CN112280880A - 用于液相芯片多重PCR鉴别猪链球菌17种新cps型的引物组合及其检测试剂盒 - Google Patents
用于液相芯片多重PCR鉴别猪链球菌17种新cps型的引物组合及其检测试剂盒 Download PDFInfo
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Abstract
本发明涉及微生物检测技术领域,具体涉及用于液相芯片多重PCR鉴别猪链球菌17种新cps型的引物组合及其检测试剂盒。本发明提供的用于液相芯片多重PCR鉴别猪链球菌17种新cps型的引物组合以wzy基因为检测靶标,包括17个特异性引物对。该引物组合及其液相芯片多重PCR检测方法具有特异性好、灵敏度高,分型结果的可重复性强、检测通量高的优势,极大地增强了现有检测方法对于猪链球菌的血清分型能力,节约了时间,提高了检测效率。
Description
技术领域
本发明涉及微生物检测技术领域,具体涉及用于液相芯片多重PCR鉴别猪链球菌17种新cps型的引物组合及其检测试剂盒。
背景技术
猪链球菌(Streptococcus suis)是一种世界性的可致猪疾病的最重要病原菌之一,同时它也是一种人兽共患病病原体。人感染猪链球菌的途径主要是接触病死猪后,致病菌经破损皮肤和黏膜侵入。猪链球菌在临床上主要分为2个类型,即败血症型和脑膜炎型。另外,猪链球菌还可侵入人体的关节、眼睛和心脏等,引起化脓性关节炎、眼内炎、心内膜炎和休克等。
血清分型不仅是用来了解猪链球菌某次特定暴发的流行情况或者监测血清型流行情况的一种很重要并且很有价值的诊断方法,而且还对疫苗的研制有重要指导意义。目前,公认的猪链球菌的血清型有33种(1-31,33,1/2)。目前,猪链球菌的血清型主要通过凝集实验和协同凝集实验检测,但是这种方法是既耗时,又费力,且较为昂贵,结果判读主观差异较大。此外,相当一部分菌株是无法通过凝集实验和协同凝集实验鉴定的。
猪链球菌的血清型主要取决于其荚膜(CPS)的抗原性,而荚膜的产生又受控于基因组中的荚膜合成基因簇(cps)。与传统的利用抗血清检测血清型的方法相比,基于血清型特异cps基因的PCR检测方法是一种简单、可靠、且经济的方法。随着监测菌株规模的不断增加,血清未分型菌株的比例逐年增加,对这些菌株的检测对于猪链球菌的诊断和流行病学监测具有重要意义。
发明内容
本发明的目的是提供一种用于液相芯片多重PCR(mPCR)鉴别猪链球菌17种新cps型的引物组合,本发明还提供含有该引物组合的检测试剂盒。
具体地,本发明提供以下技术方案:
第一方面,本发明提供用于液相芯片多重PCR鉴别猪链球菌17种新cps型的引物组合,所述引物组合以wzy基因为检测靶标,其包括含有以下核苷酸序列的引物对:SEQ IDNO.1-2,SEQ ID NO.3-4,SEQ ID NO.5-6,SEQ ID NO.7-8,SEQ ID NO.9-10,SEQ ID NO11-12,SEQ ID NO.13-14,SEQ ID NO.15-16,SEQ ID NO.17-18,SEQ ID NO.19-20,SEQ IDNO.21-22,SEQ ID NO.23-24,SEQ ID NO.25-26,SEQ ID NO.27-28,SEQ ID NO.29-30,SEQID NO.31-32,SEQ ID NO.33-34。
为便于液相芯片检测,以上所述的引物组合中,上游引物SEQ ID NO.1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33所示核苷酸序列的5’端还含有与对应编号液相芯片微球的anti-TAG序列反向互补的TAG序列,同时,所述TAG序列与SEQ ID NO.1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33所示核苷酸序列之间插入一个12碳胺基基团。
具体地,所述引物组合包括以下序列的引物对:SEQ ID NO.35-2,SEQ ID NO.36-4,SEQ ID NO.37-6,SEQ ID NO.38-8,SEQ ID NO.39-10,SEQ ID NO.40-12,SEQ ID NO.41-14,SEQ ID NO.42-16,SEQ ID NO.43-18,SEQ ID NO.44-20,SEQ ID NO.45-22,SEQ IDNO.46-24,SEQ ID NO.47-26,SEQ ID NO.48-28,SEQ ID NO.49-30,SEQ ID NO.50-32,SEQID NO.51-34。多重PCR的反应体系中会存在不同引物之间以及不同引物与模板之间的相互作用,对扩增效果产生影响,以上引物组合为发明人经大量筛选和验证得到,能够很好地保证多重PCR扩增的特异性和灵敏度。
为便于液相芯片检测,以上所述的引物组合中,下游引物SEQ ID NO.2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34所示引物的5’端含有生物素标记。
第二方面,本发明提供所述引物组合在制备用于液相芯片多重PCR鉴别猪链球菌的试剂盒中的应用。
第三方面,本发明提供用于液相芯片多重PCR鉴别猪链球菌的试剂盒,该试剂盒含有所述引物组合。
优选地,所述试剂盒还含有管家基因thrA的扩增引物,其核苷酸序列如SEQ IDNO.52-53所示。
优选地,所述试剂盒还可包含用于多重PCR扩增的其他试剂,包括但不限于PCR反应缓冲液、DNA聚合酶、dNTP、MgCl2、阳性对照等。
以上所述的试剂盒在用于猪链球菌鉴别时,各引物的在多重PCR反应体系中的终浓度为0.2-0.3μM。
以上所述的试剂盒在用于猪链球菌鉴别时,多重PCR的反应条件为:94℃预变性5min;94℃变性30s,58℃退火40s,72℃延伸50s,30个循环;最后延伸72℃,10min。
第四方面,本发明提供一种液相芯片多重PCR鉴别猪链球菌的方法,该方法为:以待测样品的DNA为模板,利用以上所述的引物组合进行多重PCR,将PCR扩增产物与液相芯片微球结合形成PCR扩增产物-液相芯片微球-SAPE复合物,检测所述复合物的荧光强度,根据荧光强度判断待测样品的猪链球菌的cps分型情况。
本发明的有益效果在于:本发明对部分血清未分型的猪链球菌菌株进行了Illumina Solexa高通量全基因组测序,发现了17种猪链球菌新的cps型别。基于此,本发明开发了以cps型特异基因wzy为靶基因、用于液相芯片多重PCR鉴别猪链球菌17种新cps型的引物组合和检测试剂盒,并建立了的基于液相芯片的多重PCR检测方法。
本发明提供的引物组合、试剂盒和检测方法的特异性好、灵敏度高,分型结果的可重复性强,便于不同检测环境的结果比对,极大地增强了现有检测方法对于猪链球菌的血清分型能力;
本发明的检测方法具有检测通量高,可在一个反应中同时检测17种新的cps型别,节约了时间,提高了检测效率。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。
实施例1猪链球菌17种新cps型的发现及其检测引物设计
1、菌株
276株血清未分型菌株用于测序。菌株于哥伦比亚平板37℃、5%CO2培养过夜,挑单克隆菌落接种于THB培养基中,37℃、5%CO2培养8h。利用以下菌株检测mPCR方法的特异性:Klebsiella pneumoniae 46117-3和62株其他链球菌属菌株,包括:6株Streptococcuspneumoniae(ATCC700657,ATCC700670,ATCC700676,ATCC700902,ATCC700906和ATCC49619)Streptococcus pyogenes 32003,Streptococcus sanguis 32214,Enterococcusfaecalis 32221,Streptococcus oralis 32231,Streptococcus bovis ATCC33317,Streptococcus lutetiensis 033,猪链球菌33种血清型标准菌株以及2株原来被认为是猪链球菌32(菌株EA1172.91)和34(菌株92-2742)型的Streptococcus orisratti。
2、全基因组测序和cps基因簇的截取
全基因组DNA用Wizard Genomic DNA Purification kit(Promega,Madison,USA)试剂盒提取和纯化。用Illumina Solexa GA IIx(Illumina,San Diego,CA)测序菌株基因组后,构建一个平均插入长度为500bp to 2000bp的paired-end(PE)文库,以SOAP denovo(Release 1.04)组装数据。根据以往对猪链球菌血清2型cps基因簇特征的描述从全基因组序列中截取各血清型的cps基因簇。Artemis program(www.sanger.ac.uk)用来预测和注释cps开放读码框可读框(ORFs),BLAST和PSI-BLAST软件(http://blast.ncbi.nlm.nih.gov/Blast.cgi)用来搜索GenBank(www.ncbi.nlm.nih.gov/GenBank),Clusters ofOrthologous Groups(COG;www.ncbi.nlm.nih.gov/COG/)和Pfam(pfam.sanger.ac.uk)蛋白数据库。cps基因的命名也参照于猪链球菌2型cps基因的命名方法,基因共同冠以cps前缀,后跟相应的血清型数字,其后以字母A到Z依次对cps基因命名。
3、cps基因簇中血清型特异wzy基因的确定及新血清型分组
猪链球菌的CPS合成和其他链球菌一样被认为是wzy决定路径。这种CPS合成途径广泛存在于肺炎链球菌中。胞内的寡糖重复单位由翻转酶(Wzx)转运至胞外,不同的Wzy可催化寡糖重复单位形成不同的糖键连接。有研究证明group B Streptococcus(GBS)的血清Ia和III型就是由多糖聚合酶(Wzy)所决定。因此,wzy基因是非常理想的建立mPCR实验引物设计的靶基因。
利用TMHMM v2.0(http://www.cbs.dtu.dk/services/TMHMM/)分析血清未分型菌株cps基因簇中蛋白的疏水性跨膜结构域。存在跨膜结构域的蛋白为wzx或wzy基因,用BLAST网站(https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&BLAST_PROGRAMS=megaBlast&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome)进行序列及功能比对,最终确定wzy基因。用本地BLAST程序(downloaded fromftp://ftp.ncbi.nlm.nih.gov/blast/executables/LATEST),将全部wzy基因及33个已知血清型标准菌株wzy基因做序列两两比对。E-value设为10-10,其余参数为默认设置。wzy基因氨基酸同源性超过95%认为属于同一血清型,经比对共发现17种新cps型,分别命名为NCL1-16和Chz。不同型别的wzy基因氨基酸同源性低于70%。
4、引物设计
为了能够使各引物在同一条件和体系中的mPCR实验中有效工作,以Primer-BLASTprogram(http://www.ncbi.nlm.nih.gov/tools/primer-blast/)进行引物设计,使每对引物拥有基本相近的共同特征。长度在20-24bp之间,退火温度在49-52℃。同时也设计了一对被用作内部对照的管家基因thrA的引物。各引物的序列如表1所示。每个wzy基因的上游引物5’端增加一个特异的24bp的“TAG”序列,该序列与相应编号液相芯片微球的“anti-TAG”序列反向互补。“TAG”序列与上游引物间插入一个12碳胺基基团,有助于“TAG”序列与“anti-TAG”序列的共价结合。每个wzy基因下游引物5’端进行生物素标记。
表1用于猪链球菌鉴别的引物及其相关信息
注:*代表引物5’端有生物素标记。
实施例2液相芯片mPCR检测方法的建立
1、mPCR扩增
以17株新型cps型参考菌株的DNA为模板对每对引物进行单重PCR扩增来检测其特异性,在此基础上建立多重PCR检测方法。
用于mPCR扩增的PCR试剂为2×Taq PCR Master Mix(Biomed,Beijing,China)。每个PCR反应体系如下:Taq DNA polymerase:0.05units/μl;MgCl2:4mM;dNTP:4mM和PCR缓冲液;表1中所示的每条鉴别引物的终浓度为0.2μM,管家基因引物的终浓度为0.3μM。
mPCR的反应条件为:94℃预变性5min;94℃变性30s,58℃退火40s,72℃延伸50s,30个循环;最后延伸72℃,10min。
2、PCR扩增产物-液相芯片微球-SAPE复合物的结合与检测
步骤1中扩增获得的标记生物素的血清特异的wzy基因扩增产物与相应编号微球的“anti-TAG”序列共价结合而被捕获。Luminex MagPlex-xTAG微球(Luminex公司)使用前振荡30s,使用1×Tm杂交缓冲液(0.2M NaCl,0.1M Tris,0.08%Triton X-100,pH 8.0,过滤除菌)将微球稀释成125个微球/μl的预混液。在每个PCR反应中,每种微球的数量为2500个。1×Tm杂交缓冲液将染料链霉亲和素(Streptavidin),R-藻红蛋白(R-Phycoerythrin)复合物(SAPE,Invitrogen公司)稀释为10μg/ml的预混液。将5μl PCR反应产物与20μl微球预混液和75μl SAPE预混液混匀后,40℃作用30分钟进行杂交反应,将扩增的PCR产物与相应的微球和SAPE特异结合,形成微球-PCR产物-SAPE复合物。
使用Luminex xPONENT 3.1软件(Luminex公司)在Bioplex 200(Bio-Rad)仪器上检测微球-PCR产物-SAPE复合物的荧光强度中位数值(median fluorescenceintensity,MFI),检测参数设置为:每种微球检测100个,门限值为7366到19262,平台温度设为40℃。每种微球的MFI值超过200即判定为阳性。
实施例3检测方法的敏感度和特异性评价
1、敏感性评价
将17株新型cps型别参考菌株的DNA分别倍比稀释为10ng,1ng,100pg,10pg,1pg,0.5pg,100fg/μl。以上述不同倍比稀释的DNA为模板,利用实施例2的液相芯片多重PCR检测方法对各鉴别引物(表1所示)的检测灵敏度进行分析。
结果表明,本发明的液相芯片多重PCR检测方法(实施例2)的检测灵敏度达到0.5-20pg,多数引物的检测灵敏度达到0.5(相当于2×102cfu)-1pg(相当于5×102cfu)。
2、特异性评价
利用分离自北京,江苏,四川屠宰场健康猪的276株猪链球菌血清未分型菌株、33个猪链球菌血清型标准菌株以及62株非猪链球菌验证本发明的液相芯片多重PCR检测方法(实施例2)的特异性。
结果表明,在其他62株链球菌属菌株以及33株猪链球菌已知血清型的参考菌株的检测中均未有任何非特异扩增条带,而276株血清未分型菌株的分型结果与根据序列分析的cps基因簇分型结果完全吻合(表2),表明本发明的检测方法具有较好的特异性。
表2 17种新cps型序列分析的分型结果与液相芯片多重PCR检测结果的比对
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
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<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ttcttgatta tgctgttctc gt 22
<210> 4
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
aaacacaaca tcctgtactt ca 22
<210> 5
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
attcaggagg tattcaacca ag 22
<210> 6
