CN108546772A - Exserohilum turcicum LAMP detection primer and its rapid detection method and application - Google Patents
Exserohilum turcicum LAMP detection primer and its rapid detection method and application Download PDFInfo
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Abstract
The present invention provides a kind of Exserohilum turcicum LAMP detection primer and its rapid detection method and applications, it is exclusively used in the specific detection of Exserohilum turcicum, belong to corps diseases detection and biotechnology, devise a kind of Exserohilum turcicum LAMP detection primer, including outer primer F3 and B3, and inner primer FIP and BIP, see sequence table.Based on the Exserohilum turcicum detection method that the primer is established, chromogenic reaction or agarose gel electrophoresis detection, can be observed green fluorescence or the characteristic trapezoid-shaped strips of LAMP occur after LAMP constant-temperature amplifications.The LAMP detection primer and its detection method invented can realize quick, sensitive, the accurate detection of infection Exserohilum turcicum plant in production practice, it can be used for the early diagnosis of field diseases and the monitoring of germ, identification simultaneously, reliable technology and theoretical foundation provided for the early warning and prevention and control of the leaf blight of corn.
Description
Technical field
The present invention relates to a kind of Exserohilum turcicum LAMP detection primer and its rapid detection method and applications, are exclusively used in jade
The rapid molecular detection of rice Exserohilum Turcicum, while can realize the early diagnosis of field corn leaf blight and the monitoring of germ, identification,
Belong to corps diseases detection, identification, prevention and biotechnology.
Background technology
Corn is wide with its purposes, yield is high, planting conditions are required with the multiple countries plantation extensively in the world of low advantage.
In China, corn is that cultivated area ranks first, yield occupies important grain and the industrial crops of second place, in addition to as grain purposes,
Modern Maize Industry also develops the multiple uses such as the energy, feed, fruits and vegetables.Maize Industry develops in a healthy way to improving China's agricultural production
Industry structure, Ensuring Food Safety, expanding gross national economy has very positive important function.
The leaf blight of corn (northern corn leaf blight, NCLB) is by Exserohilum bacterium (Exserohilum
Turcicum the withered property fungal disease of serious leaf caused by) infecting, can cause harm blade, also cause harm when serious leaf sheath and bract.
The leaf blight of corn in 1876 is reported for the first time in Italy, early in the twentieth century betides America, Europe, Asia and ocean extensively
The corn producing region in continent becomes one of main leaf diseases of corn in the world today, can also cause fusarium root rot of maize, stem rot sometimes
Other maize diseases occurring and damage.It is reported that the general time underproduction 20% or so of the leaf blight of corn, the underproduction 50% when serious
More than.Exserohilum turcicum cause of disease is overwintering with invalid body with mycelium and conidium, and the First aggression for becoming morbidity in the 2nd year comes
Source generates conidium in corn growth season overwintering bacterium source, is propagated on healthy maize leaf with rainwater or air-flow, suitable
Temperature and humidity conditions under sprout intrusion.Such as in susceptible variety, local wilting can be caused for 14 days or so by invading, tissue necrosis,
And then withered scab is formed, a large amount of conidiums can be generated again on scab, causes repeatedly to infect with air-flow propagation, causes disease
Eruption and prevalence.Therefore the initial phase for infecting initial stage to disease of Exserohilum turcicum is the best period of disease control, and big spot
The germ initial stage of infecting is difficult to from symptom distinguish with graywall, Northern leaf spot and helminthosporium maydis, and the disease based on symptom is conventional
Diagnostic techniques needs using Koch's Postulates by pathogenicbacteria separation culture, pathogen identification, connects bacterium, symptom analysis,
Time-consuming, efficiency is low, accuracy is poor, it is difficult to accomplish detection in time and the propagation of effective control pathogen and disease when disease occurs
It is popular.And Exserohilum turcicum in morphological feature with southern corn leaf blight, that is, Bipolaris maydis (Bipolaris
Maydis) extremely similar, it is difficult to identify, therefore the big spot of corn is used for there is an urgent need to establish quick, the sensitive detection method of one kind
The early diagnosis of disease, technical support is provided for the control approach of disease.
Ring mediated isothermal amplification (loop-mediated isothermal amplification, LAMP) technology is by day
The exploitations such as this scientist Notomi it is a kind of it is easy, quickly, accurately and efficiently nucleic acid constant-temperature amplification method.The technology is in isothermal
Under the conditions of large amplification is realized in the short time, realize 10 in 30min-60min9-1010Amplification again has very high sensitivity
And specificity, and it is easy to operate, testing result can visually judge.Compared to traditional round pcr, LAMP technology whole process constant temperature is anti-
It answers, is not necessarily to PCR instrument, and the big high sensitivity of amplification amount.
