CN110093446A - Recombinase-mediated isothermal duplication-Sidestream chromatography technology detection camphor tree phytophthora primer and probe combination and its application - Google Patents
Recombinase-mediated isothermal duplication-Sidestream chromatography technology detection camphor tree phytophthora primer and probe combination and its application Download PDFInfo
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- 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
- C12Q1/6804—Nucleic acid analysis using immunogens
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- 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
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- 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
Abstract
The invention discloses a kind of primer and probe combination of RPA-LFD technology detection camphor tree phytophthora and its application methods.Camphor tree phytophthora is detected using the primer and probe combinations method particularly includes: 1) extract sample to be tested DNA；2) using DNA as template, RPA amplification is carried out；3) amplified production detection is carried out using LFD；When two brown bands occur in test strips, one is located in quality control region, and one is located at detection zone, then result is the positive, shows to contain camphor tree phytophthora in sample；When test strips only have quality control region a brown band occur, detection zone does not have band, then the result is that negative, shows in sample without containing camphor tree phytophthora.Primer provided by the present invention and probe combinations RPA expanding effect are good, band high specificity, high sensitivity, provide new technological means for the detection of camphor tree phytophthora.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to recombinase-mediated isothermal duplication-Sidestream chromatography technology (RPA-
LFD the primer and probe combination and its application of camphor tree phytophthora (Phytophthora cinnamomi)) are detected.
Background technique
Camphor tree phytophthora (Phytophthora cinnamomi) belongs to oomycota (Oomycota), Oomycete
(Oomycetes), Peronosporales (Peronosporales), pythiaceae (Pythiaceae), Phytophthora (Phytophthora).Camphor tree
Phytophthora can infect cinnamon and cause the diseases such as butt rot and limb ulcer, can cause crushing blow to cinnamon.
Camphor tree phytophthora host is extensive, can infect thousands of kinds of plants.It is reported that camphor tree phytophthora has resulted in Australian southwest forest
Recurring structure and plant population variation, have seriously threatened local natural ecosystems and bio-diversity.At present to this
There are no effectively preventing measures for disease, reinforce quarantine, and preventing the Spreading and diffusion of pathogen is the most effective measure for controlling the disease.
It should reinforce epidemic monitoring thus, establish rapid detection method, foundation be provided for the risk and research decision of disease, to be conducive to
Reduce loss caused by camphor tree epidemic disease.
The taxonomic identification of traditional camphor tree phytophthora is mainly based upon morphological feature, Pathogenicity, physiological and biochemical property
Deng.The method of traditional separation phytophthora separates phytophthora from soil, irrigation water and vegetable material using mass trapping.In order to mention
High trap effect, can be added a small amount of antibiotic in the soil liquid and fungicide inhibits varied bacteria growing.Suitable for doing the plant of bait
Object material is because different phytophthoras are also different, and pine needle is suitable for trapping camphor tree phytophthora.Conventional method is detected in camphor tree phytophthora
In played important function, but it is time-consuming and laborious and require that operator has the phytophthora separation of profession, Morphological Identification is known
Know and experience abundant；Time-consuming, sensitivity is low, vulnerable to factors such as artificial and environment for traditional classification identification method simultaneously
Interference, cannot make diagnosis in disease incubation period and early stage, be difficult that disease occurs to carry out timely monitoring and effectively control
System.With the development of molecular biology, round pcr provides quick, sensitive, accurate advantage for pathogenic diagnosis.Commonly
The method of PCR has been used successfully to detection camphor tree phytophthora, although PCR method is greatly improved in specificity and sensibility,
It is that detection time is still long, general 4~5h, while PCR method relies on accurate temperature cycling device, detection sensitivity ratio
It is higher, detection process is complicated, be not able to satisfy the demand quickly detected.