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
aattcactag catcaacaaa cg 22
<210> 7
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
tgcttattat gactgttgcc tt 22
<210> 8
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atcagttgat aaggttgctg tt 22
<210> 9
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
cagatgagtc agcaagtaat ca 22
<210> 10
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
agggaagagt aagattcaag gt 22
<210> 11
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
tgatacgggt actgttgagt at 22
<210> 12
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
attactactt ctggttgggt ca 22
<210> 13
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
gttgatttat ttgcgggact ac 22
<210> 14
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
cagaaaaaca atagcagtga cc 22
<210> 15
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
aaaattttca cttcacctcg ac 22
<210> 16
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
aatcttccaa tcaatgctac ga 22
<210> 17
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
gggtagatac gttctatttg gg 22
<210> 18
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
gcgacggtat atagagctat tc 22
<210> 19
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
acaaaaatag tagacgggct tt 22
<210> 20
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
ttgcacgcca agtataaaat tc 22
<210> 21
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
gccttaataa tggtgggttt tg 22
<210> 22
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
tacctaaaac attttgccca ga 22
<210> 23
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
gtgagagatt tcggtgtagt tt 22
<210> 24
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
gcatcagcat acatctttcc ta 22
<210> 25
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
tcttgctagg tctaatcgta gt 22
<210> 26
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
ctcgcttcca attaataaac cg 22
<210> 27
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
ctcgcttcca attaataaac cg 22
<210> 28
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
tctttgctca gctattgagt tt 22
<210> 29
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
tatgctattg ttacgatgtg gg 22
<210> 30
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
cttggagagt aaaacgatag gg 22
<210> 31
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
agggttattc tttttggtgg at 22
<210> 32
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
tcttgtaagc agaaaatcgt ga 22
<210> 33
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
attttgggca gtgagtgata tt 22
<210> 34
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
tcaatacttt tcttgaaccc ga 22
<210> 35
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
ttaatacaat tctctctttc tctaattcac atagtaacat tgcgga 46
<210> 36
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
acatcaaatt ctttcaatat cttcttcttg attatgctgt tctcgt 46
<210> 37
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
cttaaactct acttacttct aattattcag gaggtattca accaag 46
<210> 38
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
acttacaata actactaata ctcttgctta ttatgactgt tgcctt 46
<210> 39
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
aatctctaca atttctctct aatacagatg agtcagcaag taatca 46
<210> 40
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
ctaaacatac aaatacacat ttcatgatac gggtactgtt gagtat 46
<210> 41
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
ctaaatcaca tacttaacaa caaagttgat ttatttgcgg gactac 46
<210> 42
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
ttcaattcaa atcaaacaca tcataaaatt ttcacttcac ctcgac 46
<210> 43
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
tacttaaaca tacaaactta ctcagggtag atacgttcta tttggg 46
<210> 44
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
tcttactaat ttcaatactc ttacacaaaa atagtagacg ggcttt 46
<210> 45
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
atctcaatta caataacaca caaagcctta ataatggtgg gttttg 46
<210> 46
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 46
ctatcattta tctctttctc aattgtgaga gatttcggtg tagttt 46
<210> 47
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 47
ctttatcaaa ttctaattct caactcttgc taggtctaat cgtagt 46
<210> 48
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 48
acactcattt aacactattt cattaaaaga aatggaaagc agtgtg 46
<210> 49
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 49
cataaatctt ctcattctaa caaatatgct attgttacga tgtggg 46
<210> 50
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 50
tctcatctat catactaatt ctttagggtt attctttttg gtggat 46
<210> 51
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 51
aataacaact cactatatca taacattttg ggcagtgagt gatatt 46
<210> 52
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 52
actacttatt ctcaaactct aatagaaaat atgaagagcc atgtcg 46
<210> 53
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 53
gacaacgaac ataacagaaa cttc 24
<210> 54
<211> 1221
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 54
agtagaaaaa taaaaatttc gtcattagag ctgtatttga atattatagc aattatacta 60
attttagtgt ctgaaataaa tttttatact cttgagagca taaaaattta ttcggtgcta 120
acaatgacag gtatgattat ttcatatagc ttaggagtgc aatattttcg ctataaagta 180
agattggatc attttattat ttggctttca gtgatttact ttatgttttg tttttatgga 240
atattttttc taagatctgg aattttttct attgattcga tattatatcg gtattttgaa 300
aatattgctt tatatttatc agtttcgtct attttacgaa agaattacat aaaaataaca 360
attccatttg tccttgctgg attgttttca attctatatc tgatttctaa agagggggca 420
gatataatat caggaggcgt acgaataggt ggttccttaa gtggaaacgt aaactctgta 480
ggatttaact ttggtttggt tagttcttta ctggcatgga gatattgtat tactaagaat 540
aagcgcgaca tgattttatt tttagtattt ataattttta tgttgctgac aggatctaaa 600
aagactttat tttttttatt gactaattta tttatttttt attatcataa tcgtcataaa 660
gcgattattt ggttaaaggt catcctatcg ttcttttttt tatggattct aatttttaaa 720
attccctatt tttattcaat tatgggggtt cgaatcgagc agatgttgct gactttactg 780
ggggattcta atgcccttgc atattcttat tcaacggata tgcgtgaaaa aatgttagtt 840
gaatcattac atttttttct ggagcacccg atttttggag gtggatggaa ttattttggt 900
ttaaaaacaa ccttgaacta tgattattca catagtaaca ttgcggaatt atcggtaaca 960
tttggcttga tggggttatt cacatattac gctagataca tatataatat atatatatct 1020
ttgaatggtt tgagggattt agcaaataag aaaaaaaata atttttctct cttatctttt 1080
tctttagtta gtacaatatt gtttattgaa tggtctgcgg taactttctc cgcacaggtt 1140
ttttggtatc tacctattat tatttcctgc gcaatgttga atgaaatttt acagaagtct 1200
gaatccaaat agaagagtaa c 1221
<210> 55
<211> 1509
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 55
atgaaggagg cacttgtgtt aagaataaag aatgaattga aaggctacat acttctgttc 60
atggtactgt ctgtagtata tttctttgca tcactctatt ttattgatat ccatacacaa 120
aactataata actgtaaatt cttgattatg ctgttctcgt ggttgggctt ttttcaatta 180
ttttttacat taagcagtat aaaaagaaat tttggtagtt atatcagtct ctacagtatc 240
ttttatatgt ttctatttat atttagttat ggccagttta ttatgtgggc attgggcttt 300
cattatgatg ctgaaatgac tgtttctagg catgttcgct tcattgatga gaacacggta 360
gttcgtatac aagtgatgag cttacattta ttatcaattt ttcatttgtt tgcattgttt 420
gcttttggaa agaataattc aataaaaaat gaagtacagg atgttgtgtt tagggacata 480
tcaaggttga tagctactcc tatgattata gtttctttcg taataaatct ttatgtttcc 540
