Disclosure of Invention
Aiming at the problems of long period, poor specificity, low sensitivity and fewer specific detection targets of a detection method for Fusarium proliferatum (F.proliferatum) in the prior art, the invention provides a new detection target FPRO-09882 of Fusarium proliferatum (F.proliferatum) and an RPA-LFD detection primer composition based on the new detection target. Another objective of the invention is to provide an RPA-LFD detection kit for Fusarium stratiotes (F.proliferum) described above.
In a first aspect, the invention provides a specific detection target FPRO-09882 of Fusarium proliferatum, wherein the protein sequence of the detection target is shown in SEQ ID NO: 1, and the following steps:
MRRVLGPIAHDKWKNKSTMIGWCLYRAEAGEFTHLLRHWNATEVETAGRLIAWGVPNFVLPALLGLPLAEARTGDDETIYRWVKDEERILEHQYDPFQEGRFNDGGSSADNGYGRSENPYRGRENISGPLGVTHVSARRGNSGSLQQDNRLQEERPGRAGNSTETSHETEAHTLGEIDGLGGFDNFGYREPSNRTRGEGPRSVGNGTKNPYKSNRDGLDR(SEQ ID NO.1)。
in another aspect, the invention provides a fusarium proliferatum-specific detection target FPRO _09882, wherein the nucleotide sequence of the detection target is set forth in SEQ ID NO: 2, as shown in the figure:
ATGCGACGCGTCCTCGGCCCCATCGCTCACGACAAATGGAAAAACAAGTCGACAATGATAGGCTGGTGTTTATATCGGGCTGAGGCCGGCGAATTTACTCACTTACTAAGGCATTGGAATGCTACAGAGGTGGAAACGGCTGGGAGGTTAATAGCATGGGGAGTACCAAATTTTGTCCTTCCAGCTCTGCTGGGTCTACCGTTGGCAGAGGCTCGAACCGGGGATGATGAGACGATTTACCGATGGGTGAAGGATGAAGAGAGGATACTGGAGCATCAATATGATCCGTTCCAAGAGGGGCGATTCAACGATGGTGGAAGCAGTGCCGACAATGGGTATGGGAGAAGTGAGAATCCGTATAGGGGACGGGAGAACATATCGGGGCCTTTGGGGGTGACTCACGTATCAGCAAGACGTGGGAACTCCGGGAGTTTGCAGCAAGACAATCGTCTTCAGGAGGAGAGACCCGGGAGGGCTGGTAACAGTACCGAGACGTCACATGAGACGGAAGCGCATACCTTGGGGGAGATAGACGGGCTAGGAGGTTTCGACAACTTTGGGTATAGGGAGCCAAGCAATCGTACCCGAGGAGAGGGACCCAGGAGTGTTGGTAACGGTACCAAGAACCCGTACAAGTCGAACCGGGATGGTTTAGACCGTTAA(SEQ ID NO.2)。
in another aspect, the invention also provides a primer and probe combination for detecting fusarium stratiotes, wherein the forward primer FPRO _09882F1 has the sequence shown in SEQ ID NO: 3, and the sequence of a reverse primer FPRO-09882R 1 is shown as SEQ ID NO: 4, the sequence of the probe sequence FPRO-09882P is shown in SEQ ID NO: 5, respectively.
Fpro_09882F1:AACGGCTGGGAGGTTAATAGCATGGGGAGTACCA(SEQ ID NO:3);
Fpro_09882R1:ATACGGATTCTCACTTCTCCCATACCCATTGTCG(SEQ ID NO:4);
Fpro _09882P:5'-GCTCGAACCGGGGATGATGAGACGATTTACGATGGGT GAAGGATG-3' (SEQ ID NO: 5), which was labeled with FAM at the 5' end, modified with C3Spacer at the 3' end, and THF-modified at 30bp apart from the 5' end in the sequence of the probe.
In another aspect, the invention provides a kit for detecting Fusarium stratifying bacteria, comprising at least 1 dose of detection solution comprising the primer and probe combination described above.
Further, the kit further comprises: the kit comprises a twist Amp reaction unit tube filled with freeze-dried enzyme powder, a Buffer, MgAc, deionized water, a HybriDetect assay Buffer and a lateral flow chromatography test strip.
