CN107043820A - The method that loop-mediated isothermal amplification detects pine wood nematode - Google Patents
The method that loop-mediated isothermal amplification detects pine wood nematode Download PDFInfo
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Abstract
The invention discloses a kind of method that loop-mediated isothermal amplification detects pine wood nematode, belong to the Molecular Detection field of pine wood nematode.SYG 2 gene of the present invention first using pine wood nematode designs as target gene and screens one group of acquisition for the high LAMP primer group of pine wood nematode specificity, the LAMP primer group being capable of specific differentiation pine wood nematode and B. mucronatus.The present invention establishes a kind of LAMP quick determination methods of pine wood nematode using the LAMP primer group, can fast and accurately differentiate in sample whether carry pine wood nematode.The present invention further discloses a kind of pine wood nematode LAMP detection kit.The LAMP detection method specificity of pine wood nematode of the present invention is high, and easy to operate, time-consuming short, cost is low, and the quick detection for the pine wood nematode that the Monochamus alternatus that is particularly suitable for use in is carried and the generation of monitoring pine nematode are with spreading.
Description
Technical field
The LAMP detection sides of pine wood nematode are carried the present invention relates to the Molecular Detection of pine wood nematode, more particularly to Monochamus alternatus
Method, belongs to the Molecular Detection field that Monochamus alternatus carries pine wood nematode.
Background technology
Pine nematode (Pine wilt disease), the pine tree wilt disease that is otherwise known as, droop, the disease is by pine line
Worm [Bursaphelenchus xylophilus (Steiner＆Buhrer) Nickle] causes, and mainly passes through black day Bos
(Monochamus) insect taken food on pine tree and lay eggs carry out natural propagation and diseased wood, diseased wood processing wood transport it is long
Propagation.The propagation of pine wood nematode can cause harm including Pinus (Pinus spp.), larch mainly by Monochamus alternatus in forest zone
Belong to (Larix spp.), Picea (Picea spp.), Pseudotsuga (Pseudotsuga spp.), Abies (Abies spp.)
With the 36 kinds of pine genus plants and 8 kinds of non-pine genus plants of Cedrus (Cedrus spp.), wherein pine genus plant is topmost posts
It is main.Lack effectively preventing method at present because its harm is extremely serious, No.1 quarantine object is classified as by multiple countries.
Morphology is the standard method of line insect types general survey, and the identification of pine wood nematode relies primarily on the form spy of adult
Levy.But, it is extremely difficult for layman by morphology precise Identification pine wood nematode adult, and larva can not be carried out
Precise Identification.Due to morphologic limitation, immunology, Physiology and biochemistry and molecular biology method are applied to the mirror of pine wood nematode
On fixed.
Monochamus alternatus is the most important communication media of pine nematode, and carrying is pine wood nematode larva, and the line carried
Worm has a variety of, it is difficult to pine wood nematode larva is distinguished and identified according to morphological feature.The pine that Accurate Diagnosis Monochamus alternatus is carried
Material nematode is urgent need to solve the problem during monitoring pine nematode spreads.The introducing of Protocols in Molecular Biology, overcomes
Some morphologic limitations, realize Rapid identification.The exploitation for the pine wood nematode molecular detection technology that Monochamus alternatus is carried, for
Early stage epidemic place is found in time, effectively by pine nematode control in certain scope, reduces the generation of pine nematode, drop
Low cost accounting, has great importance；Simultaneously foundation is provided for the overall early warning and control strategy of pine nematode.
At present, the detection of pine wood nematode and identification are main based on the method for molecular biology.Wherein, DNA probe technology
Though pine wood nematode can be effectively identified, it is costly, with radioactivity；RAPD technical Analysis pine wood nematode and the pass of allied species
System, it is as a result repeated poor；RFLP technologies distinguish pine wood nematode and B. mucronatus, and restriction endonuclease is expensive, time-consuming；Fluorescence
PCR is sensitive, quick, but costly；Round pcr is easy to operate, cost is less, but required time is also longer.
