CN108220474A - A kind of LAMP detection primer of Fusarium graminearum and its application - Google Patents
A kind of LAMP detection primer of Fusarium graminearum and its application Download PDFInfo
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Abstract
LAMP detection primer and its application the invention discloses a kind of Fusarium graminearum, the composition that the detection primer is made of 1 couple of outside primers F 3/B3,1 couple of inside primers F IP/BIP and 1 couple of ring primer Loop F/Loop B, the LAMP detection primer composition is applied to detection Fusarium graminearum and whether corn crop has infected maize kernel rot.Fusarium graminearum LAMP detection primer composition high specificity, the expanding effect that the present invention designs are good, detection method is without being isolated and purified and being cultivated to pathogen, it is simple and efficient, it is highly practical, high sensitivity, as a result accurately and reliably, the early diagnosis fallen ill early period, initial stage available for Fusarium graminearum in plant tissue.
Description
Technical field
The invention belongs to corps diseases detection, identification and Prevention Technique fields, are related to a kind of to cause maize kernel rot
Fusarium graminearum LAMP detection primer, the detection kit containing the detection primer and using the detection primer or
The LAMP detection method of detection kit.
Background technology
Maize kernel rot is one of the most common disease in corn growth later stage, is generally occurred in corn-growing regions, incidence 5
~10%.Susceptible variety incidence is up to 50% or even in indivedual serious plot up to 100%, causes heavy losses.
Maize kernel rot is caused by a variety of pathogen infections, and wherein sickle-like bacteria is the main pathogenic fungi.That has reported at present draws
The sickle-like bacteria of maize kernel rot is played at 15 kinds or more, Fusarium graminearum is dominant population.Sickle-like bacteria can generate in pathogenic course
A variety of toxin such as zeranol, the mould toxin of single-ended spore, moniliformin, fumonisins, can promote Apoptosis, to dynamic
Plant part causes a variety of pathology damages, carrys out hidden trouble to human foods safety belt.
The form of sickle-like bacteria has polymorphism and variability, and the precise Identification for carrying out pathogen only in accordance with morphological feature has
Larger difficulty, and there are the problems such as detection cycle length, heavy workload, inaccurate result for conventional identification method.Therefore, have in time
Effect ground carries out sickle-like bacteria quick, accurate detection, is to prevent the base that its host plant disease occurs, ensures agricultural product quality and safety
Plinth.
Pathogenicbacteria separation detection method is traditional Defect inspection technology, uses selective medium from soil, plant
Isolated strains in tissue carry out taxonomic identification further according to its morphological feature and biological characteristics to pathogen.And pathogen
Isolation and purification culture at least needs 3~5 days, time and effort consuming, and various miscellaneous bacterias are easily polluted during being separately cultured, and identification is accurate
Exactness is relatively low.This method is easily influenced by human factor and environment, and the experimental skill and practical experience to operator have very high want
It asks, is unable to reach the requirement quickly examined.More most important to be, this method easily omits in the incubation period or hidden disease phenomenon occurs
Pathogen so that delay treatment, causes disease to be broken out, so as to delay the timely prevention of disease.Therefore, traditional pathogeny detection
Method has been unable to meet the needs of modern plants pathological research, establishes a kind of quick, sensitive, accurate detection method to corn
The prevention and control of ear rot are of great significance.
In recent years, Protocols in Molecular Biology is fast-developing, and the quick detection for pathogenic provides new thinking.PCR
Technology is applied in the detection of maize kernel rot.Shi Yajuan etc. (causes two kinds of Fusariumsp double PCRs of maize kernel rot
Foundation [J] Plant Pathologies of rapid detection system, 2017,47 (1):It 35-39) establishes cereal reaping hook and intends taking turns
The double PCR rapid detection system of branch sickle-like bacteria, the system have stronger specific and higher sensitivity, can be disposable
It detects cereal reaping hook and intends wheel branch sickle-like bacteria.However, the reaction system need to carry out before establishing the separation of pathogen, purifying,
Culture, DNA extractions, carries out PCR amplification again later, and step is more complicated, and only being separately cultured for pathogen just needs 10 days or so,
Cannot meet the needs of quickly detecting.And round pcr detection time is longer, needs by valuable instrument and equipments such as PCR instruments, right
For simple and crude agrotechnical department of base of the plant quarantine department and condition that need to speed passage through customs, the reality of these molecular detecting methods
It is restricted with property.Therefore, it is necessary to develop novel, quick, easy, reliable and inexpensive detection means.
