Species-specific LAMP primers for detecting Thecodiplosis japonensis and their applications

Abstract

The present invention relates to the technical field of molecular detection, and particularly relates to species-specific LAMP primers for detecting Thecodiplosis japonensis and their applications. The nucleotide sequences of the species-specific LAMP primers for Thecodiplosis japonensis provided by the present invention are shown in SEQ ID NO.1-4. The present invention also provides a method for detecting Thecodiplosis japonensis, which can quickly and accurately identify Thecodiplosis japonensis, a major forestry quarantine pest, in a short time, especially the identification of other insect states (eggs, young larvae and pupae) except adults and mature larvae. The method provided by the present invention can be used for the rapid detection of Thecodiplosis japonensis in quarantine work such as the transportation of seedlings at home and abroad.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular detection, and particularly to species-specific LAMP primers for detecting Thecodiplosis japonensis and their applications. Background Art

[0002] Thecodiplosis japonensis belongs to the genus Thecodiplosis of the family Cecidomyiidae in the order Diptera, synonym: T. pinicola, and its English name is Pine needlegall midge. Thecodiplosis japonensis is a small insect that harms Pinus thunbergii, Pinus densiflora, and Pinus tabulaeformis, with a body length of 2.5 - 3 mm. This insect is an initial pest. The larvae inhabit the base of pine needles and suck the sap to damage the host, resulting in the swelling of the base of the needles to form galls. The damaged pine needles are significantly shortened, gradually turn yellow and wither, and fall off, seriously affecting the normal growth of pine trees. After being damaged by it, the tree vigor of the plant weakens, it is easily damaged by secondary pests, and the plant may die; when severely damaged, it can also cause the death of pine trees in the current year alone.

[0003] Research shows that Thecodiplosis japonensis can harm a variety of Pinus plants. The hosts that produce galls at the base of the damaged pine needles mostly belong to the subsection Sylvestres of Pinus sylvestris, including: Pinus resinosa, Pinus nigra, Pinus mugo, Pinus insularis, Pinus coulteri, Pinus sylvestris, Pinus luchuensis, Pinus radiata (Australia, South Africa), Pinus taiwanensis, Pinus thunbergii, Pinus massoniana, Pinus densiflora, and Pinus tabulaeformis. According to the prediction of the CLIMEX model and the MaxEnt niche model, most provinces in the south of China are its potential geographical distribution areas. Therefore, Thecodiplosis japonensis has potential harm to southern pine forests.

[0004] Plant quarantine is the most effective means to prevent the invasion and spread of alien harmful organisms. In order to distinguish Thecodiplosis japonensis from common gall midges in China, a large amount of morphological identification work is required. At present, the morphological identification of gall midges mainly relies on the morphological differences of the wing veins of adults and the sclerites of mature larvae. These identification methods require complex sample processing procedures and professional knowledge of insect taxonomy. Moreover, gall midges in the egg, low-instar larva, and pupa stages are very similar in morphology, and there are currently no reliable identification characteristics.

[0005] In recent years, more and more studies have proven that molecular biology methods can provide strong evidence for pest identification. Mitochondrial DNA is strictly maternally inherited, and the cytochrome oxidase I gene (COI) in it is highly conserved, has a stable structure, and no introns. Therefore, it is often used in the research of species classification, identification, and genetic relationship. There are also reports on designing specific primer pairs for the COI gene sequence of Thecodiplosis japonensis to conduct molecular identification on it, but this SS-COI method requires equipment such as a PCR instrument and a nucleic acid electrophoresis instrument, and is suitable for the detection of professional institutions. The present invention will propose a simple detection method.

[0006] Loop-mediated isothermal amplification technology (LAMP) relies on 4 specific primers and a DNA polymerase with strand displacement activity (Bst DNA polymerase), and can efficiently, rapidly, and specifically amplify the target sequence under isothermal conditions. After the reaction, it only needs to be observed with the naked eye to identify whether amplification occurs. Moreover, the equipment used in the LAMP reaction is simple, the cost is low, and it can meet the need for rapid detection, and has been increasingly widely used in the fields of scientific research of nucleic acids, diagnosis and prevention of diseases, sex identification of animal embryos, and detection of genetically modified foods. Summary of the Invention

[0007] The object of the present invention is to provide a method for rapid visual identification of Thecodiplosis japonensis, which can quickly and accurately identify Thecodiplosis japonensis, a major forestry quarantine pest in China, in a short time, especially the identification of other insect states (eggs, low-instar larvae, and pupae) except adults and mature larvae.

