CN105039331A - Peronophythora litchi LAMP primer as well as rapid detection method and application thereof - Google Patents
Peronophythora litchi LAMP primer as well as rapid detection method and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of crop disease control and plant quarantine inspection, and discloses a peronophythora litchi LAMP primer as well as a rapid detection method and application of the peronophythora litchi LAMP primer in early diagnosis, bacterial monitoring and identification. The primer comprises inner primer pairs F3 and B3 as well as outer primer pairs FIP and BIP, and the sequences of the primers are as shown in SEQ NO.1 to SEQ NO.4. The detection method is reliable in result, easy to operate, high in specificity, high in sensitivity, suitable for rapidly and reliably detecting and identifying peronophythora litchi in a disease-attacked tissue, capable of being directly applied to high-sensitivity rapid detection of a plant with bacteria and capable of being used in the early diagnosis, bacteria monitoring and identification of peronophythora litchi. A monitor technical system for the peronophythora litchi which is rapid, simple, high in specificity and high in sensitivity is established, and important significance for the early monitoring before a disease syndrome caused by the peronophythora litchi and for determining an optimum disease control period in the agricultural production can be realized.
Description
Technical field
The invention belongs to crop disease control and field of plant quarantine, particularly a kind of peronophythora litchi LAMP primer and method for quick thereof and the application in the early diagnosis of peronophythora litchi, the monitoring of germ and qualification.
Background technology
Lichee (LitchiichinensisSonn.) originates in south China and the famous tropical and subtropical zone local product fruits of North Vietnam, is mainly distributed in the provinces and regions such as Guangdong, Guangxi, Fujian, Hainan, Taiwan, Sichuan, Yunnan, Guizhou, Zhejiang in China.The cultivated area of China's lichee and output are first of the world, and export in a large number.Lichee is high because of its economic worth, is China's one of the most competitive fruit in the international market.Along with the expansion of lichee continuous cropping and cultivated area, the development of lichee industry affected primarily of the occurrence and harm of fungal disease, especially peronophythora litchi (PeronophythoralitchiiChenexKoetal.), be modal main fungal venereal disease evil on lichee, become one of limiting factor affecting lichee production.The maturescent fruit of germ main harm, also young fruit, carpopodium, Hua Sui and blade is endangered, fruit loss during often causing a large amount of decayed fruit, shedding and storing, time serious, decayed fruit rate reaches 30 ~ 80%, the serious threat yield and quality of lichee, and normal mixing with other lichee diseases occurs, difficulty is caused to the diagnosis of disease, then often because of not clear pathogenic factor in production, be difficult to prophylactico-therapeutic measures targetedly, therefore cause even more serious harm.Therefore peronophythora litchi rapid detection system is set up, early stage quick and precisely detection disease plant being carried out to peronophythora litchi, to prediction disease a situation arises, take in time the propagation of effectively preventing controlling measurement pathogenic bacteria and popular, reduce financial loss all there is important theoretical and practical significance.
Mostly still continue to use traditional separate tissue to the detection of peronophythora litchi at present to cultivate and Morphological Identification method, but, this authentication method length consuming time, and Success rate of virus isolation is not high.If multiple pathogenic bacteria Combined Infection, the disease sample be separated is stale, then more difficultly make a definite diagnosis pathogenic bacteria, is difficult to meet the actual needs to peronophythora litchi diagnosis, this to the detection of pathogenic bacteria and prevention and control very unfavorable.
Loop-mediated isothermal amplification technique (Loop-mediatedisothermalamplification is called for short LAMP) is a kind of New Cycle constant temperature nucleic acid amplification technology developed by people such as Japanese Rong Yan Co., Ltd. Notomi for 2000.LAMP reaction designs 4 primers for 6 sites of target gene, utilize a kind of archaeal dna polymerase (BstDNApolymerase) of chain type substitute activity, under constant temperature, (60 ~ 65 DEG C) insulation 30 ~ 90 minutes, can complete amplified reaction.Due to high efficiency and the isothermal rapid amplifying of LAMP reaction, in short 30 ~ 90 minutes, just can realize 10
9~ 10
10the amplification of times product.The detection of LAMP amplified production generally adopts the methods such as fluorescence dye visual observations, agarose gel electrophoresis and turbidity observation.Because LAMP reaction is simple, quick, efficient, economic dispatch feature, thus there is application prospect very widely.Current LAMP detects and is mainly used in people and animals' pathogen, food safety and sanitary detection, and in phytopathogen detects, report is few, and the detection of Peronophythora Litchii is not reported both at home and abroad.