Notomi developed a kind of easy, quick, accurate and cheap nucleic acid efficient amplification technology equal to 2000 ---
Loop-mediated isothermal amplification technique (Loop-mediated Isothermal Amplification, LAMP), the technology utilize height
Activity strand displacement archaeal dna polymerase (BstDNA polymerase) specific amplification is carried out to target DNA fragment, it depends on
It can identify the primer and the Bst DNA polymerases of a kind of tool de-rotation function and efficient amplification of 4 specific regions on target sequence,
Under constant temperature can efficiently, it is quick, specifically expand target sequence, realize the quick detection of disease.This method has following
Advantage：1）It is specific high, using 6 regions on 4 special primer identifying purpose genes；2）It is easy to operate, it is only necessary to water-bath
As thermostat, nucleic acid amplification can be both realized, do not need to the expensive equipments such as PCR instrument, plant protection technology department of especially suitable base,
Inspection and quarantine department uses；3）Detection speed is fast, without being separately cultured to pathogen, available for being deposited in detection plant tissue
In germ, it is only necessary to can both complete within 1 hour to expand, so as to fulfill the quick and precisely detection of pathogen.
The technology has been gradually known to researcher, to be widely used in the related fields such as life science at present.Researcher
While being applied to detect various pathogen by the technology, many improvement also are proposed to the technology, make its gradual perfection, energy
Enough it is successfully applied to the detection of viral pathogen, bacterial pathogens, fungal pathogens, parasite, tumour etc..But about causing jade
The LAMP detections of the Fusarium graminearum of rice ear rot, are not reported both at home and abroad.
Invention content
The object of the present invention is to provide a kind of LAMP detection primer compositions of Fusarium graminearum, cause corncob to establish
The LAMP rapid detection systems of the Fusarium graminearum of maize ear rot.
It is a further object of the present invention to provide a kind of LAMP detection kits of Fusarium graminearum.
A kind of LAMP detection method of Fusarium graminearum is provided, realizes quick, the sensitive and special molecular of Fusarium graminearum
Detection is the another goal of the invention of the present invention.
For achieving the above object, the present invention by measure Fusarium graminearum (Fusarium graminearum) and
Other Fusariumsps (FusariumSpp.)translation elongation factor 1-alpha gene(EF- 1alpha) gene order, and using Clustal X softwares to reaping hook category and other pathogen difference inter-speciesEF-1alphaGene
Sequence is compared, and utilizes online LAMP primer design software Primer Explorer V5 software (http://
Primerexplorer.jp/lampv5e the specific LAMP inspections of a set of Fusarium graminearum for causing maize kernel rot) are designed
Survey Primer composition.
Fusarium graminearum LAMP detection primer composition of the present invention is drawn by 1 couple of outside primers F 3/B3,1 pair of inside
Object FIP/BIP and 1 pair of ring primer Loop F/Loop B composition, the particular sequence of each primer are as follows：
F3：5'-TCTTCCCACAAACCATTCC-3'；
B3：5'-TGAGAATGTGATGACAGCAG-3'；
FIP：5'-CCAGGCGTACTTGAAGGAACCCAACCAGTCACTAACCACC-3'；
BIP：5'-GTGAGCGTGGTATCACCATTGACATACCAATGACGGTGACAT-3'；
Loop F：5'-GGCGGCTTCCTATTGACA-3'；
Loop B：5'-TCTGGAAGTTCGAGACTCCT-3'.
And then the present invention provides a kind of LAMP detection kit of Fusarium graminearum, every 1mL of the kit is detected
Contain in solution：Each 1 μM of each 0.25 μM of Outside primer F3 and B3, inner primer FIP and BIP, ring primer Loop F and Loop B
Each 0.5 μM, 2 × LAMP reaction mixtures 625 μ L, 8U Bst25 μ L of polymerase.
Above-mentioned LAMP detection primer composition or LAMP detection kit are applied to detect whether that there are cereal sickles
Knife bacterium.