Invention content
For in the prior art it is cumbersome to the detection and identification program of Exserohilum turcicum, time-consuming, is wanted to identification experience
Ask height, accuracy low, PCR detects the problem of needing by equipment such as amplification instruments, and the present invention provides a kind of Exserohilum turcicums
LAMP detection primer and simplicity, quick, sensitive, special visible detection method.
The purpose of the present invention is achieved through the following technical solutions：
A kind of Exserohilum turcicum LAMP detection primer, the Exserohilum turcicum LAMP detection primer include just drawing outward
Object F3, reversed outer primer B3, forward direction inner primer FIP and reversed inner primer BIP, the nucleotides sequence of each primer are classified as：
F3:5’-GGAAGGGCACCATGTTGAC-3’；
B3:5’-GCTGGTGGAGAACTCTGAC-3’；
FIP:5’-ACCTCGTCTCCGCCGTCATGT-AGAGTTAAGCTGACCAGGGA-3’；
BIP:5’-GGTTCAGGTCGCCGTACGAG-CTGCATTGACAACGAGGCT-3’。
A kind of Exserohilum turcicum LAMP rapid detection methods utilize positive outer primer F3：5’-
GGAAGGGCACCATGTTGAC-3 ', reversed outer primer B3：5 '-GCTGGTGGAGAACTCTGAC-3 ', positive inner primer FIP：
5 '-ACCTCGTCTCCGCCGTCATGT-AGAGTTAAGCTGACCAGGGA-3 ' and reversed inner primer BIP：5’-
GGTTCAGGTCGCCGTACGAG-CTGCATTGACAACGAGGCT-3 ' carries out LAMP reactions.
For the prior art, the advantage of the invention is that：
1. high specificity, accuracy are high：The present invention is to have chosen 6 according to Exserohilum turcicum β-tubulin gene orders
A specific region devises 4 LAMP primers for having specific amplified effect to Exserohilum turcicum.To different geographical next
The Exserohilum turcicum in source, southern corn leaf blight, Rhizoctonia solani Kahn, D. carbonum, Gray Leaf Spot Pathogen, corn south
The corn tissue of rust germ, the plant tissue for carrying Exserohilum turcicum and health has carried out detection verification, and only corn is big
The positive is presented in pinta bacterium and the tissue for carrying the germ, illustrates the primer sets and detection method designed by the present invention for detecting jade
Rice Exserohilum Turcicum is accurate and reliable, can the raw similar disease of symptom characteristic on corn of effective district distribution；
2. high sensitivity：LAMP reachable 100fg on DNA level to the detection sensitivity of Exserohilum turcicum, have very
High sensitivity；
3. applicability is wide, practicability is good：The detection method of the Exserohilum turcicum of the present invention, can not only be to germ mycelium
It is detected, can also be detected the early detection, it can be achieved that Exserohilum turcicum to susceptible corn tissue, i.e., it is aobvious in disease
It is detected before disease, prevents the eruption and prevalence of disease.
4. easy to operate quick：LAMP is to carry out under isothermal conditions, only one water-bath of need, result visualization,
General entire detection process can be completed in 1.5 hours, simple and efficient to handle.
Description of the drawings
Fig. 1 is specific detection result of the LAMP detection method of the present invention to Exserohilum turcicum：Upper figure is that agarose is solidifying
Gel electrophoresis testing result, figure below are visualization colour developing result.Swimming lane M is 5000bp DNA Marker in upper figure, and swimming lane 1 is sun
Property control, the Exserohilum turcicum that swimming lane 2-4 is separate sources, swimming lane 5-10 is respectively：Southern corn leaf blight, maize sheath blight
Bacterium, D. carbonum, Gray Leaf Spot Pathogen, corn southern rust germ, negative control；1- in figure below visualization colour developing result
4 display green fluorescences, other do not show green fluorescence.
Fig. 2 is sensitivity testing result of the LAMP detection method of the present invention to Exserohilum turcicum：Upper figure is that agarose is solidifying
Gel electrophoresis testing result, figure below are visualization colour developing result.Swimming lane M is 5000bp DNA Marker in upper figure, and swimming lane 1 is sun
Property control, the template DNA concentration of swimming lane 2- swimming lanes 9 is respectively：10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg,
Swimming lane 10- swimming lanes 11 are negative control；1-7 shows green fluorescence in figure below visualization colour developing result, other do not show that green is glimmering
Light.