Ring mediated isothermal amplification (loop-mediated isothermal amplification, abbreviation LAMP) is 2000
A kind of new nucleic acid amplification method of the exploitations such as the Notomi T of year Japan Rong Yan chemical strain formula meeting, this method is for target gene
6 regions devise 2 pairs of special primers, and primer is in the presence of strand displacement archaeal dna polymerase (Bst DNA polymerase)
Dozens of minutes is incubated under constant temperature (65 DEG C or so), because of its unique inverse design, is infinitely expanded itself carrying out strand displacement
Increase；It can be completed in whole 1 hour of course of reaction, the processes such as thermal denaturation, annealing, the temperature cycles of template not needed therebetween,
The characteristics of amplification has high specific, isothermal is quick.Therefore, several molecular detection technologies of the above are compared, LAMP amplifications need not
Expensive instrument and reagent, is more conducive to the application in some infrastructures.
SYG-2 genes are the genes of multifunctional coded CAF.SYG-1 and SYG-2 are present in all animal classes
In group, from the formation of cynapse to the foundation of filtering defensive barrier, a variety of main physiological functions are participated in.Therefore, with pine wood nematode
SYG-2 genes be drone design specific primer, to Monochamus alternatus carry pine wood nematode carry out LAMP rapid amplifyings, for
The Rapid identification of pine wood nematode and effective prevention and control are significant.
The content of the invention
First technical problem to be solved by this invention is to provide for detecting in sample whether carry pine wood nematode
LAMP primer group；
Second technical problem to be solved by this invention is to provide a kind of LAMP detection kit of pine wood nematode；
3rd technical problem to be solved by this invention is to set up a kind of loop-mediated isothermal amplification detection pine line
The method of worm.
In order to solve the above technical problems, the technical solution used in the present invention is：
The present invention is disclosed for detecting whether sample carries the LAMP primer group of pine wood nematode, the LAMP primer first
Any one group in following 2 groups of primers of group：
Primer sets (1)：Including a pair of outer primers and a pair of inner primers；The outer primer by nucleotides sequence to being classified as SEQ ID
Outer primer composition in downstream shown in upstream outer primer shown in No.1 and SEQ ID No.2；The inner primer by nucleotides sequence to being classified as
Inner primer composition in downstream shown in upstream inner primer shown in SEQ ID No.3 and SEQ ID No.4；
Primer sets (2)：Including a pair of outer primers, a pair of inner primers and a pair of ring primers；The outer primer is to by nucleotides
Sequence is outer primer composition in downstream shown in upstream outer primer shown in SEQ ID No.1 and SEQ ID No.2；The inner primer to by
Nucleotides sequence is classified as the composition of downstream inner primer shown in upstream inner primer shown in SEQ ID No.3 and SEQ ID No.4；The ring draws
Thing to as nucleotides sequence be classified as shown in SEQ ID No.5 lantern primer with SEQ ID No.6 shown in lower lantern primer sets into.
Wherein, described detection sample is preferably Monochamus alternatus.
The SYG-2 genetic fragments of pine wood nematode and B. mucronatus are compared through NCBI, present invention discover that this section of gene is in pine
Guarded in material nematode kind, it is extremely big with B. mucronatus interspecific difference.Therefore, the present invention using the SYG-2 genes of pine wood nematode as
Target gene, devises 16 groups of LAMP primers.Testing result to pine wood nematode Bx and B. mucronatus Bm proves, No. 8 primer sets
Ring primer (primer sets (1) i.e. of the present invention) is not added with, B. mucronatus can not be expanded, the specificity detected to pine wood nematode
It is good；And No. 8 primer sets are not added with ring primer, detection pine wood nematode Bx appearance time is 15-20min, and amplification efficiency is high.And 8
Number primer sets (primer sets (2) i.e. of the present invention) in the case where adding ring primer, can amplify B. mucronatus, to pine
The poor specificity of nematode detection.