[0008] Due to the tiny body of Thecodiplosis japonensis, morphological identification is very difficult, and it requires experts with a strong professional background and rich experience to complete the identification work. And it can only be carried out when adult and mature larva samples are obtained. Gall midges in the three insect states of eggs, low-instar larvae, and pupae are very similar in morphology, and it is very difficult to accurately identify them morphologically. The conventional PCR amplification identification method based on specific primers needs to use a PCR amplifier to perform thermal denaturation and temperature cycling amplification on the template. In addition, electrophoresis and ultraviolet observation and other processes are required to judge the reaction results. The whole process is cumbersome, highly professional, needs to be completed in a professional institution with certain conditions, and is time-consuming.

[0009] To achieve the object of the present invention, in the first aspect, the present invention provides Thecodiplosis japonensis species-specific LAMP primers, and the nucleotide sequences of the Thecodiplosis japonensis species-specific LAMP primers are shown in SEQ ID NO.1-4.

[0010] According to the understanding of those skilled in the art, the present invention also claims the application of the above-mentioned Thecodiplosis japonensis species-specific LAMP primers in the preparation of a Thecodiplosis japonensis detection kit.

[0011] In a second aspect, the present invention provides a detection kit for Thecodiplosis japonensis, wherein the detection kit contains Thecodiplosis japonensis species-specific LAMP primers with nucleotide sequences as shown in SEQ ID NO.1-4.

[0012] In the detection kit provided by the present invention, it further includes a color-changing premix WarmStart Colorimetric LAMP 2X Master Mix.

[0013] The present invention also claims the application of the above detection kit in the visual detection of Thecodiplosis japonensis.

[0014] In a third aspect, the present invention provides a method for detecting Thecodiplosis japonensis, using the DNA of a sample to be tested as a template and amplifying it with Thecodiplosis japonensis species-specific LAMP primers as shown in SEQ ID NO.1-4.

[0015] More specifically, the present invention provides a method for detecting Thecodiplosis japonensis, using the DNA of a sample to be tested as a template and detecting Thecodiplosis japonensis with the above detection kit.

[0016] In the method for detecting Thecodiplosis japonensis provided by the present invention, by volume ratio, in the amplification reaction system, the color-changing premix: SEQ ID NO.1-2: SEQ ID NO.3-4 is 25:8:1.

[0017] In the method for detecting Thecodiplosis japonensis provided by the present invention, the amplification reaction conditions are isothermal amplification at 61°C for 60 min.

[0018] In the method for detecting Thecodiplosis japonensis provided by the present invention, the judgment criterion for the detection result is: for a sample containing Thecodiplosis japonensis, the reaction system turns from red to yellow after amplification; for a sample without Thecodiplosis japonensis, the reaction system remains red after amplification.

[0019] The beneficial effects of the present invention are at least as follows:

[0020] By designing Thecodiplosis japonensis species-specific LAMP primers and optimizing the construction of a rapid molecular detection system for Thecodiplosis japonensis, the present invention can rapidly and accurately identify Thecodiplosis japonensis, a major forestry quarantine pest, in a short time, especially for other insect stages (eggs, young larvae, and pupae) except adults and mature larvae. The invention can be used for the rapid detection of Thecodiplosis japonensis in quarantine work such as the domestic and international transportation of seedlings. Description of the Drawings

[0021] Figure 1Amplification results of four gall midges using specific LAMP primers for Thecodiplosis japonensis. A shows the amplification results detected under visible light, B shows the results detected under ultraviolet light, and C shows the results detected by electrophoresis. In the figure, NC is the control; + represents Thecodiplosis japonensis; TSP represents Thecodiplosis sp. of Pinus massoniana; CSP represents Contarinia sp.; CCA represents Contarinia caryafloralis.

[0022] Figure 2 Electrophoresis diagram of adult and larval DNA samples amplified by specific LAMP primers for Thecodiplosis japonensis. A shows the amplification results detected under visible light, B shows the results detected by electrophoresis, and NC is the control.