Fig. 3 be LAMP detection method of the present invention to corn incidence tissue practicability testing result, upper figure is Ago-Gel
Electrophoresis detection is as a result, figure below is visualization colour developing result.Swimming lane M is 5000bp DNA Marker, swimming lane 1- swimming lane 5 in upper figure
Respectively Exserohilum turcicum, the corn tissue of naturally-occurring leaf blight, artificial infection hair occur the corn tissue of leaf blight, are good for
Health corn tissue, positive control, swimming lane 6- swimming lanes 7 are negative control；1-3,5 display greens are glimmering in figure below visualization colour developing result
Light, other do not show green fluorescence.
Specific implementation mode
In order to make content of the present invention easily facilitate understanding, With reference to embodiment to of the present invention
Technical solution is described further.
Test method used in following embodiments is conventional method unless otherwise specified.
Test material as used in the following examples, reagent etc., unless otherwise specified, commercially obtain.
1 Exserohilum turcicum LAMP primer group of embodiment designs
There is specific amplified effect to Exserohilum turcicum according to the design of Exserohilum turcicum β-tubulin gene orders
LAMP detection primer group, including 2 outer primers (F3 and B3) and 2 inner primers (FIP and BIP), nucleotide sequence is respectively：
F3：5’-GGAAGGGCACCATGTTGAC-3’；
B3：5’-GCTGGTGGAGAACTCTGAC-3’；
FIP：5’-ACCTCGTCTCCGCCGTCATGT-AGAGTTAAGCTGACCAGGGA-3’；
BIP：5’-GGTTCAGGTCGCCGTACGAG-CTGCATTGACAACGAGGCT-3’.
The foundation of 2 Exserohilum turcicum LAMP detection method of embodiment
1. the extraction of sample to be tested DNA：
1. for when detecting pathogen pure culture, strains tested genomic DNA is extracted using CTAB methods, specific steps are such as
Under：
(1) it takes 0.1g hypha powders in 1.5mL centrifuge tubes, 900 μ L 2wt.%CTAB extracting solutions is added, use oscillator
Mixing is vibrated, 60 DEG C of water-bath 60min, under room temperature, 12000r/min centrifuge 15min；
(2) 700 μ L of supernatant are taken, adding isometric phenol, chloroform, isoamyl alcohol mixed liquor, (each volume ratio is 25:24:1), mildly
It shakes, under room temperature, 8000r/min centrifuges 10min；
(3) 500 μ L of supernatant are taken, isometric chloroform is added and extracts again once, under room temperature, 8000r/min centrifugations
10min；
(4) 350 μ L of supernatant are taken, 1/10 volume 3mol/L NaAc and 2 times of volume absolute ethyl alcohols, -20 DEG C of precipitations are added
60min, under the conditions of 4 DEG C, 8000r/min centrifuges 5min；
(5) liquid is discarded supernatant, 700 μ L volumetric concentrations of addition are 70% ice ethyl alcohol, jog 10sec, under the conditions of 4 DEG C,
8000r/min centrifuges 10sec, dries, and 50 μ L TE buffer solutions is added, -20 DEG C save backup.
2. when for detecting plant tissue with the presence or absence of Exserohilum turcicum, being extracted using NaOH rapid cleavage methods beautiful
Rice plant tissue genomic DNA, is as follows：
A. plant tissue 0.1g to be detected is weighed, 30 μ L of 0.5mol/L NaOH are added, tissue is fully milled to paste；
B. paste tissue is transferred in 1.5mL centrifuge tubes, 12000r/min centrifuges 6min, takes 5 μ l of supernatant；
C. 495 μ L 0.1mol/L Tris-HCl (pH=8.0) are added in supernatant, is uniformly mixed, takes 1.0 μ L conducts
Pcr template is expanded；
2. carrying out LAMP amplifications as template to extract sample to be tested DNA：25 μ L of LAMP reaction systems, reaction system include
0.2mmol/L F3,0.2mmol/L B3,1.6mmol/L FIP, 1.6mmol/L BIP, Bst archaeal dna polymerases are 8U, DNA moulds
LAMP reaction mixtures (40mM Tris-HCl, the 20mM (NH of plate 50~100ng, 12.5 μ L4)2SO4, 20mM KCl, 16mM
MgSO4, 1.6mol/L glycine betaines, 2.0mM dNTPs, 0.2%Trion X-100), supply 25 μ L with sterile ultra-pure water.