attgtaagtt ttggaactgc tgcaaataac ggatatcttg ctacattcga tataaatctt 600
cctgttttgt taaaatattt tagctatatg tttatacctg caatttttct aaatcttatt 660
gtgcacaatt tttcaaaaaa atcgtttttt attttgtcgg gaatatttct catttatgct 720
attccgcttg cgattgcagg tgatagagga agttggattt attttttggg gccatggcta 780
tggtgttatt tctttagggt agatatacag aaacaggtga tgactcctga taaatataag 840
tggcaacgaa agaaaaaaat ctttcggatg ctcatcattg ttttgacagc attatatgtg 900
tcttctgtat ttgtgtcggt cagggacgtc ggctattctg ttttagtgaa agaagatttt 960
gaattcactt gggaaagctt atataagcct ttcgtaaagc ctttctttga aatgggacaa 1020
tctgcacgga ttcttgggat tattattcaa gatggtcttg ataagacata taattatgga 1080
aatactttca tagctgatgt attggggatg actttatcga gtgtaaaaac atggtttggt 1140
tatcctgaat ggtatgtaga aaactggatg tcatctgatt atcttggtat ggtgaattat 1200
ggagtaggtt tttcatcatt tgctgaagct tatttgaatg gtggagtata tttttcatgg 1260
ttttatatgg caatatttgg gtggtttatt ggtagtttat taaatgtatc ctctgtacca 1320
ggacaatcaa gttctaagag agtgtttgta gcattatcag cgacatcggt gttaggaccg 1380
tctgttaggg ccacattaga tttttggttg agacagtttt tttggggagt tttacttctc 1440
ttgcttgtat gttggggaat tggaattttt gccaaagaaa gtatatctac gagagtagca 1500
cggtattag 1509
<210> 56
<211> 1230
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 56
ttgttgataa gtaattatag tcaacccacc tctagttcta agagccttag aggttccgaa 60
ataatatttt tatgtttttt gctaggtaaa ccattttatt tttttaattc aggaggtatt 120
caaccaagtg acatactttt gatattctct tttattgcat ttttatatga aaataattgg 180
aggataccat atgataaaaa agaaaaaaat tattcaatat ttttatgttg tattattttg 240
ataaatttat tttattgggg actatatttt atatcaggaa atggttggtc attccttcta 300
tcgacagtat attatctata caatacaatt gtaattttga tgtgtcgaca attttttaaa 360
gggaaacgct ttatagaaag tttgctattt attgttaagg gaggaatata cctccaattc 420
gcattttacg gtattgattt ttttgttcca ctctcgtttg ttgatgctag tgaattaagg 480
tacattggta catttaacga cccaaatcaa cttggttttt ttctactaac tagtatattt 540
atcattatta ttttaaacag attattaggt agaaaagaaa atttatttgt tcattgtttg 600
acgttagtac ttttatttgc aacatcttct acaggtatct ttttaggttt cgctttctta 660
tatttcttta tgtttgctat ttctgaaaat aaggaaaaaa ttattaaaca aattgttatt 720
ctcgttttgg catttttcat tctattttgt ttacattttg ctggtgtttt ccaatttttt 780
gataagatac aggatagctt tctgtatgaa agggtattgc agaaatttgg aagaggtttt 840
actataaatc aaaatcaaac ggaattgtca cttattcaag aaagaggaat tgataggata 900
attttatatc cagagtatct tttatttggt gcaggagaag gttatcctca gcgctttgct 960
ttaagtggta tttcaaatct tgaaatacat tcaacactct ttggcttgct gttttattat 1020
ggtttccttc cttttataat atttgtaaga tggatttgga taaacataag aaaatcaggt 1080
aaccttattt tttatatttt tgttccatta atgtttgaaa gtttaacttt agcaaatcac 1140
aggcaacctt atttttggat gttatttctt ctggcatctt tctttgacga tgatagtaga 1200
aattttagaa aatggagaat tgtaaaatga 1230
<210> 57
<211> 1299
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 57
ttgctttaca tattacttac gtcgattttc ttgcttatta ttcttgaatt aaaattgaga 60
aagaattttg ctactccaag tgtgctttta ctattatcat ttttaatggc aagtttattg 120
atttttatca attataagaa ttgggatata tctttatcgt ttcattttgt tgtatatgtg 180
ctaacggcca ttatttcttt tatattatcg actcatttag tacatagttt tttaaaaaat 240
agtagcaaga tcccctcgtt atcattgcaa aagcttcaag tactatctag acctcgtgct 300
tcgttgatgc ttattatgac tgttgccttt tctatctata gcatattgta tattagacga 360
gtaggttttt cactagattt gacaacgata ttatctactg cctatagttc aaatgtatta 420
aatagtggct attctggtag ttttttagaa acacaaatgc taaaaataat tactggaatt 480
gcttatatta attattatca atttttatta tcaaaatttg ttgtaaagat gaatttttcg 540
caattcatta atttttataa tattgtatta tttttaataa cagcaacctt atcaactgat 600
cgaaatattc ttcttcgttt ttttatatat gcactagttc tttggatttt gtttttcttt 660
gatataaata aagagtcgat gaggaatatc aatttcaaga ttatcagaag tttagtgata 720
tatggaatcg ttatattgat atcgcttttt ttagcaggca gaataaaagg gtatgtttca 780
gattttcaac gttctattgg actttatggt ggttcaggtc ttaataattt taatcttttt 840
ttgaataatt ttaatggtca ggaattgcag tgggggagag taacttttaa cagtttacga 900
gtattagttc agacgttcct tggaatagag gaatatagtg gtttagctat gtttgatatc 960
cctatcgttt ttaaatcaag tacaggattt gtttatattt caaacgttta ttcagcttta 1020
agaccatttg tactagattt tggatatatt ggaatgttat tatttccggc gatggtaggt 1080
attttttttg aaatactgtt ttcaaccgct ataaaccgta agtttgattt ttcgtggatt 1140
ttttactcag caacggttta ttcccttatt tatttctcta tacaagaaca attttttgct 1200
cgaatgcatt tggggagcct atatgaaatt ttctggttga gtatttttta tttgtatatt 1260
aagtcaccta taaaaattaa attaaggagt aatgtatga 1299
<210> 58
<211> 1389
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 58
ttggaaaaaa atattatttt cagaatagga tttagtatct tatttctggt attaattatt 60
ttatcgcttt ggaaagggat taacgaagta tttacggcag tactaattgt ttggtttggg 120
gttgtttatt attgttttcg tgatttaccg aaacacttta cactcttctc ttttatgatt 180
acctttttca catttttatt aggtagagat gctacatatt tatttttaga tgcaagaagt 240
tttagaatta tttcagatga gtcagcaagt aatcatgcat attggtcaat attggtatcg 300
cttatatttg tatttttagc atattatttt actaaagcat cttccttgat aaggattaga 360
gaaatacagg ctcaacattc aattgattat atcgtggccg ttagagctgt agctaaatat 420
tctttttatt ttacgtacat atttttgatt attgctctga tatatcaagt tagatatgtt 480
ttgaatgttg gttaccttga atcttactct tccctagagg ctggagcagg aacgccaaat 540
tttatttcgc gtttgtcatc attttctcca tttttatttt ttatatttct agggacaaaa 600
ccaagtaaaa aagaggcttt ttggccaatt ctactgtatt ttgtttatgg catttctact 660
ttattaactg gtcaaagatt tccagttatt gcggtgactt tggttttgat gaattattat 720
gtattccgaa atagagagga gaagtggatt gaaaaatggc atattacagc gattctaatc 780
tctattccat tggtattatc tggtcttttt ttgatggatt atattcgact ttctaaagtt 840
gtagaatctg tgaatttcct tgattcaatc tttaatttct ttgccgttca gggaggttct 900
attaatgtaa taaagtatgc ttatctttat caggatagtc ttgcagaaac tcctttctat 960
tcctttaatt ctattaggag taatatatta aatattaatg agttttcagg taatactata 1020
caacatgcat tatacggttc ttcaatggca catagaatgt catacataat atacggcgag 1080
tacgcctaca ttcacggaag aggttctggc tctagctata ttgccgaatt gttccatgat 1140
tttggttatt taggaatcgc tataggaagc atgttttacg gatatctaat aaaaaaaatt 1200
agtttgatga aatttaatca tttctttaaa aatgcattcc aacttgcaat gttatctcca 1260
ctattcttgg cacccagagg cacatttgat agtgttattg gtcagccatt cagacgttca 1320
aatctttttg ttttgctcat ggtgtttatc gttgcaaaga ctttgtcaga tcgtaataga 1380
aaatactag 1389
<210> 59
<211> 1059
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 59
atggttgata gagtattgtg gttgatacgg gtactgttga gtattttcta tatttttaaa 60
gtgctttttt cttataaaat cgaaaagcta gatgtcttta ttgtggtatt tttaactagt 120
cagatactgt ctgcacagca gacggacaca ttgtatctag gatatctttc tgggcaaatt 180
acagcacttg ggctatacgc cttcttaaaa tataaattgt tgacccaacc agaagtagta 240
attaaaggta tttttagctt atttcttatt ttcattctct atcaaatttt cacccagctc 300
cgttttccaa ctggatttga tattttacat ccttatggtg atagtagaga atattttttg 360
gggcgaaaaa atgccctgac cccttatcta gtctttacaa tgggttgttt ttatctgctg 420
acaaataaaa tgagtcgaag actatcctat tctgaaataa tatttttaat tttaggtagc 480
tttgtgtcat tgttatctaa ttctagtacg accatacttt gttttgcctt atttgtcttc 540
tttagatttt ggggccttag ggacgtggtc agcaatgctt atttgaagat aacattgata 600
tcttatgcag ttttttcttt tttaatttta tctgcacaga gtcccttttt tagggttctt 660
actggtctat ttggaaagga cataacattt tctggaagac tgagtatttg gcaacaggca 720
atagcgttct ttaagcagaa tcctttgttt ggtaatgggt taaccttgaa ttttactccg 780
tggacaaatg gtgtagttgt gaatacagca cacaattatt tattggactt actagcacgt 840