Recombinase-mediated isothermal duplication (Recombinase Polymerase Amlification, RPA) technology is recognized
It is the nucleic acid detection technique that can substitute PCR.Its principle is to form Protein-DNA mixtures, energy in conjunction with primer using recombinase
Homologous sequence is found in double-stranded DNA.Once primer located homologous sequence, Exchange reaction of chain will occur and formed and started
DNA synthesis carries out exponential amplification to the target area in template.In addition, RPA technology has the matching of multiple tools enzyme to carry out effectively
Amplification, amplification efficiency are much higher than traditional round pcr, and the time used is shorter, most short to realize in 5min.Due to RPA technology
Have the characteristics that the amplification used time is short, be not required to expensive instrument, is specific good, making it using more and more extensive.The maximum of RPA technology is special
Point is the amplification for only needing 1 pair of primer that can realize template nucleic acid under 37 DEG C or so of constant temperature, does not need to pass through height
Temperature cycles realize nucleic and melting and annealing, therefore do not need expensive instrument and equipment.And 37 DEG C popular response temperature very
It readily satisfies, is suitble to quick detection of the base to pathogen.
RPA can combine application with Sidestream chromatography test strips (Lateral flow dipstick, LFD), in RPA
In amplification system, the reverse primer and fluorescein-labeled probe of biotin labeling are needed, probe is in 30 away from fluorescein bases
There is an abasic site (dSpacer) at place, which can be identified by nfo ribozyme, cutting, generates new C-terminal, and
Archaeal dna polymerase effect is lower to be extended, and is formed the RPA product for having biotin and divergent projection, is then tried product application LFD
Paper slip sample is detected.LFD sample end is coated with the nano-scale gold particle with anti-fluorescein antibody, and it is anti-that biotin is coated in detection line
Body, when reaction solution enters test strips, the amplified production with fluorescein and biotin will by antigen-antibody combination,
Biotin antibody-accounting-nano-scale gold particle complex is formed in detection line and is developed the color.Detection time is short, 5 minutes or so can mesh
It surveys as a result, naked eyes interpretation.
Currently, RPA technology has been widely used for human body, the viral diagnosis on animal or plant, but for camphor tree phytophthora
RPA detection has no that related application is reported.
Summary of the invention
Goal of the invention: being directed to the above-mentioned problems in the prior art, and the object of the present invention is to provide one kind to be based on RPA-
LFD technology detects the primer and probe combination of camphor tree phytophthora, and establishes a kind of RPA-LFD detection method of camphor tree phytophthora, high specificity,
High sensitivity；The kit for the primer and probe combination containing the camphor tree phytophthora that it is a further object of the present invention to provide a kind of, can use
In quickly detection camphor tree phytophthora.
Technical solution: to solve the above-mentioned problems, the technical solution adopted in the present invention is as follows:
To achieve the above object, the present invention adopts the following technical solutions:
A kind of primer and probe combination based on RPA-LFD technology detection camphor tree phytophthora, forward primer sequence such as SEQ ID
Shown in No.1, reverse primer sequences are as shown in SEQ ID No.2, and probe sequence is as shown in SEQ ID No.3, wherein reverse primer
The end of sequence 5 ' is marked with biotin (Biotin)；The end of probe sequence 5 ' is marked with fluorescein (FAM), and distance 5 ' holds 30 bases
Tetrahydrofuran (THF) is used to substitute a base as dSpacer at position, 3 ' ends are blocked with C3-spacer；The primer and spy
Needle sequence is as follows:
Forward primer RPA-PciRPA-F:
Reverse primer RPA-PciRPA-R:
Probe sequence are as follows: PciRPA-P:
Application of the primer and probe combination in detection camphor tree phytophthora.
Application of the primer and probe combination in the detection reagent of preparation camphor tree phytophthora or kit.
The present invention also provides a kind of kits based on RPA-LFD technology detection camphor tree phytophthora, which is characterized in that at least wraps
The above-described primer and probe combination of 1 dosage is included, forward primer sequence is as shown in SEQ ID No.1, reverse primer sequence
Column are as shown in SEQ ID No.2, and probe sequence is as shown in SEQ ID No.3.
The kit further include: equipped with the freeze-drying TwistAmp reaction member pipe of enzyme powder, Rehydration Buffer,
MgAc, deionized water, running buffer, Sidestream chromatography test strips.
Application of the kit of the detection camphor tree phytophthora in detection camphor tree phytophthora.
