CN114854875A - Species-specific LAMP (loop-mediated isothermal amplification) primer for detecting pine needle sheath gall midge and application thereof - Google Patents

Abstract

The invention relates to the technical field of molecular detection, in particular to a species-specific LAMP primer for detecting pine needle sheath gall midge and application thereof. The nucleotide sequence of the specific LAMP primer for the pine needle sheath gall mosquito species provided by the invention is shown in SEQ ID No. 1-4. The invention also provides a method for detecting the pine needle sheath gall midge, which can quickly and accurately identify major forestry quarantine pest pine needle sheath gall midge in a short time, particularly identify other insect states (eggs, young larvae and pupae) except imagoes and mature larvae.

Description

Species-specific LAMP (loop-mediated isothermal amplification) primer for detecting pine needle sheath gall midge and application thereof

Technical Field

The invention relates to the technical field of molecular detection, in particular to a species-specific LAMP primer for detecting pine needle sheath gall midge and application thereof.

Background

The coleus pinicoli theospicensis belongs to the genus coleus (theodophyllae) of the family Diptera (Diptera) cecidomyidae (ceredomyididae), synonyms: pinicola, english name Pine needle toll middle. The cecidomyiia pinicola is a small insect harmful to Pinus thunbergii, Pinus densiflora and Pinus sylvestris, and has a body length of 2.5-3 mm. The insect belongs to an initial pest, the larva inhales juice at the base of the pine needle to be a harmful host, so that the base of the needle leaf expands to form insect gall, the damaged pine needle is obviously shortened, and the pine needle gradually withers and falls off, and the normal growth of the pine is seriously influenced. The plants damaged by the pesticide are weak in tree vigor and are easily damaged by the next-stage pests, and the plants die; when seriously harmed, the pine tree can be harmed and killed in the same year.

Research shows that pine needle cecidomyiia can harm various pinus plants. The hosts which are damaged to produce gall at the base of the pine needles belong to Pinus densiflora, and the method comprises the following steps: lipo pine p.resinosa, european black pine p.nigra, european mountain pine p.mogo, island pine p.insularis, american big fruit pine p.coulter, european red pine p.sylvestris, luchauensis p.luchauenuensis, radiata p.radiata, yellow mountain pine p.taiwanensis, japanese black pine p.thunbergii, massoniana p.massoniana, red pine p.densiflora and oil pine p.tabulaeformis. According to the prediction of the CLIMEX model and the MaxEnt ecological niche model, most provinces in the south of China are potential geographical distribution areas of the southern pine, so that the pine needle gall midge has potential hazard to southern pine forests.

Plant quarantine is the most effective means for preventing invasion and spread of external pests. In order to distinguish pine needle sheath gall midge from domestic common gall midge insects, a large amount of morphological identification work is needed, the morphological identification of the gall midge insects at present is mainly based on the morphological difference of adult fin veins and mature larva sword-bone fragments, and the identification methods need complicated sample processing procedures and professional insect taxonomy knowledge. Furthermore, gall midge insects in the three stages of egg, young larva and pupa are very similar in morphology and have no reliable identification characteristics at present.

In recent years, more and more research has demonstrated that molecular biological means can provide a powerful basis for identifying pests. Mitochondrial DNA is strictly inherited as a mother, and the cytochrome oxidase I gene (COI) has the characteristics of high conservation, stable structure and no intron. Therefore, it is commonly used for species classification, identification and genetic relationship studies. There are also reports that specific primers are designed for the COI gene sequence of the cecidomyiia pine needle to carry out molecular identification, but the SS-COI method needs equipment such as a PCR instrument, a nucleic acid electrophoresis instrument and the like, and is suitable for detection of professional institutions. The invention provides a simple detection method.

The loop-mediated isothermal amplification (LAMP) relies on 4 specific primers and a DNA polymerase with strand displacement activity (BstDNA polymerase), and can amplify target sequences efficiently, quickly and specifically under isothermal conditions. After the reaction is finished, whether amplification is carried out or not can be identified only by observing with naked eyes, equipment used in LAMP reaction is simple and low in cost, the requirement of rapid detection can be met, and the LAMP reaction is increasingly widely applied to the fields of scientific research of nucleic acid, diagnosis and prevention of diseases, sex identification of animal embryos, detection of transgenic foods and the like.

Disclosure of Invention

The invention aims to provide a method for rapidly and visually identifying pine needle sheath gall midge, which can rapidly and accurately identify the pine needle sheath gall midge which is an epidemic detection pest in major forestry in China in a short time, and particularly identify other insect states (eggs, young larvae and pupae) except adults and mature larvae.

The cecidomyiia is very difficult in morphological identification due to tiny insect bodies, and the identification work can be completed by experts with strong professional backgrounds and rich experience. And can only be done when adult and mature larva samples are obtained. Gall midge insects in the three insect states of eggs, young larvae and pupae are very similar in morphology, and accurate morphological identification of the gall midge insects is difficult. The conventional PCR amplification identification method based on the specific primers needs to utilize a PCR amplification instrument to perform thermal denaturation and variable-temperature cyclic amplification on a template, and also needs to perform processes such as electrophoresis and ultraviolet observation to judge reaction results, so that the whole process is complicated, high in specificity, needs to be completed in a professional institution with certain conditions, and is time-consuming.

In order to achieve the purpose of the invention, in a first aspect, the invention provides a pine needle sheath gall mosquito species specific LAMP primer, and the nucleotide sequence of the pine needle sheath gall mosquito species specific LAMP primer is shown as SEQ ID NO. 1-4.

According to the understanding of the technical personnel in the field, the invention also claims the application of the LAMP primer specific to the pine needle sheath gall midge species in the preparation of a pine needle sheath gall midge detection kit.

In a second aspect, the invention provides a detection kit for pine needle sheath gall midge, which contains a pine needle sheath gall midge species specificity LAMP primer with a nucleotide sequence shown in SEQ ID NO. 1-4.

The detection kit provided by the invention further comprises a color-changing premix, namely WarmStart Colorimetric LAMP2X Master Mix.

The invention also claims application of the detection kit in visual detection of the pine needle sheath gall midge.

In a third aspect, the invention provides a method for detecting the pine needle sheath gall midge, which uses DNA of a sample to be detected as a template and utilizes the pine needle sheath gall midge species specificity LAMP primer shown as SEQ ID NO.1-4 to amplify.

More specifically, the invention provides a method for detecting pine needle sheath gall midge, which uses DNA of a sample to be detected as a template and utilizes the detection kit to detect the pine needle sheath gall midge.

In the method for detecting cecidomyiia according to the present invention, in the amplified reaction system, the color-changing premix: 1-2 of SEQ ID NO: SEQ ID NO.3-4 is 25: 8:1.

In the method for detecting the cecidomyiia pine needle, the amplification reaction condition is isothermal amplification at 61 ℃ for 60 min.

In the method for detecting cecidomyiia according to the present invention, the determination criteria of the detection result are: the sample containing the pine needle sheath gall midge is amplified, and the reaction system is changed from red to yellow; the sample without pine needle sheath gall midge keeps red after amplification.

The invention has the beneficial effects that:

according to the invention, by designing the LAMP primer for the specificity of the pine needle sheath gall mosquito species and optimizing and constructing the LAMP rapid molecular detection system for the pine needle sheath gall mosquito, the identification of the major forestry quarantine pest pine needle sheath gall mosquito can be realized rapidly and accurately in a short time, particularly the identification of other insect states (eggs, young larvae and pupae) except adult and mature larvae, and the invention can be used for rapid detection of the pine needle sheath gall mosquito in quarantine work such as domestic and foreign seedling regulation and transportation.

Drawings

FIG. 1 is a graph showing the amplification results of four kinds of gall midges using the LAMP primer specific to the pine needle sheath gall midge. A is the result of detecting amplification under visible light, B is the result of detecting amplification under ultraviolet light, and C is the result of detecting amplification under electrophoresis. In the figure, NC is a control; + is pine needle sheath gall midge t. japonensis; TSP is the masson pine colecidae midge Thecodiplosis sp; CSP is jujube gall midge Contarinia sp; CCA is hickory gall midge C.