tttggactga ttacgggaat tctttttatt ctattgttgg ttaattttgt tttaggtaaa 900
aatagaataa aaaataaaac tttatttgct atgttggttt gttacttata ttatatatta 960
atggaaataa gttcaaccag cttttattta gcattaatcg catgtatttt ttatgtgggt 1020
gtgcaagaaa gtagattgaa aaatgaaaaa aatatttaa 1059
<210> 60
<211> 1116
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 60
atgacaatat atgttctctt tttagcatat atcctcatat gtgccgtaat tatatacttc 60
caaaaaggcg atccagaaag aaagaataag atttttttga attatgtgtt tatagcaatg 120
tttctattgt attcactgcg agcaagttct gtaggaaggg atgtacctgg atatgaaaga 180
gcatatggat tagcaaatgt tgatttattt gcgggactac atatcgggaa ttttgaaaaa 240
ggctatctgt ttctcatgtg gatatgtaat aaattgaatc tttcttttca gtggtttttg 300
acattatgta atgcaattat tttgttcccg atatatcgct atatacgtaa atactctcag 360
aatacttttc tatccgtaat catttatgtt tgctatatat tctttgagtt taatttgacc 420
ggtcttcgcc aagcaatagc aacttcaatt gtattgatgg ctattatggt actgatagaa 480
tccaaacagg actggcttgg acttttgaaa tattgtttaa ttgtattatt ggcacccttg 540
tttcataccg gtgcatttgt tgcgctgttt tatttgccat ttcactttat gaaaaagttc 600
aaaacctata cattgtccat tatggtcact gctattgttt ttctggtagg tagaggctat 660
attatgggat ttgtcaaaaa cttctttgga aaggattcta tgaatgcaag tgccggactg 720
tatattggat tgaatatgct tttcttaatt ggacttgctg ttttatttat aattggtgga 780
aaaaacagag aaattggaga aagcaacaca tcggagggga atacttctgt aacagtcgag 840
gatactccta tgcaagttct tatggaaaag atgttttatt taagcattgc cgtactattc 900
ttgttcggtt ctgacacatc cgttagaagt ttcatgctgt tgaaccaagt aattatagtc 960
ttgcttccga atagcttgcc gtatattttt gaggaaaaat cacagcgaat agcaatctgg 1020
atttttacta ttttctttat aatatttttc tttataaata cgttacttcc aaataatttc 1080
gatattgtcc catataaatt cttctggcaa ttataa 1116
<210> 61
<211> 1206
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 61
atgaaatcaa agaacaactt agtagcagtg ttagcctggc ttctccccaa ttcttatgct 60
tttatagatg tattgaaggt attttacccg accattaatt tgcaaattgt aagttctttc 120
ttagcggcag tagctattct tctctttttt attcaaattt attataaaaa gtatgaatta 180
atgctttttc aagtagcttt gctatttatc gctttacttt actctacccg atttttttat 240
catgaaaatt ttgaattata tcaatctttt tacaattctt ttttgatacg ttcttttcca 300
gctatactag tagctatgat gttgtctcga tataatagga ttaaggaatt tataaagtgg 360
tcggaaccgt acatgttact ttatacccta gcggtattta taacaagctt tgctccccgt 420
aatagtatta tttcttatca agctttatct tactactcta tgaccgccta catgctaaca 480
atttttagtg ccgtttatcg taaaaatctt ttggcaaatg gtctaacatt tttcaaattt 540
cgttcgtgga cacctatttg ttatggtttg atgatagttc aattgtacac aatgctttat 600
ggtggtggtc ggggggcgtt tatactttgt attattttta cactaatctt tttcaagaaa 660
attttcactt cacctcgact ttttacagta tttcttatgt gtacgcttgt tttggtaata 720
ggaattattt tgactgatta tatagatatt tcccagatac taaacaatga tggtgtgcga 780
cgtctagtga atttctttgg gagatacgac cgtatttcag ggtaccaacg gcttgtactt 840
tatgaacgag cttgggaatc cattcaagaa agacctttgt taggttgggg aatcggatcg 900
actttattaa gatttaatgg ctattctcat aatatttttc tagatatctt gcatgataca 960
ggtctgatag gtctatctat tatggcattt ttgatgacga tttgtaccat tttctggtat 1020
aaaattcgta gcattgattg gaagattgcg ttgtttggat ttatgattat cgaaacaatg 1080
gtacatatga attttagcgg aagttatctt gcagatggtc gcttgtggtt ttggtttact 1140
tttatttatt gttactatag atggaagaag gatgaaaaaa atgaagttag caattctact 1200
atctga 1206
<210> 62
<211> 1245
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 62
atgaaaaaag ggaacatggc ttcggataaa atcatatgtg cattaattac ttttatgatt 60
atgtcttttt atatatttga aacagcatcc tggggtagat acgttctatt tgggattacg 120
attcttataa cattagttta tgtcgcaaat aattgtttta aattgccctt agtaataaat 180
aagtcatttc atcttcatat tgctatcttt gctctgtttt gctttgcgtc gtctatttgg 240
gcgtggaatc catttcaagc agtatcaaaa gggatgacag ttttttcaat attagtatgt 300
ttttcgttgt tctatccata ttttcaacaa aagcgaagta tagatttgtt attggatgct 360
tttatgtttt caggatatgg aatagctcta tataccgtcg catattacgg aacaagtggt 420
attgcggaaa tgctgaacgg aaatatacgt ttaggtaatg attttacaaa tgctaattct 480
attgggctta ttacggcgac atcatgcatc attcaatttt cttatctatt gagaaaagaa 540
aagaaaaaat atttgatttt cttggttccg tgcattatag ctcttgcgat ttctcagtct 600
cgtaaagcta tgattttcct tgttgttgga attgctttct taattatgtc caacggaagt 660
gttcgtggtt ctatcctaaa aaaggctttc aatatttttg ggggagtcat agcggtttat 720
cttttgatat attcgttatc gcaattagaa attttttctg gtgtatttca tagattcgag 780
actttgtttg aatcaattaa tggtacaaga gtagaagata taagatctgt atataggcga 840
attggaatgc agcaattcta taaaactcct atcttgggta taggaatagg aaactcatta 900
gaattactcg aaagtgttgg gcaaaggaga acttatttac attgtaattt tgtagaacta 960
ctttcgagtg ggggaattgt tgggtttatt atatattatt caatatattt taagctaatt 1020
gttgagctct ggaaatatag gaaatatcgt tttacaacga ccaacctgtg tatcgtccta 1080
ttgtttttga tgctaatcat ggattatgga atggtaacat actatgataa acagcaatac 1140
ctctatttta tgtgcttttt cttacaacta gaatttataa aagaagattg ctatacagat 1200
gtagatgtaa tagaaaagtc agaaaggtgc gaaaacaatg tatga 1245
<210> 63
<211> 1122
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 63
gtggctattt ttatatactt gtttttaatt tttattaatc tatttttaag tttcaaagat 60
tatatccatc cttttaacaa aaatagtaga cgggctttat gggggatagt aatattcagt 120
tttattataa tctatctatt cttatctgga tatagaaatt atagcggact ttctggtgat 180
ttatataatc tccaaactga gtatgaatat tatttaagaa ctggtaattc tatttatgaa 240
ggtggatata aaatatttat gggtgttgga aaggccttta ccctagaatt ttatacttgg 300
cgtgcaataa ttatattttt tgctttacta ttaaattttt attcaatcat caggtatgca 360
aagaatccgc acttagttat tgctttattt tttatatttc tgattattcc ctcagcggag 420
caacttcgca attttttagc tttttccttt tttcaagttg gaatgtcgca acttatatac 480
tcgactaaat caaataaata taagaatttc atattttctc tatttgtact aattgcaggt 540
tatgtacact cttcttttta tatttatatt gtaatactgc taattgataa aataacgttg 600
aaatacataa aaatattggc ttggtttgtg ttgttatttt gtgttgttat tttcctaaat 660
aacaataata tcccaggatt ttcaataata atcaattatt ttgaaaatag tagagtttct 720
aattatttaa atcagaatat tcgttttggg tttttactat atatgtttct tcacggtctt 780
tctctctata ttctttatgt tactatgtcg cttactcaaa agttgcaaat taaagattta 840
aaaattgata aagtgtttac tactaattta ttgttaataa tttttttccc cctactaatg 900
gtaaatggaa catttattcg gatatttaga aacattctat tactgaatta cttttccttt 960
ggtgaattct tatcactaag gtattttcat tcgaaaaagt taatattcgt cttaatgatc 1020
gttagtagtt gtgtgatgtg ggtctatttg acttacttct ttgcgaatac tccttatgca 1080
ctttattttc catttttttt agacaacata tactttcgtt aa 1122
<210> 64
<211> 1272
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 64
atgaaaataa atagatcatt aaatatagaa atcttattgc tttctgcagt aattattttt 60
ttgggtcaga tagcattaaa aaatgattca aatgctattc tgctcattgc cttaataatg 120
gtgggttttg cggcagctat tctcccacaa gataacttaa tgccatttat gctttgtatg 180
atagcaccaa atagattatt aacttatggt tcgatatctg caccaataat tattatatta 240
gttgccttgt ttaagggaaa tagatacctt catcaaccga aaaagttttt tttgagttcg 300
atggcgttga ttggatatac tattattaca ttatttatag gggaatcttt aatatttgat 360
gcaataaaaa ttgtagcggt attgttgttc tttattatgt atacaaattg tcaagatatt 420
cacacttcgt atgttaggta cactgttttt tgttcccttg gctgtttgtt atctgcgggc 480
ataactctac taataaataa aaattctatt tcagaagcgg ggagatttac aataaaaggc 540
tctgggcaaa atgttttagg tatcttgtgt gctattatgg caatcaatct attggttatt 600
gtattgaatt atgttaataa aaaaaagtta ttctatgttt ttctatcgac tatcttgtgt 660
ggaattggtt ttctgacagg ttcaagaagc tttttattag caattggcac ggggatattt 720
gggttagtaa taatgatgat ggtaaaacta gattttaaac gattgtttaa aattggtttc 780