[0023] Figure 3 Sensitivity results of species-specific primer LAMP for Thecodiplosis japonensis. A shows the amplification results detected under visible light, B shows the results detected under ultraviolet light, C shows the results detected by electrophoresis, and NC is the control.

[0024] Figure 4 Electrophoresis results of three groups 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).

[0025] Figure 5 Amplification curve of SEQ ID NO.1-4.

[0026] Figure 6 Amplification curves of SEQ ID NO.5-8 and SEQ ID NO.9-12.

[0027] Figure 7 Amplification efficiency of SEQ ID NO.1-4 at different temperatures.

[0028] Figure 8 Amplification efficiency of SEQ ID NO.1-4 at different primer ratios.

[0029] Figure 9 Amplification efficiency of SEQ ID NO.1-4 after adding loop primers. Detailed implementation methods

[0030] The following examples are used to illustrate the present invention, but do not limit the scope of the present invention.

[0031] Example 1 Design of species-specific LAMP primers

[0032] The present invention takes Thecodiplosis japonensis as the target, and uses three other common gall midge species in China as references, designs species-specific LAMP primers based on the mitochondrial COⅠ gene sequence, and constructs and optimizes a rapid molecular detection system for Thecodiplosis japonensis.

[0033] The design process of the species-specific LAMP primers for Thecodiplosis japonensis is as follows:

[0034] 1. Extraction of insect genomic DNA

[0035] Use a DNA microextraction kit to extract the genomic DNA of different species of Cecidomyiidae collected from 4 regions in China.

[0036] (1) Put the whole insect body into a 1.5 ml centrifuge tube, add 200 μl of buffer PBS, and grind thoroughly with a grinding rod.

[0037] (2) Add 150 μl of buffer PBS and 0.9 μl of Rnase A, and then gently grind for 30 s.

[0038] (3) Collect 350 μl of the ground tissue homogenate and transfer it to a 2 ml centrifuge tube. If the volume of the homogenate is less than 350 μl, supplement PBS to 350 μl.

[0039] (4) Add 150 μl of buffer C-L and 20 μl of proteinase K. Immediately vortex for 1 min to mix evenly. After a short centrifugation, place the centrifuge tube in a 56 °C water bath for 10 min.

[0040] (5) Add 350 μl of buffer P-D, vortex for 30 s to mix evenly, and centrifuge at 12000×g for 10 min.

[0041] (6) Place the DNA preparation tube in a 2 ml centrifuge tube, transfer the mixture from the previous step to the preparation tube, and centrifuge at 12000×g for 1 min.

[0042] (7) Discard the filtrate, place the preparation tube back into the original 2 ml centrifuge tube, add 500 μl of buffer W1, and centrifuge at 12000×g for 1 min.

[0043] (8) Discard the filtrate, place the preparation tube back into the original 2 ml centrifuge tube, add 700 μl of buffer W2, centrifuge at 12000×g for 1 min, and wash again with 700 μl of buffer W2 in the same way.

[0044] (9) Discard the filtrate, place the preparation tube back into the original 2 ml centrifuge tube, and centrifuge at 12000×g for 1 min.

[0045] (10) Discard the filtrate, place the preparation tube back into the original 2 ml centrifuge tube, add 100 μl eluent to the center of the membrane in the preparation tube, let it stand at room temperature for 1 min, and centrifuge at 12,000×g for 1 min to elute the DNA.

[0046] (11) DNA concentration detection. Use a ultra-micro spectrophotometer to detect the concentration of the extracted DNA. Use Nanodrop2000 (Thermo company). Before detection, first wash the detection well with deionized water, dry it, then aspirate 1 μl of the elution buffer Elution Buffer used during DNA extraction for calibration. After calibration, aspirate 2 μl of the sample and add it to the detection well, and measure the concentration of all DNA samples separately.

[0047] 2. Design of specific LAMP primers for Thecodiplosis japonensis

[0048] Based on the mitochondrial genome sequences of Thecodiplosis japonensis (SEQ ID NO.13) and its related species Thecodiplosis sp. of masson pine (SEQ ID NO.14), and the base sequences of 8 other gall midges publicly available in the database, a group of specific LAMP primers for Thecodiplosis japonensis were manually designed as shown in Table 1: including two outer primers (Tjap_F3 and Tjap_B3) and two inner primers (Tjap_FIP and Tjap_BIP).