Summary of the invention
In order to overcome peronophythora litchi in above-mentioned prior art biological detection method needed for cycle long, detection method poor specificity, the shortcoming that sensitivity is low and deficiency, primary and foremost purpose of the present invention is to provide a kind of peronophythora litchi (Peronophythoralitchii) LAMP primer.
Another object of the present invention is to provide a kind of method for quick based on above-mentioned peronophythora litchi LAMP primer.This detection method reliable results, easy handling, high specificity, highly sensitive.
Still a further object of the present invention is to provide the application of above-mentioned method for quick in the early diagnosis of peronophythora litchi, the monitoring of germ and qualification.
Object of the present invention is realized by following proposal:
A kind of peronophythora litchi (Peronophythoralitchii) LAMP primer, comprise inner primer to F3 and B3, and outer primer is to FIP and BIP, sequence is as follows:
F3：5’-TGAGGACGTGTACTCGTTCC-3’；
B3：5’-CGCTCATACAGTGGGTGATC-3’；
FIP：
5’-AGCTAAACTGTGACCAGGGTGGCGTCTCCTTTTGGCTTCGTG-3’；
BIP：
5’-GTTACCCGGTCAGCTATGCTGGGCGGGCTTTGAACATCTTGT-3’。
Present invention also offers a kind of method for quick based on above-mentioned peronophythora litchi LAMP primer, comprise the following steps:
With the DNA of sample to be detected for template, utilize above-mentioned primer to carry out LAMP reaction amplification, then detected by fluorescence dye visual observations method or agarose gel electrophoresis method.
Described LAMP reaction system is preferably: 25 μ L, wherein F3 and B3 concentration is respectively 0.2 μM, and FIP and BIP concentration is respectively 1.6 μMs, 20mMTris-HCl, 10mM (NH
4)
2sO
4, 10mMKCl, 2 ~ 4mMMgSO
4, 0.1%Tween-20,0 ~ 1.2MBetaine, 1mMdNTPs, 8UBstDNA polymerase Large fragment, 10 ~ 50ngDNA template, insufficient section aseptic double-distilled water is supplied; Reaction conditions is at 60 ~ 65 DEG C of incubation 30 ~ 90min, 80 DEG C of insulation 5 ~ 10min.
Described LAMP reaction system is more preferably: 25 μ L, and wherein F3 and B3 concentration is respectively 0.2 μM, and FIP and BIP concentration is respectively 1.6 μMs, 20mMTris-HCl, 10mM (NH
4)
2sO
4, 10mMKCl, 2 ~ 4mMMgSO
4, 0.1%Tween-20,0 ~ 1.2MBetaine, 1mMdNTPs, 8UBstDNA polymerase Large fragment, 25ngDNA template, insufficient section aseptic double-distilled water is supplied; Reaction conditions is at 65 DEG C of incubation 60min, 80 DEG C of insulation 10min.
Described LAMP reaction system is more preferably: 25 μ L, and wherein F3 and B3 concentration is respectively 0.2 μM, and FIP and BIP concentration is respectively 1.6 μMs, 20mMTris-HCl, 10mM (NH
4)
2sO
4, 10mMKCl, 2mMMgSO
4, 0.1%Tween-20,0.2MBetaine, 1mMdNTPs, 8UBstDNA polymerase Large fragment, 25ngDNA template, insufficient section aseptic double-distilled water is supplied; Reaction conditions is at 65 DEG C of incubation 60min, 80 DEG C of insulation 10min.
Described fluorescence dye visual observations method: add 1 μ L developer in the final amplified production of LAMP reaction, described developer is SYBRGreen I, observes display result.Colour developing result is observed green fluorescence and is judged as the positive, is orangely judged as feminine gender.