FIG. 2 is an electrophoresis diagram of DNA samples of adult and larva amplified by specific LAMP primers of pine needle cecidomyiia. A is the result of detecting amplification under visible light, B is the result of detecting amplification by electrophoresis, and NC is the control.

FIG. 3 shows the sensitivity result of specific primers LAMP for pine needle sheath gall mosquito species. A is the amplification result detected under visible light, B is the amplification result detected under ultraviolet light, C is the amplification result detected under electrophoresis, and NC is a control.

FIG. 4 shows the results of electrophoresis of three sets of LAMP primers (M: marker,1-2: SEQ ID NO.5-8,3-4: SEQ ID NO.9-12,5-6: SEQ ID NO. 1-4).

FIG. 5 is a graph showing the amplification curves of SEQ ID NO.1 to 4.

FIG. 6 is a graph showing amplification curves of SEQ ID Nos. 5 to 8 and SEQ ID Nos. 9 to 12.

FIG. 7 shows the amplification efficiencies of SEQ ID NO.1-4 at different temperatures.

FIG. 8 shows the amplification efficiency of SEQ ID NO.1-4 at different primer ratios.

FIG. 9 shows the amplification efficiency of SEQ ID NO.1-4 after addition of the loop primer.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1 design of specific LAMP primers

According to the invention, pine needle sheath gall midge is taken as a target, other three kinds of gall midge which are common in China are taken as reference, a species-specific LAMP primer based on a mitochondrion COI gene sequence is designed, and a rapid molecular detection system of the pine needle sheath gall midge is constructed and optimized.

The design process of the specific LAMP primer for the pine needle sheath gall mosquito species comprises the following steps:

1. extraction of insect genomic DNA

Genomic DNA of an unused species of the gall midge family collected from 4 regions in China is extracted by using a DNA micro-extraction kit.

(1) The whole worm bodies were placed in 1.5ml centrifuge tubes, 200. mu.l buffer PBS was added, and ground well with a grinding rod.

(2) Add 150. mu.l buffer PBS and 0.9ul RNase A and gently triturate for 30 s.

(3) 350 μ l of the ground homogenate was collected and transferred to a 2ml centrifuge tube. If the homogenate volume is less than 350. mu.l, PBS is supplemented to 350. mu.l.

(4) 150ul buffer C-L and 20ul protease K were added. Immediately vortex and shake for 1min to mix evenly. After brief centrifugation, the tube was placed in a 56 ℃ water bath for 10 min.

(5) Add 350. mu.l of buffer P-D, mix well by vortex shaking for 30s, and centrifuge at 12000 Xg for 10 min.

(6) The DNA preparation tube was placed in a 2ml centrifuge tube, the mixture from the previous step was transferred to the preparation tube and centrifuged at 12000 Xg for 1 min.

(7) The filtrate was discarded, and the preparation tube was returned to the original 2ml centrifuge tube, 500. mu.l of buffer W1 was added, and the mixture was centrifuged at 12000 Xg for 1 min.

(8) The filtrate was discarded, and the prepared tube was returned to the original 2ml centrifuge tube, and 700. mu.l of buffer W2 was added thereto, and the mixture was centrifuged at 12000 Xg for 1min, and washed once more with 700. mu.l of buffer W2 in the same manner.

(9) The filtrate was discarded, and the preparation tube was returned to the original 2ml centrifuge tube and centrifuged at 12000 Xg for 1 min.

(10) The filtrate was discarded, the preparation tube was returned to the original 2ml centrifuge tube, 100. mu.l of the solution was added to the center of the preparation tube, and the tube was left standing at room temperature for 1min and centrifuged at 12000 Xg for 1min to elute DNA.

(11) And (5) detecting the DNA concentration. The concentration of the DNA is measured by a ultramicro spectrophotometer. Using Nanodrop2000 (Thermo), the wells were washed with deionized water before the assay, 1ul of Elution Buffer used for DNA extraction was aspirated after wiping off the wells and corrected, 2. mu.l of sample was pipetted into the wells after correction was completed, and the concentration of all DNAs was measured.

2. LAMP primer design for cecidomyiia conifer specificity

Based on the comparative analysis of the base sequences of the pine needle coleoptile midge (SEQ ID NO.13) and its related species, the masson pine coleoptile midge Thercodiplosis sp. mitochondrial genome sequence (SEQ ID NO.14) and other 8 species of cecidomyiia which have been published in the database, the LAMP specific primer 1 set of the pine needle coleoptile midge is manually designed and is shown in Table 1: two outer primers (Tjap _ F3 and Tjap _ B3) and two inner primers (Tjap _ FIP and Tjap _ BIP) are included.

TABLE 1 pine needle cecidium specific LAMP primers

Example 2 species specificity and sensitivity test of pine needle cecidomyiia LAMP primer

The species specificity of the pine needle sheath gall midge LAMP primer is tested by taking DNA of domestic common gall midge insects as a template and taking the pine needle sheath gall midge DNA as a positive control. And (3) amplifying by using DNA standard products of the pine needle cecidium mosquitoes with different concentrations as templates, and checking the sensitivity of the primer.

1. Preparation of standards

After the DNA of 4 kinds of gall midges were subjected to concentration and purity measurement, a mother liquor was prepared at a concentration of 30 ng/. mu.l. And diluting the pine needle sheath gall midge DNA sample into standard solutions of 30 ng/mu l, 3 ng/mu l, 300 pg/mu l, 30 pg/mu l, 3 pg/mu l, 300 fg/mu l, 30 fg/mu l and 3 fg/mu l in sequence according to the proportion of (1: 10). All standards were stored at 4 ℃ until use.

2. Amplification of LAMP species-specific primers

LAMP amplification is carried out by using a WarmStart Colorimetric LAMP2X Master Mix (NEB company) color-changing premix, the system is specifically prepared as shown in Table 2, DNA is replaced by ultrapure water in a control group, and the reaction system is placed in a metal bath at a constant temperature of 61 ℃ for amplification for 60min during detection.

TABLE 2LAMP reaction System (25. mu.L)

3. Amplification product detection

Direct detection with naked eyes: the color of the system is changed from red to yellow after the positive sample is amplified, and the color of the negative sample is kept unchanged.

Ultraviolet lamp detection: 0.5. mu.L of GelRed dye was added to the system after amplification and placed under an ultraviolet lamp to observe whether amplification occurred.

And (3) electrophoresis detection: the amplified products were detected by electrophoresis on a 1.5% agarose gel prestained with GelRed dye (voltage 130V, time 30 min), 1 XTAE as electrophoresis buffer and DL2000DNA Marker as Marker.

4. Specificity test result of LAMP primer of cecidomyiia conifer

The amplification is carried out by using the pine needle sheath gall midge specific LAMP primer designed by the invention, only the pine needle sheath gall midge is successfully amplified, and the amplification capability is not provided for other 3 kinds of gall midge and negative control (see figure 1), which indicates that the pair of primers are the species specific primers of the pine needle sheath gall midge.

The DNA of the imago and larva of the pine needle cecidomyiia is taken as a template, the specific LAMP primer of the pine needle cecidomyiia is used for amplification, and the result shows that the DNA extracted from the imago and larva can stably amplify specific fragments (shown in figure 2).

5. Sensitivity test result of pine needle sheath gall midge LAMP primer

Specific primer sensitivity tests were performed using a gradient of DNA standards from the sheath gall midge of pine needles (see FIG. 3). The minimum detection limit of the assay was 300fg of genomic DNA.

The results show that the amplification and result judgment program can be completed by using one constant-temperature metal bath, and the invention can be used for rapidly identifying the cecidomyiia conifer on site by matching with a DNA crude extraction method operated on site.

Comparative example 1 results on specificity of different LAMP primers

The comparative example 1 provides 2 groups of pine needle sheath gall midge LAMP primers, and the sequence information is shown in SEQ ID NO.5-8 and SEQ ID NO. 9-12. The LAMP primers of the pine needle cecidomyiia are the same as the design method of the embodiment 1.