In another aspect, the invention also provides the specific detection target FPRO-09882, the primer and probe combination, and the application of the kit in detecting Fusarium proliferatum.
On the other hand, the invention also provides a method for detecting the layered Fusarium, which is characterized in that,
1) extracting DNA of a sample to be detected;
2) using DNA as a template, and carrying out RPA amplification by using the combination of the primers (SEQ ID NO.3 and SEQ ID NO.4) and the probe (SEQ ID NO.5) or the kit;
3) wherein the RPA amplification: to a 0.2ml LTwistAmp reaction unit tube (Twist enzymes nfo kits, Twist) containing lyophilized enzyme powder, 29.5. mu.L of Buffer, 2.1. mu.L of 10. mu.M upstream primer (SEQ ID NO.3), 2.1. mu.L of 10. mu.M downstream primer (SEQ ID NO.4), 0.6. mu.L of probe (SEQ ID NO.5), 2.0. mu.L of DNA, and 2.5. mu.L of MgAc were added inside the PCR tube cap, and deionized water was added to 50. mu.L; fully and uniformly mixing the RPA amplification system, centrifuging for 10s at 5,000 Xg, placing on a metal bath at 39 ℃ for reaction for 30min, incubating for 4 min, uniformly mixing the reaction tube again, centrifuging for 3-5s, and placing in a water bath kettle at 39 ℃ for further reaction for 30 min.
4) Detecting the RPA amplification product by using a lateral flow chromatography test strip;
mu.L of the RPA reaction product was diluted by adding 190. mu.L of HybriDetect assay buffer (Milenia Biotec, Giessen, Germany), and 10. mu.L of the diluted product was dropped onto a HybriDetect 1strip of a test strip. The other strip was inserted vertically into 100. mu.L of HybriDetect assay buffer and left at room temperature for 5 min.
When the test strip has two brown strips, one is positioned in the quality control area and the other is positioned in the detection area, the result is positive, and the sample contains Fusarium proliferatum (F.proliferum); when only the quality control area of the test strip has a brown strip and the detection area has no strip, the result is negative, which indicates that the sample does not contain Fusarium proliferatum (F.proliferatum).
Compared with the conventional PCR, the method has the advantages of high detection speed, no need of three steps of denaturation, annealing and extension, the optimum temperature of the RPA reaction is between 25 and 40 ℃, no need of denaturation, and the reaction can be completed at normal temperature for about 20 min. Thus, isothermal amplification is realized, thermal cycle is not necessary like a PCR method, dependence on a thermal cycle instrument is eliminated, stable heat source RPA reaction can be generated, the use range of RPA is greatly expanded, and portable field rapid nucleic acid detection can be really realized. The PCR reaction time needs 1 half hour, while the RPA reaction time only needs 15 minutes, thus greatly shortening the detection time.
Compared with the prior art, the invention has the following beneficial effects:
1) the invention provides a novel specific Fusarium stratifying molecule detection target FPRO-09882 with high reliability, and provides a novel detection way for detecting Fusarium stratifying.
2) The method can quickly, conveniently, efficiently, specifically and sensitively detect the layered fusarium under the isothermal condition, does not need complex instruments, can better meet the field detection of the layered fusarium, and has important effects on promoting the quick molecular detection research of the layered fusarium and the early diagnosis of diseases caused by the detection.
3) The detection method provided by the invention has high accuracy: according to the sequence of a new target FPRO-09882 for detecting fusarium laminariiformis, a specific detection primer and probe combination is designed, the target band is analyzed according to the detection result of an LFD lateral flow chromatography test strip, and the RPA detection concentration is 1 pg.mu.L -1 The sensitivity of the detection method of the RPA lateral flow chromatography test strip is 100 times higher than that of the PCR method.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby. It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the invention pertains.
