CN108676910A - A kind of LAMP detection primer of fusarium prolifertum and its application - Google Patents
A kind of LAMP detection primer of fusarium prolifertum and its application Download PDFInfo
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- CN108676910A CN108676910A CN201810743738.5A CN201810743738A CN108676910A CN 108676910 A CN108676910 A CN 108676910A CN 201810743738 A CN201810743738 A CN 201810743738A CN 108676910 A CN108676910 A CN 108676910A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The invention discloses a kind of LAMP detection primer of fusarium prolifertum and its applications, 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 prolifertum and whether corn crop has infected maize kernel rot.Fusarium prolifertum 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 accurate and reliable, it can be used for the early diagnosis that fusarium prolifertum is fallen ill early period, initial stage 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 prolifertum 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 causes heavy losses up to 100% in individual serious plot.
Maize kernel rot is caused by a variety of pathogen infections, and wherein sickle-like bacteria is the main pathogenic fungi.Sickle-like bacteria was being caused a disease
Cheng Zhongneng generates a variety of toxin, can promote Apoptosis, cause a variety of pathology damages to animals and plants body, brought to food security
Hidden trouble.That has reported at present causes the sickle-like bacteria of maize kernel rot at 15 kinds or more, and wherein fusarium prolifertum is sociales
Group.
Fusarium prolifertum belongs to that Fungi Imperfecti door, tumor seat are full to belong to fungi, can both endanger field crops for example corn, sorghum,
Rice etc., the various crops such as industrial crops such as mango, asparagus, onion, garlic, can also exist in soil.Fusarium prolifertum
Wind-force propagation is relied primarily on, having the corn kernel energy tegillum of wound to go out fusarium infection leads to maize kernel rot.Layer goes out sickle
Knife bacterium, which infects crop, can cause extremely serious damage, secondary metabolites such as fumonisin (Fumonisins), the list of secretion
Duan Baomei alkene race endotoxin (Trichothecenes), fusaric acid (Fusaricacid, FA) and zearalenone etc. are to people
Poultry safety also has a significant impact.
The form of fusarium prolifertum has polymorphism and variability, and the precise Identification of pathogen is carried out only in accordance with morphological feature
With larger difficulty, and conventional identification method the problems such as there are detection cycle length, heavy workload, inaccurate results.Therefore, and
When effectively to fusarium prolifertum carry out quickly, accurate detection, be to prevent its host plant disease to occur, ensure agricultural product quality
The basis of safety.
Traditional phytopathogen method for separating and detecting is that first separation obtains pathogen bacterium from diseased tissues or pedotheque
Strain, then taxonomic identification is carried out by cultural character and microscopic morphology observation, with clear Species of Pathogens.And the separation of pathogen is pure
Change culture and at least need 3~5 days, time and effort consuming, and various miscellaneous bacterias are easily polluted during be separately cultured, identification accuracy compared with
It is low.This method is easily influenced by human factor and environment, it is desirable that veteran professional could complete, and to the reality of operator
Testing technical ability and practical experience has higher requirement.
More most important, pathogenicbacteria separation detection method is easy to omit in the incubation period or occur the cause of disease of hidden disease phenomenon
Bacterium, so that delay treatment, causes disease to be broken out, to delay the timely prevention of disease.Therefore, traditional pathogeny detection method has been
The needs of modern plants pathological research cannot be met, establish a kind of quick, sensitive, accurate detection method, to corncob corruption
The prevention and control of disease are of great significance.
With the development of round pcr and sequencing technologies, Protocols in Molecular Biology be the detection of plant pathogenic fungi, identification and
Quantization provides new ways and means.One good molecular detection technology should have high specificity, high sensitivity, detection speed
Soon, simple operation and other advantages.
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
Active strand displacement archaeal dna polymerase (BstDNA polymerase) specific amplification is carried out to target DNA fragment, it depends on
It can identify 4~6 of the area Fl, F2 and F3 at the 6 different zones i.e. area B3c, B2c and Blc at the ends 3' and the ends 5' on target sequence
Specific template primer, under constant temperature can efficiently, quickly, specifically expand target sequence, realize the quick inspection of disease
It surveys.
This method is easy to operate, does not need the expensive instrument such as PCR instrument, is amplifiable target sequence under constant temperature, especially
It is suitble to plant protection technology department of base, inspection and quarantine department to use；Detection speed is fast, can without being separately cultured to pathogen
For detecting, there are germs in plant tissue, it is only necessary to which 30min can both complete to expand, to realize the quick and precisely inspection of pathogen
It surveys.