Further, above-mentioned LAMP detection primer composition or LAMP detection kit are applied to detection corn crop
Whether maize kernel rot has been infected.
The present invention also provides a kind of LAMP detection methods of Fusarium graminearum, and the method includes extracting micrometer life to be checked
The DNA of object using the DNA of extraction as template, is carried out using the LAMP detection primer composition or LAMP detection kit
LAMP is expanded, and amplified production trapezoid-shaped strips occurs and be judged as the positive, there are Fusarium graminearums, do not have with agarose gel electrophoresis
There is band and be judged as feminine gender, there is no Fusarium graminearums.
And then in the above-mentioned detection method of the present invention, the reaction condition of the LAMP amplifications is incubates 30 at 61~63 DEG C
~75min.
The LAMP amplification reaction conditions are preferably 62 DEG C of incubation 60min.
Further, detection method of the present invention is for when detecting pathogen pure culture, it is preferred to use Biospin is true
Bacterium genome DNA extracting reagent kit extracts the DNA of germ bacterial strain.For detecting Fusarium graminearum cause of disease present in plant tissue
During bacterium, it is preferred to use NaOH rapid cleavages method extraction Fusarium graminearum DNA.
More specifically, when combining analyte detection Fusarium graminearum using LAMP detection primer, the LAMP detections preferably established are anti-
The system is answered to be：Each 0.8 μM of each 0.2 μM of Outside primer F3 and B3, inner primer FIP and BIP, ring primer Loop F and Loop B are each
0.4 μM, 2 × LAMP PCR isothermal amplifications mixed liquor 12.5 μ L, 8UBst0.5 μ L of polymerase, 1.0 μ L of DNA profiling, to go out
Bacterium ultra-pure water complements to 25 μ L.
And then when using LAMP detection kit detection Fusarium graminearum, 1.0 μ L DNA profilings are preferably taken, are added in
Kit detection solution described in 20.0 μ L and 4.0 μ L sterilizing ultra-pure waters carry out LAMP amplifications.
Preferably, the present invention takes 2.0 μ L LAMP amplified productions, is detected using 2.0% agarose gel electrophoresis, detection
Condition is voltage 80V, electric current 110A, detection time 30min.
One of an important factor for selection of target gene is LAMP detections.For Fusarium fungi, Standard PCR detection is common
Target gene include Internal Transcribed Spacer (rDNA-ITS)、EF-1alpha、mitochondrial small
subunit rDNA(mtSSU) and beta-tubulin (TUB2) etc..The present invention analyzes Fusarium fungi Fusarium graminearumEF-1alphaGene and the gene order difference of other Fusariumsps, are based onEF-1alphaGene chooses 6 specific regions, design
3 pairs of specific LAMP primers, any one region is mismatched with primer in 6 regions, cannot carry out nucleic acid amplification.
Therefore, the Fusarium graminearum LAMP detection primer composition high specificity designed by the present invention, expanding effect are good, can
For the quick detection of high sensitivity of Fusarium graminearum.The Fusarium graminearum LAMP detection method established on this basis is equally
With very strong specificity, and testing result is accurately and reliably.
Maize kernel rot morbidity is universal and harm is serious, the traditional biological detection method of pathogen there are period length,
The problems such as program is cumbersome, poor specificity, sensitivity are low, and the PCR molecular detection technologies modernized are needed by thermal cycler
Wait expensive instruments, and being separately cultured of pathogen, DNA extractions, detection time are long, it can not be in base's popularization and application.The present invention
Loop-mediated isothermal amplification technique is applied to the quick detection of Fusarium graminearum for causing maize kernel rot, establishes and causes corn
The LAMP new detecting methods of the Fusarium graminearum of ear rot.
Detection method directly can extract disease without pathogen is isolated and purified and cultivated from site of pathological change
LAMP detections are directly carried out after opportunistic pathogen DNA；Detection method does not need to complex instrument, a thermostatic equipment is only needed, 62
Under DEG C isothermy just can it is quick, efficiently, it is high it is special, detect Fusarium graminearum with sensitivity；Detection method is in 2h
Whole detection process inside can be completed, detection cycle is short.Therefore, the Fusarium graminearum for causing maize kernel rot that the present invention establishes
LAMP Fast Detection Technique systems are highly practical, and method is simple and efficient, as a result accurately and reliably, can preferably meet scene to cereal
The needs of the fast and reliable detection and identification of sickle-like bacteria.