[0049] Table 1 Specific LAMP primers for Thecodiplosis japonensis

[0050]

[0051] Example 2 Species specificity and sensitivity test of LAMP primers for Thecodiplosis japonensis

[0052] Using the DNA of common gall midge insects in China as the template and the DNA of Thecodiplosis japonensis as the positive control, the species specificity of the LAMP primers for Thecodiplosis japonensis was tested. Using DNA standards of Thecodiplosis japonensis at different concentrations as templates for amplification, the sensitivity of the primers was tested.

[0053] 1. Preparation of standards

[0054] After measuring the concentration and purity of the DNA of 4 species of gall midges, a mother liquor with a concentration of 30 ng / μl was prepared. And the DNA sample of Thecodiplosis japonensis was serially diluted into standard solutions of 30 ng / μl, 3 ng / μl, 300 pg / μl, 30 pg / μl, 3 pg / μl, 300 fg / μl, 30 fg / μl, 3 fg / μl according to the ratio of (1:10). All standard solutions were stored at 4°C for later use.

[0055] 2. Amplification of LAMP species-specific primers

[0056] LAMP amplification was carried out using the WarmStart Colorimetric LAMP 2X Master Mix (NEB). The specific formulation of the reaction system is shown in Table 2. In the control group, DNA was replaced with ultrapure water. During detection, the reaction system was placed in a 61 °C constant-temperature metal bath for 60 min of amplification.

[0057] Table 2 LAMP reaction system (25 μL)

[0058]

[0059] 3. Detection of amplification products

[0060] Direct visual detection: After amplification, the color of the reaction system of positive samples changed from red to yellow, while that of negative samples remained red.

[0061] UV lamp detection: 0.5 μL of GelRed dye was added to the amplified system, and then it was observed under a UV lamp to check whether amplification occurred.

[0062] Electrophoresis detection: The amplified stock solution was used to detect the amplification products by 1.5% agarose gel electrophoresis pre-stained with GelRed dye (voltage 130 V, time 30 minutes). 1×TAE was used as the electrophoresis buffer, and DL2000 DNA Marker was used as a marker.

[0063] 4. Specificity test results of the LAMP primers for Thecodiplosis japonensis

[0064] Using the specific LAMP primers for Thecodiplosis japonensis designed in the present invention for amplification, only Thecodiplosis japonensis was successfully amplified, and it had no amplification ability for the other 3 species of gall midges and the negative control (see Figure 1 ), indicating that this pair of primers is a species-specific primer for Thecodiplosis japonensis.

[0065] Using the DNA of Thecodiplosis japonensis adults and larvae as templates and the specific LAMP primers for Thecodiplosis japonensis for amplification, the results showed that specific fragments could be stably amplified from the DNA extracted from both adults and larvae (see Figure 2 ).

[0066] 5. Sensitivity test results of the LAMP primers for Thecodiplosis japonensis

[0067] Specific primer sensitivity tests were carried out using Thecodiplosis japonensis DNA standard products with different concentration gradients (see Figure 3 ). The minimum detection limit of the test was 300 fg of genomic DNA.

[0068] The above results of the present invention indicate that the amplification and result judgment procedures can be completed using a single constant temperature metal bath. In combination with a DNA crude extraction method for on-site operation, the present invention can be used for the rapid on-site identification of Thecodiplosis japonensis.

[0069] Specificity Results of Different LAMP Primers in Comparative Example 1

[0070] This Comparative Example 1 provides 2 sets of Thecodiplosis japonensis LAMP primers, and the sequence information is shown in SEQ ID NO.5-8 and SEQ ID NO.9-12. The design method of the Thecodiplosis japonensis LAMP primers in this comparative example is the same as that in Example 1.

[0071] SEQ ID NO.5: TTATCTGATTATTTAATCCTCCTGAAATTCG;

[0072] SEQ ID NO.6: CTGCTTTATCAAATCGAATTGGAGATGTATG;

[0073] SEQ ID NO.7: CGGCTCCTACTCCTGTTTCAGCCTCCAGCGGTAACTAAAGTTGAAG;

[0074] SEQ ID NO.8: CCATAGCAGCAGGTAACCAAGAAGAAATTATAGCAGCTATAACTAAAAGAGCAC.