Described agarose gel electrophoresis method: the final amplified production getting 6 μ LLAMP reactions detects with 1% agarose gel electrophoresis, observations.Be judged as the positive if there is the distinctive trapezoid belt of LAMP, do not occur that amplified band is judged as feminine gender.
The DNA of described sample to be detected extracts preferably by DNA extraction agent box method.
When described sample to be detected is thalline, fungal DNA extraction agent box method is preferably used to extract.
When described sample to be detected is plant tissue, plant tissue DNA's extraction agent box method is preferably used to extract.
Detection method reliable results, easy handling, high specificity, highly sensitive, be applicable to fast and reliable detection and the qualification of peronophythora litchi in incidence tissue, the plant highly sensitive rapid detection of carrying disease germs can be directly used in, can be used in the early diagnosis of peronophythora litchi, the monitoring of germ and qualification.The present invention establishes that peronophythora litchi is quick, easy, high specificity, highly sensitive Monitoring techniques system, for peronophythora litchi in agriculture production cause disease show disease before early monitoring, determine that disease control best period tool is of great significance.
The present invention, relative to prior art, has following advantage and beneficial effect:
1, reliable results: LAMP primer of the present invention and LAMP reaction system, has carried out repeatedly repeated authentication, reliable results to the plant tissue of peronophythora litchi and band Peronophythora Litchii.
2, high specificity: LAMP primer of the present invention designs 4 Auele Specific Primers for 6 different zones in Peronophythora Litchii M90 gene order, in 6 regions, any region and primer do not mate and all can not carry out nucleic acid amplification, therefore specificity is high.
3, highly sensitive: LAMP detection method of the present invention can reach 1pg to the detection sensitivity of peronophythora litchi on DNA level, detect high 1000 times than Standard PCR.
4, fast easy and simple to handle: application the inventive method, carry out detection to the tissue of band peronophythora litchi to complete in 1 hour, and LAMP nucleic acid amplification carries out under isothermal conditions, only need a water-bath, do not need complicated plant and instrument and expensive molecular agents, result naked eyes are directly visible.
Accompanying drawing explanation
Fig. 1 is the special LAMP agarose gel electrophoresis detected result figure of peronophythora litchi.Wherein: swimming lane M is DL2000DNAmarker, swimming lane 1 is Peronophythora Litchii, and swimming lane 2 is lichee anthrax-bacilus, and swimming lane 3 is cucumber phytophthora, and swimming lane 4 is cucumber pythium spp, and swimming lane 5 is negative control, and swimming lane 6 is blank.
Fig. 2 is the LAMP susceptibility agarose gel electrophoresis detected result figure of Peronophythora Litchii.Wherein: swimming lane M is DL2000DNAmarker, swimming lane 1 ~ 9 template concentrations is respectively 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg, and the 10th swimming lane is negative control.
Fig. 3 is the agarose gel electrophoresis detected result figure to inoculation blade.Wherein, swimming lane M is DL2000DNAmarker, and the 1st swimming lane is positive control, swimming lane 2 ~ 3 is the incidence of leaf of Peronophythora Litchii, 4th ~ 5 swimming lanes are the blade of Peronophythora Litchii and the morbidity of lichee anthrax-bacilus compound, and the 6th swimming lane is negative control, and the 7th swimming lane is blank.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
The following example agents useful for same: BstDNA polymerase Large fragment is purchased from NEB company of Britain; DNAmarker and dNTPs reagent is purchased from precious biotechnology Dalian company limited; Trimethyl-glycine (Betaine) and SYBRGreen I reagent are purchased from prosperous company limited of Beijing ancient cooking vessel state.