The research report for quickly detecting LAMP for fusarium prolifertum is not found at present.
Invention content
The object of the present invention is to provide a kind of LAMP detection primer compositions of fusarium prolifertum, go out reaping hook to establish layer
The LAMP rapid detection systems of bacterium.
It is a further object of the present invention to provide a kind of LAMP detection kits of fusarium prolifertum.
A kind of LAMP detection method of fusarium prolifertum is provided, realizes quick, the sensitive and special molecular of fusarium prolifertum
Detection is the another goal of the invention of the present invention.
For fusarium prolifertum (Fusarium proliferatum) translation elongation factor (EF-1alpha) gene sequence
Row, the present invention utilize online LAMP primer design software Primer Explorer V5 software (http://
Primerexplorer.jp/lampv5e the specific LAMP inspections of a set of fusarium prolifertum for causing maize kernel rot) are designed
Survey Primer composition.
Fusarium prolifertum 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'-AGTACCAGTGATCATGTTCTTG-3'.
B3：5'-TCCTGTCCACAACCTCAA-3'.
FIP：5'-AAGTTCGAGACTCCTCGCTACTGAGGAAGTAGGATGAGGTATGA-3'.
BIP：5'-CTCGGCCTTGAGCTTGTCAACAATAGGAAGCCGCTGAG-3'.
Loop F：5'-TCACCGTCATTGGTATGTTGT-3'.
Loop B：5'-AGGCGTACTTGAAGGAACC-3'.
In turn, the present invention provides a kind of LAMP detection kit of fusarium prolifertum, 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 layers to go out sickle
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 prolifertum, and the method includes extracting crop to be detected
Or the DNA of microorganism utilizes the LAMP detection primer composition or LAMP detection kit using the DNA of extraction as template
LAMP amplifications are carried out, detect whether that there are fusarium prolifertums using any one following method.
By amplified production with agarose gel electrophoresis, there are trapezoid-shaped strips and be judged as the positive, there are fusarium prolifertums, do not have
There is band and be judged as feminine gender, fusarium prolifertum is not present.
Alternatively, I color developing agents of SYBR green and mixing is added in LAMP reacts amplified production, under 254nm ultraviolet lamps
Visualization color developing detection is carried out, observes that it is the positive to generate green fluorescence, there are fusarium prolifertums, and it is the moon not generate green fluorescence
Property, fusarium prolifertum is not present.
In turn, in the above-mentioned detection method of the present invention, the reaction condition of the LAMP amplifications is to incubate 30 at 60~65 DEG C
~75min.
The LAMP amplification reaction conditions are preferably 62 DEG C of incubation 30min.
Further, when detection method of the present invention is for when detecting pathogen pure culture, it is preferred to use Biospin
Fungal genomic DNA extracts kit extracts the DNA of germ bacterial strain.For detecting the disease of fusarium prolifertum present in plant tissue
When opportunistic pathogen, it is preferred to use NaOH rapid cleavage methods extract fusarium prolifertum DNA.
More specifically, when detecting fusarium prolifertum using LAMP detection primer composition, 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.
In turn, when detecting fusarium prolifertum using LAMP detection kit, 1.0 μ L DNA profilings are preferably taken, are added
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.
Maize kernel rot morbidity is universal and harm is serious, the traditional biological detection method of pathogen there are period length,
Program is cumbersome, poor specificity, low sensitivity the problems such as, and the PCR molecular detection technologies modernized are needed by thermal cycler
Equal 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 is based on LAMP technology, according to the translation elongation factor of fusarium prolifertum (EF-1alpha) sequence, it designs
Specific detection primer composition, and by the optimization to reaction system and reaction condition, fusarium prolifertum is established respectively
Agarose gel electrophoresis detects and with the Visual retrieval that SYBR Green I are fluorescence developing indicator, constructs and cause corn
The LAMP new detecting methods of the fusarium prolifertum of ear rot.
Detection method directly extracts pathogen it is not necessary that pathogen is isolated and purified and cultivated from site of pathological change
DNA carries out LAMP detections；LAMP detection primer composition high specificity, the expanding effect that the present invention designs are good, can be used for layer and go out
The quickly detection of the high sensitivity of sickle-like bacteria；Detection method is easy to operate, does not need complex instrument, and detection cycle is short.
