CN109439792B - The detection target Pcin100006 and its special primer for checking and rapid detection method of a kind of camphor tree phytophthora - Google Patents
The detection target Pcin100006 and its special primer for checking and rapid detection method of a kind of camphor tree phytophthora Download PDFInfo
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Abstract
The invention discloses a kind of detection target Pcin100006 of camphor tree phytophthora and its special primer for checking and rapid detection method, and the DNA sequence dna of detection target Pcinn100006 is as shown in SEQ ID NO.1.Special primer for checking is made of positive inner primer FIP, reversed inner primer BIP, positive outer primer F3, reversed outer primer B3, forward direction ring primer LF and reversed ring primer LB.Detection method accuracy height of the invention, high specificity, easy to operate, practicability is good, realizes constant-temperature amplification, new technology platform also is provided for the detection of camphor tree phytophthora simultaneously, it can be used for the high sensitivity quickly detection of camphor tree phytophthora, pathogen is identified at disease infestation initial stage simultaneously, the camphor tree phytophthora in field soil can be detected.The present invention is blindly used to epidemic prevention and control caused by camphor tree phytophthora and to pesticide is reduced, and reduces production cost, the environmental pollution for reducing pesticide is also of great significance.
Description
Technical field
The invention belongs to the technical field of gene detection of camphor tree phytophthora, and in particular to a kind of detection new target drone of camphor tree phytophthora
The quick LAMP detection primer composition and its LAMP detection kit and LAMP detection method of Pcin100006 and camphor tree phytophthora.
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.
Nucleic acid amplification technologies are an important research means of molecular biology field, and nucleic acid amplification technologies mainly include
Alternating temperature amplification technique and isothermal amplification technique two major classes based on Standard PCR.Currently, based on the change based on Standard PCR
Warm amplification technique mainly includes regular-PCR, nest-type PRC, multiplex PCR, real-time fluorescence quantitative PCR etc., and domestic and international researcher uses
These detection techniques (Wang, et al., 2006) detection phytophthora germ, many researchs at present both for single phytophthora,
But in practice examining quarantine, it is desirable that primary to examine the high-throughput detection skill that detected simultaneously to a variety of pathogens
The demand of art is higher and higher.Katarzyna Sikora etc. (Sikora, et al., 2012) is combined using Padlock probe
Microarray chip technology has carried out the high-throughput detection of 23 kinds of phytophthoras, but diagnostic method program is cumbersome, the period is long, cost
Height, while being also required to not be suitable for base, less developed country and area using expensive instrument and reagent etc.；Multiple PCR technique
Although can detect simultaneously to several pathogens, amplification is easy to cause non-specific and missing inspection etc. existing while several pairs of primers
As.Loop-mediated isothermal amplification technique (LAMP, Loop-mediated isothermal developed in recent years
Amplification) be expected the method to overcome drawbacks described above to provide, the technology have high specificity, sensitive height, isothermal,
Easy to operate, product easily detects visual feature.LAMP technology is by Notomi etc. (Notomi, et al., 2000) report
A kind of novel nucleic acids amplification technique, this method be mainly using can identify conserved sequence DNA 6 specific fragments 4
Primer, a kind of isothermal duplication carried out amplification reaction using a kind of strand displacement archaeal dna polymerase (Bst DNA polymerase)
Technology.The amplification of the gene of LAMP detection architecture and the detection of product can a step complete, amplification efficiency is high, can be in 30~60min
Amplification 109~1010Times, specificity is higher, while in LAMP reaction process, from the dNTP pyrophosphate ion being precipitated and instead
Answer the Mg in solution2+In conjunction with, by-product magnesium pyrophosphate is generated, milky white precipitate can be formed, the detection to target-gene sequence
It need to observe by the naked eye that whether there is or not milky white precipitates, thus whether judging amplification.LAMP detection technique has been used successfully to bacterium
(Pan, et al., 2011), virus, fungi (Niessen&Vogel, 2010) detection in, in the application of oomycetes mainly by
It wears graceful equal for the first time using LAMP technology detection soyabean phytophthora (Dai, et al., 2012, Dai Tingting, et al., 2013).It is whole
A detection process only needs 60min, can directly estimate experimental result by naked eyes, the LAMP detection architecture established can be used for mouth
Bank and field are used for quickly detecting soyabean phytophthora.With the perfect and development of the technical system, examined in phytophthora bacterium molecule
The application in survey field also will be more and more extensive and be goed deep into.
In conclusion excavating high reliability specific molecular detection target and establishing sensitive, accurate, high pass based on new target drone
The LAMP detection technique system of amount examines disease early stage caused when promoting the rapid molecular detection research and its detection to Phytophthora
It is disconnected to play a significant role.Currently, existing camphor tree phytophthora LAMP detection technique system can't fully meet use demand.