SEQ ID NO.5：TTATCTGATTATTTAATCCTCCTGAAATTCG；

SEQ ID NO.6：CTGCTTTATCAAATCGAATTGGAGATGTATG；

SEQ ID NO.7：CGGCTCCTACTCCTGTTTCAGCCTCCAGCGGTAACTAAAGTTGAAG；

SEQ ID NO.8：CCATAGCAGCAGGTAACCAAGAAGAAATTATAGCAGCTATAACTAAAAGAGCAC。

SEQ ID NO.9：CCTTTTATTTCAATTAAATCAATTGGACATCAATG；

SEQ ID NO.10：GAGAAATTCTTTCTAATGAAATAGGTATAAATGAGTG；

SEQ ID NO.11：GAATGTAATACATCTGCTGCTGTTACTATAATTCGCGATTATTAGATACTGATAATAAAATTATCATCCC；

SEQ ID NO.12：AGTAGATGCAACTCCTGGACGATTAAATCGAACATTGACCAAAAAATATTCTTGATCG。

Comparing the amplification efficiencies of SEQ ID NO.1-4, SEQ ID NO.5-8 and SEQ ID NO.9-12 to screen a primer group with the highest amplification efficiency, and finding out that: three sets of primers were able to amplify the ladder-shaped band characteristic of LAMP amplification (FIG. 4), but the primer sets of SEQ ID Nos. 1-4 peaked within 30 minutes (FIG. 5), whereas neither of SEQ ID Nos. 5-8 and 9-12 peaked within 35 minutes, and only peaked after 15 minutes of amplification (FIG. 6). The amplification efficiency of the primer sets of SEQ ID NO.1-4 was higher than that of the other two sets.

Comparative example 2 amplification results of different amplification reaction systems

The comparative example provides the amplification results of the pine needle cecidomyiia LAMP primer shown as SEQ ID No.1-4 in different amplification reaction systems.

This comparative example set a temperature gradient in the range of 55 ℃ to 65 ℃ and had the highest reaction efficiency when the reaction temperature was 61 ℃ (FIG. 7). When the concentration ratio of the inner primer to the outer primer (Tjap _ F3/Tjap _ B3: Tjap _ FIP/Tjap _ BIP) was 1:4, 1:8, 1:6, and 1:10, the highest reaction rate was observed when the concentration ratio of the inner primer to the outer primer was 1:8 (FIG. 8). When the upstream and downstream loop primers (Tjap _ FL (SEQ ID NO.15) CCTGTTCCTGTTTCAACTATTCTTCTAATTAATAAT and Tjap _ BL (SEQ ID NO.16) GTTTATCCTCCTCTTTCTTCAATTATTGCTCATA) are added simultaneously or when one loop primer is added alone (Tjap _ FL or Tjap _ BL), the amplification efficiency is not significantly improved, so the loop primer is not added finally in the present invention (FIG. 9).

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Beijing university of forestry

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gtttatcctc ctctttcttc aattattgct catactagaa catctgtaga tttttcaatt 1620

ttttctttac atattgcagg aatttcatca attttaggag caattaattt tatttcaaca 1680

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taaataagaa tttcatcatg attaaattta aacttacaaa atagaaattc acctttaata 2820

gaacaattaa ttttttcatg atcattcaat attaatttta attttaatta ttaatttagt 2880

aagatatata ataatttcta taatattaaa taattattat aattattttt tattatcagg 2940

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tccatcatta aaattactat atttattaga agaaacaaat aaacctttta tttcaattaa 3060

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atgaattaat aattttttat aaattgactg ataaaagtat tagtttttta aactaaaaat 3480

tttagttaat atataatatt aatttgtcaa attaaagtta ttcctcaaat aaaaccaata 3540

aattgaataa ttattttttt atctttaaat attattttaa tttttataaa tattttaaat 3600

tattttttaa aaattaattt taaaatttta aaatataata aaatttataa aaataaaaat 3660

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ttaatttatc attaaattga ctttctataa ttttattttt attattaatc ccaaaattat 3780