Embodiment 1: the design of peronophythora litchi LAMP primer
The Peronophythora Litchii M90 gene order obtained with this laboratory, the method that Peronophythora Litchii M90 gene order obtains: in ncbi database, sequence analysis is carried out by the M90 gene order (accession number XM_002909036) of phytophthora infestans, obtain and download the M90 gene order of other allied specieses multiple (accession number is respectively AF507056, XM_008903240, XM_009531244), find out its conserved sequence and devise 4 primers, sequence is respectively:
F1：5’-AAGTCGGAGAAATGGGG-3’；
R1：5’-CTCAAAACGTGCTTCCC-3’；
F2：5’-ATCTGGACGAACCCTCACAAC-3’；
R2：5’-TAGTCCTCGTTGTGTCGAAT-3’；
Again with the DNA of peronophythora litchi for template carries out pcr amplification, after order-checking, the partial sequence of Peronophythora Litchii M90 gene can be obtained, PrimerExplorerV4 software design is adopted to go out a set of LAMP detection primer, comprise outer primer to (F3 and B3) and inner primer to (FIP and BIP), primer sequence is respectively:
F3：5’-TGAGGACGTGTACTCGTTCC-3’；
B3：5’-CGCTCATACAGTGGGTGATC-3’；
FIP：
5’-AGCTAAACTGTGACCAGGGTGGCGTCTCCTTTTGGCTTCGTG-3’；
BIP：
5’-GTTACCCGGTCAGCTATGCTGGGCGGGCTTTGAACATCTTGT-3’。
The foundation of embodiment 2:LAMP rapid detection system and the specific detection of peronophythora litchi LAMP primer
In order to verify the specific primer sequences of peronophythora litchi, with cucumber phytophthora (Phytophthoramelonis), cucumber pythium spp (Pythiumaphanidermatum) and lichee anthrax-bacilus (Colletotrichumgloeosporioides) (all can buy the Microbial resources center in Institute of Microorganism, Academia Sinica) for contrast material.
When described sample to be detected is thalline, use fungal DNA extraction agent box method to extract, fungi DNA extraction agent box is the FungalDNAkit of Omega company, and article No. is D3390, and concrete grammar is as follows:
(1) get freeze-drying mycelia to be about 100mg and to put into mortar and be ground to powdery, add 800 μ LBufferFG1 immediately, fully add in 2mL centrifuge tube after mixing, 65 DEG C of water-bath 10min, put upside down mixing sample during water-bath twice.
(2) add 140 μ LBufferFG2, whirlpool mixes, centrifugal 10min under 10,000rpm.
(3) get in the new 1.5mL centrifuge tube of supernatant liquor to, add the isopropanol precipitating DNA of 0.7 times of volume, centrifugal 10min under 10,000rpm.
(4) pour out supernatant liquor, then centrifuge tube is upside down in back-off 1min on which floor clean filter paper or thieving paper, to blot remaining moisture content.
(5) adding 300 μ L sterilized waters (preheating 65 DEG C) makes DNA suspend, and adds 4 μ LRNase (final concentration 20 μ g/mL) and mixes, not needing incubation during RNA ferment treatment.
(6) add 150 μ LBufferFG3 and 300 μ L dehydrated alcohols, mixing, obtains a uniform mixed solution.
(7) the DNA sample purification column collected is placed in 2mL collection tube, centrifugal 1min under 10,000rpm, discards and leaches liquid and collection tube.
(8) pillar is set on another collection tube, adds the 750 μ LDNAWashBuffer dehydrated alcohol dilution of 96 ~ 100% (with) centrifugal 1min under 10,000rpm, discard filtrate.
(9) wash once with 750 μ LDNAWashBuffer, under 10,000rpm, centrifugal 1min, discards filtrate again.
Under (10) 10,000rpm, the centrifugal 2min of void column is to dry the membrane matrix of pillar.
(11) purification column is placed in a new 1.5mL centrifuge tube, add 60 μ LElutionBuffer or aseptic deionized water (preheating 65 DEG C) the film central authorities to pillar, at room temperature leave standstill 3 ~ 5 minutes, 10, under 000rpm, centrifugal 1min is with eluted dna, obtains detected sample DNA.