Summary of the invention
Goal of the invention: special for the length of period needed for camphor tree phytophthora biological detection method in the prior art, detection method
Property poor, problem that sensitivity is low, the object of the present invention is to provide a kind of detection target Pcinn100006 of new camphor tree phytophthora and
Its LAMP detection primer composition.It is a further object of the present invention to provide the LAMP detection kits of above-mentioned camphor tree phytophthora.This hair
Bright another object is to provide the LAMP detection method of above-mentioned camphor tree phytophthora.
Technical solution: in order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention are as follows:
The detection target Pcinn100006 of camphor tree phytophthora, DNA sequence dna is as shown in SEQ ID NO.1.
The detection target Pcinn100006 of the camphor tree phytophthora, protein sequence is as shown in SEQ ID NO.2.
Application of the detection target Pcinn100006 in detection camphor tree phytophthora.
The special primer for checking of the detection target Pcinn100006 of the camphor tree phytophthora, by positive inner primer FIP, instead
To inner primer BIP, positive outer primer F3, reversed outer primer B3, forward direction ring primer LF and reversed ring primer LB composition；Each primer sequence
It arranges specific as follows:
FIP:5 '-CGAAGGACGAGGTGAAGGTGGACGCCCATACATCACATACG-3 ':
BIP:5 '-AAGGCCGGCTACATGTACTCGTCTCGGGCAAGATGACTTC-3 ':
F3:5 '-GTTCTGCGCGATTTGGTTAG-3 ':
B3:5 '-GCGGATCTTCCGATCTGGTA-3 ':
LF:5 '-AACGTGACGCCGGCAACAA-3 '；
LB:5 '-TGTTGCCTCCGGGCTGT-3 '.
Application of the primer special in detection camphor tree phytophthora.
A kind of kit detecting camphor tree phytophthora, containing one-time detection dosage with the detection solution of upper volume, the inspection
It surveys solution to include at least: 1) primer special solution as claimed in claim 4.
The detection solution is containing including: 2) necessary LAMP reaction dissolvent；3) DNA synzyme；4) hydroxynaphthol blue.
Application of the kit in detection camphor tree phytophthora.
A method of detection camphor tree phytophthora: the DNA of microorganism to be checked being extracted, using the DNA of extraction as template, using right
It is required that primer special described in 4 or kit as claimed in claim 6, carry out corresponding LAMP detection reaction；Observing response is molten
Liquid color change, sky blue indicate test positive, and microorganism to be checked is camphor tree phytophthora；Purple indicates that testing result is feminine gender,
Microorganism to be checked is not camphor tree phytophthora.
The method of the detection camphor tree phytophthora, LAMP response procedures are as follows: 60 DEG C~65 DEG C, 60~80min.
The utility model has the advantages that compared with prior art, the advantages and positive effects of the present invention are shown:
1) excavate high reliability specific molecular detection target Pcinn100006 and based on new target drone establish it is sensitive, accurate,
High-throughput LAMP detection technique system is early to caused disease when promoting the rapid molecular detection research and its detection to Phytophthora
Phase diagnosis plays a significant role.Easy to operate: the LAMP method of detection camphor tree phytophthora provided by the invention overcomes the prior art
The problem of period needed for the biological detection method of middle camphor tree phytophthora is long, time-consuming and laborious, cumbersome, poor specificity and PCR detect skill
The problem of art needs thermal cycler instrument, can not quickly detect camphor tree phytophthora.Detection method is under 64 DEG C of isothermys, energy
Quickly, conveniently, efficiently, Gao Teyi, detect camphor tree phytophthora with sensitivity, do not need complex instrument, can preferably meet to camphor tree epidemic disease
The on-site test of mould.
2) accuracy is high: since traditional camphor tree phytophthora detection technique only determines test object, nothing according to morphological feature
Method excludes the interference of human factor, is difficult to distinguish close kind of form, detection accuracy only has 60-80%；And the present invention is according to camphor tree
Phytophthora detects the sequence of new target drone Pcinn100006, designs the LAMP primer of specificity.LAMP reaction passes through 4 primers
6 isolated areas on (FIP, BIP, F3, B3) specific recognition target sequence, specificity and sensitivity are relatively high.In addition,
Positive ring primer and reversed ring primer LB can be improved reaction rate and other four primers together, ensure to react accuracy
In the case where, it enables the invention to be rapidly performed by the detection of camphor tree phytophthora.