Present invention also provides a kind of methods based on RPA-LFD technology detection camphor tree phytophthora, comprising the following steps:
1) sample to be tested DNA is extracted；
2) using DNA as template, RPA expansion is carried out using the kit of primer and probe combination or the detection camphor tree phytophthora
Increase；
3) detection of RPA amplified production is carried out using Sidestream chromatography test strips；When two brown bands occur in test strips
(Control line andTest line), one is located in quality control region, and one is located at detection zone, then result is the positive, shows
Contain camphor tree phytophthora in sample；When test strips only have quality control region a brown band (Control line) occur, detection zone does not have
Band, then the result is that it is negative, show in sample without containing camphor tree phytophthora.
The changeable host plant cell structure and metabolic pathway that effector (effector) is pathogen secretion are to promote
Into a kind of exocrine protein molecule that host's defense response is successfully infected or caused to host plant.Many phytopathy originals can
Secrete effector albumen.The detection target of the application is effector gene, is derive specifically from camphor tree phytophthora (Phytophthora
Cinnamomi) bacterial strain, entitled RxLR effector Phyci587572, the nucleotide sequence of the gene are stored in database
Https: //fungidb.org/fungidb/app/record/gene/PHYCI_587572, which becomes in camphor tree phytophthora difference
Inter-species sequence has specificity, also has specificity in oomycetes and other fungies.The present invention is first against camphor tree phytophthora
Phyci587572 gene is as target sequence, using a series of RPA primer of 5.0 software design of Primer Premier,
And screening is optimized to designed primer by experiment, obtain a pair of primer special for RPA detection camphor tree phytophthora with
Probe.Different from routine PCR reaction, the length of primer needed for RPA reacts is usually 30-35bp, and the length of probe sequence is 46-
52bp, formed inside primer when design of primers and between secondary structure, the increase of length also makes design of primers
And the increase of selection difficulty, therefore, the design and selection of primer are most important to the result of RPA.RPA technology is in starting and grinds
Study carefully the stage, there is no special primer, probe design software, also provides foundation without a large amount of data for its design of primers principle.
Therefore, primer and probe of the invention combination is to need to design multipair primer from target sequence both ends to optimize, screen ability
It obtains.
The utility model has the advantages that compared with prior art, present invention has the advantage that
1) gene primer selected of the present invention is to screen to obtain through many experiments, and specificity is good, with other pathogens without
Cross reaction.Primed probe expanding effect used in the present invention is good, band high specificity, can be in detection zone (Test
Line the primer-probe heterodimer of higher concentration) is formed, thus makes test strips that strong positive reaction be presented, increases the spirit of detection
Sensitivity, the established detection method of the present invention can detect 10pg μ L-1Camphor tree phytophthora genome.
2) RPA technology of the invention and the method for combining Sidestream chromatography technology detection camphor tree phytophthora, both have molecular biology
That detects is highly sensitive, high-throughput, and has the advantages that the specificity of immunology detection is good, easy to operate, is further without complex instrument,
Particularly suitable for the camphor tree phytophthora rapid screening detection of laboratories and quarantine scene.
3) for method of the invention compared with Standard PCR, detection speed is fast, it is not necessary to by denaturation, annealing, extend three steps
Suddenly, the optimum temperature of RPA reaction is between 25 DEG C -40 DEG C, and without denaturation, reaction is can be completed in 20min or so at normal temperature.No
Complicated instrument and equipment is needed, on-site test is suitable for.Constant-temperature amplification is realized, unlike PCR method has to thermal cycle, thus
The dependence to thermal cycler instrument is got rid of, as long as there is stable heat source RPA reaction, greatly extending RPA makes
Range can really realize portable live Rapid nucleic acid detection.
4) present invention can be used for carrying disease germs the quick detection of camphor tree phytophthora in plant tissue, only needs an about 1h can be completed and detected
Journey is a kind of fast and effective means for detecting camphor tree phytophthora.Detection method can also be used in detection morbidity early period of camphor tree phytophthora
With the prediction of disease, had a very important significance for determining proper control time, effectively preventing disease.