When described sample to be detected is plant tissue, use plant tissue DNA's extraction agent box method to extract, plant tissue DNA used extraction agent box is Omega company PlantDNAKit, article No. D3485, and concrete grammar is as follows:
(1) get 100mg plant tissue of falling ill to put into mortar and be ground to powdery, add 500 μ LBufferCPL immediately, add 10 μ L beta-mercaptoethanols, fully add 1.5mL centrifuge tube after mixing, 65 DEG C of water-bath 15min, spin upside down mixing twice during water-bath;
(2) 500 μ L chloroform/Virahols (24:1) are added, concuss, centrifugal 10min under 10,000rpm room temperature;
(3) get supernatant liquor 300 μ L in new 1.5mL centrifuge tube, add 10 μ LRnase enzymes;
(4) add 150 μ LBufferCXD and 300 μ L dehydrated alcohols, mixing obtains a uniform mixed solution;
(5) the DNA sample purification column collected is placed in 2mL collection tube, centrifugal 1min under 10,000rpm, discards and leaches liquid and collection tube;
(6) add 650 μ LSPWWashBuffer, under 10000rpm, centrifugal 1min, discards and leaches liquid;
(7) wash once with 650 μ LSPWWashBuffer, under 10,000rpm, centrifugal 1min, discards filtrate again;
Under (8) 10,000rpm, the centrifugal 2min of void column is to dry the membrane matrix of pillar;
(9) purification column is placed in new 1.5mL centrifuge tube, add 50 ~ 100 μ LElutionbuffer or aseptic deionized water (preheating 65 DEG C) the film central authorities to pillar, at room temperature leave standstill 3 ~ 5 minutes, 10, under 000rpm, centrifugal 1min is with eluted dna, obtains detected sample DNA.
The foundation of LAMP reaction system: 25 μ L, wherein F3 and B3 concentration is respectively 0.2 μM, and FIP and BIP concentration is respectively 1.6 μMs, 20mMTris-HCl, 10mM (NH
4)
2sO
4, 10mMKCl, 2mMMgSO
4, 0.1%Tween-20,0.2MBetaine, 1mMdNTPs, 8UBstDNA polymerase Large fragment, 25ngDNA template, insufficient section aseptic double-distilled water is supplied; Reaction conditions is at 65 DEG C of incubation 60min, 80 DEG C of insulation 10min.
Detect with fluorescence dye visual observations method and agarose gel electrophoresis method.Described fluorescence dye visual observations method: add 1 μ L developer in the final amplified production of LAMP reaction, described developer is SYBRGreen I, observations, colour developing result is observed green fluorescence and is judged as the positive, is orangely judged as feminine gender.Described agarose gel electrophoresis method: get 6 μ L amplified production 1% agarose gel electrophoresis and detect, observations, is judged as the positive if there is the distinctive trapezoid belt of LAMP, can judge to there is peronophythora litchi in described lichee tissue; Do not occur that amplified band is judged as feminine gender, in namely described lichee tissue, there is not peronophythora litchi.
Result is as Fig. 1 (Fig. 1 is the DNA sample extracted for mycelia).As seen from Figure 1, except peronophythora litchi colour developing result can be observed green fluorescence or the distinctive trapezoid belt of LAMP appears in agarose gel electrophoresis detection, the display result of other bacterial strains (lichee anthrax-bacilus, melon phytophthora, cucumber pythium spp) is orange or agarose gel electrophoresis does not occur amplified band.This illustrates that the LAMP primer of the present invention's design has high specific for peronophythora litchi, can be used to detection that in production practice, in incidence tissue, peronophythora litchi is fast and reliable and qualification.
The susceptibility of embodiment 3:LAMP primer pair peronophythora litchi detects
Adopt 10 times of concentration series dilution methods that the peronophythora litchi DNA extracted in embodiment 2 is diluted to 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg be totally 9 different concns gradients.
The foundation of LAMP reaction system: 25 μ L, wherein F3 and B3 concentration is respectively 0.2 μM, and FIP and BIP concentration is respectively 1.6 μMs, 20mMTris-HCl, 10mM (NH
4)
2sO
4, 10mMKCl, 2mMMgSO
4, 0.1%Tween-20,0.2MBetaine, 1mMdNTPs, 8UBstDNA polymerase Large fragment, 25ngDNA template, insufficient section aseptic double-distilled water is supplied; Reaction conditions is at 65 DEG C of incubation 60min, 80 DEG C of insulation 10min.
In the final amplified production of LAMP reaction, add 1 μ L developer, described developer is SYBRGreen I, and colour developing result is observed green fluorescence and is judged as the positive, is orangely judged as feminine gender.Or get 6 μ L amplified production 1% agarose gel electrophoresis and detect, be judged as the positive if there is the distinctive trapezoid belt of LAMP, do not occur that amplified band is judged as feminine gender.