[0075] SEQ ID NO.9: CCTTTTATTTCAATTAAATCAATTGGACATCAATG;

[0076] SEQ ID NO.10: GAGAAATTCTTTCTAATGAAATAGGTATAAATGAGTG;

[0077] SEQ ID NO.11: GAATGTAATACATCTGCTGCTGTTACTATAATTCGCGATTATTAGATACTGATAATAAAATTATCATCCC;

[0078] SEQ ID NO.12: AGTAGATGCAACTCCTGGACGATTAAATCGAACATTGACCAAAAAATATTCTTGATCG.

[0079] The amplification efficiencies of SEQ ID NO.1-4, SEQ ID NO.5-8, and SEQ ID NO.9-12 were compared to screen the primer set with the highest amplification efficiency. The detection results showed that: all three primer sets could amplify the characteristic ladder bands of LAMP amplification ( Figure 4 ), but the primer set of SEQ ID NO.1-4 peaked within 30 minutes ( Figure 5 ), while SEQ ID NO.5-8 and SEQ ID NO.9-12 did not peak within 35 minutes and only started to peak after continued amplification for 15 minutes ( Figure 6 ). The amplification efficiency of the primer set of SEQ ID NO.1-4 was higher than the other two groups.

[0080] Amplification results of different amplification reaction systems in Comparative Example 2

[0081] This comparative example provides the amplification results of the LAMP primers of the pine needle gall midge shown in SEQ ID NO.1-4 under different amplification reaction systems.

[0082] In this comparative example, a temperature gradient was set in the range of 55°C - 65°C, and the highest reaction efficiency was obtained when the reaction temperature was 61°C ( Figure 7 ). When the concentration ratio of the inner and outer primers (Tjap_F3 / Tjap_B3:Tjap_FIP / Tjap_BIP) was 1:4, 1:8, 1:6, 1:10, the highest reaction rate was obtained when the concentration ratio of the inner and outer primers was 1:8 ( Figure 8 ). When the upstream and downstream loop primers (Tjap_FL: (SEQ ID NO.15) CCTGTTCCTGTTTCAACTATTCTTCTAATTAATAAT and Tjap_BL: (SEQ ID NO.16) GTTTATCCTCCTCTTTCTTCAATTATTGCTCATA) were added simultaneously, or when only one loop primer (Tjap_FL or Tjap_BL) was added, the amplification efficiency did not increase significantly. Therefore, the loop primers were not finally added in the present invention ( Figure 9 ).