Example 1
The present study was carried out on the basis of the whole genome sequence analysis of 17 Fusarium species such as Fusarium partially belonging to the genus Fusarium (F.proliferum, Fusarium circinatum, F.oxysporum, F.graminearum, F.solani, F.fujikuroi, F.odorationssimum, F.verteticiloides, F.odorationssimum, F.vannettenii, F.virgulforme, F.clavum, F.mangiferae, F.cerealis, F.redoles, F.sacchara, F.flexiforme, F.sporotrotrichioides, etc., the whole genome sequence search, sequence extraction, alignment and analysis were carried out, a large-scale genome database was excavated to discover the detection target of Phytophthora, F.proliferum specific detection target was obtained by whole genome alignment, F.proliferum specific detection target was obtained, and more than 1000 genes were selected as a candidate gene specific primer (1.1000 gene target gene) from the list, and the list was selected as a candidate gene (1.1000).
TABLE 1F sequence listing of 6 specific genes of proliferum pathogenic bacteria
Different species (Fusarium proliferatum), Fusarium verticillium, pine gum ulcer pathogen (f.circinatum), Fusarium granatum (f.fujikuroi), Fusarium solani (f.solani), Fusarium asiae (f.asiticum), and Fusarium oxysporum (f.oxysporum) were selected from among different species (Fusarium proliferatum), Fusarium verticillium, Fusarium oxysporum, and Fusarium oxysporum) to design each target primer for RPA-LFD detection.
Sample detection: to a 0.2mL Twist reaction unit tube (Twist p Basic kits, Twist) containing lyophilized enzyme powder, Buffer 29.5. mu.L, 10. mu.M upstream primer 2.1. mu.L, 10. mu.M downstream primer 2.1. mu.L, probe 0.6. mu.L, DNA 2.0. mu.L, MgAc 2.5. mu.L was added inside the PCR tube cap, and deionized water was made up to 50. mu.L; fully and uniformly mixing the RPA amplification system, centrifuging for 10s at 5,000 Xg, placing on a metal bath at 39 ℃ for reaction for 30min, incubating for 4 min, uniformly mixing the reaction tube again, centrifuging for 3-5s, and placing in a water bath kettle at 39 ℃ for further reaction for 30 min.
Negative control: the operation steps are the same as those of sample detection, 2.0 mu L of template DNA is changed into 2.0 mu L of sterilized ddH 2 And O. And after the RPA reaction is finished, detecting the amplification product by using a lateral flow chromatography test strip. When two brown strips appear on the Test strip, one is positioned in a Control line (quality Control area) and the other is positioned in a Test line (Test line), the result is positive, and the sample contains the layered fusarium; when only the quality control area of the Test strip has a brown strip and the Test line (Test line) has no strip, the result is negative, which indicates that the sample does not contain Fusarium stratified.
The remaining 5 primers and probes designed to detect the target, exemplified by target FPRO — 14873, have the primer sequences: Fpro-14873F: AAACAGAGCGAACAAGTAAGACTAGGGTCGACGA (SEQ ID NO: 6); Fpro-14873R: GTGGCACATTTTGCAACTCCCTAAGCGTGTCTTT (SEQ ID NO:7), different species from Fusarium stratifying (Fusarium stratifying), Fusarium verticillium (F.verticillioides), Fusarium pini ulcerans (F.circinatum), Fusarium gambosum (F.fujikuroi), Fusarium solani (F.solani), Fusarium asiae (F.asiticum), Fusarium oxysporum (F.oxysporum), and the results of RPA-LFD detection show poor primer specificity based on FPRO _14873 target design, as shown in FIG. 1.
Considering the detection specificity and sensitivity of the molecular target comprehensively, the molecular detection target FPRO-09882 obtained by screening is a new detection target for fusarium solani and a specific primer and probe combination, wherein the forward primer sequence is shown as SEQ ID NO: 3, the reverse primer sequence is shown as SEQ ID NO. 4, and the probe sequence is shown as SEQ ID NO: 5, respectively.