Detailed description of the invention
Fig. 1 is that RPA-LFD detects camphor tree phytophthora in the specific detection result figure of inter-species；1 in figure: camphor tree phytophthora
(P.cinnamima)；2: Phytophthora capsici (P.capsici)；3: Phytophthora drechsleri (P.drechsleri)；4: cloves phytophthora
(P.syringae)；5: phytophthora hibernalis (P.hibernalis)；6: soybean phytophthora (P.sojae)；7: phytophthora infestans
(P.infestans)；8: Phytophthora cactorum (P.cactorum)；9: negative control；
Fig. 2 is that RPA-LFD detects specific detection result figure of the camphor tree phytophthora between category；1: camphor tree phytophthora (P.cinnamima)；
2: Pythium ultimum (Globisporangium ultimum)；3: scouring rush's Fusariumsp (Fusarium equiseti)；4: tack charcoal
Subcutaneous ulcer bacterium (Colletotrichum truncatum)；5: verticillium dahliae (Verticilium dahliae)；6: Rhizoctonia solani Kuhn
(Rhizoctonia solani)；7: negative control；
Fig. 3 is the sensitivity test result figure that RPA-LFD detects camphor tree phytophthora (P.cinnamima).
Fig. 4 is that artificial infection camphor tree phytophthora (P.cinnamima) carries out sample detection result figure: 1-4: artificial infection camphor tree phytophthora
(P.cinnamima) the deodar needle sample RPA-LFD testing result of epidemic disease caused by；5-8: healthy deodar needle RPA-LFD
Testing result；9: negative control.
Specific embodiment
The present invention is further described below combined with specific embodiments below.Do not make point illustrated in following embodiment
Sub- biological experimental method can refer to side listed in one book of " Molecular Cloning:A Laboratory guide " (third edition) J. Pehanorm Brooker
Method or the conventional method of this field carry out, or carry out according to kit and product description.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment 1: the genomic DNA for trying cause of disease bacteria strain is extracted
Specific extraction process is as follows:
A small amount of hypha powder is taken, adds 900 μ L 2%CTAB extracting solutions and 90 μ L 10%SDS, whirlpool mixes, in 60 DEG C of water-baths
1h, intermediate every 10min turn upside down several times.12000rpm is centrifuged 10min, takes supernatant that isometric phenol/chloroform/isoamyl alcohol is added
(25: 24: 1), are mixed by inversion, and 12000rpm is centrifuged 10min；Supernatant is transferred in new pipe, isometric chloroform is added, gently
It is gently mixed by inversion, 12000rpm is centrifuged 5min.It takes supernatant to be transferred in new pipe, adds the dehydrated alcohol and 1/10 body of 2 times of volumes
Long-pending 3M NaAc (pH5.2), -20 DEG C of precipitatings (> 1h).12000rpm is centrifuged 10min, and incline supernatant, precipitates 70% second
Alcohol washes twice, and room temperature is dried.Add appropriate sterilizing ultrapure water or TE (pH8.0) dissolution precipitating (containing 20 μ g mL-1RNase), 37 DEG C
After handling 1h, -20 DEG C are saved backup.
Each soil sample is dried pulverize first by the extraction for DNA in soil, is then weighed the conduct of 0.5g soil sample respectively and is mentioned
Sample is taken, is usedThe extraction of SPIN kit (Q-Biogene Ltd, USA) progress DNA.What soil DNA extracted
Specific steps are referring to kit specification.
When, there are when camphor tree phytophthora, using the DNA of NaOH rapid cleavage method extraction camphor tree phytophthora, detailed process is such as in incidence tissue
Under: the tissue sample of one section of morbidity is taken, every milligram of tissue is added 10 μ L 0.5M NaOH, is transferred to after being fully ground in mortar
In the EP pipe of 1.5mL, 12000rpm is centrifuged 5min, takes 5 μ L supernatants that 495 μ L 0.1mM Tris (pH 8.0) are added, after mixing
1 μ L is taken to be directly used in RPA reaction.Each reaction is at least in triplicate.
Embodiment 2: the design of camphor tree phytophthora (P.cinnamima) RPA primer and probe and RPA-LFD detection method are established
The RPA primer length is generally 30 to 35 nucleotide.The too short activity that can seriously affect recombinase of primer is long
Primer may not be able to improve amplification capability, will increase a possibility that forming secondary structure instead, increase the noise from primer.
In addition, should avoid secondary structure easy to form, primer-primer interaction, the sequence of hairpin structure when design of primers as far as possible, reduction is drawn
The formation of object dimer.Amplified production size is no more than 500bp.