3) realize constant-temperature amplification, unlike PCR method has to thermal cycle, thus get rid of to thermal cycler instrument according to
Rely, as long as there is stable heat source LAMP reaction, greatly extends the range that LAMP is used, LAMP why can
React under constant heat source is so that double-stranded DNA is in the dynamic of unwinding because being added to glycine betaine in LAMP reaction solution
In state balance, amplification is realized under the action of Bst archaeal dna polymerase.
4) practicability is good.Common PCR reaction carries out gel electrophoresis to product and product is easily caused to spread, this is laboratory
One main source of pollution；And ethidium bromide (EB) has huge poison, can accumulate carcinogenic；Long-term observation ultraviolet lamp also can be to experiment
Personnel cause a degree of injury.And LAMP reaction need to only carry out in thermostat water bath, reaction passes through HNB's after terminating
Color change can direct judging result, to increase its application value detected in the plant and soil to carry disease germs.
5) present invention provides the method for excavation and technology platform of new detection target for the detection of camphor tree phytophthora, can be used for
The quickly detection of the high sensitivity of camphor tree phytophthora, while pathogen is identified at disease infestation initial stage, it also can be in field soil
Pathogen detected.The present invention reduces production cost, the environmental pollution for reducing pesticide also has to pesticide blindly use is reduced
It is significant.
Detailed description of the invention
Fig. 1 is the specific chromogenic figure that the color judgement LAMP detection of target Pcinn100006 is newly detected based on camphor tree phytophthora
And regular-PCR specificity verification electrophoretogram；It shows that P pipe P shows sky blue in figure a, is positive；Remaining manages aobvious purple, is negative；
Scheme regular-PCR in b and shows that camphor tree phytophthora generates more than 200 bp of purpose band, and remaining phytophthora does not generate purpose band；Wherein, P: camphor tree
Phytophthora (P.cinnamomi)；2: soybean phytophthora (P.sojae)；3: phytophthora parasitica (P.parasitica)；4: phytophthora infestans
(P.infestans)；5: radix aucklandiae phytophthora (P.tentaculata)；6: strawberry phytophthora (P.fragariae)；7: ramie mould
(P.boehmeriae)；7: Phytophthora drechsleri (P.drechsleri) 9: Pythium ultimum (Pythium ultimum)；10: scouring rush's sickle
Spore bacterium (Fusarium equiseti)；11: tack anthrax-bacilus (Colletotrichum truncatum)；12: Pyricularia oryzae
(Magnaporthe grisea)；13: Rhizoctonia solani Kuhn (Rhizoctonia solani)；13: verticillium dahliae
(Verticilium dahliae)；N: negative control；
Fig. 2 is that the HNB color change detection LAMP sensitivity of target Pcinn100006 and common is newly detected based on camphor tree phytophthora
Electrophoretogram is verified in PCR sensitivity；Fig. 2 a is that color determines that LAMP detects the sensitivity colour developing figure of camphor tree phytophthora；The reaction system of 25 μ L
The middle reaction tube respectively containing 10ng, 1ng camphor tree phytophthora DNA shows sky blue, is positive, in the reaction system of 25 μ L respectively
Reaction tube containing 100pg, 10pg, 1pg, 100fg, 10fg camphor tree phytophthora DNA shows purple, negative.Develop the color result table
The sensitivity of bright LAMP reaction reaches 100pg.Fig. 2 b is that electrophoretogram is verified in regular-PCR sensitivity；With the camphor tree phytophthora of same concentration
The genomic DNA of the reference culture of bacterium carries out pcr amplification reaction, compares the sensitivity of two methods as amplification template.It repeats
Experiment 3 times, to determine the sensitivity of PCR method detection camphor tree phytophthora genomic DNA.The result of 3 repetition experiments is consistent, amplification knot
Fruit is as shown in Figure 2 b.It is 1ng μ L in genomic DNA concentration-1When, PCR detection is capable of detecting when specific band, it was demonstrated that occurs
Specific amplification, testing result are judged to the positive.It and is 100pg μ L in genomic DNA concentration-1When, PCR product does not have through detection
It was found that specific band, it was demonstrated that there is no specific amplification, testing result is judged to feminine gender, i.e. PCR method detects camphor tree phytophthora base
Because the sensitivity of group DNA is 1ng μ L-1.It can be seen that the sensitivity of LAMP method is higher by 10 times (Fig. 2 b) than PCR method.
Fig. 3 is the LAMP testing result figure of the morbidity pine needle of artificial infection camphor tree phytophthora；P in figure: camphor tree phytophthora
(P.cinnamomi)；1-6: the camphor tree phytophthora of artificial infection pine needle；N: negative control.It is artificial using NaOH alkaline lysis method of extracting
It is inoculated with the DNA of the morbidity pine needle of camphor tree phytophthora, is expanded as template for LAMP.1uL DNA solution is taken, by embodiment 4
Method, carry out LAMP reaction.It is inoculated in the disease plant of camphor tree phytophthora as the result is shown and carries out LAMP, color reaction is also presented
Positive sky blue；And purple (Fig. 3) is presented in healthy plant and negative control.