Detected result: peronophythora litchi LAMP susceptibility detects, display result can be observed green fluorescence or the distinctive trapezoid belt of LAMP appears in agarose gel electrophoresis, and detection sensitivity can reach 1pg (the results are shown in Figure 2).
Embodiment 4: the detection of Peronophythora Litchii inoculation blade
Blade inoculation: take just to turn green band branch lichee young leaflet tablet.Arrange three groups of inoculation blades, after first group of blade acupuncture, inoculation does not have the culture block of bacterium, as negative control; The mycelia block of Peronophythora Litchii is only inoculated after second group of blade acupuncture; The mycelia block of Simultaneous vaccination Peronophythora Litchii and lichee anthrax-bacilus after 3rd group of blade acupuncture.Second group and the 3rd group is repeated two samples.
Sample DNA isolation and determination: incidence of leaf adopts plant tissue DNA to extract test kit DNA rapid extraction, and concrete grammar is with embodiment 2.
Carry out LAMP detection as follows:
(1) foundation of LAMP reaction system: 25 μ L, wherein F3 and B3 concentration is respectively 0.2 μM, and FIP and BIP concentration is respectively 1.6 μMs, 20mMTris-HCl, 10mM (NH
4)
2sO
4, 10mMKCl, 2mMMgSO
4, 0.1%Tween-20,0.2MBetaine, 1mMdNTPs, 8UBstDNA polymerase Large fragment, 25ngDNA template, insufficient section aseptic double-distilled water is supplied; Reaction conditions is at 65 DEG C of incubation 60min, 80 DEG C of insulation 10min.
(2) in the final amplified production of LAMP reaction, add 1 μ L developer, described developer is SYBRGreen I, and colour developing result is observed green fluorescence and is judged as the positive, is orangely judged as feminine gender.Or get 6 μ L amplified production 1% agarose gel electrophoresis and detect, be judged as the positive if there is the distinctive trapezoid belt of LAMP, do not occur that amplified band is judged as feminine gender.
Detected result: as shown in Figure 3, with the incidence of leaf of peronophythora litchi, display result can be observed green fluorescence or the distinctive trapezoid belt of LAMP appears in agarose gel electrophoresis, and the health tissues display result not inoculating Peronophythora Litchii then observes fluorescent orange or the distinctive trapezoid belt of LAMP does not appear in agarose gel electrophoresis.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1. peronophythora litchi (Peronophythoralitchii) LAMP primer, it is characterized in that comprising inner primer to F3 and B3, and outer primer is to FIP and BIP, sequence is as follows:
F3：5’-TGAGGACGTGTACTCGTTCC-3’；
B3：5’-CGCTCATACAGTGGGTGATC-3’；
FIP：
5’-AGCTAAACTGTGACCAGGGTGGCGTCTCCTTTTGGCTTCGTG-3’；
BIP：
5’-GTTACCCGGTCAGCTATGCTGGGCGGGCTTTGAACATCTTGT-3’。
2., based on a method for quick for peronophythora litchi LAMP primer according to claim 1, it is characterized in that comprising the following steps:
With the DNA of sample to be detected for template, the peronophythora litchi LAMP primer described in utilization carries out LAMP reaction amplification, then is detected by fluorescence dye visual observations method or agarose gel electrophoresis method.
3. method for quick according to claim 2, is characterized in that: described LAMP reaction system is: 25 μ L, and wherein F3 and B3 concentration is respectively 0.2 μM, and FIP and BIP concentration is respectively 1.6 μMs, 20mMTris-HCl, 10mM (NH
4)
2sO
4, 10mMKCl, 2 ~ 4mMMgSO
4, 0.1%Tween-20,0 ~ 1.2MBetaine, 1mMdNTPs, 8UBstDNA polymerase Large fragment, 10 ~ 50ngDNA template, insufficient section aseptic double-distilled water is supplied; Reaction conditions is at 60 ~ 65 DEG C of incubation 30 ~ 90min, 80 DEG C of insulation 5 ~ 10min.