Example 2
In order to verify the specific primer sequence of the layered fusarium (f.proliferatum), this example uses the layered fusarium (f.proliferatum) strain and pathogenic fungi and other oomycetes as test materials (table 2), and extracts the DNA of the layered fusarium (f.proliferatum) in the diseased tissue by the CTAB method. The specific method comprises the following steps: adding 900 μ L2% CTAB extractive solution and 90 μ L10% SDS into a small amount of mycelium powder, mixing, and turning upside down every 10min in 60 deg.C water bath for 1 h. Centrifuging at 12000rpm for 10min, collecting supernatant, adding equal volume of phenol/chloroform/isoamyl alcohol (25: 24: 1), mixing by inversion, and centrifuging at 12000rpm for 10 min; the supernatant was transferred to a new tube, added with an equal volume of chloroform, mixed by gentle inversion and centrifuged at 12000rpm for 5 min. The supernatant was transferred to a new tube, 2 volumes of absolute ethanol and 1/10 volumes of 3M NaAc (pH 5.2), precipitated at-20 ℃ (>1 h). Centrifuging at 12000rpm for 10min, decanting the supernatant, washing the precipitate with 70% ethanol twice, and air drying at room temperature. Adding appropriate amount of sterilized ultrapure water or TE (pH 8.0) to dissolve precipitate (containing 20 μ g/mL RNase), treating at 37 deg.C for 1h, and storing at-20 deg.C for use.
TABLE 2 fungal and oomycete strains for detection of stratified Fusarium (F. proliferum) RPA-LFD
The assay was carried out according to the method of example 1, and the assay results are shown in FIGS. 2 to 3.
FIG. 2 is a graph showing the results of an RPA-LFD lateral flow chromatography test strip specific assay based on the novel discovery of a novel target FPRO-09882 by Fusarium delaminate (F. proliferatum); 1, 2, Fusarium proliferatum (Fusarium proliferatum); 3, pine resin ulcer pathogenic bacteria (f. circinatum); 4, fusarium granatum (f. fujikuroi); fusarium solani (f.solani); 6, fusarium verticillium (f. verticillium iodides); fusarium asiaticum (f.); 8, N negative control. Fig. 2 shows that there are 2 bands in No. 1, one of which is a Control Line (Control Line), one of which is a detection Line (Test Line), and thus is positive, and the other bands having only one Control Line (Control Line) are negative, indicating that the sample contains fusarium laminarium (f.proliferum); the newly discovered detection target FPR O _09882 has interspecies specificity.
FIG. 3 is a chart showing the detection results of the intergeneric specificity detection of an RPA-LFD lateral flow chromatography test strip based on the high reliability specificity molecule detection new target FPRO _ 09882; 1: fusarium sporophorum (f. proliferatum); 2: pythium ultimum (Pythium ultimum); 3: phytophthora syringae (Phytop hthora syringae); 4: colletotrichum truncatum (Colletotrichum truncatum); 5: verticillium dahliae (Verticillium dahliae); 6: rhizoctonia solani (Rhizoctonia solani); 7: rice blast (Magnaporthe grisea); 8: negative control; FIG. 3 shows 2 bands in lane 1, one of which is a Control line and the other is a detection line, and thus is positive, indicating that the sample contains Fusarium proliferatum; the remaining bands with only one Control line (Control line) were negative. The new discovery detection target FPRO-09882 is shown to have intergeneric specificity. Therefore, after the primer designed in the embodiment 1 is used for carrying out the RPA amplification reaction, the detection result of the LFD lateral flow chromatography test strip shows 2 brown bands, and the target band is analyzed, so that Fusarium proliferatum can be effectively detected. While other fungi and oomycetes only have a brown band in the quality control area, and the negative control only has a brown band in the quality control area.
Example 3
After the amplification reaction of RPA of the present invention using the genomic DNA of the standard strain of Fusarium stratified at the same concentration as the amplification template, the detection result of LFD lateral flow chromatography test strip was analyzed as the target band shown in FIG. 4. Therefore, the detection result of the RPA lateral flow chromatography test strip detection method based on the newly discovered target FPRO _09882 is shown in FIG. 4, and the sensitivity is 1 pg. mu.L -1 The PCR detection result is shown in FIG. 5, and the detection concentration is 100 pg. mu.L -1 Target bands were seen indicating that the sensitivity of RPA detection was higher than that of PCR. The RPA detection time is only 30min, expensive instruments such as a PCR instrument and the like are not needed, the operation procedure is simple and convenient, and the method is more favorable for popularization and application in production.