LAMP reaction conditions are 63-65 DEG C of incubation 45-60min, 85 DEG C of inactivation 5-10min.
3.LAMP reaction results measure：Using fluorescent dye visual observations method or agarose gel electrophoresis method.Described is glimmering
Photoinitiator dye visual observations method：It waits for LAMP after reaction, fluorescent dye color developing agent is added in the amplified production of LAMP reactions
1.0 μ L of SYBR green I, colour developing result observe that the judgement of green fluorescence is the positive, and there are Exserohilum turcicums；It is orange or
Crocus is judged as feminine gender, and Exserohilum turcicum is not present.The agarose gel electrophoresis method：Take 2.0 μ L LAMP amplification productions
Object is detected with 2% agarose gel electrophoresis, trapezoid-shaped strips is such as occurred and is judged as the positive, there are Exserohilum turcicums；Do not occur
Band is then judged as feminine gender, and Exserohilum turcicum is not present.
3 Exserohilum turcicum LAMP of embodiment detects specific assay
1. extracting 3 plants of separate sources Exserohilum turcicums, southern corn leaf blight, Rhizoctonia solani Kahn, jade using CTAB methods
Rice round spot germ, Gray Leaf Spot Pathogen, corn southern rust germ genomic DNA.
2. carrying out LAMP amplifications as template for trying the DNA of bacterium to extract：25 μ L of LAMP reaction systems, reaction system include
0.2mmol/L F3,0.2mmol/L B3,1.6mmol/L FIP, 1.6mmol/L BIP, Bst archaeal dna polymerases are 8U, DNA moulds
LAMP reaction mixtures (40mM Tris-HCl, the 20mM (NH of plate 50~100ng, 12.5 μ L4)2SO4, 20mM KCl, 16mM
MgSO4, 1.6mol/L glycine betaines, 2.0mM dNTPs, 0.2%Trion X-100), supply 25 μ L with sterile ultra-pure water.
LAMP reaction conditions are 63-65 DEG C of incubation 45-60min, 85 DEG C of inactivation 5-10min.
3.LAMP reaction results measure：Using fluorescent dye visual observations method or agarose gel electrophoresis method.Described is glimmering
Photoinitiator dye visual observations method：It waits for LAMP after reaction, fluorescent dye color developing agent is added in the amplified production of LAMP reactions
1.0 μ L of SYBR green I, colour developing result observe that the judgement of green fluorescence is the positive, and there are Exserohilum turcicums；It is orange or
Crocus is judged as feminine gender, and Exserohilum turcicum is not present.The agarose gel electrophoresis method：Take 2.0 μ L LAMP amplification productions
Object is detected with 2% agarose gel electrophoresis, trapezoid-shaped strips is such as occurred and is judged as the positive, there are Exserohilum turcicums；Do not occur
Band is then judged as feminine gender, and Exserohilum turcicum is not present.
4. specificity verification result
As shown in Figure 1, green fluorescence, Ago-Gel can be observed in 3 plants of separate sources Exserohilum turcicum colour developing results
There are LAMP characteristic trapezoid-shaped strips in electrophoresis, and is orange, agar for trying other crop pathogens and negative control colour developing result
Sugared gel electrophoresis does not occur LAMP characteristic trapezoid-shaped strips, show the present invention LAMP primer group can by Exserohilum turcicum with
Other pathogens distinguish, and there is very strong specificity, detection method of the invention can be used for the special of Exserohilum turcicum
Property detection and identification.
4 Exserohilum turcicum LAMP detection sensitivities of embodiment measure
1. using the genomic DNA of CTAB methods extraction Exserohilum turcicum；
2. by the genomic DNA of the Exserohilum turcicum of extraction, after spectrophotometric determination concentration, with sterile ultra-pure water
Dilution, is configured to series concentration, spare；
3. carrying out routine LAMP amplifications as template at series concentration DNA using preparation：25 μ L of LAMP reaction systems, reactant
System includes 0.2mmol/L F3, and 0.2mmol/L B3,1.6mmol/L FIP, 1.6mmol/L BIP, Bst archaeal dna polymerases are
LAMP reaction mixtures (40mM Tris-HCl, the 20mM (NH of 8U, DNA profiling 1fg~10ng, 12.5 μ L4)2SO4, 20mM
KCl, 16mM MgSO4, 1.6mol/L glycine betaines, 2.0mM dNTPs, 0.2%Trion X-100), it is mended with sterile ultra-pure water
25 μ L of foot.LAMP reaction conditions are 63-65 DEG C of incubation 45-60min, 85 DEG C of inactivation 5-10min.