tttgaataat tcctaataat attaacttaa tttattttaa attaattaat aatttattta 3840

atgaatttaa aattattatc tataaaaaaa ataataattt tattattttt tttatttcat 3900

tatttgtatt tattataact aataattttt taggattatt tccatatatt tttactagaa 3960

caagtcattt aattattaat ttttcattag ctttaccatt atgattaaga ttaataattt 4020

ttggatgaat taataaaact aatcatatat ttactcattt agttcctcaa ggaactccta 4080

atattttaat cccatttata attttgattg aaattattag aagaataatt cgacctatta 4140

ctcttactat tcgattaaca gcaaatataa ttgcaggaca tttactatta acattattag 4200

gaaatatagg atctaaatta ttatttcttt taccattttt aataattact caaataattt 4260

tattaatttt agaaatagct gtctcaatta ttcaatctta tgtattttca attttaatta 4320

ctttatatac aagagaaatt taataaatta taatcataat tttcatttag taaattatag 4380

accttgacct ttaattggat cattatctac tataataata ttattaagaa ttattaaatt 4440

atttaatcaa aaaaatttaa ttatatatat tatatcaatt ttttctattt tattaattat 4500

atatcaatga tgacgagata tttcccgaga aagaacattt caaggattac actcatgaaa 4560

tgttttaaag ggaataaaat taggaatatt attatttatt atttctgaaa tttttttttt 4620

tatttctttt ttttgaagat tttttcattt aagattaact caattaatag aattaggaat 4680

aaaatgacct cctataggaa ttaaaacatt taatcctttt ataattcctt tattaaatac 4740

aattatttta ctttcatcag gaatttctat tacttgagca catcatagat taattaatat 4800

aaattatata caatttttaa aaagaatttt attaacaatt attttaggta tttatttttc 4860

tattttacaa atttatgaat atttagaagc atcttttaat attgcagatt cttcttatgg 4920

gtcaattttt tttttagcaa ctggatttca tggaattcat gtttttattg gaactaattt 4980

tttattaata tgtttaattc gaaatataat atttcatttt tctaataaac atcattttgg 5040

atttgaagct gctgcttgat actgacattt tgttgatatt gtttgacttt tcctttatat 5100

atcaatttat taataaatta agtaaatatt ttattataaa actagtaaat ataatttcca 5160

attataaaga aataaattaa aaccaaaata taggtatatt actgttaata atattattga 5220

attaataaat tctaatttaa tttatttatt tattaatttt ttttttattt ttataattat 5280

ttcaatatta attattaaat tatctattat aatctcttta aaaataaaat taaatttaga 5340

aaagatatcc ccatttgaat gtggatttaa tccttttttt tataaacgaa tgccattttc 5400

aatacgattt tttttaatta cagtaatttt tttaattttt gatattgaaa ttgctctttt 5460

attaccaatt atttattcta taaaattttc aaataaattt aattgaattt tatcaagaat 5520

tttatttatt tgtattttaa ttataggatt attttatgaa tgaaaaaaca aagctttaaa 5580

ttgaatatta aaaattaatt taataaaaat atatatattg ctaatataaa ttaaaattaa 5640

atttttatca taactaatga aattatcttc gaaataattt atataaattt agtttataaa 5700

aacattatat tttcaatata aaatataatt ttttttatta agcttctaac ttaatttata 5760

ataattaaaa ttattaaaat tattaattaa attgaatatt tataaatttt caatttatta 5820

cttttatcag tcattaaata atatataaat tatcaattta ttacttttat cagtcattaa 5880

ataatatata aattatcaat ttattacttt tatcagtcat taaataatat ataaattaat 5940

aataataata aaattataaa aaaaaaatta ttaaaattaa ataaactaaa ataataatta 6000

ttaaaaaaat atattttttt tttaaaaaaa taacccccta aaaattctat tcaacctata 6060

tctaaaataa aattatattt tttaataaaa tctaaaataa ttttattaaa aaatatacaa 6120

aaaataaaag gtataaatca tattatacta ataaaataaa agtttatttt ttttcaattt 6180

aaattaaaaa aaaataaact taataatatg cctaaaaaac aaattattaa aataattatt 6240

tttaaataaa ttcttaatat aattaatcta aaaaaaggaa aaattaatca tattaaaaaa 6300

gaaccaccaa ttacagaaaa aattattaaa atttttatag aaaataatat attaatcatt 6360

tcatttataa ataaaatcga aaatcctata taatttaata aacaaaaact aattaaacga 6420

aaagaatata taacagttaa tccagtagaa aaaaaaaaaa aaaaattaaa ataattagta 6480

tttgatatta atacaaattc taaaattata tctttagaat aaaatccagc taaaaaaggt 6540

atcccacaaa agctatcttg aaatattaaa aaaattaaag ttaaaattat ctgattattt 6600

aatcctcctg aaattcgaat atcttgaaaa tttattaatc tatggataaa aattccagca 6660

cacaaaaata acaaagcttt aaataaagca tgagttaata aatgaaaaaa agctaacttt 6720

ataaacccta ttaataaaat tcttattatt aaacctaatt gtcttaaagt agaaaaagca 6780

ataatttttt ttaaatctat ctcataatta gctcctaaac ctgatataaa tatagttatt 6840

ctacttaaaa ataataaaat aaataaaaac ttactattca atattaaata ataattaaat 6900

cgaattaata aataaactcc agcggtaact aaagttgaag aatgaactaa agctgaaaca 6960

ggagtaggag ccgccatagc agcaggtaac caagaagaaa aaggaatttg tgctctttta 7020

gttatagctg ctataattaa taagattaaa aaaaataaat ttaaatttat aaattctaaa 7080

taaaaataaa atctaaaact tccaatacta aatataaaaa aaatacaaat taataaacat 7140

acatctccaa ttcgatttga taaagcagtt aatatcccag cattaaatga tttataattt 7200

tgaaaataaa ttactaaaca ataagaaatt aatcctaaac catctcatcc taaaagaatg 7260

gaaataaaat ttggagaaat aattaataat attattgata aaataaataa aaaaactaaa 7320

taaataaatc gaattttata tttatcattt tttatataat caattctata aaaaattact 7380

atagaagaaa ttaataaaac aaaagatata aataatagtc ttattcaatc aaataaaaaa 7440

ataattgata aattacaaga attaaaattt attatttctc actcaaaaaa atatattaaa 7500

tctttaaata taaaaaataa aaataaaaaa aaaaataata ttctaaaaaa aaatgtaata 7560

aaacaaaaat ataaataaat tattatcttt aaattcacaa tttaaaattt tattaaacta 7620

ataaaaaaaa aacaaaataa taattttaaa aaaataatat ttaaaggaaa tcaatgaaat 7680

attattaata taaattctcg ttgattatta ttagaaaatc tataaactct aaaaaaaaaa 7740

attccatgat taactattga atataaataa aaactataag tagctctaaa aaaacataat 7800

aatattaata taattataaa aaataaagat caagatatta aactattaat taatttaatt 7860

tctgaaatta aatttaaaga taaaggagct gatatatttc ttgaagataa taaaaatcat 7920

cataaagtta atgaaggtat aaaatttaat atacctttat taattaatat tcttcgtctt 7980

aataaacgtt cataagagac attagctaaa caaaataaac cagatgaaca taatccatga 8040

gcaattatta ttaaaaaacc acctcttatt cctcaaaaag ttaaagtaaa tatacttctt 8100

aaaactattc ttatatgagc tacagaagaa taagcaatta atacttttaa atctaattgt 8160

cgtaaacaat ttaatctaat taatacacct cctaataatc taaaaattat aaaaaatata 8220

tttaaatttt taaatttata tatataaata aaaattcgaa ataaaccata acctcctaat 8280

tttaataaaa tagcagctaa aattatagaa ccaaaaattg gagcttctac atgagctttt 8340

ggtaatcata aatgaacaaa aaatattggt atttttacta aaaatgctat aattaaacct 8400

atataaatta atttattaaa aatttcatta ttaaaattaa aaataattaa acttttaaaa 8460

taaaaattta aatataaaat tcttattaat aaaggcaaag aagctaataa agtataaatt 8520

aataaatata atcctgctat aattcgttca gattgataac cccaccctaa aattatataa 8580

ataatgggaa ttaatcttat ttcaaaaaaa atataaaata ataaaaaatt attaataaaa 8640

aatctaaaaa ataaaaatat atatataaaa aatattataa aaataattaa aaataaattt 8700

ccattatatt ttattatatt aaatgacaaa attattaaaa aaataattca tatagtaagt 8760

aaaattaaaa aataatttaa attatctaaa aaaattaaat tattaaaata ataaatattt 8820

aaagaaggat aataaataaa tataaaaatt aaatatataa aaattataaa atatataaaa 8880

ttccaaaaat ttataaaaaa aaataaaata atataataaa taaaatttta aacatttaaa 8940

tttttattaa atttttatta aatttttatt aaatttaaat tttgaatatt tgaatttccc 9000

aaaatacgaa ttaaagaaat taataaagat aaacctaaca caccttcaca aactaaaaaa 9060