[0083] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention. Sequence Listing <110> Beijing Forestry University <120> Species-specific LAMP primers for detecting the pine needle gall midge and their applications <130> KHP221114436.4 <160> 16 <170> SIPO Sequence Listing 1.0 <210> 1 <211> 35 <212> DNA <213> Artificial Sequence <400> 1 cgtttagaat taagttctgt atctaattta attgg 35 <210> 2 <211> 33 <212> DNA <213> Artificial Sequence <400> 2 ctgctaatac aggtaaagaa agtaaaagta gaa 33 <210> 3 <211> 56 <212> DNA <213> Artificial Sequence <400> 3 cctgttcctg ttcctgtttc aactattctt cgattacttc ccccctctat ttcttt 56 <210> 4 <211> 68 <212> DNA <213> Artificial Sequence <400> 4 gaactgttta tcctcctctt tcttcaatta ttgctcagaa aaaattgaaa aatctacaga 60 tgttctag 68 <210> 5 <211> 31 <212> DNA <213> Artificial Sequence <400> 5 ttatctgatt atttaatcct cctgaaattc g 31 <210> 6 <211> 31 <212> DNA <213> Artificial Sequence <400> 6 ctgctttatc aaatcgaatt ggagatgtat g 31 <210> 7 <211> 46 <212> DNA <213> Artificial Sequence <400> 7 cggctcctac tcctgtttca gcctccagcg gtaactaaag ttgaag 46 <210> 8 <211> 54 <212> DNA <213> Artificial Sequence <400> 8 ccatagcagc aggtaaccaa gaagaaatta tagcagctat aactaaaaga gcac 54 <210> 9 <211> 35 <212> DNA <213> Artificial Sequence <400> 9 ccttttattt caattaaatc aattggacat caatg 35 <210> 10 <211> 37 <212> DNA <213> Artificial Sequence <400> 10 gagaaattct ttctaatgaa ataggtataa atgagtg 37 <210> 11 <211> 70 <212> DNA <213> Artificial Sequence <400> 11 gaatgtaata catctgctgc tgttactata attcgcgatt attagatact gataataaaa 60 ttatcatccc 70 <210> 12 <211> 58 <212> DNA <213> Artificial Sequence <400> 12 agtagatgca actcctggac gattaaatcg aacattgacc aaaaaatatt cttgatcg 58 <210> 13 <211> 14208 <212> DNA <213> Artificial Sequence <400> 13 atattatata aatttttact taataattca aaattattta tgcataaaca ttattttaag 60 ctaaattaag ctaatggact catatttcat ttataaaaaa aattaaattg aaatataatt 120 ttttttttat ttattaatat ttttttcaat tattatattt ttatcttcta attcatgatt 180 taatatatga ttatgattag aaattaattt aatatcattt atttatttaa taattaataa 240 taaaaataaa atttctattg aatcatcatt aatttatttt ttaattcaaa ctatctcatc 300 tattatttta ttattttcaa ttttaatatt aatatttaaa aataatttta ttttaatttt 360 aattatttca attataatta aaattggaag atcaccattt catttttgaa ttccattaat 420 tattgaaaga ttaaattgaa atataatttt ttttctttta acttgacaaa aaattaatgc 480 attatatata ttattaatta ataattataa taaatattta atttttttta taattttatc 540 tttaattatt ggatctatta caggattaaa ttatatttct ttaaaaaaaa ttatttcttt 600 ttcttctatt aatcaattaa gatgattaat aataagatta ataaataata aaattattaa 660 aatttatttt ttaatttata tatttttaat atgattaatt acaaaatctt ttaatttttt 720 aaatttaaat tttattaatc aaatttttaa attaaaatta aaaatcaatt attttaaatt 780 atttattttt acaaatttac tttctttagg aggattacca ccatttattg gatttttttc 840 taaaataatt ttaattttaa acttttataa tttaactatt ttatttttta taattatttt 900 ttctttatta acattatata tatatatacg aattattttt tctttattaa tattaaattc 960 tataaaaatt aataaaattt ttaaaaatat aaattttaaa aatataaatt atttaataaa 1020 tttatcaatt tttaataatt tatttttaat aatttatatt tttattttta aaaataagtt 1080 aaatttaaac tataaatctt caaaatttaa aattaaattt gcaatttaat tataaataaa 1140 tttatttata aatttacaat ttattacttt taatcagtca ttaaaataaa aaaatgaata 1200 ttttctacta atcataaaga tattggaact ttatatttta tatttggaat ttgagcagga 1260 atattaggaa catcattaag aattttaatt cgtttagaat taagttctgt atctaattta 1320 attggtaatg atcaaattta taatgtaatt gttacagctc atgcttttat tataattttt 1380 tttatagtaa tacctatttt aattggagga tttggaaatt gattaattcc cattatatta 1440 ggagctcctg atatggcatt tccacgaata aataatataa gattttgatt acttcccccc 1500 tctatttctt tattattaat tagaagaata gttgaaacag gaacaggaac aggatgaact 1560 gtttatcctc ctctttcttc aattattgct catactagaa catctgtaga tttttcaatt 1620 ttttctttac atattgcagg aatttcatca attttaggag caattaattt tatttcaaca 1680 ataataaata taaaaattaa atttttaaaa tttgatcaaa tttcattatt tatttgatct 1740 attataatta caacaattct acttttactt tctttacctg tattagcagg agcaattact 1800 atattattaa ctgatcgaaa ttttaatact tcattttttg atcctttagg aggaggagat 1860 ccaattcttt atcaacattt attttgattt tttggacatc cagaagttta tattttaatt 1920 cttccaggat ttggaataat ttcacatatt atttttcaag aaagaagaaa aaaagaaaca 1980 tttggtattt taggaataat ttatgcaata atttctattg gatttttagg atttattgtt 2040 tgagcacatc atatatttac agtaggaata gaagtagaca cacgagctta ttttacaaca 2100 gcaactataa ttattgcaat tcctactgga attaaagttt ttagatgatt agcaactatt 2160 caaggtagac aaataaattt taatccttca ataatttgag cattaggatt tattttttta 2220 tttacaatag gaggaattac aggagttatt ttagctaatt cttctattga tattattctt 2280 catgatactt attatgtagt agcccatttt cattatgttt tatctatagg agctgtattt 2340 gctattataa taagatttat tcattgatac cctttattta ccggacttta tttaaataat 2400 ttaatgttaa atatacaatt tattataata tttattggag taaatttaac cttttttcct 2460 