atcatttctt tagttacttt tggtgctata tttgcagtta aatttagtag ttttataaat 840
aactattggg atttgataat gtatcgggta acgaaactac agaatgtgga tgtatctaat 900
ggtaggttcg atttatggga acaatatatt acgatattca gagaacaacc aatttatttt 960
tggtttggtg gattaagtat agatgattcg caagtgcaat atgtagcaca taatatgatc 1020
atagaacaaa tcgcatcatt tggggtaatt ggttcaattt ttattttgaa tttatatatt 1080
tcagcagttt tagttattaa aaaatattct aaatgtgaca ttaaaatatt ttcttttaca 1140
gtagtaccat tgattgcctt tttgggttct agtatggttt cacatacaat acttggtatt 1200
ccgcaaacaa caatgttatt tttgtgtata ttttcattat ttagttcaaa aagaacaggg 1260
ggagaattat ga 1272
<210> 65
<211> 825
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 65
ttgttatgta agatactatt atttacagaa acatcaaata tattggtgag agatttcggt 60
gtagttttaa acgctaatac cattggtatc cagtttgcaa tagcaattgt acttatgatt 120
tggttacaaa aagaaaaaca gattggattg acattggcaa gcgtatcaat ggtgattttt 180
ggtgcgggag tgattttaac aggttcgaag aaagcactat tatctatagc agtaggttta 240
gtagcaagct atttattcac atctgaaaat tttgttcgta agattaggaa tgcctctcta 300
ttattaatcg tagggcttgg aatattctat ctgataaagg ataatgagtt tatttattct 360
ataattggta ggcgattcca aggaatgttc aatggttttt ttctaggaaa gatgtatgct 420
gatgcaagta cttatgcacg aattagtgtt tcggaagtcg cattaatgta ttggagagag 480
cgtccttttt tgggatatgg tttaaatagc ttcgaggcaa ttacacaatt tggattctat 540
actcataata attacattca actacttttt gatgtaggat tgataggttt atttatatat 600
tacagtttgt ttgtttggat aatttggaaa ggaatccgta aaactattat ttactcagta 660
tctgaaatta ttctaccgtt ggttttgata atagtcgttt gtgatgtagg ctatgttagt 720
tatggagata ctttttttca aggaatgtta gcgtttatta gtgctataat agttttatta 780
aataataatc acagtattgt tgaaagtaat ttggaaaggt cataa 825
<210> 66
<211> 1443
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 66
atgcttgcta ttagattttc aaggaatagt ttatttcata cactatcttt ttcgattgta 60
tcaataattt tattacaagc tcttaacctc tatgtatcag attcggcaat taatttttcg 120
atcgttcaaa ttctctcagt tataactacc ttgtattttt tttattcatt atatacaatt 180
tctcgtgtag aaatgagaat gtttagtctg actagcatat taatgctcat tacttattta 240
tttaatttcg gacaaatgtt tttatggaca tttggtattc atacagaatt tgaattaggg 300
aaagttcctt tgttttccaa ctttccaact ccaagttcgg agcaaatata taatgctcta 360
ttcttttcat tatactgtta ctttgcaatg gttttaggaa tttgtactat tagagttttt 420
caaactaaag agaaaactag aactgttggg actaaaaata tgaatgaaga gcttttgaag 480
cagagtattt ataaagttag tatcttgcta ggtctaatcg tagtgccatt aactttttat 540
aaaatatttt taacgttatc ccaatctata agttatggat atatctcttt gtattattct 600
gatttttcaa tgaatcctct aatagcacgt tcagaagatt acttttttcc tgtaatagtc 660
ggtttattaa ttggaagcga gtataaaaag atcagaattg catatagttt attcgcatta 720
tatatgattt tattcctttt agctggggag cgtggtaatt ggttttacaa gcttattatt 780
ttagtgtata tgcataatat ttactataga aagattagtt ggcgtaaatt cttgagattt 840
gctgctatta gctatgtatt tttaattttt gttgggttta ttattaatgt acgaaatgag 900
ggattatcaa acatttcttt aaatgaacta gtgaacgcat acactattaa tcaaagtcct 960
attattcgat tcgtgcaaga aatggggact agtttaggta taacaataat ggttatgaca 1020
tttggacgag gagttttttc acaatttgga aatacatata tctcatcagc tctgacatct 1080
ttctcatcag cactggctac tcaattagga ataaatcatg tttacttggg taattatttg 1140
tcgcaaaatc tgctcaagat tcgctgggga acgggattta attttttttc tgaggccttt 1200
attaatgggg gatggtttgg tgctttttat ctgtacttat ttggtatgct gtttgcaatg 1260
cttattaata ataaaagtac aaaactaaat caatattcct caccagttgc ttgtgcgatt 1320
gttttatcta tgatgcgtga taactcattg aatggtttcc gtatgcttgt acaggtttta 1380
ttatttacaa tattactgat tatagtagta aagaatacgt tatataaaag aatctcaact 1440
taa 1443
<210> 67
<211> 1263
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 67
atgatatata ttgttgtagc aatttttcta ttgttttcat ttattagagt tttaaacatt 60
ccattgacat cacctcctgc aattttttct gttatttgga gtgttttcat attcttctca 120
tacttgattt taagcgagag atatccattc actttttatg gtataacttg gatattctgt 180
agttgttttc tcctgtcttt aggatatgct tttagaattg agacaaaaag tacaaaaaca 240
aaagaaatgg aaagcagtgt gaattcagcc aatatttctt ggagtttgtt agcagtcttt 300
atttttctag gattgactgg ttttattttg agtgctgcaa agtatggtgt ttcattgaat 360
gtattttctg attttgatag tttacaatcg acgtcaaact caatagctga gcaaagatat 420
tcaggatcaa gtgttggttc aagcacttta gaacaaattt taaattcatt tgtttatgtt 480
ttacccctat gtggagggta tagtttagtt tatgcaaatt ctgttaggaa aaaaatactt 540
ataatattga cagcagttcc gtcactatta tccatgctat taactagtgc aaagttggct 600
gttgtagctt acactatatt attttttgtc tcatactatg tctcgtattt atatctaaac 660
aaaaaattta tctctattaa ttataggaaa attttccaat ctttgttttt aggctatatt 720
ttgtatcaat tgttttatct aagtttttta ctgagaattg gtagtacctc tgcaagcatt 780
agtagaataa ttgttactaa attgggaatc tacgcctttg gacatgttca agcttttgat 840
ttatggctcc caatatatat gtcatcagat gccgaacttg gatttggagg aaatacattc 900
ttagcatttt catctttaat tggggtattg gataaaaagc aagggattta tgaattaata 960
agtggaagtt caactaatgt ttttactcaa ttccgaagtt tgattagtga ttttggaata 1020
ctatttggct tattagttat ttttatacta ggtatagtat taagaatttt ttataaaaat 1080
gttattaatt atagtagtaa attaattgta ctacaacagg tattgtttat ctctatactt 1140
ttttatctta tttattttat tgtttctcca tgggtctatc taacatttgt gttgactttt 1200
tttatctttt atttttattt agtagttgca tttcatttta gaattaagat aaaatttcgt 1260
taa 1263
<210> 68
<211> 1434
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 68
atgcagaaaa atctagtttt gaaaaatagt tttattttac tttcagcttt gatgattgta 60
gaaatgatat tagttattac tgcttatgtt aatcaatcag aaaatcttta tgtatatgct 120
attgttacga tgtggggaat tggaattatt tattgtctct tagatattta taatagagta 180
ttctttttta tttacctttt ggcttttttt aattttttaa ttggaagaag tcttttaata 240
attcttggta ttttcaagga accctatcgt tttactctcc aagagagcat ttcaggacaa 300
caaatagtat tatttagttt aattgtttta gtttcatcct atattctact aggtaaatcg 360
atatctagga atatgcaaat tatgaatgca gatagtgtta gtatacaaaa agttcgctta 420
gctagtaaag tgttattttt atcaactttt gtatttttag ttatttctgt tttaggaaca 480
atattttatg ttattcaaaa tggttatgcc agtatatatg ttgatgttga ggcgaaagtt 540
ccaacaatca taaaaaaaat aggagacata gcgccatttg ctttctatct atttttagga 600
acattaccta ccaaaaaaga agtcaggctt ccattgatgt tatatactat ttatttaatt 660
ttaacattag gaacaggggc tcgatttgaa tttgttattg gtattttatc aatattgact 720
tattttattc ttaggaatag tttggctaag tctgagtctg aaaaatggat tcgtaaaaga 780
agtttaaaat attatgttat attgggattc attatcattg ctatgctatc agcttataat 840
aatcttcgct ttaatgagtc agttgagaca acgagtccaa ttcaattaat aacgaagttt 900
agctttgatc aaggtattag cattaatata cctaaaagga tttttaaata tagtaactat 960
atacccacag atagatttta ttcgttagac tcaacaatat cgttttttaa taaaaatatt 1020
ggtagtaaat tatttggaac gatatattat agcggtgcta gcgttgaaaa ggcaattaac 1080
gggaatagtt tatcagatgt tttatcatat attttgtaca aatctcaata tcttttggga 1140
atcggaggag gttccagtta tattgcagaa gtatatttgg attttggttg gctcggatta 1200
tttattatta acataattta ttcgttttta tttgttaagc tagaagattt ttcattaaaa 1260
tctccttata aatttttaat tgcctttgtt atatttatgg catttttacg tacaccaagg 1320
ggaaggacag atggtgttat tctaaagata tttgacccaa tgttatggat tagtttgata 1380
atcatatatt ttataactaa atatatgatc aaagtaaaga gggtaagaaa atag 1434
<210> 69
<211> 1218
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 69
atgtttaata tattactttt gtttttttat attttttctt ttagttttta taatggtggt 60
ttttttgatt cagcattgtt agttttaatt tttctttcta ttcacattat tataaatcaa 120
agttatagaa aaatggtgca aaggtatatt tctacaaaat atttcgttaa tatatttttg 180
ttgtacattg tattgaatat atggttttta ataattttat ttattaatga ttctgtagat 240