4. method for quick according to claim 2, it is characterized in that: described LAMP reaction system is: described LAMP reaction system is: 25 μ L, wherein F3 and B3 concentration is respectively 0.2 μM, and FIP and BIP concentration is respectively 1.6 μMs, 20mMTris-HCl, 10mM (NH
4)
2sO
4, 10mMKCl, 2 ~ 4mMMgSO
4, 0.1%Tween-20,0 ~ 1.2MBetaine, 1mMdNTPs, 8UBstDNA polymerase Large fragment, 25ngDNA template, insufficient section aseptic double-distilled water is supplied; Reaction conditions is at 65 DEG C of incubation 60min, 80 DEG C of insulation 10min.
5. method for quick according to claim 2, it is characterized in that: described LAMP reaction system is: described LAMP reaction system is: 25 μ L, wherein F3 and B3 concentration is respectively 0.2 μM, and FIP and BIP concentration is respectively 1.6 μMs, 20mMTris-HCl, 10mM (NH
4)
2sO
4, 10mMKCl, 2mMMgSO
4, 0.1%Tween-20,0.2MBetaine, 1mMdNTPs, 8UBstDNA polymerase Large fragment, 25ngDNA template, insufficient section aseptic double-distilled water is supplied; Reaction conditions is at 65 DEG C of incubation 60min, 80 DEG C of insulation 10min.
6. method for quick according to claim 2, it is characterized in that: described LAMP reaction system is: described fluorescence dye visual observations method: in the final amplified production of LAMP reaction, add 1 μ L developer, described developer is SYBRGreen I, observe display result, colour developing result is observed green fluorescence and is judged as the positive, is orangely judged as feminine gender.
7. method for quick according to claim 2, it is characterized in that: described LAMP reaction system is: described agarose gel electrophoresis method: the final amplified production getting 6 μ LLAMP reactions detects with 1% agarose gel electrophoresis, observations, occur that the distinctive trapezoid belt of LAMP is judged as the positive, do not occur that amplified band is judged as feminine gender.
8. method for quick according to claim 2, is characterized in that: described LAMP reaction system is: the DNA of described sample to be detected is extracted by DNA extraction agent box method.
9. method for quick according to claim 2, is characterized in that: described LAMP reaction system is: when described sample to be detected is thalline, uses fungal DNA extraction agent box method to extract; When described sample to be detected is plant tissue, plant tissue DNA's extraction agent box method is used to extract.
10. the application of the method for quick according to any one of claim 2 ~ 9 in the early diagnosis of peronophythora litchi, the monitoring of germ and qualification.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105331714A (en) * | 2015-11-24 | 2016-02-17 | 福建省农业科学院植物保护研究所 | Peronophythora litchii LAMP (loop-mediated isothermal amplification) primer and rapid detection method thereof |
| CN111088386A (en) * | 2020-01-09 | 2020-05-01 | 北京林业大学 | LAMP primer and kit for detecting Lasiodipia paraphysoides |
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| CN102899416A (en) * | 2012-10-24 | 2013-01-30 | 福建省农业科学院植物保护研究所 | Cucumber phytophthora LAMP (Loop-mediated Isothermal Amplification) primer and rapid detection method thereof |
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| CN102899416A (en) * | 2012-10-24 | 2013-01-30 | 福建省农业科学院植物保护研究所 | Cucumber phytophthora LAMP (Loop-mediated Isothermal Amplification) primer and rapid detection method thereof |
| CN103525913A (en) * | 2013-09-22 | 2014-01-22 | 福建省农业科学院植物保护研究所 | Phytophthora capsici LAMP (Loop-mediated isothermal Amplification) primer and rapid detection method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105331714A (en) * | 2015-11-24 | 2016-02-17 | 福建省农业科学院植物保护研究所 | Peronophythora litchii LAMP (loop-mediated isothermal amplification) primer and rapid detection method thereof |
| CN105331714B (en) * | 2015-11-24 | 2018-11-09 | 福建省农业科学院植物保护研究所 | A kind of peronophythora litchi LAMP primer and its rapid detection method |
| CN111088386A (en) * | 2020-01-09 | 2020-05-01 | 北京林业大学 | LAMP primer and kit for detecting Lasiodipia paraphysoides |
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| CN105039331B (en) | 2018-01-26 |
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