Specific embodiment
The present invention is described further combined with specific embodiments below.
Embodiment 1
A camphor tree phytophthora specific gene more than 1000 is obtained by whole genome sequence comparison, and therefrom excavates out high trust
It spends specific molecular and detects target Pcinn100006, DNA sequence dna is as shown in SEQ ID NO.1, protein sequence such as SEQ ID
Shown in NO.2.And sensitive, accurate LAMP detection technique system is established based on the new target drone.
LAMP detection primer composition used in the LAMP detection technique system: by positive inner primer FIP, it is reversed in draw
Object BIP, positive outer primer F3, reversed outer primer B3, forward direction ring primer LF and reversed ring primer LB composition；Each primer sequence is specific
It is as follows:
FIP:5 '-CGAAGGACGAGGTGAAGGTGGACGCCCATACATCACATACG-3 '；
BIP:5 '-AAGGCCGGCTACATGTACTCGTCTCGGGCAAGATGACTTC-3 '；
F3:5 '-GTTCTGCGCGATTTGGTTAG-3 '；
B3:5 '-GCGGATCTTCCGATCTGGTA-3 '；
LF:5 '-AACGTGACGCCGGCAACAA-3 '；
LB:5 '-TGTTGCCTCCGGGCTGT-3 '.
Using the method for Primer composition detection camphor tree phytophthora: extracting the DNA of microorganism to be checked, be with the DNA of extraction
Template carries out LAMP using the LAMP primer composition object；LAMP response procedures are as follows: 60 DEG C~65 DEG C, 60~80min.Hydroxyl
Base naphthol blue (hydroxylnaphthol blue, HNB) belongs to one kind of Metal ion indicator.HNB is Mg2+Titrant,
Its color changes with solution PH and is changed, therefore can pass through Mg in monitoring LAMP reaction system2+The variation of concentration and pH value of solution
And play the role of color indicator.HNB is added in reaction solution before reaction, reaction system is purple, Mg in reaction process2+
The by-product P reacted with LAMP2O7 4-It is largely precipitated in conjunction with generating, Mg in solution2+Concentration reduces, and pH changes, to make
The color of HNB becomes sky blue from purple.Therefore, after reaction by the color change of reaction system, to judge camphor tree phytophthora
The presence or absence of bacterium: sky blue indicates test positive, and microorganism to be checked is camphor tree phytophthora；Purple indicates that testing result is feminine gender, to
Examining microorganism is not camphor tree phytophthora.
The LAMP detection kit of camphor tree phytophthora is detected as made of LAMP detection primer combination of compositions: being examined comprising 1mL
Solution is surveyed, which includes: reversed inner primer BIP, 8mM forward direction outer primer F3, the 8mM of 32mM forward direction inner primer FIP, 32mM
Reversed outer primer B3,8mM forward direction ring primer LF, 8mM ring primer LB, 48mM dNTPs, 0.8M Tris-HC, 0.4mM KCl,
0.4mM(NH4)2SO4、0.24mM MgSO4, 4%Triton X-100,320 unit of Bst DNA polymerase, 200mM hydroxyl
Base naphthol blue.
Using the method for kit detection camphor tree phytophthora: extracting the DNA of microorganism to be checked, take 1 μ L DNA solution, be added
Detection solution and 1 μ L sterile deionized water in 23 μ L LAMP kits carry out LAMP, LAMP response procedures are as follows: 60 DEG C~65
DEG C, 60~80min.Amplified production observes LAMP reaction solution color change, and sky blue indicates positive findings, and microorganism to be checked is
Camphor tree phytophthora；Purple indicates that testing result is feminine gender, and microorganism to be checked is not camphor tree phytophthora.
In order to compare LAMP detection method and specificity and sensitivity when regular-PCR detection method detects camphor tree phytophthora
Difference, the present embodiment is using the specific primer F3/B3 in camphor tree phytophthora LAMP primer, for carrying out common PCR reaction.Commonly
The mixed liquor total volume of PCR reaction is 25 μ L, comprising: the template DNA of respective concentration, 0.5 μM of primer, and each 50 μM of 4 kinds of dNTP,
2.5 μ L 10 × PCR reaction buffers, 2mM Mg2+, 2.5 μ L 1%BSA, 1.25 unit Taq enzymes (TaKaRa), in PTC200
It is carried out amplification reaction in (MJ company) PCR instrument.Experiment is required to be added 1 part of positive control and 1 part of blank control (with double every time
Steaming water is template) parallel laboratory test is carried out, it is whether effective to confirmatory experiment result.Response procedures are as follows: 94 DEG C of initial denaturation 5min；So
Enter circulation afterwards, 94 DEG C of denaturation 30sec, 57 DEG C of annealing 30sec, 72 DEG C of extension 30sec, totally 35 recycle；Last 72 DEG C of extensions
7min.7 μ L amplified productions electrophoresis 30min (100V) in 1.0% Ago-Gel is taken after reaction, in gel imaging system
Upper detection is simultaneously taken pictures.Each experiment is at least repeated 3 times.