The detection sensitivity of detection method is high, can be on DNA level to the detection sensitivity of Fusarium graminearum
Reach 100pg/ μ L, there is very high sensitivity.
Fusarium graminearum of the detection method that the present invention establishes available for causing ear rot in the plant tissue that carries disease germs
Special highly sensitive quick real-time Molecular Detection, is particularly suitable for the early diagnosis that Fusarium graminearum falls ill early period, initial stage, especially
It is that ear rot shows germ early monitoring identification before disease, the best prevention of prediction and determining disease for pathogen
Period has a very important significance.
Description of the drawings
Fig. 1 is the gel electrophoresis figure under different LAMP reaction temperatures, wherein swimming lane M is Marker2000,1 be 61 DEG C, 2 are
62 DEG C, 3 be 63 DEG C, 4 be 64 DEG C, 5 be 65 DEG C.
Fig. 2 is the gel electrophoresis figure under the different LAMP reaction time, and wherein swimming lane M is Marker2000,1 is 15min, 2
It is 45min for 30min, 3,4 be 60min, 5 is 75min.
Fig. 3 is the gel electrophoresis figure of LAMP of the present invention detection specificity, and wherein swimming lane M is Marker2000,1 is that layer goes out sickle
Knife bacterium, 2 be Fusarium equiseti, 3 be fusarium acuminatum, 4 be Fusarium oxysporum, 5 be Fusarium graminearum, 6 be dendritic cladosporium, 7
It is to intend disk stey for Botrytis cinerea, 8.
Fig. 4 is LAMP reaction sensitivities detection gel electrophoresis figure, and wherein swimming lane M is Marker2000,1 is 10ng/ μ L, 2
It is 100pg/ μ L for 1ng/ μ L, 3,4 be 10pg/ μ L, 5 is 1pg/ μ L.
Fig. 5 is the LAMP detection gel electrophoresis figures of actual sample, and wherein swimming lane M is Marker2000,1 is healthy fringe grain, 2
For fringe grain of falling ill.
Specific embodiment
Following embodiments are only the preferred technical solution of the present invention, are not used to carry out any restrictions to the present invention.For
For those skilled in the art, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made
Any modification, equivalent substitution, improvement and etc., should all be included in the protection scope of the present invention.
Unless otherwise specified, following embodiment is operated according to conventional laboratory conditions, technology used in embodiment
The conventional means that means are well known to those skilled in the art.
Embodiment 1.
The Fusarium graminearum LAMP of maize kernel rot is caused to detect the design of specific primer sets object：By measuring cereal
Sickle-like bacteria (Fusarium graminearum) and other Fusariumsps (FusariumSpp.)EF-1alphaGene order makes
With Clustal X softwares to reaping hook category and other pathogen difference inter-speciesEF-1alphaGene order is compared, and utilizes
Online LAMP primer design software Primer Explorer V5 software (http://primerexplorer.jp/
Lampv5e), the specificity of the Fusarium graminearum of nucleotide sequence shown in a set of SEQ ID NO.1~SEQ ID NO.6 is designed
LAMP primer composition object, the composition is specifically by 1 couple of outside primers F 3/B3,1 couple of inside primers F IP/BIP and 1 pair of ring primer
Loop F/Loop B are formed.
Specific primer sequence is as follows.
F3：5'-TCTTCCCACAAACCATTCC-3'.
B3：5'-TGAGAATGTGATGACAGCAG-3'.
FIP：5'-CCAGGCGTACTTGAAGGAACCCAACCAGTCACTAACCACC-3'.
BIP：5'-GTGAGCGTGGTATCACCATTGACATACCAATGACGGTGACAT-3'.
Loop F：5'-GGCGGCTTCCTATTGACA-3'.
Loop B：5'-TCTGGAAGTTCGAGACTCCT-3'.
Embodiment 2.
Genomic DNA for examination cause of disease bacteria strain is extracted using Biospin fungal genomic DNAs extracts kit.Specifically
Extraction process is as follows.