caacattttt taggtttatc tggaatacca cgacgatatt ctgatttccc tgattcattt 2520 ttaatatgaa atataatttc atcaattgga tcatcaattt caataattag aattatattt 2580 ttaattttta ttatttgaga aagaataatt aattttaaaa aaattatttt tcctaatcaa 2640 ttaaatactt caattgaatg attccaaaat tatcctcctg aaactcatag atataaagaa 2700 attcctttaa tttctaaata ttaaaaatat gtcagatcaa tgaaatgaat ttaaaattca 2760 taaataagaa tttcatcatg attaaattta aacttacaaa atagaaattc acctttaata 2820 gaacaattaa ttttttcatg atcattcaat attaatttta attttaatta ttaatttagt 2880 aagatatata ataatttcta taatattaaa taattattat aattattttt tattatcagg 2940 acaaactatt gaaattattt gaactattat acctactatt attctaatat ttattgcttt 3000 tccatcatta aaattactat atttattaga agaaacaaat aaacctttta tttcaattaa 3060 atcaattgga catcaatgat attgaaaata tgaatattca aattttaata tagaatttga 3120 ttcttatata aataaagaaa taataaataa tagatttcga ttattagata ctgataataa 3180 aattatcatc ccatataatt attttattcg aattatagta acagcagcag atgtattaca 3240 ttcatgaact attccttcat taggaattaa agtagatgca actcctggac gattaaatca 3300 attaaatttt ttaattaatc gatcaagaat attttttggt caatgttcag aaatttgtgg 3360 agcaaatcac tcatttatac ctatttcatt agaaagaatt tctcttaaat attttttaaa 3420 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 ttaaattgaa aatgataact aatctatttt ctatatttga tccatcaact aaacttatta 3720 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 tcaaaatact gcagcctttt aaaaattcaa tgggcagatt atatttttta attaataaaa 12540 aaactatgtt tttgataaac aaatgaataa ttttttttgc tgaattatta aattttattt 12600 aaactatgtt tttgataaac aaatgaataa ttttttttgc tgaattatta aattttattt 12600 ttaatatttt actaatttat ttattatttt attacaaaat ttattaatat aataattttt 12660 ttaatatttt actaatttat ttattatttt attacaaaat ttattaatat aataattttt 12660 ataaattata aaatttattt ataaatttat ttacaaatta aaataaatta taaaattttt 12720 ataaattata aaatttattt ataaatttat ttacaaatta aaataaatta taaaattttt 12720 tttaatttta aatacttaaa aaattaaatt attaacttta aatatataaa tttttataat 12780 tttaatttta aatacttaaa aaattaaatt attaacttta aatatataaa tttttataat 12780 tttaaattaa gataatccct aatttaaatt aatataatat ataaattttt atataattat 12840 tttaaattaa gataatccct aatttaaatt aatataatat ataaattttt atataattat 12840 aaaaattaaa aattatatta taaattaaat ttatttttaa aaaaactaaa tataataaaa 12900 aaaaattaaa aattatatta taaattaaat ttatttttaa aaaaactaaa tataataaaa 12900 atcgaaaaat attttatttc tatttaaaaa ttatttataa ttataaaata ttaaaattta 12960 atcgaaaaat attttatttc tatttaaaaa ttatttataa ttataaaata ttaaaattta 12960 taattaaata gatcttttta ttttgagaaa attaaaataa ataattaatt ttaaataacc 13020 taattaaata gatcttttta ttttgagaaa attaaaataa ataattaatt ttaaataacc 13020 ctaatacaaa aggtactaaa aattaattat tttttaaaaa aatttttaaa attatttttc 13080 ctaatacaaa aggtactaaa aattaattat tttttaaaaa aatttttaaa attatttttc 13080 aattaattta tatttataat aaaaattttt tttaaaatta ctttttttaa aattactttt 13140 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 <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 tgacgagata tttctcgaga aagaactctt caaggattac attcttttaa tgtttttaaa 4620 ggaataaaaa taggaatatt attatttatt atttcagaaa ttttattttt tacttcattt 4680 ggaataaaaa taggaatatt attatttatt atttcagaaa ttttattttt tacttcattt 4680 ttttgaagat tttttcattt aagacttacc caattaattg aattaggtat aaaatgacct 4740 ttttgaagat tttttcattt aagacttacc caattaattg aattaggtat aaaatgacct 4740 cctataggaa ttacaccatt taatccattt tcaattccat tattaaatac tattatttta 4800 cctataggaa ttacaccatt taatccattt tcaattccat tattaaatac tattatttta 4800 ttaacatcag gaatttctat tacatgaact catcatagtt taataaataa taattttaat 4860 ttaacatcag gaatttctat tacatgaact catcatagtt taataaataa taattttaat 4860 caatttttta atagactttt attaacaatt attttaggta tttacttttc aacattacaa 4920 caatttttta atagactttt attaacaatt attttaggta tttacttttc aacattacaa 4920 gtttttgaat atttagaagc tccttttaat atagctgatt catcttatgg atcaacattt 4980 gtttttgaat atttagaagc tccttttaat atagctgatt catcttatgg atcaacattt 4980 tttttagcta caggatttca tggtttacat gtacttattg gaacaatttt tttactaatt 5040 tttttagcta caggatttca tggtttacat gtacttattg gaacaatttt tttactaatt 5040 tgttttttcc gaaatttatt aattcatttt tcaaataatc atcattttgg atttgaagct 5100 tgttttttcc gaaatttatt aattcatttt tcaaataatc atcattttgg atttgaagct 5100 gctgcttgat attgacattt tgtagacatt gtatgacttt ttttatttat atcaatttat 5160 gctgcttgat attgacattt tgtagacatt gtatgacttt ttttatttat atcaatttat 5160 tgatgaggta attaaaattt aaattaatat aatatagtat atataatttc caattataaa 5220 tgatgaggta attaaaattt aaattaatat aatatagtat atataatttc caattataaa 5220 gaaatttatt aaaaccaaat agaggtatat tattgttaat aataaaattg aattaaaaaa 5280 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 <400> 15 cctgttcctg tttcaactat tcttctaatt aataat 36 <210> 16 <211> 34 <212> DNA <213> Artificial Sequence <400> 16 gtttatcctc ctctttcttc aattattgct cata 34