Embodiment 2
In order to verify the specific primer sequence of camphor tree phytophthora, the present embodiment is other with 10 plants of camphor tree phytophthora bacteria strains and 14 kinds
Oomycetes and 17 kinds of disease fungus are material to be tested (table 1), and the DNA of camphor tree phytophthora in incidence tissue is extracted using CTAB method.Tool
Body method is as follows: taking a small amount of hypha powder, adds 900 μ L 2%CTAB extracting solutions and 90 μ L 10%SDS, whirlpool mixes, in 55 DEG C of water
1h is bathed, intermediate every 10min turns upside down several times.12000rpm is centrifuged 10min, takes and resets and add isometric phenol/chloroform/isoamyl alcohol
(25:24:1), is mixed by inversion, and 12000rpm is centrifuged 10min；Supernatant is transferred to new pipe, adds isometric chloroform, is gently overturned mixed
Even, 12000rpm is centrifuged 5min.Supernatant is transferred in new pipe, adds the dehydrated alcohol of 2 times of volumes and the 3M NaAc of 1/10 volume
(pH 5.2), -20 DEG C of precipitatings (> 1h).12000rpm is centrifuged 10min, and incline supernatant, precipitates with 70% ethanol washing twice, room
Temperature is dried.Appropriate sterilizing ultrapure water or TE (pH 8.0) dissolution is added to precipitate (containing 20 μ g/mL RNase), after 37 DEG C of processing 1h, -20
It DEG C saves backup.All pedotheques useSPIN kit (Q-Biogene Ltd, USA) carries out DNA's
It extracts.Soil DNA extraction step is referring to kit specification.This commercial soil microbial DNA extracts kit can be
The microorganism in soil is extracted in 0.5h.
Table 1 is used to detect fungi and the oomycetes bacterial strain of camphor tree phytophthora specificity
When LAMP amplified reaction occurs, generating a large amount of magnesium pyrophosphate white precipitate causes the turbidity of reaction solution to rise,
Shown in chromogenic reaction result by HNB, it is in sky blue in the reaction tube of camphor tree phytophthora, is positive findings, and other epidemic diseases
Mould kind, fungi, rotten mould and negative control bacterium reaction tube it is purple, be negative findings, it was demonstrated that designed LAMP specificity is drawn
Object has the specificity of kind.This illustrates that the primer sets can be used for the fast of incidence tissue and Phytophthora Cinnamomi In Soil bacterium in production practices
The reliable Testing and appraisal of speed.When for, there are when camphor tree phytophthora, extracting camphor tree phytophthora using NaOH rapid cleavage method in incidence tissue
The DNA of bacterium, detailed process is as follows: taking the plant tissue of one section of morbidity, 10 μ L 0.5M NaOH are added in every milligram of tissue, in mortar
In be fully ground after be transferred in the EP pipe of 1.5mL, 12000rpm be centrifuged 5min, take 5 μ L supernatants be added 495 μ L 0.1mM
Tris (pH 8.0) takes 1 μ L to be directly used in PCR reaction after mixing.Each reaction at least in triplicate, while being to determine in plant
No PCR mortifier exists.