1) pathogen that 7~10d is cultivated in PDA culture medium is chosen at, the fresh mycelia of picking is pulverized, and is packed into
In 1.5mL centrifuge tubes.
2) 500 μ L LE Buffer are added in, are uniformly mixed.
3) it incubates 60min (interval 15min overturns mixing 1 time) for 65 DEG C, then removes.
4) 130 μ L DA Buffer, mixing, 65 DEG C of water-bath 5min are added in.
5) 14,000 × g centrifuges 3min.
6) supernatant is taken, is moved into new 1.5mL centrifuge tubes.
7) 500 μ L E Binding Buffer are added in, are uniformly mixed.
8) mixed liquor is moved into Spin Column, in 6,000 × g centrifuges 1min.
9) 500 μ L G Binding Buffer, 10,000 × g centrifugation 30s are added in Spin Column, discards and connect liquid
Liquid in pipe.
10) 600 μ L Wash Buffer are added in Spin Column, in 10,000 × g centrifugation 30s discard adapter
Middle liquid.
11) repetitive operation 10), 10,000 × g centrifugations 1min.
12) Spin Column are put into new 1.5mL centrifuge tubes, place 10~15min, add in 40 μ L ddH2O is put
After putting 1min, 1min is centrifuged.Add in 30 μ L ddH2O, 10,000 × g centrifuge 1min, discard Spin Column, centrifuge tube liquid
In i.e. contain DNA.
Embodiment 3.
Based on the LAMP primer composition object that embodiment 1 designs, the DNA extracted using embodiment 2 establishes cereal reaping hook as template
The LAMP detection reaction systems of bacterium.
Include each 0.2 μM of Outside primer F3 and B3, each 0.8 μ of inner primer FIP and BIP in LAMP detection reaction systems
Each 0.4 μM of M, ring primer Loop F and Loop B, 2 × LAMP PCR isothermal amplifications mixed liquor (universal, raw work biology work
Journey (Shanghai) limited company) 12.5 μ L, 8UBst0.5 μ L of polymerase, 1.0 μ L of DNA profiling, are complemented to sterilizing ultra-pure water
25μL。
The reaction temperature of initial setting LAMP reactions is 63 DEG C, time 30min.
After reaction, 2.0 μ L LAMP amplified productions is taken to carry out 2.0% agarose gel electrophoresis detection, electrophoresis detection item
Part is voltage 80V, electric current 110A, detection time 30min.
Such as there are trapezoid-shaped strips in detection electrophoretogram, be judged as the positive, there are Fusarium graminearums, band do not occur and then sentence
Break as feminine gender, there is no Fusarium graminearums.
Embodiment 4.
According to embodiment 1 design primer, using embodiment 2 extract DNA as template, according to 3 reaction system of embodiment into
Row LAMP reacts, and it is respectively 61 DEG C, 62 DEG C, 63 DEG C, 64 DEG C and 65 DEG C to set reaction temperature, and detection different temperatures ties reaction
The influence of fruit.The result shows that most suitable reaction temperature is 62 DEG C, experimental result is as shown in Figure 1.
According to embodiment 1 design primer, using embodiment 2 extract DNA as template, according to 3 reaction system of embodiment into
Row LAMP reacts, and sets reaction temperature as 62 DEG C, the reaction time is respectively 15min, 30min, 45min, 60min and 75min, is examined
Survey influence of the different time to reaction result.The result shows that band cannot be amplified during 15min, with the increase of proliferation time,
LAMP characteristic trapezoid belt brightness gradually increases, but band degree of analysis clearly declines during 75min.Thus the optimum reaction time is
60min, experimental result are as shown in Figure 2.
Embodiment 5.
In order to verify the LAMP detection method of the invention established for the specificity of Fusarium graminearum, the present embodiment is with difference
Pathogen carries out LAMP reactions as material to be tested.