No. 16 primer sets designed by the present invention are in the case where being not added with ring primer, although also can not expand and intend pine line
Worm, but the appearance time that No. 16 primer sets are not added with ring primer detection pine wood nematode Bx is 25-30min, amplification efficiency is poor.4
Number primer sets add ring primer or are not added with ring primer, can not all amplify B. mucronatus, can amplify pine wood nematode, but its
Amplification efficiency is not optimal；Specifically, No. 4 primer sets add ring primer detection pine wood nematode Bx appearance time to be 20-25min；4
The appearance time that number primer sets are not added with ring primer detection pine wood nematode Bx is 30-35min.Therefore, visible by comparing, No. 8 are drawn
The amplification efficiency that thing group is not added with ring primer is optimal.In addition, No. 1, No. 10, No. 13 and No. 15 primer sets designed by the present invention add/
B. mucronatus can be amplified by being not added with ring primer, the poor specificity detected to pine wood nematode；No. 3 and No. 5 primer sets are adding ring
In the case of primer, it is amplifiable go out B. mucronatus, and be not added with ring primer, pine wood nematode and B. mucronatus can not expanded；
Remaining 7 groups of primer adds/is not added with ring primer can not amplify pine wood nematode；Above-mentioned primer sets cannot be used for Monochamus alternatus carrying
The detection of pine wood nematode.
Therefore, the LAMP primer group that the present invention is used to detect pine wood nematode is preferably that No. 8 primer sets are not added with ring primer, that is, is drawn
Thing group (1)：Including a pair of outer primers and a pair of inner primers；The outer primer as nucleotides sequence to being classified as shown in SEQ ID No.1
Outer primer composition in downstream shown in upstream outer primer and SEQ ID No.2；The inner primer by nucleotides sequence to being classified as SEQ ID
Inner primer composition in downstream shown in upstream inner primer shown in No.3 and SEQ ID No.4.
LAMP primer group of the present invention, which is particularly suitable for use in, detects whether Monochamus alternatus carries pine wood nematode.
The present invention further discloses a kind of pine wood nematode LAMP detection kit, including：LAMP primer group, 2 × RM (bags
Include：Tris-HCl, KCl, MgSO4, (NH4)SO4, Tween20, Betaine, dNTPs), SYBR Green I, Bst DNA polymerizations
Enzyme and ddH2O；Wherein, the LAMP primer group be selected from LAMP primer group of the present invention, i.e., No. 8 primer sets add ring primer or
Person is not added with ring primer, and preferably No. 8 primer sets are not added with ring primer.
LAMP detection kit of the present invention can be applied to the pine wood nematode that detection Monochamus alternatus is carried.
The invention also discloses a kind of LAMP detection method of pine wood nematode, comprise the following steps：(1) testing sample is extracted
The DNA of (being preferably Monochamus alternatus)；(2) using the DNA of extraction as template, with LAMP primer group of the present invention (i.e. No. 8 primers
Group plus ring primer are not added with ring primer) it is amplimer, set up LAMP reaction systems and be placed in carrying out in real-time fluorescence PCR instrument
PCR reacts；(3) reaction result is judged：1. fluorescence signal is collected, if there is " S " type amplification curve, is then determined as sun
Property (having nucleic acid amplification, i.e., Monochamus alternatus to be measured carries pine wood nematode)；If without " S " type amplification curve, being determined as negative (nothing
Nucleic acid amplification, i.e., Monochamus alternatus to be measured does not carry pine wood nematode)；Or, 2. PCR reaction products are inactivated, nitrite ion is added and mixes
It is even；If green is presented, it is determined as positive (having nucleic acid amplification, i.e., Monochamus alternatus to be measured carries pine wood nematode)；If presented
It is orange, then it is determined as negative (free nucleic acid is expanded, i.e., Monochamus alternatus to be measured does not carry pine wood nematode).