gttaaaaaca ttattaaaaa aaatatttca taattaaaaa ataataaatt taaataaata 9120

ttaaataata tattaagtat tataaattca attcttatta aattaattaa taaatattta 9180

aaattaaaaa aaaactataa ttccacaaaa aaaaaaatat aattataaaa taataattaa 9240

aatataaaaa aaaaatttaa aatctttaat ttccaaaatt aaaattttaa taaactataa 9300

tttaaattaa aataataatt ttgtaattta taaattttaa aatttattat ttaatttatt 9360

taattagaat actattttta aattttaata accctttaat tataggaatt atattattat 9420

tacaaacaat aaatgtagta ttaattatag gaaaaattaa ttcatattga ttttctttca 9480

ttattttttt agtatttgta ggaggtttat ttattttatt tatttatata atttcattaa 9540

catataacaa taaatttttt tctattaatt ataaatattt atatataata attttattta 9600

ttttattctt atatataaat aattttaatt ttatttttat aaaaaatgaa tctaaaaaaa 9660

aattaattga aaaagaaaat ataattaatt tattaaaatt ttttaatttt cctaataatt 9720

taattaatat tttattaatt atttatttat taattttaat aattattaca gtaaaaatta 9780

ctaattttta taaaggtccc ttaaaaatta aaaattatta ataaaaataa attattaatt 9840

aataatttct ataattcatt aaatttacct acacctttaa atattaactt cttttgaaat 9900

tttggttctt tattaggatt atgtttaatt attcaaattt taagaggatt atttttagca 9960

tttcgatata ctcctgatac aattttagct tttaatagag taaataatat tataaatgaa 10020

gcttgaaatg ggtttatttt tcgaaattta catgctaata gagcatcaat attttttttc 10080

tctttatatt ttcatattgg tcgaggaatt tattttaaat catttttatt taaaaatact 10140

tgaactattg gagtaattct attattaaca actataggaa cagctttttt aggatatgtt 10200

ttaccatgag gacaaatatc attttgagga tcaacagtta ttactaattt attatcttca 10260

attccttatt taggaaatta tttagttcaa tgaatttgag gaggattttc agttaataat 10320

ccaactttaa ctcgattctt ttgtcttcat tttattttgc cttttttaat tttattttta 10380

accattattc atttattttt tttacattta acaggatcaa gaaatccttt aggaattaat 10440

agaaatataa aaaaaattac atttaatcct tttttttctt taaaagattt tttaggaatt 10500

ttttcattat ttattattat tttatttatt atttctttta aaccttatat attaggagat 10560

agagataatt ttattttagc taacccatta ataactccta aacatattca accagaatga 10620

tattttttat ttgcttatgc aattttacgt tctattccaa gaaaattagg aggtgtaata 10680

gctttaataa tatctattat aattttattt tttttaccat tattaaatat aagaaaaata 10740

aaaagtaatc aattttatcc tttcagaaaa tttatttttt gaatattaat ttttacaatt 10800

attttattaa cttgaattgg aatacgacct gttgaatacc cttttataat tttaggacaa 10860

attttatcta tatactattt tttaatttat tttattaatc caattttaca aaaatattat 10920

gataatttta tttattaatt tattatctta aaaaaagaat ttgttttgaa aacaaaaaat 10980

ataattttaa atcttaaaaa taaaataaga aaaaataaag aaattggtaa ataaattttt 11040

caagacaaat acattaattt atcataacga taacgaggaa aagaaccacg aactcaaata 11100

aaaaaaaaag ttaaaaatac taattttaag taaaaaataa aataaatata agaattaaat 11160

aaaaatataa atctataaat tattcttaaa tataaaatta ttgaatattc agataaaaaa 11220

attaaagcaa accctcctct tctatattca atattaaatc cagaaactaa ttcagattct 11280

ccttcagcaa aatcaaaagg agctcgatta gtttcaatta aaaaaataat taatataatt 11340

atactaatta atgtattaat aaaaataaaa tttaatatat tcttgaaata ataaaaagtt 11400

tttattatta acctaaaaat taaaataaaa taacataaaa aaaataaaat tattcttact 11460

tcataagaaa ttgtttgagc aattgaacgt aatcttccta atattgaata attagaatta 11520

gaagatcaac ctgaaaatat aataaaataa actcctactc ttatacatct aaatataaat 11580

aaaaatatta aatttaaaga aaaaaaatta tataaaaaag gaaaacctaa tcaaataaac 11640

aaagaaacta taaaagaaat aattggacaa aaataataaa ttatataatt agaataaata 11700

ggaaacgtaa tttcttttaa aaataactta attgcatcac aaaaaggttg taataaacct 11760

attaaaccta ctttattagg tccttttcga atttgaatat atcttaaaat tttttgttct 11820

aataaagtta aaaaagtaac tctaattaaa attaaaataa taaaaattaa aactattaat 11880

atttttttaa ttaattataa ttaaattcta aatttaatac atttattctg ccaattaata 11940

taaaactaaa ataaaatttt taaaatcaat cctttcgtac taaatttaaa cttttttaaa 12000

aaagatagaa accaatctgg cttacaccga tttgaactca aatcatgtaa aaatttaaaa 12060

gtcgaacaga cttattattt atatttcttc atataaaaat aattttaatt caacatcgag 12120

gtcataaact tttttaaaaa taagaacttt ttaaaattat tatgctgtta tccctaaggt 12180

aatttaattt tttaatctat ataaaaagat caaataaaca tttataaatg attaaaatat 12240

aaaaagtttt ataaatttat ttatctcccc aataaaataa atttttaata ataaaataaa 12300

tatttacaaa ttaataaaaa attataaaat tctatagggt cttctcgtcc ctttttattt 12360

aattagcttt ttaactaata aataaaattc aattattaaa attaaaaaag attttatttc 12420

atttaatcat tcattctaga ttctaattaa aaatctaatt attatgctac ctttgtacag 12480

tcaaaatact gcagcctttt aaaaattcaa tgggcagatt atatttttta attaataaaa 12540

aaactatgtt tttgataaac aaatgaataa ttttttttgc tgaattatta aattttattt 12600

ttaatatttt actaatttat ttattatttt attacaaaat ttattaatat aataattttt 12660

ataaattata aaatttattt ataaatttat ttacaaatta aaataaatta taaaattttt 12720

tttaatttta aatacttaaa aaattaaatt attaacttta aatatataaa tttttataat 12780

tttaaattaa gataatccct aatttaaatt aatataatat ataaattttt atataattat 12840

aaaaattaaa aattatatta taaattaaat ttatttttaa aaaaactaaa tataataaaa 12900

atcgaaaaat attttatttc tatttaaaaa ttatttataa ttataaaata ttaaaattta 12960

taattaaata gatcttttta ttttgagaaa attaaaataa ataattaatt ttaaataacc 13020

ctaatacaaa aggtactaaa aattaattat tttttaaaaa aatttttaaa attatttttc 13080

aattaattta tatttataat aaaaattttt tttaaaatta ctttttttaa aattactttt 13140

tttaaaatta cttttttaaa aaaaaaatat tttaaaattt aaaatttaat ttaacctaat 13200

taaataattt ttttattgta aataaaaatt aataaattta tttctaaata taatttccaa 13260

tatatttact atgtttcgac ttattttaat aaattaaaag cgacgggcaa tttgtacaca 13320

tttttaaatt attcaaatat taaatttata aaattttaat tttaaatcca attattaata 13380

tattttacaa tatattaata aaaaataatt taaatgtatt tcattttaaa atcttatata 13440

taaaatgtat tatgatttga tataaaattt aaataaatta aaataatttt ttaaaaaatt 13500

atttttaaac aacaatatac aatttaatta ataataagta aattttattg tgtattatca 13560

attaataaac aaaatcctct aatttttaaa aatactgcca ttttatttaa ttttaatata 13620

aaaattaata attaaaatac tataaataaa ataatagggt atctaatcct agtttttaaa 13680

atttatataa aaaatattaa tatttcacta aataatattt aaattaataa taactttata 13740

atttatttta atattaaagt ataaccgcaa atgctggcac tttattattt ataattaatt 13800

tttactaata ataaatttaa ataataataa taaatttaaa tactaataaa tttaaatatt 13860

aataatattt atttatattt taaaatttta aaataatatg ttaaataata ataatttttt 13920

tttttttttt ttaataaatt taaataacaa ttatttattc taatttattt ctatatatat 13980

attatatata catattatat aaatttttta tgtaaatcat ttataattat ttattctaat 14040

ttatttctat atatatatta tatatacata ttatataaat tttttttata attatttatt 14100

ctaatttatt tctatatata tattatatat acatattata taaatttttt atgtaaatca 14160

tttataatta tttattctaa tttatttcta tatatatatt atatatac 14208

<210> 14

<211> 14252

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ttattaaaaa cttaataatt caaaattatt tatgcatata acatttataa aaatttaagc 60