Claims

1. Specific LAMP primers for Thecodiplosis japonensis, characterized in that, The nucleotide sequences of the specific LAMP primers for Thecodiplosis japonensis are shown in SEQ ID NO.1-4.

2. Use of the specific LAMP primers for Thecodiplosis japonensis according to claim 1 in the preparation of a detection kit for Thecodiplosis japonensis.

3. A detection kit for Thecodiplosis japonensis, characterized in that, Specific LAMP primers for Thecodiplosis japonensis containing nucleotide sequences shown in SEQ ID NO.1-4.

4. The detection kit according to claim 3, characterized in that, In the detection kit, a color-changing premix is further included.

5. Use of the detection kit according to any one of claims 3-4 in the visual detection of Thecodiplosis japonensis.

6. A method for detecting Thecodiplosis japonensis, characterized in that, Using the DNA of the sample to be tested as a template, amplification is carried out using the specific LAMP primers for Thecodiplosis japonensis shown in SEQ ID NO.1-4.

7. The method according to claim 6, characterized in that, Using the DNA of the sample to be tested as a template, the detection kit according to claim 4 is used for the detection of Thecodiplosis japonensis.

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

1.

9. The method according to claim 8, wherein The reaction conditions for the amplification are constant temperature amplification at 61 °C for 60 min.

10. The method according to claim 9, wherein The judgment criteria for the detection results are as follows: for a sample containing Thecodiplosis japonensis, the reaction system turns from red to yellow after amplification; for a sample without Thecodiplosis japonensis, the reaction system remains red after amplification.

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