Example 4
The DNA of the diseased pine needles inoculated with fusarium is extracted by adopting an NaOH alkaline lysis method and is used as a template for PCR amplification. Using 1uL of the DNA solution, the RPA-LFD assay was performed in the same manner as in example 3. The results are shown in fig. 6, which shows the test results as 1 from the left to the right, and the DNA extracted from fusarium solani (f. proliferatum) was used as a positive control; 2-4 artificially inoculating DNA extracted from pine needles (Cedrus dedara) of Fusarium (F.proliferum); 5-7 DNA extracted from pine needles (Cedrus deodara) artificially inoculated with agar blocks; 8 DNA extracted from pine needles (Cedrus deodara); 9 sterile water (negative control).
After RPA amplification reaction, the detection result of the LFD lateral flow chromatography test strip shows that the diseased pine needles (2, 3 and 4) inoculated with the layered fusarium can display 2 brown bands, and the target bands are analyzed and can effectively detect the layered fusarium. While other fungi and oomycetes only have a brown band in the quality control area, and the negative control only has a brown band in the quality control area.
Unless specifically stated otherwise, the numerical values set forth in these examples do not limit the scope of the invention. In all examples shown and described herein, unless otherwise specified, any particular value should be construed as merely illustrative, and not restrictive, and thus other examples of example embodiments may have different values.
Sequence listing
<110> Nanjing university of forestry
<120> specific detection target FPRO-09882 of Fusarium proliferatum and application thereof
<130> 2021
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 220
<212> PRT
<213> Fusarium proliferatum (Fusarium proliferatum)
<400> 1
Met Arg Arg Val Leu Gly Pro Ile Ala His Asp Lys Trp Lys Asn Lys
1 5 10 15
Ser Thr Met Ile Gly Trp Cys Leu Tyr Arg Ala Glu Ala Gly Glu Phe
20 25 30
Thr His Leu Leu Arg His Trp Asn Ala Thr Glu Val Glu Thr Ala Gly
35 40 45
Arg Leu Ile Ala Trp Gly Val Pro Asn Phe Val Leu Pro Ala Leu Leu
50 55 60
Gly Leu Pro Leu Ala Glu Ala Arg Thr Gly Asp Asp Glu Thr Ile Tyr
65 70 75 80
Arg Trp Val Lys Asp Glu Glu Arg Ile Leu Glu His Gln Tyr Asp Pro
85 90 95
Phe Gln Glu Gly Arg Phe Asn Asp Gly Gly Ser Ser Ala Asp Asn Gly
100 105 110
Tyr Gly Arg Ser Glu Asn Pro Tyr Arg Gly Arg Glu Asn Ile Ser Gly
115 120 125
Pro Leu Gly Val Thr His Val Ser Ala Arg Arg Gly Asn Ser Gly Ser
130 135 140
Leu Gln Gln Asp Asn Arg Leu Gln Glu Glu Arg Pro Gly Arg Ala Gly
145 150 155 160
Asn Ser Thr Glu Thr Ser His Glu Thr Glu Ala His Thr Leu Gly Glu
165 170 175
Ile Asp Gly Leu Gly Gly Phe Asp Asn Phe Gly Tyr Arg Glu Pro Ser
180 185 190
Asn Arg Thr Arg Gly Glu Gly Pro Arg Ser Val Gly Asn Gly Thr Lys
195 200 205
Asn Pro Tyr Lys Ser Asn Arg Asp Gly Leu Asp Arg
210 215 220
<210> 2
<211> 663
<212> DNA
<213> Fusarium proliferatum (Fusarium proliferatum)
<400> 2
atgcgacgcg tcctcggccc catcgctcac gacaaatgga aaaacaagtc gacaatgata 60