The detection sensitivity of detection method is high, can be on DNA level to the detection sensitivity of fusarium prolifertum
Reach 10 μ g/mL.
The LAMP detection method that the present invention establishes can be used for the rapid molecular detection of fusarium prolifertum in morbidity corn tissue,
Be particularly suitable for fusarium prolifertum morbidity early period, initial stage it is quick detection and Accurate Diagnosis, especially show disease before pathogen
Early monitoring has very strong specificity, and testing result is accurate and reliable, for maize kernel rot caused by fusarium prolifertum
Disease early prediction is forecast and the real-time monitoring of pathogen is of great significance.
Description of the drawings
Fig. 1 is testing result of the fusarium prolifertum under the different LAMP reaction time, and Figure 1A is the agar after LAMP amplifications
Sugared detected through gel electrophoresis is as a result, Figure 1B is the visualization color developing detection results of SYBR green I after LAMP amplifications.
Wherein：It 1 is 15min that swimming lane M, which is in DL 2000 Marker, Figure 1A and Figure 1B, in Figure 1A, 2 be 30min, 3 be
45min, 4 be 60min, 5 be 75min.
Fig. 2 is testing result of the fusarium prolifertum under different LAMP reaction temperatures, and Fig. 2A is the agar after LAMP amplifications
Sugared detected through gel electrophoresis as a result, Fig. 2 B be LAMP amplification after SYBR green I visualize color developing detection as a result,
Wherein：It 1 is 60 DEG C that swimming lane M, which is in DL 2000 Marker, Fig. 2A and Fig. 2 B, in Fig. 2A, 2 be 61 DEG C, 3 be 62 DEG C, 4 are
63 DEG C, 5 be 64 DEG C, 6 be 65 DEG C, 7 be 66 DEG C.
Fig. 3 is the LAMP specific detection results of fusarium prolifertum.Fig. 3 A are the agarose gel electrophoresis after LAMP amplifications
Testing result, Fig. 3 B are that the SYBR green I after LAMP amplifications visualize color developing detection result.
Wherein：It 1 is fusarium prolifertum that swimming lane M, which is in DL 2000 Marker, Fig. 3 A and Fig. 3 B, in Fig. 3 A, 2 is rod method
Bacterium, 3 be sharp spore cladosporium, 4 be Botrytis cinerea, 5 be Fusarium graminearum, 6 be Fusarium oxysporum.
Fig. 4 is the LAMP reaction sensitivity testing results of fusarium prolifertum.Fig. 4 A are the Ago-Gel after LAMP amplifications
Electrophoresis detection is as a result, Fig. 4 B are the visualization color developing detection results of SYBR green I after LAMP amplifications.
Wherein：It 1 is 90.65 μ g/mL that swimming lane M, which is in DL 2000 Marker, Fig. 4 A and Fig. 4 B, in Fig. 4 A, 2 is 10.16 μ
G/mL, 3 be 1.13 μ g/mL, 4 be 93.35ng/mL, 5 be 8.75ng/mL.
Fig. 5 is the LAMP testing results of actual sample.Fig. 5 A are the agarose gel electrophoresis testing result after LAMP amplifications,
Fig. 5 B are that the SYBR green I after LAMP amplifications visualize color developing detection result.
Wherein：It 1 be healthy fringe grain that swimming lane M, which is in DL 2000 Marker, Fig. 5 A and Fig. 5 B, in Fig. 5 A, 2 is fringe grain of falling ill.
Specific implementation mode
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 design of fusarium prolifertum LAMP detection specific primer sets objects：Using Clustal X softwares, by going out to layer
Sickle-like bacteria and other sickle-like bacteria (FusariumSpp.)EF-1alphaGene order is compared, and utilizes online LAMP
Primer-design software Primer Explorer V5 software (http://primerexplorer.jp/lampv5e), if
Count out the fusarium prolifertum specificity LAMP primer composition of nucleotide sequence shown in a set of SEQ ID NO.1~SEQ ID NO.6
Object, the composition is specifically by 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
Composition.
Specific primer sequence is as follows.
F3：5'-AGTACCAGTGATCATGTTCTTG-3'.
B3：5'-TCCTGTCCACAACCTCAA-3'.
FIP：5'-AAGTTCGAGACTCCTCGCTACTGAGGAAGTAGGATGAGGTATGA-3'.
BIP：5'-CTCGGCCTTGAGCTTGTCAACAATAGGAAGCCGCTGAG-3'.