taaatataag ctaatggatt catattccat ttataaataa tttatttaaa aaaaattaag 120

ctaaaatcta atttattttt ttatttatta atttattttt ctatttttat atttttatct 180

tcaaattcat gatttaatat atgattatga ttagaagtta atttaatttc attcatttca 240

ttaattaata ataataaaaa taaaatatct aatgaatctt cattaattta ttttttaatt 300

caatctattt cttcaattat tttattattt tcaattttaa tattaatatt taaaaataat 360

tttattttta ttttaatttt ttctatcata attaaaattg gaagatctcc tttccattat 420

tgaattccat taataattga aggattaaat tgaaataaaa tttttattct tttaacttga 480

caaaaaatta atcctttata tatattattt ttaaataatt tttattatta tttaaaaatt 540

tttataattt tatctttaat tattggatca atttctggat taaattttaa ttctttaaaa 600

aaaattatat cattttcttc tatcaatcaa ttaagatgaa taattataag tttaataaat 660

aataaattaa ttaagattta tattttaata tatataattt taatatatat aattttaaaa 720

ttatttaaat tatttaattt aaatttttta aatcaattat ttaatttaaa attaaaaaat 780

aaatatttta aattatttat atttataaat ttattatctt taggaggttt accaccattt 840

attggatttt ttccaaaaat aattttatta ataaatttaa ataacttatt tattatattt 900

ataataatta ttttttcatt attaacttta tttatatatt tacgaattat tttttctata 960

tttatattaa attcaattaa aataaataaa attataaaat ataaaaattt tttaaaaact 1020

aatttattaa taaaattatc tatatttaat aatttatttt taattccttt aattttttta 1080

attttaaaaa ttaagttaaa taaactataa atcttcaaaa tttaaatttt aaattaattt 1140

ttaattttaa ttaaatttgc aatttaacta aataaaatta tttataaatt tacaatttat 1200

tacttttatc agtcattaat tatttttaaa aaatgattat tttctacaaa tcataaagat 1260

attggaactt tatattttat atttggaatt tgatcaggaa tattaggaac atctttaaga 1320

attttaatcc gtttagaact tagatcaaca tgtaatctaa ttggaaatga tcaaatttat 1380

aatgttattg taacagctca tgcttttatt ataatttttt ttatagttat accaatttta 1440

attggaggat ttggaaattg attaattcca attatattag gagctcctga tatagctttt 1500

ccacgaataa ataatataag attttgatta ttacctccct ctcttacttt attattaatt 1560

agaagaatag tagaaacagg aacaggaaca ggttgaactg tttatccacc tctttcttca 1620

attattgctc atactggttc atcagtagac ttttcaattt tttcattaca tattgcagga 1680

atttcatcaa ttttaggagc tattaatttt atttctacta tattaaatat aaaaattaat 1740

tatttaaaat ttgatcaaat ttctttattt atttgatcaa tttttattac tacaattctt 1800

cttctacttt ctttacctgt tttagctgga gcaattacta tattattaac agatcgaaat 1860

ttaaatactt cattttttga tccaatagga ggaggagatc ctattcttta tcaacattta 1920

ttttgatttt ttggacatcc tgaagtatat attttaattc ttcctggatt tggaataatt 1980

tctcatatta tctttcaaga aagaagaaaa aaagaaacat ttggaatttt aggaataatt 2040

tatgcaataa tctctattgg atttttagga tttattgttt gagctcatca tatatttaca 2100

gtaggaatag aagttgatac tcgagcttat tttacaacag caactataat tattgcaatc 2160

cctacaggaa ttaaagtatt tagatgatta gcaactattc aaggtagaca attaaatttt 2220

aatccatcta tattatgagc actaggattt atttttttat ttactatagg aggaattaca 2280

ggagtaattt tagctaattc ttctattgat attattttac atgatactta ttatgttgta 2340

gctcattttc attacgtttt atctatagga gctgtattcg ctattataat aagattcatt 2400

cattgattcc ctttatttac aggtttatat ataaataata tattattaaa aattcaattt 2460

ttaataatat ttattggagt taatttaact tttttccctc aacatttttt aggattatca 2520

ggtatacctc gacgatactc tgattttcct gattcttttt tattatgaaa tataatttct 2580

tcaattggat cttctatttc tataattaga attttatttt ttatttttat tatttgagaa 2640

agaataatta atataaaaaa aattattttt ccaaatcaat taaacacttc tattgaatga 2700

tttcaagaat atcctcctaa aaatcataga tacacagaaa ttcctataat ttcatattaa 2760

aaaaaatgtc agataaatga aatgaattta aaattcataa atgatttata atatcttgat 2820

taaatttaaa tttacaaaat agtaattctc ctttaataga acaattaatt ttttttcatg 2880

atcattctat attaatttta attttaatta ctaatttagt gggttatata ataatttcaa 2940

tattatttaa taattacaat aattattttt tattatcagg acaaactatt gaaattattt 3000

gaactattat cccagccatt attctattat ttattgcatt tccttcacta aaattattat 3060

atttattaga agaaacaaat aaacccttta tttctatcaa atcaattggt catcaatgat 3120

attgaaaata tgaatattca aattttaata tagaatttga ttcttatata tttaataata 3180

atgaaataat taataatttt cgtttattag atacagataa tcgaattact attccattta 3240

attattttat tcgaattata gtaacagcaa ctgatgtttt acactcttga actatccctt 3300

ctttaggaat caaagtagat gctactcctg gacgattaaa ccaattaaat tttttaacca 3360

atcaatcaag aatttttttt ggtcaatgct cagaaatttg tggagcaaat cattcattta 3420

tacctatttc tatagaaaga atttcactaa aatatttttt aaaatgacta aataattaat 3480

aaattgactg attaaagtat tagtttttta aactaaatat ttagttaatc aataacatta 3540

atttgtcata ttaaaattta ttcctcaaat aaaaccaata aattgaattt tattattttt 3600

aattttaaat ataatattaa ctttaataaa tattataaat tatttttttt ttataaatat 3660

aaaaaataaa aaaaatcata aaaatattaa taatttaaat aacctaaatt gaaaatgata 3720

actaatttat tttctatatt tgatccaaca acaaatttta ttataaaatt ttcaataaat 3780

tgaatatcaa taattttatt tttaattatt attccaaatt tattttgaac aatgcctaat 3840

aatattaatt ttttatatta tatattaatt aataatttat ttaaagaact aaaaattatt 3900

attaaaaaaa aaaataataa ttttatcatt atatttattt ctttatttat attcattata 3960

attaataatt ttttaggatt atttccatat atttttacca gaacaagtca tttagtaatt 4020

aatatatcat tagctttacc tttatgatta agattaataa tttttggttg aattaattta 4080

actaatcata tacttattca tttagttcct caaggaacac ctaatatttt aattccattt 4140

ataattttaa ttgaaactat cagaagaatt attcgaccta ttactttaac tattcgatta 4200

acagcaaata taattgctgg acatttacta ataactttat taggaaacat aggaccaaaa 4260

ttaattattt tttttatacc ttttttacta tcaattcaaa taattttatt aattttagaa 4320

tcagctgtat caattattca atcctatgtt tttgctatct taataatttt atatacaaga 4380

gaaatttaaa ttataaatta taatcataat ttccatttag taaattacag accttgacca 4440

ttaattagat ctttatctac aatattatta ttattaagga ctattaaaat atttaatcaa 4500

aaaaatttaa taatatatat attatcaatt ataattattt tattaattat atatcaatga 4560

tgacgagata tttctcgaga aagaactctt caaggattac attcttttaa tgtttttaaa 4620

ggaataaaaa taggaatatt attatttatt atttcagaaa ttttattttt tacttcattt 4680

ttttgaagat tttttcattt aagacttacc caattaattg aattaggtat aaaatgacct 4740

cctataggaa ttacaccatt taatccattt tcaattccat tattaaatac tattatttta 4800

ttaacatcag gaatttctat tacatgaact catcatagtt taataaataa taattttaat 4860

caatttttta atagactttt attaacaatt attttaggta tttacttttc aacattacaa 4920

gtttttgaat atttagaagc tccttttaat atagctgatt catcttatgg atcaacattt 4980

tttttagcta caggatttca tggtttacat gtacttattg gaacaatttt tttactaatt 5040

tgttttttcc gaaatttatt aattcatttt tcaaataatc atcattttgg atttgaagct 5100

gctgcttgat attgacattt tgtagacatt gtatgacttt ttttatttat atcaatttat 5160

tgatgaggta attaaaattt aaattaatat aatatagtat atataatttc caattataaa 5220

gaaatttatt aaaaccaaat agaggtatat tattgttaat aataaaattg aattaaaaaa 5280

attctaataa tatatattat tttactataa attttttaat attttcttct atttcattat 5340

taattataaa aatatctata ttaatttcat ataaattaaa attaaactta gaaaaaatat 5400