Wherein, when the LAMP primer group is that No. 8 primer sets are not added with ring primer, LAMP reaction systems include：Reactant
The cumulative volume of system is 25 μ l；Wherein, the μ l of 2 × RM 12.5 (including：PH8.8 Tris-HCl 40mM, KCl 20mM, MgSO4
16mM, (NH4)SO420mM, Tween200.2%, Betaine 1.6M, dNTPs (2.8mM is every kind of)), SYBR Green I
0.5 μ l, 40 μM of upstream inner primer 1 μ l, 40 μM of downstream inner primer 1 μ l, 10 μM of upstream outer primer 0.5 μ l, under 10 μM
μ l, the 8U/ μ L of the outer primer 0.5 μ l of Bst archaeal dna polymerases 1, the μ l of DNA profiling 1 is swum, surplus is ddH2O.Or, as the LAMP
When primer sets are that No. 8 primer sets add ring primer, the LAMP reaction systems include：The cumulative volume of reaction system is 25 μ l；Wherein,
2 × RM, 12.5 μ l, SYBR Green I 0.5 μ l, 40 μM of upstream inner primer 1 μ l, 40 μM of downstream inner primer 1 μ l, 10 μM
The μ l of upstream outer primer 0.5,10 μM of the μ l of downstream outer primer 0.5,20 μM of the μ l of upper lantern primer 0.5,20 μM of lower lantern draws
μ l, the 8U/ μ L of the thing 0.5 μ l of Bst archaeal dna polymerases 1, the μ l of DNA profiling 1, surplus is ddH2O。
In LAMP detection method of the present invention, the program of the PCR reactions includes：63 DEG C of 15s, 63 DEG C of 45s are used as one
Individual circulation；Fluorescence signal is collected at 63 DEG C of 45s.The inactivation is 85 DEG C of inactivation 3min.
The Monochamus alternatus set up using the present invention carries the LAMP detection method of pine wood nematode, and Nanjing is gathered
The recall rate for carrying the Monochamus alternatus of pine wood nematode is respectively 28.60% and 43.75%.
Technical solution of the present invention compared with prior art, has the advantages that：
The present invention is using conservative SYG-2 genes in pine wood nematode kind as target gene, and designing and screen acquisition can be special
Property distinguish the LAMP primer group of pine wood nematode and B. mucronatus.The specific LAMP that the present invention is further obtained using screening draws
Thing group establishes the LAMP detection method that a kind of Monochamus alternatus carries pine wood nematode, the pine that this method can be carried to Monochamus alternatus
Material nematode is fast and accurately detected that not only specificity is high, and easy to operate, time-consuming short, and cost is low.The inventive method
The pine wood nematode carried for Accurate Diagnosis Monochamus alternatus, monitors the generation of pine nematode and spreads and to pine nematode
Overall early warning and control strategy etc. be respectively provided with important meaning.
Brief description of the drawings
Fig. 1 is that No. 1, No. 4 and No. 15 primer sets are not added with ring primer, to pine wood nematode Bx and B. mucronatus Bm detection knot
Really；
Fig. 2 is that No. 8 and No. 16 primer sets are not added with ring primer, to pine wood nematode Bx testing result；
Fig. 3 is that No. 8 and No. 16 primer sets are not added with ring primer, the result detected to longicorn；Wherein, NJ1 represents Congjiang southern Jiangsu
The longicorn of capital collection carries pine wood nematode (June 21 batch)；NJ2 represents the longicorn gathered from Nanjing and carries pine wood nematode (6
The moon 24 days batches)；
Fig. 4 is that No. 10 and No. 13 primer sets are not added with ring primer, to pine wood nematode Bx and B. mucronatus Bm testing result；
Fig. 5 is that No. 1, No. 8, No. 13 and No. 15 primer sets add ring primer, the inspection to pine wood nematode Bx and B. mucronatus Bm
Survey result；
Fig. 6 is that No. 3 primer sets add ring primer, to pine wood nematode Bx and B. mucronatus Bm testing result；
Fig. 7 is that No. 4 primer sets add ring primer, to pine wood nematode Bx and the testing result of longicorn；Wherein, NJ1 represents the Congjiang
The longicorn of southern Jiangsu capital collection carries pine wood nematode (June 21 batch)；NJ2 represents the longicorn gathered from Nanjing and carries pine line
Worm (June 24 batch)；SD-1 represents the pine wood nematode gathered from Shandong；JS represents the pine wood nematode gathered from Jiangsu；JST-10
Represent the pine wood nematode gathered from Jiangsu；JS and JST-10 represent different strains；
Fig. 8 is that No. 5 and No. 16 primer sets add ring primer, to pine wood nematode Bx and B. mucronatus Bm testing result；
Fig. 9 is that No. 10 primer sets add ring primer, to pine wood nematode Bx and B. mucronatus Bm testing result.