Loop F：5'-TCACCGTCATTGGTATGTTGT-3'.
Loop B：5'-AGGCGTACTTGAAGGAACC-3'.
Embodiment 2.
Using the extraction of Biospin fungal genomic DNA extracts kits for the gene of examination fusarium prolifertum cause of disease bacteria strain
Group DNA.Specific extraction process is as follows.
1) it is chosen at the fusarium prolifertum pathogen that 7~10d is cultivated in PDA culture medium, the fresh mycelia of picking is ground into
Powder is fitted into 1.5mL centrifuge tubes.
2) 500 μ L LE Buffer are added, 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.
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, 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 to Spin Column, discard and connects liquid
Liquid in pipe.
10) 600 μ L Wash Buffer are added to Spin Column, in 10,000 × g centrifuges 30s, discards adapter
Middle liquid.
11) repetitive operation 10), 10,000 × g centrifuges 1min.
12) Spin Column are put into new 1.5mL centrifuge tubes, place 10~15min, and 40 μ L ddH are added2O is put
After setting 1min, 1min is centrifuged.30 μ L ddH are added2O, 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 is established layer and goes out reaping hook as template
The LAMP of bacterium detects reaction system.
It includes each 0.2 μM of Outside primer F3 and B3, each 0.8 μ of inner primer FIP and BIP that LAMP, which is detected in reaction system,
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 liability 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 62 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, is judged as the positive, there are fusarium prolifertums, band do not occur and then sentence
Break as feminine gender, fusarium prolifertum is not present.
At the same time it can also carry out visualization color developing detection to LAMP amplified productions using I color developing agents of SYBR green.
I color developing agents of SYBR green and mixing are added in LAMP amplified productions, is observed under 254nm ultraviolet lamps, if it is observed that green is glimmering
Light is judged as the positive, and there are fusarium prolifertums, does not generate green fluorescence and is then judged as feminine gender, and fusarium prolifertum is not present.
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 reactions set reaction temperature as 63 DEG C, and the reaction time is respectively 15min, 30min, 45min, 60min and 75min, inspection
Survey influence of the different time to reaction result.
Agarose gel electrophoresis the result shows that, when 15min, cannot amplify band, and 30~75min can amplify ladder
Shape band, and with the increase of proliferation time, LAMP characteristic trapezoid belt brightness gradually increases, but band degree of analysis clearly when 75min
Decline.The result is shown in Figure 1 A.
SYBR green I visualize color developing detection the result shows that, reaction 15min products colour developing be shallow orange, show no expansion
Increase reaction, 30~75min products develop the color it is observed that green fluorescence, shows there is amplified reaction.Concrete outcome such as Figure 1B.
Thus, amplified reaction time range is 30~75min, and the optimum amplified reaction time is 30min.
According to embodiment 1 design primer, using embodiment 2 extract DNA as template, according to 3 reaction system of embodiment into
Row LAMP reactions set reaction temperature as 60 DEG C, 61 DEG C, 62 DEG C, 63 DEG C, 64 DEG C, 65 DEG C and 66 DEG C, and detection different temperatures is to anti-
Answer the influence of result.
The agarose gel electrophoresis of Fig. 2A the result shows that, 60~65 DEG C can amplify trapezoid-shaped strips, wherein 62 DEG C of whens expand
It is most clear to increase band；66 DEG C cannot amplify clear band.
The SYBR green I of Fig. 2 B visualize color developing detection the result shows that, 60~65 DEG C of products are it is observed that green is glimmering
Light shows there is amplified reaction；66 DEG C of product redgreen fluorescence displays, amplified reaction does not occur.
Thus, amplified reaction temperature range is 60~65 DEG C, and optimum amplified reaction temperature is 62 DEG C.
Embodiment 5.
In order to verify the LAMP detection method of the invention established for the specificity of fusarium prolifertum, the present embodiment is with difference
Pathogen carries out LAMP reactions as material to be tested.
Include for examination pathogen：Fusarium prolifertum (Fusarium proliferatum), alternaric bacteria (Alternaria alternata), sharp spore cladosporium (Cladosporium oxysporum), the pathogen of Botrytis cinerea (Botrytis cinerea), standing grain
Paddy sickle-like bacteria (Fusarium graminearum), Fusarium oxysporum (Fusarium oxysporum)。
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, and experimental result is as shown in Figure 3.