ccccatttga atgtggattt aatccttttt ttttaaacgt ataccatttt ctttacaatt 5460

ttttttaatc tctgtaattt ttttaatttt tgacattgaa attgctttaa tatttccaat 5520

aattaaatct ataaatttct ctaataaatt aatttgaata ttaactagaa ttatatttat 5580

tataatttta atttttggac tattttatga atgaaaaaat ggagccttaa attgaattta 5640

aaaaattaat ttaatttaaa atatatatat tgctaatata aatttatatc ataaataatg 5700

aaattatctt cgaaataata tattaataat taatttaaaa aaaaaatatt atattttcaa 5760

tataaaatta tataattttt atattaagct tctaacttaa tttataataa ttaatattat 5820

taaattatag aattacatat ataaattttc aatttattac tttaatcagt catttttttt 5880

tttaaaaaaa atgacaatat atcatatata aaatatcaat ttattacttt aatcagtcat 5940

ttttttttta aaaaataaaa aataataaaa ataataataa aaaaaaattt ttaaaattaa 6000

aaaaattaaa aaaataaata ttaaaaaaac tatttttaaa aaaccttccc cctaaaaatt 6060

ctattcatcc taaatctaaa aaaaaattat attttttaat aaaatttaaa ataattttat 6120

taaaaaataa acaaaaaata aatattaaaa atcatattga agaaaaaaaa aatatagaat 6180

aaattttatt aaacataaaa aaatcatatt taaaaaatac tccaaaaatt aaacctataa 6240

aacaaataaa taaagtaaat aattttataa aaattgttaa aacaattaaa ttaaaaaaag 6300

gaaaaattaa ccatattaaa aaagatcctc caaaaacaga aaaaatcatt aaaaatttta 6360

tagaaaataa tataattact ctttcaccta tacacaaaat agaaaatcct atataattaa 6420

ttaaacaaaa cctaattaaa cgaaaagaat atataacagt tattttaacc agtagaaaaa 6480

aaaaaaaaaa aattaaaata ttaatattag atattattac aaattctaaa attatatctt 6540

tagaataaaa tccagctaaa aatggtatac cacataatgc taatctagaa atattaaaaa 6600

ctaaagttaa aactatttga ttactaaatc ccccagaaaa tcgaatatct tgaaaattta 6660

ttaaactatg aataaaaatt ccagcacata aaaataataa agctttaaat aaagcatgag 6720

ttaataaatg aaaaaaagct agttttataa aacctaaaga taaaattctc attatcaaac 6780

ctaactgtct taaagtagat aaagcaataa ttttttttaa atctgtttca taattagctc 6840

ctaatccaga tataaatata gttattctac ctaaaaataa taaaaaaatt aaaaaacttg 6900

aatttaataa taaataataa ttaaatcgaa ttaataaata tactccagca gttactaatg 6960

tagaagaatg aactaatgca gaaacaggag taggagctgc tatagcagct ggtaatcaag 7020

aagaaaaagg aatctgtgct cttttgttat agctgctata attaatatta ataaaaaaaa 7080

aatttaaatt tataaattct aaataaaaat aaaatctaaa tcttccaata ttaaatatta 7140

aagaaataca aattaataaa ccaacatctc ctactcgatt agataaagct gtaagtattc 7200

cagcattaaa tgatttataa ttttgaaaat aaattactaa acaataagaa actaaaccta 7260

atccatctca tcctaaaaga atagaaataa aattaggaga aataattaaa aatattatag 7320

atataataaa taaaaaaatt aaatagataa atcgaatttt atatttatca ttaattatat 7380

aatcaattct ataaaaaact actattgaag aaataaataa aacaaaagat ataaataaaa 7440

atcttattca atcaaatata aaattaattg ataatctaca agaatttaaa tttattaatt 7500

ctcattcaaa aaaatatact aaatctttaa ataaaaaata taaaaatata aaaaaagttc 7560

taaatctcga aaacaaaata aataaatata tatatataaa tatttatttt taaattcaca 7620

aattaaaatt tttattaaat taatataaaa aaatataaat aaagatttta aaaaaataat 7680

atttaaagga aatcaatgaa ataatattaa taaaaattct cgttgattat ttatagaaaa 7740

tctataaatt ctagaaaaaa atactccatg ttgactaatt gaatataaat aaaatctata 7800

agatgctcta aaaaacataa taatattaat aaaataatat taattaaaga tcaagatatt 7860

aatctattga ttaatttaat ttcagaaatt aaatttaaag ataaaggagc tgatatattt 7920

ctagaagata aaagaaatca tcaaaaagct atagaaggta aaaaattaat tattccttta 7980

ttaattaata tacttcgtct aaataaacgt tcataagaaa tattagctaa acaaaataaa 8040

ccagaagaac acaatccatg agcaattatt atcaaaaatc tcccacttat acctcaaaaa 8100

gtaatagtaa acaatccccc taacactaaa cctatatgag ctacagaaga ataagcaatt 8160

aatattttta aatctaattg tcgtaaacaa tttaatctaa ttataattcc accgaataaa 8220

cttaaaatta taaaaaataa atttaaattt ttaaatttta ataaaaacat taaaactcga 8280

aataatccat atcctcctaa ttttaataaa atagcagcta aaactattga acctgaaata 8340

ggtgcttcaa catgagcttt aggcaaccat aaatgaacaa aaaatattgg tattttaact 8400

aaaaaagcta taattaaact aaaatataat aaattataaa aaattttatt attaaaattt 8460

ataataatta accttttaaa ataataatta atatacaaaa tagctattaa taaaggaaga 8520

gaagctaata aagtataaaa taataaatat aacctagcta taattcgttc aggttgatac 8580

cctcatccta taattaaata aataatagga ataagtctta tttcaaaaaa aatataaaaa 8640

aataaaaaat tattcataaa aaatctaaaa aataaaaaaa taaatataaa aaataatata 8700

aaaattatta aaaaaatatt attttttaaa aatataatta tatttataga taataatatt 8760

aaaaaaataa ctcaaatagt taataaaatt aaaaaataat ttaaattatc caaaaaaatt 8820

aaattattaa gactataaat aaaaaaagaa ggataaaaaa aaattataaa aatttgataa 8880

ataataatta ttaagtatat aaaatttcaa aaatttaaaa aaaaaataaa atattataat 8940

aaaaataatt ttaaacattt aaatttttat taaatttaaa ttttgaatat tagaatttcc 9000

taaaatacga attaatgaaa ttaataaaga taaccctaaa gctccttcac aaactataaa 9060

agttaaaaat attattaaaa aaaatatttc ataattaaaa atcaataaat taaaaaaaat 9120

atttattaat aatcttaata ttataaattc aatccttaat aaattaatca ataaatattt 9180

aaaattaaaa aaaaactata ataccacaaa aaaaaaatat ttaataaaaa ataataatta 9240

aaatataaat aaattatttt aaaatcttta atatccaaaa ttaaaatttt aattaaacta 9300

taatttaatt taaaataata attttgtaaa ttatttttaa aaaattttta tattaatata 9360

tataataaat attttatttt taaatattaa taatccttta ctaataggca ttttaatttt 9420

attacaaaca ttaaatatta ttataataat aggaaaaatt aattattcat tttgattttc 9480

ttatattatt tttttagttt ttgtaggagg tttatttatt ttatttattt atataatttc 9540

tttaacttat aataataaat ttttttcaat taattataaa tatatatata tattattttt 9600

tttaattttt attataattt taaaaaaaaa taatatatct tatttaaatg aagaaacaaa 9660

aaaaactttt aaagaaattt taataaattt tataaaattt tttaattttc ctaataattt 9720

tttaaacatt ttattaatta tttatttatt aattttaata attattatcg taaaaattac 9780

taatttttac aaaggtcctt taaaaacatt ttataaaaaa aaataaacta ttaattaata 9840

atttaaataa ttcattaaat cttcctactc caattaatat taatttattt tgaaattttg 9900

gatcactatt aggattatgt ttaattattc aaattttaac aggaatattt ttagcttttc 9960

gttattctcc tgatattatt ttagcatttt atagagtaaa taatatcata aatgaaacat 10020

gaagtggatt tatatttcgt aatatacatg ctaatggagc ttctatattt tttatgttta 10080

tattttcata ttggacgagg aatttactta agtcttttat ttttaaaaaa acatgaacta 10140

ttggagtaat tttattatta ataacaatag gaacagcatt tcttggatat gtattaccat 10200

gaggacagat atcattttga ggatcaacag taattacaaa tcttctatcc tcaattcctt 10260

atttaggaaa ttatttagtt caatgaatct gaggaggatt ttcaattaat aatccaacat 10320

taactcgatt tttttgtcta cattttattt tacctttttt aattataaca ataacaattg 10380

tacatttatt ttttttacat ttaacaggat caaataatcc attagggata aatagtaata 10440

aaataaaaat tccatttaat ccattttttt cactaaaaga tttaatagga tttttatttt 10500

tatttattat tattttttat attatttctt ttaaacccta tatattagga gatagagata 10560

attttatttt agctaatcct ttagtaactc cagaacatat tcaaccagaa tgatattttt 10620

tatttgcata tgctatttta cgctctattc caagaaaatt aggaggagtt attgcattaa 10680

taatatccat tataatttta ttttttttac cattattaaa tataagaaaa ttaaaaagta 10740