ggctggtgtt tatatcgggc tgaggccggc gaatttactc acttactaag gcattggaat 120
gctacagagg tggaaacggc tgggaggtta atagcatggg gagtaccaaa ttttgtcctt 180
ccagctctgc tgggtctacc gttggcagag gctcgaaccg gggatgatga gacgatttac 240
cgatgggtga aggatgaaga gaggatactg gagcatcaat atgatccgtt ccaagagggg 300
cgattcaacg atggtggaag cagtgccgac aatgggtatg ggagaagtga gaatccgtat 360
aggggacggg agaacatatc ggggcctttg ggggtgactc acgtatcagc aagacgtggg 420
aactccggga gtttgcagca agacaatcgt cttcaggagg agagacccgg gagggctggt 480
aacagtaccg agacgtcaca tgagacggaa gcgcatacct tgggggagat agacgggcta 540
ggaggtttcg acaactttgg gtatagggag ccaagcaatc gtacccgagg agagggaccc 600
aggagtgttg gtaacggtac caagaacccg tacaagtcga accgggatgg tttagaccgt 660
taa 663
<210> 3
<211> 34
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 3
aacggctggg aggttaatag catggggagt acca 34
<210> 4
<211> 34
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 4
atacggattc tcacttctcc catacccatt gtcg 34
<210> 5
<211> 45
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 5
gctcgaaccg gggatgatga gacgatttac gatgggtgaa ggatg 45
<210> 6
<211> 660
<212> DNA
<213> Fusarium proliferatum (Fusarium proliferatum)
<400> 6
atgagccaag agctaaatga caagcagttt taccaagctt cggccccatt attaacgatc 60
gaaaggaaag catttatcaa tgctacatgg aagtcttcca aagccttcaa cgaatttgaa 120
ttgtcaggct actttaggtt ccttgaaaca gagcgaacaa gtaagactag ggtcgacgat 180
cccgcatact ctcacctttg ttttaaacac attttcgaca ttcaaagtat tctctacgaa 240
aacctcaatg aacctccgct acgcctacaa gacttagtcc aaagaagctt agtcttgtgg 300
gaactagagc tcgaagtcgt tcaggactta atcattctcg ttattaagtt aacatttatg 360
gttcgggccg agttcccaaa ctcctactcg catccttcgc cattccaaat gcagatgcaa 420
gacaaccaaa gccttaaaga cacgcttagg gagttgcaaa atgtgccacc tttacagaat 480
ttgggtacac aaaatgagct gccgtcgtgg tttaatgtta ttgatctgga gaaaaaggct 540
aacctgagaa ttggttggac tgaccatctt gatgaacatc tgactttcca aagtggaact 600
cttgtgatat ttcgacatat cgcagtcctc atgtatatga gagaatcagg aatcttgtga 660
<210> 7
<211> 34
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 7
aaacagagcg aacaagtaag actagggtcg acga 34
<210> 8
<211> 34
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 8
gtggcacatt ttgcaactcc ctaagcgtgt cttt 34
<210> 9
<211> 660
<212> DNA
<213> Fusarium proliferatum (Fusarium proliferatum)
<400> 9
atgactcgat tcagcaccac ggtagaagat gttcagaaca tcccgcaagg cggtgttagg 60
ctactcttac gtagagagaa tgctggagaa acagacactt ggtatgagga ggatttcgat 120
catcttgtcg tcgcgacggg ccacaacagt gtcccccgcg tgcccaagat acccggcctc 180
gaggcctgga acggaagcct gcaacatgcc tccacatggc gatcggcaca agagttcaag 240
ggcaagaaaa tcttagtggt cggtaccagt gagagtgcca tcgatctcgt tcttcagtct 300
cttcctcatg ccaagggcga tattcatgtg tctcaaagaa aaccgcaccc ccggtatcct 360
aacgtgtttg atcggcctgg cgtcaaactc gtgaccacga ttgaccattt taccgaagac 420
tctattcatc tagatgacgg ttctgtcttg ggaggcatcg atgtggtagt ttttgcgacg 480
gggtacttct acacgtaccc cttcctctcg aatgtccgcc cacctgtatc ctgcaggagt 540
ttagtctgtc ctgagagaga aaaacaagta gggtcgccaa tgcgtccaaa tttaacctgc 600
aaagcaacaa caaagggaaa ctggagctat ttcttaatcc gatggggttc aatcaactag 660
<210> 10
<211> 34
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 10
caacagtgtc ccccgcgtgc ccaagatacc cggc 34