tttacttttt taaagacatt tgctaacatg tttcagactt gggtgactgg aggattacta 300
ttccaatatt attgtattca gaataaagaa aaagaaattg ttaatcaatt aatgataatt 360
tttgctgtac aaacactgat tcagtggtta gcattttctg tcccatccat aaaaacatta 420
ataaattata ctaaaagcta ttcgacgata cttaaaggga tgtcgtattc tggaattaga 480
gcgaatgcat tatcaggaag ttcatttttt ggattatctg cagcttatgc tattatatta 540
atactgtttg tttcgaataa aaatcaatta tttgtgaaaa aaccaacttt gaaatttgga 600
ctgtttttgt ttattttaac aggaggattt tttgctggac gtaccacttt tatgggtctt 660
tttattgcga taattgtatt tttgtgtgtg aggaaattca aaccaacaaa agaacagata 720
ctatcagtga gtgtcggtat acctagtctg tttgtcatgc tatttatatt gaactcctat 780
tttggaaagt tagagagtat tattaattta aaaaattttg catttcaatt tttctataaa 840
tatagtactc aagggactat agttatttct agtgttgaga gtctatcaga aatgtatttc 900
attccagaac tatcaacttg gttagtaggt gatggaaaat attctgtggc tactggttat 960
tatatgagta ctgatgtagg ttatcttagg gttattcttt ttggtggagt actctcttta 1020
ttggtattat ttacaatgca atttttattg atgcaggtaa ataaaggtaa agaaaagttt 1080
attaaaattt cgtgtcttat tgcattattg atgttaaatc taaaaggtga agtgatattc 1140
tggagtcaaa atgttcttgc tattatcacg attttctgct tacaagactt gtttacatta 1200
gatagaaaat ctcgttag 1218
<210> 70
<211> 26876
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 70
atgaaaaaga gaagcggacg aagtaagtcg tccaagttca aattggtaaa ttttgcgctt 60
ttgggacttt atgccattac tctatgtttg ttcttagtga ctatgtatcg ctataacatc 120
ctagatttcc ggtatttaaa ctatattgtg acgcttttgc tagtcggagt ggcagtattg 180
gctggattat tgatgtggcg taagaaagcg cgcatattta cagcgctctt acttgttttt 240
tcactggtca tcacgtctgt cggaatctat ggaatgcaag aagttgtaaa attttcaaca 300
cggctaaatt caaattcgac gttttcagaa tatgaaatga gcatccttgt cccagcaaat 360
agtgagatta cagatgttcg tcagcttact agtattcttg ctccagccga atacgaccaa 420
gataacatca ccgctttatt ggatgacata tccaaaatgg aatctactca actagcaact 480
agccccgcaa cctcttacct gacagcatat caatctatga taaatggcga gagtcaagcg 540
atggtcttca atggagtttt caccaatatt ttagaaaatg aagatccaga cttttcttca 600
aaagtaaaaa aaatatatag tttcaaagtg actcagactg ttgaaacagc tactgagcag 660
gtgagtggag atagctttaa tatctatatt agtggtatag atacttacgg tccaatttca 720
tctgtttcac gttcagatgt caatatcatt atgactgtca atcgtgcgac acataagatt 780
ttattgacaa ctactccacg agattcatac gttgctattg cagatggtgg gcaaaatcaa 840
tacgataaac taacacatgc tgggatttat ggtgttaacg cctcagtgca caccttagaa 900
aatctttatg gtattgacat tagtaactat atccgactca actttacttc cttcttgcag 960
ttgattgatt tggttggtgg tattgatgtt gaaaataccc aagaatttac aagcggaggg 1020
tataatttcc ctgttgggac agtacatcta gacgcagagc aagcgctaat ctttgttcgt 1080
gagcggtatt cgttggctaa tggtgataat gaccgtggta aaaaccaaga aaaagtcatt 1140
gcagccctca tcaaaaaatt gagttcacca gagaatttac gcaattatca agctattcta 1200
actggtttgg aagggtctat ccaaacagat ttgagcttag aaacaattat aggcttggta 1260
aatacccaat tagaatcagg aacacaattt acagtagagt cacaagcatt gacaggaaca 1320
ggacgctcag atttatcttc ttatgcgatg cctggttcac aactttatat gatggaaatt 1380
aaccaagata gtctggagca agcaaaggca gcgattcagt cagtattgga tggaaattaa 1440
aaaaatagga gaaaacatga ataatcaaga aatgaataca atcgaaattg atattttact 1500
cttactgaaa acgatttgga gaaagaaatt tcttattatc ctcacagctc tttttggagc 1560
agggattgct tttgtctata gtagtttttt agtaacccct caatttgact ccactacacg 1620
gatttatgta gtcagtcaaa atgttgaagc cggtgcggga ttgactagcc aagagttaca 1680
agcgggtacc tatttggtaa aagactataa ggaaattatc ctatcacaag atgtattgac 1740
acaagtagca acggaattga atctgaaaga gagtttgaaa gaaaaaattt cagtttctat 1800
tcctgttgat actcgtatcg tttctatctc cgtgcgtgat gcggatccaa atgaagcggc 1860
acgtattgca aatagccttc gcacctttgc agcgcaaaag attgttgagg tcaccaaggt 1920
aagcgatgtg acgacacttg aagaagcagt cccagcggaa gaaccaacca ctccgaatat 1980
aaaacgcaat atcttgcttg gtttattagc tggaggtatc ttggcaacgg gaattatctt 2040
ggctatggaa gttttggatg accgtgtcaa acgccctcag gatattgaag aggtaatggg 2100
cttgaccttg cttggtgtag taccagattc gaagaaattg aaataggaga acaatatggc 2160
aacgttagaa attgcgcgta caaaaagaga gatagtaaat aaaaccgagg agtatttcaa 2220
tgccatccgt accaacattc agcttagcgg agcggatatt aaggttgttg gcattacctc 2280
tgttcaatca aatgaaggta agagtacaac tgcggctagt ctcgctattg cctatgctcg 2340
ttcaggttat aagaccgttt tggtggatgc agatatccga aattcagtca tgcctggttt 2400
cttcaagcca attacaaaga ttacaggctt gacggattac ctagcaggga caacagactt 2460
gtctcaagga ttatgcgata cagatattcc aaacttgacc gtcattgagt caggaaaggt 2520
ttctcccaac cctactgccc ttttacaaag taagaatttt gaaaatctac ttgcgactct 2580
tcgtcgctat tatgattatg ttatcgttga ctgtccacca ttaggactgg taattgatgc 2640
agctatcatt gcacaaaaat gtgatgcgat ggttgcagta gtagaagcag gcagtgttaa 2700
gcgctcatct ttgaaaaaag taaaagagca gttggatcaa acaggcacac cgttctcagg 2760
cgttatcttg aacaaatatg atattgccaa tgagaagtat agtgaatacg gaaactacgg 2820
acattacggc aaaaaagcct aatttctcag ataacataag tttgataagt aggtattagt 2880
atgattgata ttcattcgca tatcatattt ggtgtggatg acggccccaa aactattgaa 2940
gaaagcctga gtttgatagg cgaagcttat cgtcaaggtg ttcgctatat cgtagcgaca 3000
tctcatagac gaaaagggat gtttgaaaca ccagaaaaag ttatcatgac taactttctt 3060
caacttaaag cagcagtagc agaagtttat cctgaaatac gattgtgcta tggtgctgaa 3120
ttgtattata gtaaagatat cttaagcaaa cttgaaaaaa agaaagtacc aacgcttaat 3180
ggctcacgct atattctctt ggagttcagt atgaatactc cttggaaaga gattcaagaa 3240
gcagtgaacg aagtgacgct acttgggcta actcctgtac ttgcccatat agaacgatat 3300
gatgcactag cgtttcatgc agagagagta gaagagctaa ttgacaaggg atgctacact 3360
caggtaaata gtaaccatgt gctgaagccc gctttaattg gcgatcgagc aaaagaattt 3420
aaaaaacgca ctcggtattt tttagagcag gacttagtac attgtgttgc tagcgatatg 3480
cataatttat ctagtagacc tccgtttatg agggaggctt ataagttaat aacagaggaa 3540
tttggcaaag ataaagcgaa agcgttgcta aaaaagaatc ctcttatgct attaaaaaac 3600
caggcgattt aaactggtta ctctagattg tggagaaaaa atggatttag gaactgttac 3660
tgacaaactg ttagagcgca acagtaaacg attgatactc atgtgcatgg atacgtgtct 3720
tcttatagtt tccatgattt tgagcaggct gtttttggat gttattattg acattccaga 3780
tgaacgcttc attcttgcag ttttatttgt tttaatattc tacctgatta tctcagttag 3840
actaaaggtt ttttcgctaa ttaccaggta tactggatat caaagttatg tgaaaatagg 3900
actgagtttg atatccgctt attcattatt tttaatcatt tcaatgattc tatggcagac 3960
ctttagttat cgtttcatct tagtgtcctt atttttgtcg tatgtaatgc tcattactcc 4020
gaggattgtt tggaaagtct tacatgagac gagaaaaaat gtcattcgaa agaaggatag 4080
cccactaaga atcttagtag taggtgctgg agatggtggg aatatgttta tcaataccgt 4140
cgaagatcga aaattgaatt ttgaaattgt cggtatagtt gaccgtgatc caaataaact 4200
tggaacattt atccgtactg ccaaagtttt aggaaaccgt aatgatattc cacgattggt 4260
agaagaatta gctgttgacc aagtgacgat tgccatccct tctttaaatg gtaaggagag 4320
agagaagatt gttgaaatct gtaacactac aggagtgacc gtcaataata tgccgagcat 4380
tgaagacatt atggcgggta acatgtctgt cagtgccttt caggaaattg acgtagcaga 4440
ccttcttggt cgaccagagg ttgttttgga tcaggatgga ttgagtcagt ttttcaaagg 4500
gaaaacaatc cttgtcacag gagcaggtgg ctctatcggt tcagagctat gtcgtcaaat 4560
tgctaagttt acgcctaaac gcttgttgtt gcttggacat ggagaaaatt caatctatct 4620
cattcatcga gagttactgg aaaagtacca aggtaagatt gagttggtcc ctctcattgc 4680
agatattcaa gatagagaat tgatttttag cataatggct gaatatcaac ccgatgttgt 4740
ttatcatgct gcagcacata agcatgttcc tttgatggaa tacaatccac atgaagcagt 4800
gaagaataat atttttggaa cgaagaatgt ggctgaggcg gctaaaactg caaaagttgc 4860
caaatttgtt atggtttcaa cagataaagc tgttaatccg ccaaatgtta tgggagcgac 4920