Agarose gel electrophoresis the result shows that, except fusarium prolifertum detection there are the characteristic trapezoid belts of LAMP in addition to, other
Bacterial strain agarose gel electrophoresis does not occur amplified band (Fig. 3 A).
Color developing detection the result shows that, in addition to the amplified production of fusarium prolifertum is observed that green fluorescence, remaining colour developing
Result is orange (Fig. 3 B).
The above the results show, the LAMP detection method that the present invention establishes have fusarium prolifertum special well
Property.
Embodiment 6.
Go out institute's extract layer using the ultramicron protein nucleic acid analysis-e/or determining of BioDrop models 80-3006-51 and goes out reaping hook
It, is diluted to 100 μ g/mL, 10 μ g/mL, 1 μ g/mL, 100ng/mL by a concentration of 676.2 μ g/mL of bacterium genomic DNA successively
And 10ng/mL, measure diluent concentration again, be followed successively by from high to low 90.65 μ g/mL, 10.16 μ g/mL, 1.13 μ g/mL,
93.35ng/mL and 8.75ng/mL.
According to the LAMP reaction systems of embodiment 3 and optimal reactive temperature and the time of embodiment 4, to various concentration
DNA carries out LAMP reactions, and experimental result is as shown in Figure 4.
Agarose gel electrophoresis the result shows that, DNA concentration does not have the characteristic trapezoid-shaped strips of LAMP below 1.33 μ g/mL
(Fig. 4 A)；Colour developing the result shows that, when a concentration of 90.65 μ g/mL, 10.16 μ g/mL and 1.33 μ g/mL, it is observed that green is glimmering
Light, remaining colour developing result is orange (Fig. 4 B).
Therefore, the fusarium prolifertum DNA minimum concentrations that LAMP detection method of the present invention can detect are 1.33 μ g/mL,
I.e. sensitivity is 1.33 μ g/mL.
Embodiment 7.
The present embodiment is tested from the field selection natually morbid corncob grain of plant and healthy corncob grain.
The pathogen in NaOH rapid cleavage method rapid extractions morbidity corncob grain and healthy corncob grain is used first
DNA。
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 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, 495 μ L 0.1mol/L Tris-HCl (pH=8.0) are added, are uniformly mixed, as DNA moulds
Plate carries out LAMP amplifications.
Specific LAMP reactions are with embodiment 5, as a contrast with healthy corncob grain.Experimental result is as shown in figure 5, LAMP is anti-
Induction method can effectively amplify the DNA of pathogen in morbidity corncob grain, and agarose gel electrophoresis is the result shows that morbidity corn
There are LAMP characteristic trapezoid-shaped strips in fringe grain, and healthy corncob grain does not amplify band (Fig. 5 A), and colour developing is the result shows that hair
Green fluorescence can be observed in sick corncob grain, illustrates there are fusarium prolifertum, and healthy corncob grain colour developing result is orange (figure
5B), illustrate that fusarium prolifertum is not present.
The above results demonstrate again that the method for the present invention testing result is accurate and reliable, there is very strong practicability.
SEQUENCE LISTING
<110>Agricultural University Of Shanxi
<120>A kind of LAMP detection primer of fusarium prolifertum and its application
<160> 6
<170> SIPO Sequence Listing 1.0
<210> 1
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<223>Outside primer F3 sequences
<400> 1
AGTACCAGTG ATCATGTTCT TG 22
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<223>Outside primer B3 sequences
<400> 2
TCCTGTCCAC AACCTCAA 18
<210> 3
<211> 44
<212> DNA
<213>Artificial sequence
<220>
<223>Inner primer FIP sequences
<400> 3
AAGTTCGAGA CTCCTCGCTA CTGAGGAAGT AGGATGAGGT ATGA 44
<210> 4
<211> 38
<212> DNA
<213>Artificial sequence
<220>
<223>Inner primer BIP sequences
<400> 4
CTCGGCCTTG AGCTTGTCAA CAATAGGAAG CCGCTGAG 38
<210> 5
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>Ring primer Loop F sequences
<400> 5
TCACCGTCAT TGGTATGTTG T 21
<210> 6
<211> 19
<212> DNA
<213>Artificial sequence
<220>
<223>Ring primer Loop B sequences
<400> 6
AGGCGTACTT GAAGGAACC 19
Claims (10)
1. a kind of LAMP detection primer of fusarium prolifertum 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'-AGTACCAGTGATCATGTTCTTG-3'；
B3：5'-TCCTGTCCACAACCTCAA-3'；
FIP：5'-AAGTTCGAGACTCCTCGCTACTGAGGAAGTAGGATGAGGTATGA-3'；
BIP：5'-CTCGGCCTTGAGCTTGTCAACAATAGGAAGCCGCTGAG-3'；
Loop F：5'-TCACCGTCATTGGTATGTTGT-3'；
Loop B：5'-AGGCGTACTTGAAGGAACC-3'.