LAMP testing result shows that sapphire positive reaction or agarose can be observed in 10 plants of camphor tree phytophthora bacteria strains
There is the stair-stepping band of LAMP in gel electrophoresis, remaining 14 kinds of oomycetes and 17 kinds of disease fungus colour developing results are the feminine gender of purple
There is not amplified band in reaction or agarose gel electrophoresis.Selection and camphor tree phytophthora (soybean phytophthora not of the same race；Parasitic epidemic disease
It is mould；Phytophthora infestans；Radix aucklandiae phytophthora；Strawberry phytophthora；Ramie mould etc.) and bacterium (the tack anthrax-bacilus that does not belong to；Eggplant sickle-like bacteria；Rice
Seasonal febrile diseases bacterium；Rhizoctonia solani Kuhn；Verticillium dahliae；Pythium ultimum) DNA as template, take 1 μ L DNA solution, it is real that 23 μ L be added
The detection solution and 1 μ L sterile deionized water for applying the preparation of example 1 carry out LAMP reaction, response procedures are as follows: 64 DEG C of 60min.Display is expanded
When increasing the DNA profiling of camphor tree phytophthora, sky blue is presented；The DNA profiling and yin of amplification and the bacterium that camphor tree phytophthora is not of the same race, does not belong to
Property control all present purple (Fig. 1 a).Regular-PCR verifying is carried out using upstream and downstream primer F3/B3, testing result is shown, in addition to camphor tree
Phytophthora can produce target stripe, amplified band (figure do not occur in remaining oomycetes and disease fungus agarose gel electrophoresis
1b)。
The sensitivity test of 3 camphor tree phytophthora LAMP of embodiment reaction and PCR reaction
It is in order to determine the sensitivity of LAMP detection method, the DNA spectrophotometric determination of the camphor tree phytophthora of extraction is dense
Degree (1 μ g/ μ L) carries out 10 doubling dilutions with DEPC water afterwards, and -70 DEG C save as template.It is each dense after taking 10 doubling dilutions respectively
1 μ L of DNA dilution is spent as template, and the detection solution and 1 μ L sterile deionized water that the preparation of 23 μ L embodiments 1 is added carry out LAMP
Reaction, response procedures are as follows: 64 DEG C of 80min.2 μ L amplified production loadings are taken, agarose gel electrophoresis and HNB colour developing are anti-as the result is shown
It should indicate that the sensitivity of LAMP reaction reaches the DNA (Fig. 2 a) of the camphor tree phytophthora of 100pg.With the mark of the camphor tree phytophthora of same concentration
The genomic DNA of quasi- bacterial strain carries out pcr amplification reaction, compares the sensitivity of two methods as amplification template.Repeat experiment 3
It is secondary, to determine the sensitivity of PCR method detection soyabean phytophthora genomic DNA.The result of 3 repetition experiments is consistent, amplification
As shown in Figure 2 b.It is 1ng μ L in genomic DNA concentration-1When, PCR detection is capable of detecting when specific band, it was demonstrated that occurs special
Specific amplification, testing result are judged to the positive.It and is 100pg μ L in genomic DNA concentration-1When, PCR product is not sent out through detecting
Existing specific band, it was demonstrated that there is no specific amplification, testing result is judged to feminine gender, i.e. PCR method detects soyabean phytophthora base
Because the sensitivity of group DNA is 1ng μ L-1.It can be seen that the sensitivity of LAMP method is higher by 10 times (Fig. 2 b) than PCR method.
4 camphor tree phytophthora LAMP of embodiment reacts primer specificity verifying and sensitivity verifying
For the LAMP primer group of camphor tree phytophthora, 8 groups of qualified primers are devised altogether, and finishing screen selects 1 group the most
Primer composition used in the special and high primer, that is, embodiment 1 of sensitivity (including positive inner primer FIP, it is reversed in
Primer BIP, positive outer primer F3, reversed outer primer B3, forward direction ring primer LF and reversed ring primer LB).Using remaining of design
Primer (selects 1 group from remaining 8 groups of primers at random), and primer sequence is as follows: FIP:5 '-CGAAGGACGAGGTGAAGGTGGACGC
CCATACATCACATACG-3′；BIP:5 '-AAGGCCGGCTACATGTACTCGTCTCGGGCAAGATGACTTC-3 '；F3:5 '-
GTTCTGCGCGATTTGGTTAG-3′；B3:5 '-GCGGATCTTCCGATCTGGTA-3 '；LF:5 '-
AACGTGACGCCGGCAACAA-3′；LB:5 '-TGTTGCCTCCGGGCTGT-3 '
Using bacterial strain employed in embodiment 2 as material to be tested (10 plants of camphor tree phytophthora bacteria strains and 14 kinds of other oomycetes and
17 kinds of disease fungus), LAMP testing result shows that the specificity of primer selected by residue is not high, and sensitivity is also poor, illustrates to implement
Example 1 is for Primer composition of the present invention specificity with higher and sensitivity.
The LAMP detection of camphor tree phytophthora in 5 living tissue of embodiment
Using the DNA of the morbidity pine needle of NaOH alkaline lysis method of extracting inoculation camphor tree phytophthora, LAMP is used for as template
Amplification.1uLDNA solution is taken, as described in Example 4, carries out LAMP reaction.It is inoculated with the disease plant of camphor tree phytophthora as the result is shown
Positive sky blue is also presented in middle carry out LAMP, color reaction；And purple (Fig. 3) is presented in healthy pine needle and negative control.