taaacgtgtt gcagaaatga ttgtaacagg tttaaacgag ccaggtcaga ctcaatttgc 4980
tgcagttcgt tttgggaatg ttctaggtag tcgtggaagt gttgttccgc tattcaaaga 5040
gcaaattaga aaaggtggtc ctgttacggt caccgacttt aggatgactc gttatttcat 5100
gacgatccct gaggcaagtc gtttggttat acaagctgga catttggcaa aaggcggaga 5160
aatctttgtc ttggatatgg gtgagcctgt acaaattttg gaattggcaa gaaaagttat 5220
tctgctgagt ggtcatactg aggaagaaat cgggattgta gaatctggaa tcagaccagg 5280
cgagaaactc tacgaggaat tactatcaac agaagaacgt gtcagcgaac agatttatga 5340
aaaaatattt gtgggtcgcg ttacaaataa gcaatcggac attgtcaatt catttatcaa 5400
tggattactc caaaaagata gaaatgaatt aaaagatatc ttgattgaat ttgcaaaaca 5460
agaataagaa agtaaaaaat atttttactt tcctagagtc taaatagtat ttagattcta 5520
ggaaggttga aatactttcc tgaggtaaga tatgctaggt cataggttag taagagaaaa 5580
agaaaatagg atcaaaggtg gtctttacca taagattcaa atcgagctgg ctttcaactc 5640
aaaccacatt gaaggcagtc aactgacgta tgaccagaca aggtatatct atgagaccaa 5700
tacgattggt tttgaagaaa attcaaccat aaaggtggat gatattatcg aaacgaccaa 5760
tcattttacg gcttttgatt ttctcttaga tagctttcaa gatgatttga atgaaacttg 5820
gataaaggat attcatgcta tcttgaagag aggaacctct gattctcgga agtcatggtt 5880
tcaagtcgga gactataagt cgtacccaaa tgaagttggc ggacgtgaaa cgactttgcc 5940
agaaaaagta gaatccgata tgcaagaatt gctaaacagc tatcaccgct tgcctgagat 6000
ttcctttgag gatattcttg actttcatgt tcgatttgaa cgcattcatc ccttccaaga 6060
tggtaacggg cgagtaggtc gattgattat gtttaaagaa tgtctcaaac atggccatgt 6120
cccctttatc atcggagatg acatgaagta tttttactat cgtggcttag ccgagtgggg 6180
agaacagaat ggtttcttat tagatacttg tctagccgca caagatcgat ttaaggacta 6240
tttagagtat tttaggattg aggattaagg attaaggaga ttggaatgta tccgattatt 6300
aagcgtattt tatctgttat tatttcaggg tgttccatta ttgttttaag tccagttcta 6360
ttattagttg cactggcaat caaactggat tcaaaaggtc cagtactatt taaacaaaaa 6420
cgagttggta agaataagaa atacttcatg atttataagt tccgttcaat gtatgtggat 6480
gcaccaagtg atatgccaac tcatttatta aaagatccga atgctatgat tactaaagta 6540
ggaggctttc ttcgaaaaac gagtttagat gagcttccgc aactatttaa cattttaaaa 6600
ggtgaaatgg ctattgtcgg accacgtcct gctttatgga atcaatatga ccttattgaa 6660
gaacgtgata agtacggggc aaatgatatc cgacctggat taactggttg ggctcaaatt 6720
aatggtcgtg atgaattgga aatcgatgag aagtcaaaat tggatggata ttatgttcaa 6780
aatatgagtc tagggttgga tattaaatgt ttctttggaa ctttccttag tgttgctaag 6840
agcgaaggag tagtcgaagg cggaacgggg cagaaaggaa agtaatgaaa agagtattga 6900
taacaggagc aaactcatat atcgggacat ctttcgaaaa atatgttttg gaaaaaggta 6960
ttgattttga aatagatacc ttggatttat tagagcctaa ctggaaagac tacgattttt 7020
cagggtatga ttctgttttt catgttgcag ggatagcaca tttttctaaa gacgaaagta 7080
aaaaagaact atattataaa gtaaatactg aactaactga gcatgttgcg aagattgcta 7140
agcaagcagg tgttggtcag tttatattta tgagtagcat tatcgtttat ggtgatagta 7200
cgagtgggga acgagttata actaaggata cccaacctgc accttctgac ttttatggcg 7260
acagtaaatt gcaagctgaa cagagactgg aaaagttagt gggcgataat tttagagttg 7320
tcattctcag accaccgatg atttatggta aaggttctaa gggaaattat cctaaattat 7380
caaaagtagc tcagaaaact ccaatttttc caaaaatttc gaattcaaga agcatgttgc 7440
atatcgataa tctttgcgaa tttgttaaac aagttatcaa gttggaattg agtggtgtgt 7500
attgtccaca aaattcagaa tatgttaata cttctgaatt agttaaagct atagctgaag 7560
tacatggaaa gaaagtatgt ttgacaaaaa tatttaaccc aatcataaag tttttcttcc 7620
atttagatac agtgaaaaaa gtatttggaa atcttactta taatcaatca ctgagtcaat 7680
atgaatttga gtatcaaatt gtagatttta atgaatccat taggttgaca gaggggtact 7740
tatgaaaatt gtaattgtaa attgctttga cacatatgaa cacagagtcg acctcttgct 7800
tgcttatttt ttatctaggg gagacgaagt ttcagttata gcctcaaatt ttaggcacat 7860
tgataaggta aaaagatttg ataaaaagaa acattatact tttgttgacg ctattcctta 7920
ccgtaagaat ttatcttata cgcgtttaaa gtcacactgg aagatagcaa aacaaatttt 7980
ttcagaattg ccttgtgata tagatttact gtgggtgctt gttcccccta actcatttgt 8040
aaagcaagca gcagaatata agaaaagaaa tccttgcacc aaattagtgt ttgatattat 8100
tgatatgtgg ccggaaacaa taccggccaa caaatttaag aatttctttt tgttaaaatt 8160
ctggggcaac attagagatt cctatttgaa gtattctgat tatgttgtga cggagtgtgg 8220
tctttatcag actaaactac cattagagaa tttcggaaat agaatctcaa ctctttactt 8280
atcttttggt gatgttaata tcgctcgaga taattcttta ttaaatttga gtggaaaaga 8340
aatagtttta tgttatcttg gttcgatgaa caatattgtt gatataaata ttatttcaaa 8400
tataatcgct tcgctttcta tgaaatacgc tgttacactt catctagtag gcgatggtga 8460
aaagaaagaa aatttgcttt cgttacttaa atctcaaaag attaaaataa tcgatcatgg 8520
aaagatattt gatcaagaag ttaaatccca aattttaagt aagtgtcatt ttggattgaa 8580
cataatgaag aaggaagtgt ttgttgggtt aacgatgaag agtttggatt atttaaaaaa 8640
tggtctgcct cttattaata atttaaaggg ggatacgtgg ggatttgtag agagggacgg 8700
tataggaata aacactgagg atggagtaat tactgcagaa gtacttactg attattttga 8760
aaaatattat catattcagt ctttttatag taatcacttt tcggaacaaa aattttattc 8820
ggatttaagt ttaattatga agaaaataga gtgtgactaa tgaatagaag ttctaattat 8880
aacatgatag aaaaaataga aaacttgttt tttttaatcc tgactaaatt gctcttcaaa 8940
tctgcaagac ttattcgatt tccggtttat ctacgaggag gtcaatatat ggaccttgga 9000
aatcagttaa ccttgggaag atactgtaga tttgatgtgc ttggaaatca taaatctaag 9060
aaactcattt tcggaagaaa tgtttgcata ggagaccatg ttaggatttc ttgtgttgaa 9120
aaaattgaag ttggtaacga tgttttgatt ggttcaagag tgctaataat cgataattct 9180
cacggaaatt atgcggggga aaatcaagat agtccattag tacctccgaa tcatagagga 9240
gtgcactcct ctccaatttt aataggggat aatgtttgga ttggtgaggg agttgttatt 9300
caaaaaggag tgactattgg tagtggaagt attatcgcag caaatactgt tataacgaaa 9360
aatgttcctt caaattgtat tgttgcagga cttccgggaa aagtgataaa ggaatataat 9420
tttgaaaaga gagtatggat ttaagtatga gtgaaaataa tatttgtaat aaaccaaaat 9480
tttctatcat tgttcctgtt tataatgttg aatcaactat atcacgatgt ctagatagta 9540
ttttaaatca gacatatgaa gattatgaag ttattttaat taatgatggt tcaacagatt 9600
ctagtttaga catttgtaat caatacaaaa gtattgacag acgtataaaa gtttttacta 9660
aaattaacgg tggtttaagc tctgcaagaa atacagggat aacttatgca attggagact 9720
atctctgttt tgttgatagc gatgattgga tagccaatga tatgttacaa cattttgaag 9780
tattgctgaa agaacaccaa ccagatatca tatccacatc gtataaaatt acaaaaaaaa 9840
taacagattc tgaaattgat actagcttag ttaataccaa aataatgact aggacggaag 9900
ctttagaata ttttttattt gagggaatga atagtcgagt atctgattat ccagtttgga 9960
taaagtgtta caaaaaagaa ttgtttagga atatcaaatt tccaatgggt gttctatatg 10020
aagattatgc aacaaatatt agattgataa aaaaaacaaa aaagtatgta aaaagctcaa 10080
aaatctgtta tttttattat caaggtggca gtagcatagt tcgaagcgga tttaagtata 10140
aggacagtga cctaattcat caaagtatgc ttgtagaaaa acttgttagt ggggaatcat 10200
tgctttgtca aaagtttgca aaggaaaaga cggcacgttc atatttttct ttattactaa 10260
agattgcagt ttatggattt gatgaatcag tgagtgccag tgttcaaaaa gaacttgtca 10320
aaaatttttc aaaacaagtt cgtactcatt taaagttatt gttgaggtca tctatgccat 10380
tgaatcgtaa attattgatg ttattaatgt gtattgacta taggttgcta ttgccattaa 10440
gaataggagt aaaaaaatga atgttatgtt tgtttctagt tcattaacag gagggggagc 10500
agaacgtgtc atatccgtta tagcaaatga attttctaaa attcctgcta tagataacat 10560
aactttagtt tcaattattg aagataaagt ctcttatgaa ttgaacaaac aaattaaata 10620
tattccatat aagggtatta atgaaaataa actgattcga gtcataaaac gtttctcatt 10680
tttacgtaaa acgattataa aagaaaaacc agatgtaatc atatcatttg ctacacaggt 10740
taacatttat tcaatagtgg ctaatctcgg tttaaaaact aaagtaatta tttccgagag 10800
gaatgaccct aatagagatc ctattcaaag cacagttagg aaaatccgag atcaagtata 10860