atcaatttta tcctataaga aaattaattt tttgaacatt aatttttaca attattttat 10800

taacttgaat tggaataaaa cctattgaat atccttttat aattttagga caaattatat 10860

ctataattta tttttttatt tattttttta atccattatt acaaaaatat tttgataatt 10920

ttatttatta attatttatc ttaaataaag aatttgtttt gaaaacaaat aatataaaaa 10980

taaaaattta aaccttaaaa ataaaagtaa aaaaaataaa gaaataggta aataaatttt 11040

tcaagttaga tatatcaatt tatcataacg atatcgagga aaagatcctc gtactcaaat 11100

aaaaataaaa gttaaaaaaa taaactttaa ataaaaaatt aaataaaata tagaattaaa 11160

taaaaatata aaactaaaaa ttatacttaa aaataaaatt atagaatatt cagataaaaa 11220

aattaaagca aatcctcctc ttctatattc tacattaaat ccagaaacta actcagattc 11280

tccttcagca aaatcaaaag gtgatcgatt agtttcagct aaaaaaataa taaataaaat 11340

tgctctaata aataaattta ataataaaaa tcttataaat tcttgatata ataaaaattt 11400

tttaatatta aatctaaaaa ttaaaataaa ataacaaata aaaaataaaa ttatacaaac 11460

ttcataagaa atagtttgag caatagctcg taatcttccc aataaagaat aattagaatt 11520

agaagatcat cctgaaaata taataaaata aacacctata cttatacaac taaataaaaa 11580

taaaaatata aaatttaaag taataaaatt aaaaaataaa ggaaatgtta accaaataaa 11640

taaagaaatt aaaaaagata ataaaggaca actataataa actaaataat tagaataaat 11700

tggaaaaata aactctttta aaaataattt aattgcatca caaaaaggtt gtattaatcc 11760

taaaaatcct actttattag gaccttttcg aatctgaata taacttaaaa ttttttgttc 11820

taataaagtt aaaaaagtaa ttcttactaa aataaaaatt aaaaaaataa aattaacaac 11880

aattttaata atttattata attaaattct aaatttaata cattatattc tgtcaattta 11940

ttataaaata taaaatttta aaaattaatc ctttcgtact aaatttttaa aaataaaaaa 12000

agatagaaac caatctggct tacaccgatt tgaactcaaa tcatgtaaaa atttaaaagt 12060

cgaacagact taatatttat atatcttcat ataaaaataa ttttaattca acatcgaggt 12120

cataaacatt attaaaaata agaacttttt aaaactatta tgctgttatc cctaaggtaa 12180

tttaaatttt taatcaataa aaaaagatca aaaatacatt tataaatgaa ttaaatatat 12240

aaagtttata aaatttattt atctccccaa taaaataaaa atttaataat taaaataata 12300

ataataataa aaaattaata ataatttata aaattctata gggtcttctc gtcccatttt 12360

aaaacattag ctttttaact aataaataaa attcaattat taaaattaaa aaagatttta 12420

tttcatttta ccattcattc tagatttaaa ttaaaaacct atttattatg ctaccttaat 12480

acagtcaaaa tactgcagcc ttttaaaaaa attcaatggg caggttatat ttttaattaa 12540

atataaaaac tatgtttttg ataaacaaat gaataatttt ttttgctgaa ttaataaatt 12600

ttatttatat ttttttacta atttatttat tattttatta tataaattat taatataatt 12660

attttaataa aaattaaaat ttaattataa ataaaatata aaaatataaa ttataaaatt 12720

ttttaaaatt ataattactt aaaaatctaa ataaattaat tattcaataa aatttttata 12780

ttttaaatta agataatccc taatttaatt aaataataat taaaaattta aataaaacta 12840

taaaaatttt attaatatta taaattaaat ttaattctaa aaaaactaaa tataataaaa 12900

atcgaaaaac attttattac tatttaaaaa ttattaataa ttttataata ttaaatttta 12960

tatttaaata gatcttttta ttttgagaaa aataaataat taattaattt taaataaccc 13020

taatacaaaa ggtactaaaa attaattatt ttattattta aatttaaata aattattcaa 13080

tttatttaaa tttaatataa aaatttctta aataatactt taaattaaat tatttttttt 13140

tattattaaa aaaaaaaaaa tttttataaa ttaaaaattt aatttaaata tattaaataa 13200

taattttatt gtaaataaaa taaataaatt tttttttcta aatataactt ccattatatt 13260

tactatgttt cgacttattt taataaaatt aaaagcgacg ggcgatttgt gcatattttt 13320

aaattaatta atatttaata tttattaaat attaatttta aatccaattt taaaaatttt 13380

ttacaaaatt atatccaaaa atatttttaa tgtattccat tttaaaattt tatatattta 13440

atatattatg atttgataaa aaataaaaat aaataaaaat aaattttcaa aaaattattt 13500

taacaacaat atataattta attaatataa ataaatttta ttgtgtatta tcaattaata 13560

aatagaatcc tctaattttt aaaaatacta ccaatttatt taacttttta aaaaaaaaat 13620

attaattaat aaaattataa aaaaaataat agggtatcta atcctagttt ttaaaattta 13680

taaataaaat aaaaatattc cactaactta tattttaatt aataaaaatc aataaaattt 13740

ataattaaat taaagtataa ccgcaaatgc tggcacttta ttatttattt aatttattac 13800

tataaataat ttcattaata aaaataattt aaaatattaa attaaattat atttattata 13860

tttatttata ttataatttt acttatatta taattttaaa taataataat ttttttttat 13920

atatatttaa atttattttc attttaaaat ttaaaatata tatttaaatt tattttatat 13980

atttatttaa tatatatata tttaaattta ttttcatttt aaaatttaaa atatatattt 14040

aaatttattt tatataaata aaaaaaaacc aataaaaatt aataaggaaa aaaaaaacca 14100

ataaaaatta ataaggaaaa aaaaagttat taaaaataaa aaaaaaccaa taaaaattaa 14160

taaggaaaaa aaaaaaccaa taaaaattaa taagaaaaaa aaaagttatt aaaaataaaa 14220

aaaaaccaat aaaaattaat aaggaaaaaa ag 14252

<210> 15

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

cctgttcctg tttcaactat tcttctaatt aataat 36

<210> 16

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

gtttatcctc ctctttcttc aattattgct cata 34

Claims (10)

1. The specific LAMP primer for the pine needle sheath gall mosquito species is characterized in that the nucleotide sequence of the specific LAMP primer for the pine needle sheath gall mosquito species is shown in SEQ ID NO. 1-4.

2. The application of the LAMP primer specific to the pine needle sheath gall mosquito species in the preparation of the pine needle sheath gall mosquito detection kit in claim 1.

3. A kit for detecting pine needle sheath gall midge is characterized by comprising a pine needle sheath gall midge species specificity LAMP primer with a nucleotide sequence shown in SEQ ID No. 1-4.

4. The detection kit according to claim 3, further comprising a color-changing premix.

5. Use of the test kit according to any one of claims 3 to 4 for the visual detection of pine needle colecidomyiia.

6. A method for detecting pine needle cecidomyiia is characterized in that DNA of a sample to be detected is used as a template, and pine needle cecidomyiia species-specific LAMP primers shown as SEQ ID No.1-4 are used for amplification.

7. The method according to claim 6, wherein the detection of the pine needle sheath gall midge is performed by using the detection kit according to claim 4 by using DNA of a sample to be detected as a template.

8. The method according to claim 7, wherein in the reaction system for amplification, the ratio of the color-changing premix: 1-2 of SEQ ID NO: SEQ ID NO.3-4 is 25: 8:1.

9. The method according to claim 8, wherein the amplification reaction condition is isothermal amplification at 61 ℃ for 60 min.

10. The method of claim 9, wherein the determination criteria of the detection result are: the sample containing the pine needle sheath gall midge is amplified, and the reaction system is changed from red to yellow; the sample without pine needle sheath gall midge keeps red after amplification.

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