According to the Ypt1 gene (DQ162954.1) of camphor tree phytophthora, forward primer is devised using Primer Premier 5.0
Sequence RPA-PciRPA-F, reverse primer sequences RPA-PciRPA-R, probe sequence PciRPA-P.Wherein reverse primer sequences
5 ' the ends of RPA-PciRPA-R are marked with biotin (Biotin)；It is marked with fluorescein (FAM) at the 5 ' ends of probe sequence PciRPA-P
Remember, use tetrahydrofuran (THF) to substitute a base as dSpacer at the position of 30 bases in the end of distance 5 ', C3- is used at 3 ' ends
Spacer is blocked；Particular sequence is as follows, and particular sequence is as follows:
RPA-PciRPA-F:
RPA-PciRPA-R:
PciRPA-P:
Using the DNA of extraction as template, using the RPA primer of design, RPA reaction is carried out in following reaction systems:
Sample detection: to 0.2mL TwistAmp reaction member pipe (the TwistAmp Basic equipped with freeze-drying enzyme powder
Kits, Twist) in be added Rehydration Buffer (TwistAmp Basic kits, Twist) 29.5 μ L, 10 μM upper
Swim primer 2 .1 μ L, 10 μM of 2.1 μ L, 280mM probe of downstream primer, 0.6 2.0 2.5 μ L of μ L, MgAc of μ L, DNA, deionized water
Complement to 50 μ L；RPA amplification system is mixed well, 5,000 × g is centrifuged 10s, and it is placed on 39 DEG C of metal baths and reacts 30min, temperature
It educates 4 minutes, reaction tube is mixed again, be centrifuged 3-5s, be put into 39 DEG C of water-bath the reaction was continued 30min；It is tried using Sidestream chromatography
Paper slip (LFD strip (Milenia Biotec, Giessen, Germany)) carries out amplified production detection.10 μ L are pipetted to sample
On product pad, sample pad end is placed in the running buffer (Milenia Biotec, Giessen, Germany) of 200 μ L.2-5
After minute, positive sample will appear two bands, and negative sample only has a band.When test strips occur two brown bands, one
In quality control region (Control line), one is located at detection zone (Test line), then result is the positive, shows in sample
Contain soybean phytophthora (P.sojae)；When test strips only have quality control region a brown band occur, detection zone (Test line) does not have
There is band, then the result is that it is negative, show in sample without containing camphor tree phytophthora (P.cinnamima).
Negative control: 2.0 μ L template DNAs are changed to that 2.0 μ L deionized waters are added by the same sample detection of operating procedure.RPA is anti-
After answering, amplified production detection is carried out using LFD Sidestream chromatography test strips.When test strips occur two brown bands, one
In quality control region (Control line), one is located at detection zone (Test line), then result is the positive, shows in sample
Contain camphor tree phytophthora；When test strips only have quality control region a brown band occur, detection zone (Test line) does not have band, then ties
Fruit is feminine gender, is shown in sample without containing camphor tree phytophthora.
Embodiment 3:RPA-LFD detects the specificity verification of camphor tree phytophthora (P.cinnamima)
In order to verify the specificity of RPA Sidestream chromatography test strips detection method, with camphor tree Mtr mutant and other phytophthoras and
Pathogen is material to be tested (table 1), and the test strips of the phytophthora of camphor tree as the result is shown of RPA Sidestream chromatography test strips detection method occur two
Brown band, one is located in quality control region (Control line), and one is located at detection zone (Test line), then result is
Positive (+), the test strips of other pathogens only have quality control region a brown band occur, and detection zone (Test line) does not have item
Band, then the result is that negative (-), shows in sample without containing camphor tree phytophthora.