Embodiment
The invention will now be further described with reference to specific embodiments, advantages of the present invention and feature will be with description and
It is apparent.It should be understood that the embodiment is only exemplary, any limitation is not constituted to the scope of the present invention.This area
Technical staff should be understood that without departing from the spirit and scope of the invention can to the details of technical solution of the present invention and
Form is modified or replaced, but these modifications or substitutions each fall within protection scope of the present invention.
The Monochamus alternatus of embodiment 1 carries the foundation of the LAMP quick determination methods of pine wood nematode
1st, materials and methods
1.1 Monochamus alternatus DNA extraction
Take Monochamus alternatus imago head and breast tissue 0.2g to be put into 2mL EP pipes, be put into steel ball beveller grinding 5min
(60Hz)；600 μ L 3M guanidinium isothiocyanate Extraction buffers are added, during which lysis at room temperature 1h overturns and mix；Add 600 μ
LTris- phenol chloroform isoamyl alcohol (25:24:1), 12000r/min centrifuges 10min；Aspirate supernatant, adds 600 μ L chloroform isoamyls
Alcohol (24:1), 12000r/min centrifuges 10min；Aspirate supernatant, adds 600 μ L isopropanols -20 DEG C of precipitations 20min, 12000r/
Min centrifuges 10min；Abandon supernatant, 70% ethanol cleaning precipitation 2 times, each 12000r/min centrifugations 3min；Ethanol is blotted thoroughly to do
It is standby with 60 μ LBuffer TE dissolving DNAs after dry.1.2LAMP design of primers
The SYG-2 genetic fragments of pine wood nematode and B. mucronatus are compared through NCBI, find this section of gene in pine wood nematode
It is conservative in kind, it is extremely big with B. mucronatus interspecific difference.Therefore, the new target that SYG-2 can be detected as pine wood nematode LAMP
Gene.
Using SYG-2 genes as target gene, pass through the LAMP primer Photographing On-line software http of Rong Yan companies://
Primerexplorer.jp/e/ has carried out LAMP primer design.Designed LAMP primer group is shown in Table 1.
The LAMP primer that table 1 is designed using SYG-2 as target gene
1.3LAMP reaction
LAMP reaction methods are with reference to loopamp DNA cloning kit (Eiken Chemical), and according to the form below reaction solution is matched somebody with somebody
Than the LAMP reaction systems (table 2) for building 25 μ l.
Table 2LAMP reaction systems
After mentioned reagent is mixed, 20 μ l sealing fluids are added, centrifugation is mixed, covers tightly lid.
1.4LAMP reaction results are detected
1.4.1 real-time fluorescence method
Reaction tube is put into real-time fluorescence PCR instrument, response procedures are 63 DEG C of 15s, and 63 DEG C of 45s are as a circulation, in 63
Fluorescence signal is collected at DEG C 45s, if there is " S " type amplification curve, is judged as positive (having nucleic acid amplification), if without the amplification of " S " type
Curve, then be judged as negative (free nucleic acid amplification).
1.4.2 decoration method
Reaction tube is taken out from real-time fluorescence PCR instrument, then 85 DEG C of inactivation 3min drop in 1 μ l nitrite ions in PCR pipe lid
Between, cover tightly lid.Reverse reaction tube for several times, makes reaction mixture fully be mixed with nitrite ion, observing response liquid color.If presenting
Green then be positive (having nucleic acid amplification), it is then negative (free nucleic acid amplification) to present orange.
2nd, result and analysis
2.1LAMP primer pair pine wood nematode Bx and B. mucronatus Bm testing result
The LAMP primer designed using SYG-2 as target gene, the testing result to pine wood nematode Bx and B. mucronatus Bm is shown in
Table 3.