4.LAMP reaction results measure：Using fluorescent dye visual observations method or agarose gel electrophoresis method.Described is glimmering
Photoinitiator dye visual observations method：It waits for LAMP after reaction, fluorescent dye color developing agent is added in the amplified production of LAMP reactions
1.0 μ L of SYBR green I, colour developing result observe that the judgement of green fluorescence is the positive, and there are Exserohilum turcicums；It is orange or
Crocus is judged as feminine gender, and Exserohilum turcicum is not detected.The agarose gel electrophoresis method：Take 2.0 μ L LAMP amplifications
Product is detected with 2% agarose gel electrophoresis, trapezoid-shaped strips is such as occurred and is judged as the positive, there are Exserohilum turcicums；Do not go out
Existing band is then judged as feminine gender, and Exserohilum turcicum is not detected.
5. testing result
As shown in Fig. 2, green fluorescence can be observed in colour developing result, it is characteristic trapezoidal that LAMP occurs in agarose gel electrophoresis
Band, detection sensitivity is up to 100fg.
The LAMP detections of Exserohilum turcicum in the morbidity plant of embodiment 5
1.. CTAB methods are used to extract Exserohilum turcicum genomic DNA；Plant is extracted using NaOH rapid cleavage methods
Tissue gene group DNA.
2. to carry out LAMP amplifications as template using the DNA for extracting test sample：25 μ L of LAMP reaction systems, reaction system packet
0.2mmol/L F3 are included, 0.2mmol/L B3,1.6mmol/L FIP, 1.6mmol/L BIP, Bst archaeal dna polymerases are 8U, DNA
LAMP reaction mixtures (40mM Tris-HCl, the 20mM (NH of template 50~100ng, 12.5 μ L4)2SO4, 20mM KCl, 16mM
MgSO4, 1.6mol/L glycine betaines, 2.0mM dNTPs, 0.2%Trion X-100), supply 25 μ L with sterile ultra-pure water.
LAMP reaction conditions are 63-65 DEG C of incubation 45-60min, 85 DEG C of inactivation 5-10min.
3.LAMP reaction results measure：Using fluorescent dye visual observations method or agarose gel electrophoresis method.Described is glimmering
Photoinitiator dye visual observations method：It waits for LAMP after reaction, fluorescent dye color developing agent is added in the amplified production of LAMP reactions
1.0 μ L of SYBR green I, colour developing result observe that the judgement of green fluorescence is the positive, and there are Exserohilum turcicums；It is orange or
Crocus is judged as feminine gender, and Exserohilum turcicum is not present.The agarose gel electrophoresis method：Take 2.0 μ L LAMP amplification productions
Object is detected with 2% agarose gel electrophoresis, trapezoid-shaped strips is such as occurred and is judged as the positive, there are Exserohilum turcicums；Do not occur
Band is then judged as feminine gender, and Exserohilum turcicum is not present.
4. testing result
As shown in figure 3, Exserohilum turcicum, the corn tissue of the naturally-occurring leaf blight of corn, artificial infection generation corn
Green fluorescence can be observed in the corn tissue of leaf blight, positive control colour developing result, and LAMP features occurs in agarose gel electrophoresis
Property trapezoid belt, and healthy corn tissue, negative control colour developing result are orange, and agarose gel electrophoresis does not occur LAMP features
The trapezoid belt of property, shows that LAMP primer group and detection method of the present invention can be additionally used in the inspection of field corn leaf blight disease plant
It surveys.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification should all belong to the covering scope of the present invention.