Include for examination pathogen：Fusarium graminearum (Fusarium graminearum), fusarium prolifertum (Fusarium proliferatum), Fusarium equiseti (Fusarium equiseti), fusarium acuminatum (Fusarium acuminatum)、
Fusarium oxysporum (Fusarium oxysporum), Botrytis cinerea (Botrytis cinerea), dendritic cladosporium
(Cladosporium cladosporioides), intend disk stey (Pestalotiopsis paeoniicola)。
The genomic DNA of above-mentioned various cause of disease bacteria strains is extracted according to 2 method of embodiment, the LAMP according to embodiment 3 is anti-
The optimal reactive temperature and time for answering system and embodiment 4 carry out LAMP reactions.Experimental result is as shown in figure 3, except cereal reaping hook
Bacterium detection occurs outside the characteristic trapezoid belts of LAMP, other bacterial strain agarose gel electrophoresis do not occur amplified band, explanation
The method of the present invention has specificity well.
Embodiment 6.
Go out extracted cereal using Eppendorf BioSpectrometer fluorescence spectrophotometric determinations
A concentration of 57.9ng/ μ L of sickle-like bacteria genomic DNA.
It is diluted to 10ng/ μ L, 1ng/ μ L, 100pg/ μ L, 10pg/ μ L and 1pg/ μ L successively, according to embodiment 3
The optimal reactive temperature and time of LAMP reaction systems and embodiment 4 carry out LAMP reactions for the DNA of various concentration.Experiment
The result shows that the Fusarium graminearum DNA minimum concentrations that LAMP detection method of the present invention can detect are 100pg/ μ L, experiment knot
Fruit is as shown in Figure 4.
Embodiment 7.
The present embodiment is tested from the fruit of field selection plant natural occurrence and healthy fruit.
First using the pathogen DNA in NaOH rapid cleavages method rapid extraction disease fruit and healthy fruit.
1) fruit in 75% ethanol solution is impregnated into 3min, with aseptic water washing 3 times, dried.
2) 10 μ L 0.5mol/L NaOH solutions are added in into every mg plant tissues.
3) tissue is fully milled to paste in mortar, is transferred in 1.5mL centrifuge tubes.
4) 11,000 × g centrifuges 6min.
5) 5 μ l of supernatant are taken, add in 495 μ L 0.1mol/L Tris-HCl (pH=8.0), are uniformly mixed, as DNA moulds
Plate carries out LAMP amplifications.
Specific LAMP reactions are with embodiment 5, using healthy fruit as control.Experimental result is as shown in figure 5, LAMP reaction sides
Method can effectively amplify the characteristic trapezoid belts of LAMP of pathogen in morbidity fruit, and healthy fruit does not expand shaping
Band demonstrates again that the method for the present invention testing result accurately and reliably, there is very strong practicability.
SEQUENCE LISTING
<110>Agricultural University Of Shanxi
<120>A kind of LAMP detection primer of Fusarium graminearum and its application
<160> 6
<170> SIPO Sequence Listing 1.0
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence
<220>
<223>Outside primer F3 sequences
<400> 1
TCTTCCCACA AACCATTCC 19
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>Outside primer B3 sequences
<400> 2
TGAGAATGTG ATGACAGCAG 20
<210> 3
<211> 40
<212> DNA
<213>Artificial sequence
<220>
<223>Inner primer FIP sequences
<400> 3
CCAGGCGTAC TTGAAGGAAC CCAACCAGTC ACTAACCACC 40
<210> 4
<211> 42
<212> DNA
<213>Artificial sequence
<220>
<223>Inner primer BIP sequences
<400> 4
GTGAGCGTGG TATCACCATT GACATACCAA TGACGGTGAC AT 42
<210> 5
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<223>Ring primer Loop F sequences
<400> 5
GGCGGCTTCC TATTGACA 18
<210> 6
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>Ring primer Loop B sequences
<400> 6
TCTGGAAGTT CGAGACTCCT 20
Claims (9)
1. a kind of LAMP detection primer of Fusarium graminearum is by 1 couple of outside primers F 3/B3,1 couple of inside primers F IP/BIP and 1
To the Primer composition of ring primer Loop F/Loop B compositions, each primer sequence is as follows：
F3：5'-TCTTCCCACAAACCATTCC-3'；
B3：5'-TGAGAATGTGATGACAGCAG-3'；
FIP：5'-CCAGGCGTACTTGAAGGAACCCAACCAGTCACTAACCACC-3'；
BIP：5'-GTGAGCGTGGTATCACCATTGACATACCAATGACGGTGACAT-3'；
Loop F：5'-GGCGGCTTCCTATTGACA-3'；
Loop B：5'-TCTGGAAGTTCGAGACTCCT-3'.