Table 3LAMP primer pair pine wood nematode Bx and B. mucronatus Bm testing result
16 groups of primers designed by the present invention, wherein 9 groups of primer pair pine wood nematode Bx and B. mucronatus Bm carry out LAMP
Detection, as a result for：
No. 1, No. 10, No. 13 and No. 15 primer sets add/are not added with ring primer can amplify B. mucronatus；
No. 3 and No. 5 primer sets in the case where adding ring primer, it is amplifiable go out B. mucronatus, and be not added with ring primer, expand
Do not go out pine wood nematode and B. mucronatus；
No. 8, No. 16 primer sets in the case where adding ring primer, it is amplifiable go out B. mucronatus, and be not added with ring primer, expand
Do not go out B. mucronatus；Wherein, the amplification efficiency that No. 8 primer sets are not added with ring primer is optimal.
No. 4 primer sets add/are not added with ring primer can not all amplify B. mucronatus, but amplification efficiency is not very optimal.
Specifically, No. 1, No. 4 and No. 15 primer sets are not added with ring primer, the detection to pine wood nematode Bx and B. mucronatus Bm
As a result (real-time fluorescence amplification curve) is shown in Fig. 1；
No. 8 and No. 16 primer sets are not added with ring primer, and the testing result to pine wood nematode Bx is shown in Fig. 2；No. 8 and No. 16 primer sets
Ring primer is not added with, the testing result to longicorn is shown in Fig. 3；
No. 10 and No. 13 primer sets are not added with ring primer, and the testing result to pine wood nematode Bx and B. mucronatus Bm is shown in Fig. 4；
No. 1, No. 8, No. 13 and No. 15 primer sets add ring primer, to pine wood nematode Bx and B. mucronatus Bm testing result
See Fig. 5；
No. 3 primer sets add ring primer, and the testing result to pine wood nematode Bx and B. mucronatus Bm is shown in Fig. 6；
No. 4 primer sets add ring primer, and the testing result to pine wood nematode Bx and longicorn is shown in Fig. 7；
No. 5 and No. 16 primer sets add ring primer, and the testing result to pine wood nematode Bx and B. mucronatus Bm is shown in Fig. 8；
No. 10 primer sets add ring primer, and the testing result to pine wood nematode Bx and B. mucronatus Bm is shown in Fig. 9.
Using other 7 groups of primers (i.e.：No. 2, No. 6, No. 7, No. 9, No. 11, No. 12 and No. 14 primer sets) to pine wood nematode Bx
LAMP detections are carried out with B. mucronatus Bm, are as a result found, this 7 groups of primers, which add ring primer or are not added with ring primer, can not all expand
Increase and pine wood nematode.
2.2 carry the Monochamus alternatus recall rate of pine wood nematode
Ring primer is not added with using No. 8 primer sets of the present invention, 2 batches of Monochamus alternatus are detected respectively, it is as a result loose
The recall rate of pine wood nematode is respectively 28.60% and 43.75% (table 4) in black longicorn.
The recall rate of pine wood nematode in the Monochamus alternatus of table 4
SEQUENCE LISTING
<110>RES INST OF FOREST ECOLOGY ENV
<120>The method that loop-mediated isothermal amplification detects pine wood nematode
<130> BJ-2003-170423A
<160> 6
<170> PatentIn version 3.5
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gctggtctat gtcctattgc 20
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aataggcagc agaagttaga c 21
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tgtaaggcat tacctaatcg tc 22
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Claims (10)
1. the LAMP primer group that pine wood nematode whether is carried in sample for detecting, it is characterised in that the LAMP primer group choosing
Any one group from following 2 groups of primers：
Primer sets (1)：Including a pair of outer primers and a pair of inner primers；The outer primer by nucleotides sequence to being classified as SEQ ID
Outer primer composition in downstream shown in upstream outer primer shown in No.1 and SEQ ID No.2；The inner primer by nucleotides sequence to being classified as
Inner primer composition in downstream shown in upstream inner primer shown in SEQ ID No.3 and SEQ ID No.4；
Primer sets (2)：Including a pair of outer primers, a pair of inner primers and a pair of ring primers；The outer primer is to by nucleotide sequence
The outer primer composition in downstream shown in upstream outer primer and SEQ ID No.2 shown in SEQ ID No.1；The inner primer is to by nucleosides
Acid sequence is inner primer composition in downstream shown in upstream inner primer shown in SEQ ID No.3 and SEQ ID No.4；The ring primer pair
As nucleotides sequence be classified as shown in SEQ ID No.5 lantern primer with SEQ ID No.6 shown in lower lantern primer sets into.