2. a kind of LAMP detection kit of fusarium prolifertum, 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'-AGTACCAGTGATCATGTTCTTG-3'；
B3：5'-TCCTGTCCACAACCTCAA-3'；
FIP：5'-AAGTTCGAGACTCCTCGCTACTGAGGAAGTAGGATGAGGTATGA-3'；
BIP：5'-CTCGGCCTTGAGCTTGTCAACAATAGGAAGCCGCTGAG-3'；
Loop F：5'-TCACCGTCATTGGTATGTTGT-3'；
Loop B：5'-AGGCGTACTTGAAGGAACC-3'.
3. a kind of LAMP detection method of fusarium prolifertum is the DNA of extraction crop to be detected or microorganism, with the DNA of extraction
For template, carried out using LAMP detection kit described in LAMP detection primer composition described in claim 1 or claim 2
LAMP is expanded, and amplified production trapezoid-shaped strips occurs and be judged as the positive with agarose gel electrophoresis, and there are fusarium prolifertums, do not have
There is band and be judged as feminine gender, fusarium prolifertum is not present.
4. a kind of LAMP detection method of fusarium prolifertum is the DNA of extraction crop to be detected or microorganism, with the DNA of extraction
For template, carried out using LAMP detection kit described in LAMP detection primer composition described in claim 1 or claim 2
LAMP is expanded, and I color developing agents of SYBR green and mixing are added in amplified production, visualization colour developing is carried out under 254nm ultraviolet lamps
Detection observes that it is the positive to generate green fluorescence, and there are fusarium prolifertums, and it is feminine gender not generate green fluorescence, and there is no layers to go out
Sickle-like bacteria.
5. the LAMP detection method of fusarium prolifertum according to claim 3 or 4, it is characterized in that LAMP amplifications is anti-
It is that 30~75min is incubated at 60~65 DEG C to answer condition.
6. the LAMP detection method of fusarium prolifertum according to claim 5, it is characterized in that incubating 30min at 62 DEG C.
7. the LAMP detection method of fusarium prolifertum according to claim 3 or 4, it is characterized in that utilizing claim 1 institute
State the reaction system that LAMP detection primer carries out 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.
8. the LAMP detection method of fusarium prolifertum according to claim 3 or 4, it is characterized in that utilizing claim 2 institute
When stating LAMP detection kit and carrying out LAMP amplifications, take 1.0 μ L DNA profilings, be added kit detection solution described in 20.0 μ L and
4.0 μ L sterilizing ultra-pure waters carry out LAMP amplifications.
9. LAMP amplimer of the LAMP detection primer of fusarium prolifertum described in claim 1 as detection fusarium prolifertum,
Application in detecting fusarium prolifertum.
10. application of the LAMP detection kit of fusarium prolifertum described in claim 2 in detecting fusarium prolifertum.
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| CN110551797A (en) * | 2019-08-20 | 2019-12-10 | 临沂大学 | Method for carrying out double-round signal amplification visual detection on Fusarium proliferatum based on T5 exonuclease |
| CN111304357A (en) * | 2020-04-21 | 2020-06-19 | 广西壮族自治区农业科学院 | Fusarium oxysporum bitter gourd specialized LAMP visual detection method and kit thereof |
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| CN110551797B (en) * | 2019-08-20 | 2022-09-13 | 临沂大学 | Method for carrying out double-round signal amplification visual detection on Fusarium proliferatum based on T5 exonuclease |
| CN111304357A (en) * | 2020-04-21 | 2020-06-19 | 广西壮族自治区农业科学院 | Fusarium oxysporum bitter gourd specialized LAMP visual detection method and kit thereof |
| CN114480722A (en) * | 2022-03-18 | 2022-05-13 | 湖南农业大学 | LAMP (loop-mediated isothermal amplification) detection primer, kit and LAMP rapid detection method for pathogenic bacteria of phyllosticta |
| CN116004878A (en) * | 2022-07-07 | 2023-04-25 | 云南省烟草农业科学研究院 | Detection method for fusarium layering and application thereof |
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