Embodiment 6 detects camphor tree phytophthora from soil sample of carrying disease germs
The method that the camphor tree phytophthora detection kit of embodiment 1 is used to detect camphor tree phytophthora, comprising:
1) in soil egg spore enrichment: take 40~80 grams of pedotheque to be checked, grind, successively gone using 200 mesh screens
Locate larger grogs, then passes through 400,500,800 mesh net filtrations, while with 3~10 liters of water repeated flushing, from 800 mesh screens
Upper collection egg spore, with 1mL aqueous suspension.Since egg spore cannot penetrate 800 mesh screens, processing, which can achieve, in this way makes egg spore
The effect of enrichment.
2) DNA is extracted from micro egg spore: by the centrifuge tube for being transferred to 1.5mL with the egg spore of sterile aqueous suspension,
In 12000r.min-1It is centrifuged 5 minutes under revolving speed, pours out liquid；50 μ L CTAB buffer are added, grinds, adds 500 μ L
CTAB buffer, water-bath 30 minutes；Isometric chloroform is added, in 12000r.min-1It is centrifuged 10 minutes, draws under revolving speed
Supernatant；Be added the 3M NaAc of 1/10 volume, 2 times of volumes without water-ice ethyl alcohol, precipitation at room temperature 30 minutes, 12000r.min-1Revolving speed
Lower centrifugation 10 minutes, dry liquids；Add 1mL 70% (V/V) ethanol washing, 12000r.min-1It is centrifuged 10 minutes under revolving speed,
Dry liquids are dried to alcohol-free taste；10 μ L aseptic double-distilled waters are added to dissolve, the template for LAMP amplification.
3) camphor tree phytophthora LAMP is detected, comprising: the LAMP of camphor tree phytophthora is detected: taking 1 μ L DNA solution, the inspection of 23 μ L is added
Solution and 1 μ L sterile deionized water are surveyed, total volume is 25 μ L；Response procedures are as follows: 64 DEG C of 60min；With HNB (hydroxynaphthol blue) work
For reaction indicator, sky blue is presented in the color of LAMP reaction system after amplification, and being judged with this can from soil sample of carrying disease germs
It generates positive reaction and contains camphor tree phytophthora.
To 13 parts of camphor tree pedotheque of the doubtful generation butt rot from Shandong, Anhui and Jiangsu, biography is respectively adopted
The LAMP detection method that system pathogenicbacteria separation method and this research are established detects the camphor tree phytophthora of above-mentioned sample.Regular-PCR disease
Opportunistic pathogen partition method positive rate is 6/13 (46.2%), and the positive rate for the LAMP method that the application establishes is 13/13 (100%), inspection
Quantity is high out.It is all simultaneously to isolate the sample of object bacteria, the LAMP that the application establishes in traditional pathogenicbacteria separation method kind
Detection method can detect the positive, illustrate that the detection specificity of this method is very strong.So this research establish based on new target drone
LAMP detection method can greatly improve detection efficiency.
Sequence table
<110>Nanjing Forestry University
<120>a kind of detection target Pcin100006 of camphor tree phytophthora and its special primer for checking and rapid detection method
<130> 100
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 301
<212> DNA
<213> Phytophthora cinnamomi
<400> 1
atgcgccttc tgattcccgg cgctctatgc gcgttgaggt tctgcgcgat ttggttagaa 60
atacgcccat acatcacata cgacagcccg gaggcaacac ggccaccttc acctcgtcct 120
tcgtcaaggc cggctacatg tactcgtcac ggccaacgtg acgccggcaa caactacttg 180
gtcgagatgg aagtcatctt gcccgaggat ttggtgtacc agatcggaag atccgctaca 240
ccggtactga gctatttgct atgctgcaac aaaattgctc tcgcatttaa gggatcacta 300
a 301
<210> 2
<211> 100
<212> PRT
<213> Phytophthora cinnamomi
<400> 2
Met Arg Leu Leu Ile Pro Gly Ala Leu Cys Ala Leu Arg Phe Cys Ala
1 5 10 15
Ile Trp Leu Glu Ile Arg Pro Tyr Ile Thr Tyr Asp Ser Pro Glu Ala
20 25 30
Thr Arg Pro Pro Ser Pro Arg Pro Ser Ser Arg Pro Ala Thr Cys Thr
35 40 45
Arg His Gly Gln Arg Asp Ala Gly Asn Asn Tyr Leu Val Glu Met Glu
50 55 60
Val Ile Leu Pro Glu Asp Leu Val Tyr Gln Ile Gly Arg Ser Ala Thr
65 70 75 80
Pro Val Leu Ser Tyr Leu Leu Cys Cys Asn Lys Ile Ala Leu Ala Phe
85 90 95
Lys Gly Ser Leu
100
<210> 3
<211> 41
<212> DNA
<213>FIP sequence (Artificial)
<400> 3
cgaaggacga ggtgaaggtg gacgcccata catcacatac g 41
<210> 4
<211> 40
<212> DNA
<213>BIP sequence (Artificial)
<400> 4
aaggccggct acatgtactc gtctcgggca agatgacttc 40
<210> 5
<211> 20
<212> DNA
<213>F3 sequence (Artificial)
<400> 5
gttctgcgcg atttggttag 20
<210> 6
<211> 20
<212> DNA
<213>B3 sequence (Artificial)
<400> 6
gcggatcttc cgatctggta 20
<210> 7
<211> 19
<212> DNA
<213>LF sequence (Artificial)
<400> 7
aacgtgacgc cggcaacaa 19
<210> 8
<211> 17
<212> DNA
<213>LB sequence (Artificial)
<400> 8
tgttgcctcc gggctgt 17
Claims (10)
1. the detection target of camphor tree phytophthoraPcinn100006, DNA sequence dna is as shown in SEQ ID NO.1.