2. according to the LAMP primer group described in claim 1, it is characterised in that the LAMP primer group is primer sets (1)：Including
A pair of outer primers and a pair of inner primers；The outer primer to be classified as nucleotides sequence upstream outer primer shown in SEQ ID No.1 and
Downstream outer primer shown in SEQ ID No.2 is constituted；The inner primer as nucleotides sequence to being classified as shown in SEQ ID No.3 in upstream
Inner primer composition in downstream shown in primer and SEQ ID No.4.
3. application of the LAMP primer group in pine wood nematode whether is carried in detecting sample described in claim 1 or 2；It is preferred that
, the sample is Monochamus alternatus.
4. a kind of LAMP detection kit of pine wood nematode, including：LAMP primer group, 2 × RM, SYBR Green I, Bst DNA
Polymerase and ddH2O；It is characterized in that：The LAMP primer group is selected from the LAMP primer group described in claim 1 or 2.
5. according to the LAMP detection kit described in claim 4, it is characterised in that：The LAMP primer group is claim 2
Described LAMP primer group.
6. application of the LAMP detection kit in detection sample in carrying pine wood nematode described in claim 4 or 5；It is preferred that
, the sample is Monochamus alternatus.
7. a kind of detect the LAMP detection method that pine wood nematode whether is carried in sample, it is characterised in that comprises the following steps：
(1) DNA of testing sample is extracted；(2) using the DNA of extraction as template, using the LAMP primer group described in claim 1 or 2 as expansion
Increase primer, set up LAMP reaction systems and enter performing PCR reaction in real-time fluorescence PCR instrument；(3) reaction result is judged：
1. fluorescence signal is collected, if there is " S " type amplification curve, is then determined as the positive；If without " S " type amplification curve, judged
For feminine gender；Or, 2. PCR reaction products are inactivated, nitrite ion is added and mixes, if green is presented, be determined as the positive；If
It is presented orange, then is determined as feminine gender.
8. according to the LAMP detection method described in claim 7, it is characterised in that the sample is Monochamus alternatus；
The LAMP reaction systems include：The cumulative volume of reaction system is 25 μ l；Wherein, 2 × RM12.5 μ l, SYBR Green I
0.5 μ l, 40 μM of upstream inner primer 1 μ l, 40 μM of downstream inner primer 1 μ l, 10 μM of upstream outer primer 0.5 μ l, under 10 μM
μ l, the 8U/ μ L of the outer primer 0.5 μ l of Bst archaeal dna polymerases 1, the μ l of DNA profiling 1 is swum, surplus is ddH2O；
Or, the LAMP reaction systems include：The cumulative volume of reaction system is 25 μ l；Wherein, 2 × RM 12.5 μ l, SYBR
Green I 0.5 μ l, 40 μM of upstream inner primer 1 μ l, 40 μM of downstream inner primer 1 μ l, 10 μM of the μ l of upstream outer primer 0.5,
10 μM of downstream outer primer 0.5 μ l, 20 μM of upper lantern primer 0.5 μ l, 20 μM of μ l, the 8U/ μ L of lower lantern primer 0.5 Bst
The μ l of archaeal dna polymerase 1, the μ l of DNA profiling 1, surplus is ddH2O。
9. according to the LAMP detection method described in claim 7, it is characterised in that the program of the PCR reactions includes：63℃
15s, 63 DEG C of 45s are used as a circulation；
The collection fluorescence signal is the collection fluorescence signal at 63 DEG C of 45s.
10. according to the LAMP detection method described in claim 7, it is characterised in that：The inactivation is 85 DEG C of inactivation 3min.
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