.Soil sample that all pcr amplifications are positive adopts leaf dish mass trapping all to elicit soybean phytophthora, does not then elicit as the soil sample of negative control, prove the special primer that the present invention designs can be used for detecting whether carry soybean phytophthora in the soil.
The nucleotides sequence tabulation
＜110〉Agricultural University Of Nanjing
＜120〉be used to detect primer, test kit and the method for soybean phytophthora molecule
<160>4
<210>1
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: the sequence of synthetic
<400>1
CGGTGGCTCT?CGGCATTCGT?G 21
<210>2
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: the sequence of synthetic
<400>2
CACCCTACTG?TTATAGACAC?G 21
<210>3
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: the sequence of synthetic
<400>3
ATCTGACGGT?GGCTCTCGGC 20
<210>4
<211>22
<212>DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: the sequence of synthetic
<400>4
ATCTAATCAA?CCATCACTCA?CC 22
Claims (10)
1. primer that is used to detect soybean phytophthora is characterized in that: its upstream primer nucleotide sequence is as described in the SEQ ID No.1, and the downstream primer nucleotide sequence is as described in the SEQ ID No.2.
2. a test kit that detects soybean phytophthora is characterized in that: comprise the described primer of claim 1.
3. test kit according to claim 2 is characterized in that: also comprise 4 kinds of dNTP, 10 * PCR reaction buffer, 2mM Mg
2+, 1%BSA and Taq enzyme.
4. method of utilizing the described primer of claim 1 to detect soybean phytophthora, it is characterized in that: the DNA that extracts testing sample is as template, utilize described primer to carry out the PCR reaction, getting PCR reaction amplified production detects, if exist molecular weight to be about the DNA band of 267bp, then prove in institute's sample product and contain soybean phytophthora.
5. method according to claim 4 is characterized in that: the detection to PCR reaction amplified production is a detected through gel electrophoresis.
6. primer that is used to detect soybean phytophthora, it is characterized in that: comprise two pairs of primers, first pair of its upstream primer nucleotide sequence of primer is as described in the SEQ ID No.1, the downstream primer nucleotide sequence is as described in the SEQ ID No.2, second pair of its upstream primer nucleotide sequence of primer be as described in the SEQ ID No.3, and the downstream primer nucleotide sequence is as described in the SEQ ID No.4.
7. a test kit that detects soybean phytophthora is characterized in that: comprise the described primer of claim 6.
8. test kit according to claim 7 is characterized in that: also comprise 4 kinds of dNT, 10 * PCR reaction buffer, 2mM Mg
2+, 1%BSA, tween 20 and Taq enzyme.
9. method of utilizing the described primer of claim 6 to detect soybean phytophthora, it is characterized in that: the DNA that extracts testing sample carries out the sleeve type PCR reaction, the template of first round PCR reaction is for extracting the DNA of testing sample, primer is described first pair of primer, second template of taking turns the PCR reaction is a first round PCR reaction product, primer is described second pair of primer, getting second takes turns PCR reaction amplified production and detects, if exist molecular weight to be about the DNA band of 267bp, then prove in institute's sample product and contain soybean phytophthora.
10. method according to claim 9 is characterized in that: the detection to PCR reaction amplified production is a detected through gel electrophoresis.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101942520A (en) * | 2010-10-14 | 2011-01-12 | 南京农业大学 | Marker of avirulence gene PsAvr3b of phytophthora sojae |
| CN102220415A (en) * | 2011-04-15 | 2011-10-19 | 南京农业大学 | Markers of phytophthora sojae avirulence gene Avr5 |
| CN102643925A (en) * | 2012-05-16 | 2012-08-22 | 南京农业大学 | Loop-mediated isothermal amplification (LAMP) primer composition for detecting phytophthora rot and application thereof |
| CN107674923A (en) * | 2017-11-06 | 2018-02-09 | 东北农业大学 | The specific molecular detection primer of Germ To Soybean Frogeye Leaf Spot and its application |
| CN109943624A (en) * | 2019-03-14 | 2019-06-28 | 南京林业大学 | A kind of method and its primer special and probe combinations based on RPA- Sidestream chromatography technology detection soybean phytophthora |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1453367A (en) * | 2003-05-21 | 2003-11-05 | 南京农业大学 | Soybean phytophthora testing reagent kit and its test method |
| CN101519697A (en) * | 2009-04-07 | 2009-09-02 | 福建省农业科学院植物保护研究所 | Phytophthora sojae molecule detecting primer and detection reagent kit thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1453367A (en) * | 2003-05-21 | 2003-11-05 | 南京农业大学 | Soybean phytophthora testing reagent kit and its test method |
| CN101519697A (en) * | 2009-04-07 | 2009-09-02 | 福建省农业科学院植物保护研究所 | Phytophthora sojae molecule detecting primer and detection reagent kit thereof |
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| 《Phytopathology》 20061231 Yuanchao Wang et al. Rapid and Sensitive Detection of Phytophthora sojae in Soil and Infected Soybeans by Species-Specific Polymerase Chain Reaction Assays 1315-1321 1-10 第96卷, 第12期 2 * |
| 《Science》 20060901 Brett M. Tyler et al. Phytophthora Genome Sequences Uncover Evolutionary Origins and Mechanisms of Pathogenesis 1261-1266 1-10 第313卷, 2 * |
| 《南京农业大学学报》 20040831 王立安等 大豆疫霉的 ITS 分子检测 38-41 1-10 第27卷, 第3期 2 * |
| 《微生物学报》 20100204 殷丽华 大豆疫霉菌一个 DNA 指纹分析重复序列探针的鉴定 524-529 1-10 第50卷, 第4期 2 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101942520A (en) * | 2010-10-14 | 2011-01-12 | 南京农业大学 | Marker of avirulence gene PsAvr3b of phytophthora sojae |
| CN101942520B (en) * | 2010-10-14 | 2012-10-17 | 南京农业大学 | Marker of avirulence gene PsAvr3b of phytophthora sojae |
| CN102220415A (en) * | 2011-04-15 | 2011-10-19 | 南京农业大学 | Markers of phytophthora sojae avirulence gene Avr5 |
| CN102643925A (en) * | 2012-05-16 | 2012-08-22 | 南京农业大学 | Loop-mediated isothermal amplification (LAMP) primer composition for detecting phytophthora rot and application thereof |
| CN107674923A (en) * | 2017-11-06 | 2018-02-09 | 东北农业大学 | The specific molecular detection primer of Germ To Soybean Frogeye Leaf Spot and its application |
| CN107674923B (en) * | 2017-11-06 | 2020-12-01 | 东北农业大学 | Specific molecular detection primer for soybean botrytis cinerea and application thereof |
| CN109943624A (en) * | 2019-03-14 | 2019-06-28 | 南京林业大学 | A kind of method and its primer special and probe combinations based on RPA- Sidestream chromatography technology detection soybean phytophthora |
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