2. the detection target of camphor tree phytophthora described in claim 1Pcinn100006, the protein sequence such as SEQ ID of coding
Shown in NO.2.
3. detection target of any of claims 1 or 2Pcinn100006Application in detection camphor tree phytophthora.
4. the detection target of camphor tree phytophthora described in claim 1Pcinn100006Special primer for checking, it is characterised in that:
By positive inner primer FIP, reversed inner primer BIP, positive outer primer F3, reversed outer primer B3, forward direction ring primer LF and reversed ring
Primer LB composition；Each primer sequence is specific as follows:
FIP:5 '-CGAAGGACGAGGTGAAGGTGGACGCCCATACATCACATACG-3 '；
BIP:5 '-AAGGCCGGCTACATGTACTCGTCTCGGGCAAGATGACTTC-3 '；
F3:5 '-GTTCTGCGCGATTTGGTTAG-3 '；
B3:5 '-GCGGATCTTCCGATCTGGTA-3 '；
LF:5 '-AACGTGACGCCGGCAACAA-3 '；
LB:5 '-TGTTGCCTCCGGGCTGT-3 '.
5. application of the primer special as claimed in claim 4 in detection camphor tree phytophthora.
6. a kind of kit for detecting camphor tree phytophthora, it is characterised in that: containing detection solution, the detection solution is at least wrapped
It includes: 1) solution of primer special as claimed in claim 4.
7. the kit of detection camphor tree phytophthora according to claim 6, it is characterised in that: the detection solution also wraps
It includes: 2) LAMP reaction dissolvent；3) DNA synzyme；4) hydroxynaphthol blue.
8. application of the kit described in claim 6 or 7 in detection camphor tree phytophthora.
9. a kind of method for detecting camphor tree phytophthora, it is characterised in that: the DNA for extracting microorganism to be checked, using the DNA of extraction as mould
Plate carries out corresponding LAMP detection reaction using primer special as claimed in claim 4 or kit as claimed in claim 6；
The variation of observing response solution colour, sky blue indicate test positive, and microorganism to be checked is camphor tree phytophthora；Purple indicates detection knot
Fruit is feminine gender, and microorganism to be checked is not camphor tree phytophthora.
10. the method for detection camphor tree phytophthora according to claim 9, it is characterised in that: LAMP response procedures are as follows: 60 DEG C ~
65 DEG C, 60 ~ 80min.
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| CN106868164B (en) * | 2017-03-23 | 2020-09-22 | 福建省农业科学院植物保护研究所 | Primer for detecting phytophthora camphorata and nested PCR detection method |
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Application publication date: 20190308 Assignee: Nanjing Forest Protection Bird Biotechnology Co., Ltd. Assignor: Nanjing Forestry University Contract record no.: X2019320000149 Denomination of invention: Phytophthora cinnamomi detection target Pcin100006 and special detection primer and fast detection method thereof Granted publication date: 20190820 License type: Common License Record date: 20191022 Application publication date: 20190308 Assignee: MAANSHAN Panshi Biotechnology Co., Ltd. Assignor: Nanjing Forestry University Contract record no.: X2019320000150 Denomination of invention: Phytophthora cinnamomi detection target Pcin100006 and special detection primer and fast detection method thereof Granted publication date: 20190820 License type: Common License Record date: 20191022 Application publication date: 20190308 Assignee: Nanjing Dawson Biotechnology Co., Ltd Assignor: Nanjing Forestry University Contract record no.: X2019320000151 Denomination of invention: Phytophthora cinnamomi detection target Pcin100006 and special detection primer and fast detection method thereof Granted publication date: 20190820 License type: Common License Record date: 20191022 |
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