1 strains tested of table and RPA-LFD testing result
Selection and camphor tree phytophthora (Phytophthora capsici not of the same race；Phytophthora drechsleri；Cloves phytophthora；Phytophthora hibernalis；Soybean phytophthora；It causes a disease
Phytophthora；Phytophthora cactorum etc.) and the bacterium (Pythium ultimum that does not belong to；Scouring rush's Fusariumsp；Tack anthrax-bacilus；Verticillium dahliae；Miliary damping-off
Bacterium etc.) DNA as template, carry out RPA-LFD detection.It is detected according to the method for embodiment 1-2, testing result such as Fig. 1
With shown in Fig. 2, as seen from the figure, after carrying out RPA amplified reaction with the primer that embodiment 1 designs, expanded containing camphor tree phytophthora sample RPA
The LFD Sidestream chromatography test strips testing result of product shows 2 brown bands, and target stripe is analysed clearly, can effectively detect camphor tree epidemic disease
It is mould.And only there is a brown band in quality control region in other fungies and oomycetes, negative control also only occurs one in quality control region
Brown band.The result shows that designed primer and probe combination and the RPA-LFD detection camphor tree phytophthora method energy established
Camphor tree phytophthora is specifically detected, can be used for the detection application of deodar phytophthora.
Embodiment 4:RPA-LFD detects the sensitivity determination of camphor tree phytophthora (P.cinnamima)
Measuring embodiment 1 to extract the concentration of camphor tree phytophthora DNA using 2000 micro-spectrophotometer of Nanodro is 100ng
μL-1.It is successively diluted to 10ng μ L-1、1ng·μL-1、100pg·μL-1、10pg·μL-1、1pg·μL-1、100fg·μ
L-1、10fg·μL-1, according to the primer of embodiment 1-2 use, reaction system and reaction condition, the DNA of various concentration is carried out
RPA-LFD detection.
As a result as shown in figure 3, containing 100ng μ L in reaction system respectively-1、10ng·μL-1、1ng·μL-1、
100pg·μL-1、10pg·μL-1There are two brown bands in the test strips of camphor tree phytophthora DNA, are positive, in reaction system
Contain 1pg μ L respectively-1、100fg·μL-1、10fg·μL-1There is a brown band in the paper slip of camphor tree phytophthora DNA, is negative
Reaction；Colour developing is the result shows that the sensitivity of RPA- Sidestream chromatography Lateral Flow Strip reaches 10pg μ L-1(Fig. 3)；RPA detection time
The expensive instrument and equipment such as 20min is only needed, and does not need PCR instrument, operation sequence is easy, more favorably promotes and applies in production.
Embodiment 5: actual sample experiment detection
The present embodiment carries out actual sample experiment detection from artificial infection cedar needles.The method of embodiment 1 is used first
Rapidly extracting is inoculated with the DNA of the cedar needles of pathogen.Then, anti-using DNA progress RPA of the method for embodiment 2 to extraction
Should and amplified production detection be carried out using LFD (Sidestream chromatography test strips).As a result as shown in figure 4, when there are two palm fibres in test strips
Vitta band, one is located in quality control region (Control line), and one is located at detection zone (Test line), then result is the positive,
Show to contain camphor tree phytophthora in inoculation sample 1-4；When test strips only have quality control region a brown band, detection zone (Test occur
Line) there is no band, then the result is that negative, the only band of healthy plant No. 5-8 and negative control sample 9 does not contain camphor tree
Phytophthora.It is accurate and reliable to again demonstrate RPA- Sidestream chromatography test strips detection method testing result, there is very strong practicability.
Unless specifically stated otherwise, the numerical value otherwise illustrated in these embodiments is not limit the scope of the invention.?
In all examples shown and described herein, unless otherwise prescribed, any occurrence should be construed as merely illustratively, and
Not by way of limitation, therefore, other examples of exemplary embodiment can have different values.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme should all cover within the scope of the claims and the description of the invention.
Sequence table
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<213>PciRPA-P primer sequence (Artificial)
<400> 3
agtcgacgat gatgactcct ccgaagattc tccgaagaga ggacg 45
Claims (7)
1. a kind of primer and probe combination based on RPA-LFD technology detection camphor tree phytophthora, which is characterized in that its forward primer sequence
As shown in SEQ ID No.1, reverse primer sequences are as shown in SEQ ID No.2, and probe sequence is as shown in SEQ ID No.3.
2. application of the primer and probe combination described in claim 1 in detection camphor tree phytophthora.
3. application of the primer and probe combination described in claim 1 in the detection reagent of preparation camphor tree phytophthora or kit.