Sequence table
<110>Inst. of Plant Protection, fujian Academy of Agricultural Science
<120>Exserohilum turcicum LAMP detection primer and its rapid detection method and application
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 1
ggaagggcac catgttgac 19
<210> 2
<211> 19
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 2
gctggtggag aactctgac 19
<210> 3
<211> 41
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 3
acctcgtctc cgccgtcatg tagagttaag ctgaccaggg a 41
<210> 4
<211> 39
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 4
ggttcaggtc gccgtacgag ctgcattgac aacgaggct 39
Claims (8)
1. a kind of Exserohilum turcicum LAMP detection primer, it is characterised in that：The Exserohilum turcicum LAMP detection primer packet
Include positive outer primer F3, reversed outer primer B3, forward direction inner primer FIP and reversed inner primer BIP, the nucleotide sequence of each primer
For：
F3:5’-GGAAGGGCACCATGTTGAC-3’；
B3:5’-GCTGGTGGAGAACTCTGAC-3’；
FIP:5’-ACCTCGTCTCCGCCGTCATGT-AGAGTTAAGCTGACCAGGGA-3’；
BIP:5’-GGTTCAGGTCGCCGTACGAG-CTGCATTGACAACGAGGCT-3’。
2. a kind of Exserohilum turcicum LAMP rapid detection methods, it is characterised in that：Utilize positive outer primer F3：5’-
GGAAGGGCACCATGTTGAC-3 ', reversed outer primer B3：5 '-GCTGGTGGAGAACTCTGAC-3 ', positive inner primer FIP：
5 '-ACCTCGTCTCCGCCGTCATGT-AGAGTTAAGCTGACCAGGGA-3 ' and reversed inner primer BIP：5’-
GGTTCAGGTCGCCGTACGAG-CTGCATTGACAACGAGGCT-3 ' establishes LAMP detection method, to Exserohilum turcicum into
Row detection.
3. Exserohilum turcicum LAMP rapid detection methods according to claim 2, it is characterised in that：LAMP reaction systems
For 25 μ L, reaction system includes 0.2mmol/L F3,0.2mmol/L B3,1.6mmol/L FIP, 1.6mmol/L BIP, Bst
Archaeal dna polymerase is 8U, 50~100ng of DNA profiling, and the LAMP reaction mixtures of 12.5 μ L supply 25 μ L with sterile ultra-pure water,
Wherein, DNA profiling is extracted from corn sample to be detected.
4. Exserohilum turcicum LAMP rapid detection methods according to claim 3, it is characterised in that：LAMP reaction mixing
Liquid Tris-HCl containing 40mM, 20mM (NH4)2SO4,20mM KCl,16mM MgSO4, 1.6mol/L glycine betaines, 2.0mM
DNTPs, 0.2%Trion X-100.
5. Exserohilum turcicum LAMP rapid detection methods according to claim 4, it is characterised in that：LAMP reaction conditions
For 63-65 DEG C of incubation 45-60min, 85 DEG C of inactivation 5-10min.
6. Exserohilum turcicum LAMP rapid detection methods according to claim 2, it is characterised in that：Wait for LAMP reaction knots
Shu Hou, is added 1.0 μ L of color developing agent SYBR green I in the amplified production of LAMP reactions, and colour developing result observes that green is glimmering
The judgement of light is the positive, and orange or crocus is judged as feminine gender.
7. Exserohilum turcicum LAMP rapid detection methods according to claim 2, it is characterised in that：Wait for LAMP reaction knots
Shu Hou takes 2.0 μ L LAMP amplified productions to be detected with 2% agarose gel electrophoresis, trapezoid-shaped strips occurs and be judged as the positive, do not have
There are trapezoid-shaped strips and is then judged as feminine gender.
8. a kind of application of the LAMP detection primer described in claim 1, it is characterised in that：LAMP detection primer is applied to jade
The early diagnosis and germ identification of rice leaf blight.
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| CN111088393A (en) * | 2020-03-06 | 2020-05-01 | 河南科技大学 | LAMP (loop-mediated isothermal amplification) detection primer group for rhizoctonia cerealis and application of LAMP detection primer group |
| CN111088394A (en) * | 2020-03-06 | 2020-05-01 | 河南科技大学 | LAMP (loop-mediated isothermal amplification) detection primer group for Helminthosporium funiculosum of rhizoctonia solani and application of LAMP detection primer group |
| CN112251363A (en) * | 2020-10-27 | 2021-01-22 | 唐山师范学院 | Corn northern leaf blight attenuated strain STAM-226 and application thereof |
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| CN111088393A (en) * | 2020-03-06 | 2020-05-01 | 河南科技大学 | LAMP (loop-mediated isothermal amplification) detection primer group for rhizoctonia cerealis and application of LAMP detection primer group |
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| CN112251363A (en) * | 2020-10-27 | 2021-01-22 | 唐山师范学院 | Corn northern leaf blight attenuated strain STAM-226 and application thereof |
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