2. a kind of LAMP detection kit of Fusarium graminearum, every 1mL of the kit is detected to be contained in solution：Outside primer
Each 1 μM of each 0.25 μM of F3 and B3, inner primer FIP and BIP, each 0.5 μM of ring primer Loop F and Loop B, 2 × LAMP reaction
Mixed liquor 625 μ L, 8U Bst25 μ L of polymerase, wherein, the Outside primer F3/B3, inner primer FIP/BIP and ring primer
The primer sequence of Loop F/Loop is as follows：
F3：5'-TCTTCCCACAAACCATTCC-3'；
B3：5'-TGAGAATGTGATGACAGCAG-3'；
FIP：5'-CCAGGCGTACTTGAAGGAACCCAACCAGTCACTAACCACC-3'；
BIP：5'-GTGAGCGTGGTATCACCATTGACATACCAATGACGGTGACAT-3'；
Loop F：5'-GGCGGCTTCCTATTGACA-3'；
Loop B：5'-TCTGGAAGTTCGAGACTCCT-3'.
3. a kind of LAMP detection method of Fusarium graminearum is the DNA for extracting microorganism to be detected, using the DNA of extraction as template,
LAMP amplifications are carried out using LAMP detection kit described in LAMP detection primer composition described in claim 1 or claim 2,
There are trapezoid-shaped strips and is judged as the positive, there are Fusarium graminearums, band do not occur and sentence with agarose gel electrophoresis in amplified production
Break as feminine gender, there is no Fusarium graminearums.
4. the LAMP detection method of Fusarium graminearum according to claim 3, it is characterized in that the reaction of LAMP amplifications
Condition is that 30~75min is incubated at 61~63 DEG C.
5. the LAMP detection method of Fusarium graminearum according to claim 4, it is characterized in that incubating 60min at 62 DEG C.
6. the LAMP detection method of Fusarium graminearum according to claim 3, it is characterized in that using described in claim 1
LAMP detection primer carries out the reaction system of LAMP amplifications：Each 0.2 μM of Outside primer F3 and B3, inner primer FIP and BIP is each
0.8 μM, each 0.4 μM of ring primer Loop F and Loop B, 2 × LAMP PCR isothermal amplifications mixed liquor 12.5 μ L, 8U Bst
0.5 μ L of polymerase, 1.0 μ L of DNA profiling complement to 25 μ L with the ultra-pure water that sterilizes.
7. the LAMP detection method of Fusarium graminearum according to claim 3, it is characterized in that using described in claim 2
When LAMP detection kit carries out LAMP amplifications, take 1.0 μ L DNA profilings, add in kit detection solution described in 20.0 μ L and
4.0 μ L sterilizing ultra-pure waters carry out LAMP amplifications.
8. LAMP amplimer of the LAMP detection primer of Fusarium graminearum described in claim 1 as detection Fusarium graminearum,
Application in Fusarium graminearum is detected.
9. application of the LAMP detection kit of Fusarium graminearum described in claim 2 in Fusarium graminearum is detected.
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| CN108676910A (en) * | 2018-07-09 | 2018-10-19 | 山西农业大学 | A kind of LAMP detection primer of fusarium prolifertum and its application |
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| CN110643730A (en) * | 2019-09-09 | 2020-01-03 | 安徽省农业科学院植物保护与农产品质量安全研究所 | RPA primer, probe and detection method for detecting wheat scab pathogen fusarium graminearum |
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| CN110643730B (en) * | 2019-09-09 | 2022-08-26 | 安徽省农业科学院植物保护与农产品质量安全研究所 | RPA primer, probe and detection method for detecting wheat scab pathogen fusarium graminearum |
| CN111602630A (en) * | 2020-06-24 | 2020-09-01 | 山西农业大学 | Method for establishing broiler chicken small intestine mechanical injury model caused by vomitoxin |
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| CN114395557B (en) * | 2022-02-17 | 2023-06-20 | 河南农业大学 | Application of corn miRNA |
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