<210> 11
<211> 34
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 11
cacatcgatg cctcccaaga cagaaccgtc atct 34
<210> 12
<211> 651
<212> DNA
<213> Fusarium proliferatum (Fusarium proliferatum)
<400> 12
atgtttgtat cagaaatcat cagccctctc ctcctcgtcg ccagcgccgc actggcggct 60
gccaatcctc agccctcctt tgcggctgtt gtggctcccc gtctgcaggt ttccgtcgaa 120
gcagtcgacg aactcctgat gagcaacact actgagcatc tcacaagcat tgatatcgcc 180
aaatgggctc ctgctctcgg gcagccagtc gaggttctca ccagcttgga ctctgaaacc 240
aagttcaagg tgttctattt cctccaccaa gccatttccg aggggcctgg atcgctttct 300
aagcgtgacc aggcgactgc tgataaagat gccgccatca tcagggagaa ggcagaatcc 360
taccaaagca cagtcacagt tgaggcagcg gaagatgagc ttcgttgcca gaataccgct 420
tcttgtgttc tctgtatcgg tgcagctgca actaccgcgg gaggtctcat ttcttcatgc 480
tctgcagttg cactacgagc caacaacctt cgtgttgcag gaaacgctag cccaacagct 540
gccgaggtta gcggtgctgt cattatcgcg gagctcttag catgcgccgc caagcctctt 600
accggtttcc tcgttgctac tggtgtctgc ctcaaggtta caggccatta a 651
<210> 13
<211> 34
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 13
acactactga gcatctcaca agcattgata tcgc 34
<210> 14
<211> 34
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 14
gacctcccgc ggtagttgca gctgcaccga taca 34
<210> 15
<211> 507
<212> DNA
<213> Fusarium proliferatum (Fusarium proliferatum)
<400> 15
atggcaactc cgatttccca agaaccctct gggccgtgtt cagaacaaga acaaatgaat 60
gaaatgtcta gaaatttcta ttacgttaga aagagagaag tccgtatgtt tccaggcgca 120
acggctttgc taaaaatgtc gattcagaag tacatggcga agtacgaggt agaatttatg 180
gatgacgacc aaaagcttcg cgttttgctg cctctggatg tgaataggga ggactctgat 240
aagaaattcc aactgctgat ggaaatgccc gataatatga agaaggcgaa gcttttgacc 300
ttctttcgtg tcgatacgat caaggaattc gaaaaacatg tccagatgac agagatctct 360
atcttaggcc tcgaaaggag aaatacctgc acaagtcgga gaggcaggac ctcaaagatc 420
aacgagattt ctagcaatac acaaggcaga actggcaaaa cagaaacaaa ttcagggaga 480
atagaagagc tgagactcag gagctag 507
<210> 16
<211> 34
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 16
aatttctatt acgttagaaa gagagaagtc cgta 34
<210> 17
<211> 34
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 17
gatcgtatcg acacgaaaga aggtcaaaag cttc 34
<210> 18
<211> 558
<212> DNA
<213> Fusarium proliferatum (Fusarium proliferatum)
<400> 18
atgcctcgta attcgtctcg cagaaagtct cgccgcagcg agaggtctgc ttcccagtct 60
acacctgagt ctacatcccc atctgcaacc ccgcctactt cacaggattc cccgcgccct 120
cggtcttcga gaccgaccac tcccggctct tcaggccctc agtcttcgag accgaccact 180
cccggctctt caggccctca gtctccctgg tcaagcggct ttgaggaaca gggtgtaact 240
ggtctgccca gtcctccagc atcacaatct ccgtctcttg gaacctcgag ccctgctaat 300
gtcgctctat catcctctgg aatctcaaca ccatcaattg aggaacatga accttatcgc 360
tccgccactc cgatagagga tatgactgca cccccatcta cgactgctag tcagtcgtcg 420
gccaaatcta ttaaggctga ccctttggag gatgaaactc tacccaacga tcaggataca 480
ggctacgtga tcgagacaat ttaccttctc cattccaaag ccatctggag ttacatccag 540
ttcatacttg ggaactag 558
<210> 19
<211> 34
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 19
gctcttcagg ccctcagtct tcgagaccga ccac 34
<210> 20
<211> 34
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 20
agatgggggt gcagtcatat cctctatcgg agtg 34