4. a kind of kit based on RPA-LFD technology detection camphor tree phytophthora, which is characterized in that more than 1 dosage
Primer and probe combination described in claim 1, the primer and probe combination, forward primer sequence such as SEQ ID No.1
Shown, reverse primer sequences are as shown in SEQ ID No.2, and probe sequence is as shown in SEQ ID No.3.
5. detecting the kit of camphor tree phytophthora described in claim 4, which is characterized in that the kit further include: lyophozyme is housed
The TwistAmp reaction member pipe of powder, Rehydration Buffer, MgAc, deionized water, running buffer, Sidestream chromatography examination
Paper slip.
6. detecting application of the kit of camphor tree phytophthora in detection camphor tree phytophthora described in claim 4.
7. a kind of method based on RPA-LFD technology detection camphor tree phytophthora, which comprises the following steps: 1) extract to be measured
Sample DNA；2) using DNA as template, detection camphor tree epidemic disease described in the combination of primer and probe described in claim 1 or claim 4 is utilized
Mould kit carries out RPA amplification；3) detection of RPA amplified production is carried out using Sidestream chromatography test strips；When test strips occur two
Brown band, one is located in quality control region, and one is located at detection zone, then result is the positive, shows to contain camphor tree phytophthora in sample；
When test strips only have quality control region a brown band occur, detection zone does not have band, then the result is that negative, shows to be free of in sample
There is camphor tree phytophthora.
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| CN201910467152.5A CN110093446B (en) | 2019-05-31 | 2019-05-31 | Primer and probe combination for detecting phytophthora cinnamomi by recombinase-mediated isothermal amplification-lateral flow chromatography technology and application of primer and probe combination |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112852997A (en) * | 2021-03-30 | 2021-05-28 | 南京林业大学 | Detection target Pcinn11345 of phytophthora camphora, detection primer and rapid detection method thereof |
| CN113105533A (en) * | 2021-04-08 | 2021-07-13 | 南京林业大学 | Phytophthora camphora effector protein Avh49 and application thereof |
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| CN104372104A (en) * | 2014-12-08 | 2015-02-25 | 南京林业大学 | LAMP (loop-mediated isothermal amplification) detection primer composition, LAMP detection kit and LAMP detection method for phytophthora cinnamomi |
| WO2016097726A1 (en) * | 2014-12-16 | 2016-06-23 | Fungialert Limited | Device and method for detecting plant pathogens |
| CN106868164A (en) * | 2017-03-23 | 2017-06-20 | 福建省农业科学院植物保护研究所 | A kind of primer and nested PCR detection method for detecting camphor tree phytophthora |
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| CN104372104A (en) * | 2014-12-08 | 2015-02-25 | 南京林业大学 | LAMP (loop-mediated isothermal amplification) detection primer composition, LAMP detection kit and LAMP detection method for phytophthora cinnamomi |
| WO2016097726A1 (en) * | 2014-12-16 | 2016-06-23 | Fungialert Limited | Device and method for detecting plant pathogens |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112852997A (en) * | 2021-03-30 | 2021-05-28 | 南京林业大学 | Detection target Pcinn11345 of phytophthora camphora, detection primer and rapid detection method thereof |
| CN112852997B (en) * | 2021-03-30 | 2021-09-07 | 南京林业大学 | Detection target Pcinn11345 of phytophthora camphora, detection primer and rapid detection method thereof |
| CN113105533A (en) * | 2021-04-08 | 2021-07-13 | 南京林业大学 | Phytophthora camphora effector protein Avh49 and application thereof |
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| CN110093446B (en) | 2022-09-30 |
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Application publication date: 20190806 Assignee: Yulinwei (Nanjing) Biotechnology Co.,Ltd. Assignor: NANJING FORESTRY University Contract record no.: X2022320000315 Denomination of invention: Recombinase Mediated Isothermal Amplification Side Flow Chromatography for Detection of Phytophthora camphora and Its Application Granted publication date: 20220930 License type: Common License Record date: 20221210 Application publication date: 20190806 Assignee: MAANSHAN Panshi Biotechnology Co.,Ltd. Assignor: NANJING FORESTRY University Contract record no.: X2022320000333 Denomination of invention: Recombinase Mediated Isothermal Amplification Side Flow Chromatography for Detection of Phytophthora camphora and Its Application Granted publication date: 20220930 License type: Common License Record date: 20221210 |
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