taaattatgt gatggagcag tatttcaaac tacagatgca atgaattatt ttgaaaaggt 10920
gggtagattt cctagaacag tgatattaaa tccattgaaa gcaaatttac cagagccata 10980
cgaagggata agagagagta gaattgttac ggttgctaga ttacatccag caaaaaattt 11040
acctttattg ataagtagtt ttaaaaacat cattagaaag tttccgaacc ttactcttga 11100
aatatatgga gaaggagagg agaaaaatca tttaatagaa ttagttaaga ctaaaggaat 11160
tacagagagg gtatttttta aaggttttag taattcaata cataatgata taaatgcggc 11220
tgcttgtttt gttttgtcat caaactatga gggcatgtca aatgcaatgc ttgaatcact 11280
tgcgttgggt attccgacta tatgtacaga ttgtccgata gggggtgctc gaatggtaat 11340
tgatcatatg gaaaatggtt tgttatttcc tgttggaaat caagaaaaat tagaacaata 11400
tttagaatta attcttgagg atttttcgtt aagagaaaaa tttaaagaaa attctataaa 11460
aataagagag aggttgtcgt cagaaaaaat atcaaaacaa tggttcgaat ttgtaaagtt 11520
aatattgaca gagtagaaaa aaatgaaata tagattaaag ccttttgcag catatttttt 11580
attgttgttt atgtttagtt taatgactgt taattacagc tcagtgacaa tttttagagt 11640
aaatttattt tttttaattg ttatatttat gtttggatat actattttga acataatata 11700
taatcgcaaa atatatttgg acggattttt tagaatatat ttgctgtttt ttttatggag 11760
ctgcttgaca aaattttggg cagtgagtga tatttattat tcgttgggaa tcaatacaat 11820
gagccttact ttaataatac tatttatatt tagtaatttg ataaaaagta aagaagatat 11880
ttatcataca atttgggcga tagcttgggc aggtgtaggt atggtattat tttatggttt 11940
ttattttgga ttccgtacta tgatttatat tcggttgaat aataacataa caactgtcaa 12000
tgcaaataca tatggattgt gtgcattttt ttcagcaata gggttcttgt atatgtatat 12060
gacaaatcaa aaaaggttaa tcaatttgat actgatgttg tctatgttag ggattagtat 12120
attatcgggt tcaagaaaag tattgattct ttttttaatt ataatattag gaacctatct 12180
atttacagaa ttgaaaaaaa gttttcatag atttttgaat ataacgatca tgatttttgt 12240
tttttattta ttgatacaaa acaatcaatt tttatataat actattggtc tacgaatttt 12300
agagacagtg aattatttgt cagaagatgg tgtagctagt aatacaagta ctggaattcg 12360
atcttcgatg attgaattgg gtatgagatt gtttaggtta aaaccatggc taggatatgg 12420
tgtaatgggg tcgtatattt ataatggtaa tacttatttt cataataatt ttgttgaact 12480
tttagttaat acaggcatag taggtactgt tatttattat tccggtttct ttatattact 12540
aaagcacaat attaaagcgt atctatatag ttttaatttt gaaacactat ttttcttgtt 12600
attacttatt tctattattt ttatggatta ttcattagtt tcgtataatg aaagaatagt 12660
gttggtgtgg ctaattttga tgtttaaaat gtataaaatg ggatttttgt ttgagggagg 12720
ccgtatatga aaaggttatt aaaagttatt agtaacagag gatatcgttt caaagtattg 12780
actaaattag gattttataa taatctacca gatgatgtct ttttgaagag attatataaa 12840
tttgaaatga ataaggaact tgaactagat aatccaaaaa catataatga gaaattgcaa 12900
tggttaaaaa ttaatgatag aaatccctat tatacagatc tttcagataa atataaagtg 12960
aaagatattg ttgctaatgc aataggaggg aaatacatag ttcctacact tgggatttgg 13020
gataacgtac aggatattga tttcgaagca ttacctaatc aatttgtttt aaaatgtacg 13080
catgattcag gtagtgttat tatttgtaga aataaagaag agttggatat aaataaagtt 13140
aaaaaaactc ttgataaagc attgaaggga aatttttatt actattctag agaatggtgt 13200
tataaaaatg tacaacctag gattattgct gaagaatatt tagaagattt agctaataca 13260
gatgatacgg tagattataa atttttcttt tttaacggga gaccagaatt tatatatata 13320
tctagaggat tggaagatca ttccactgct caaatttcat tttatgattt ggaagggcaa 13380
gaattggact tttgtagaga tgattatcgt cgtttaggtg attttagcct acctaaaaat 13440
tataaagaaa tgagagaagt agcagaaaaa ctttataaga tatgtaacgc tccatttgtg 13500
cggatagatt tgtattctgt taatggtagg atatattttt cggaatttac cttttatcca 13560
gcaggaggct ttttaccatt ttcacctaaa gaaacagatg aaaaacttgg agagttgtta 13620
gatttgacac agattgatta aacttctcct tgattaatca tcgactatga ggatatataa 13680
ttgataaaaa agttaacgac aaaagttata tttaaaaaca ccattattta catggtttgt 13740
ggggtgttaa ttagaggatt tagttttttt ctacttccgc tctatacttc ttatttaacc 13800
actgttgatt atggtattac aagtcttgta agtagttttt taataaccgg ttcattttta 13860
gtcgctgcat cgttatattc agcaatattt cgcttttatg tagatttaaa acatgacaca 13920
gttaggttaa gtcgctttta tgggactata attttatttg ttttaataat gagtattatt 13980
gctagtgtag tcatgatact tggaaaggag ctagttggtc attatttttt tcccaacatc 14040
gacttttttc cgacgatatt catttccatt ttatcactag ttttttattg tcagtatact 14100
atttatgaaa atattttaaa aagtcaacag caagctggaa agtatgcaat tattacactg 14160
atatttttta ttcttaccct agttttaaat atattatttg tagtacattt taaactggga 14220
gcgaatggtg tcttacttgc caatctcata acatatgctg tgtacttctt tgtaatgtta 14280
gttgatttat ggaaatctag tatgataatg tggtgtttgg ataaaagctt actaaaggaa 14340
gctttgctat attcgatacc aataatccct cataatttat caacccatat cacagttttt 14400
ttgtcgaagc tttttatcgg agacattggg aatctaggag aggtaggctt gtttgcgatt 14460
gcctcccaat tcggtggtat tgctgatata gttcaaggtt atgtgaatag tgcttacggt 14520
ccatggctct atgagcagat gtcgatgtta aacgattcag ttcggaagaa tattgcgaaa 14580
atttcagaaa ttttaacgta tgtaattggt ttttttcttt tgggcattgc actattttct 14640
catgattata ttattttgtt attagataaa ggctatgctt cttcttggaa gtatattcct 14700
ctgatagtaa gtgtattttt gataaagata ctttactatt tttatgtaga aattctcttc 14760
tatcataaga aagcatcaag attgattttt gttgcaactt tatcaagtag cttggtgaat 14820
gtagtaattt cttattattt gattggaatc ttaggtataa ctggttccat cgttgcagat 14880
tttatttcaa tgctcatacg agtcgggatt gtcataacga ttagcaaacg atttgaagat 14940
ataggattaa agctatcaaa acttatttat atcatcatgg tagttgttgt ttttatgggg 15000
attgggctgt attttaccta tcttaactat cagtttacat ttagttatat tgagtttgtg 15060
tacagagttg ttgtgatgtt tacttaccta ggttttgtac tttataaatt ccgttcagag 15120
tttaaacaat tcttaaatca gagaaaaaat cctttgggag gaaaaaatga atagaaatga 15180
atttttggaa gaggttcgta agagttcata ttatcgtaaa gattttgaac aaatttttga 15240
agagttcaga tcctatcaag atttaatttt gaatgctttg agagaatttc ataaggtttg 15300
tgtcacgaac tcaattccat atcaattagc gtatggttcc ttattaggtg ctgtgagaga 15360
taatggtcaa attccatggg attatgatgt ggatgttttt gttccatttt cgaaaaaaga 15420
ggagcttatt gaggcactga agaatgattt gaacgacgag ttttattttt actgtccaga 15480
agtcgataag aactgtcgtc attatatgat gcggattgca ccaaaaggtt attctacgga 15540
gttatttcat ttagatgtat tttatttgat tggtatgcca gaagagtcag aagaaaggaa 15600
gagattttct aaagaagtac aacaactggc ctttatcaga tactataaat tgatttctct 15660
tataaaatca actaattcat taaaaggtgt agtaaaaatc agtttattta agctgagata 15720
cttatttcga aatgttgaaa gagattataa gaaatttgaa gaattatgta gtatgtatgg 15780
actgaaggag tcaagttacg ccacaacagc agacattttt gccacatggt atgagattcc 15840
aaccaaaaat gtactatcga tagaaaaaat gcaaacttta gaaggtgaat tttgtgtgcc 15900
aacatcttac gagcagttac tttcgttact ctatggtgat tatttaaagg tacctgtatt 15960
agaaaatagg attaaagaat tactacatca tcatagttta cagtctaaga acatttaatg 16020
aggttgcatg gatgatacta aaaaatgcta ttattctcgc ggcgggctta ggtagacgaa 16080
ctatcccttt aaattttgag actcacaagg cttttctaga agtgaatggt gagatattga 16140
ttgaacgatt aatcgtacaa ctcaaagaag ctggggtttc tgaaattatt attgttattg 16200
gatacaaaaa agaacaattc cgatatttaa tagataagta cgaagttgaa ttaattgaaa 16260
atgatgattt tgctaactcc aatacactat actccctaag tctagcagaa tcctatttgt 16320
cgaattccta tataattcca tgtgacatct ggtgcgcaac aaatcctttt actagcaaga 16380
aagatgactc ttcttggtat atgattggtg atacttcaaa aagcgtaaca aagttagata 16440
atttgagcga aagattagct gttgctttta tagagcaatc agattcaatt tggataaaac 16500
agaggcttcg agagttggca aataatccta gtcaacaaat gctagcttgg gaagagcttt 16560
tggttacaga tggggaacta gcaataccta cattcaaaaa ttgcgagcat tttatacaag 16620
atattaatac atttgaagat ttgatatact tagatgctat gtctaatcat ttaagagtag 16680
aagcaataga tattatttgt acaacttttg atattgctcc taaagaaata aagaatgtat 16740
ttgccttgaa aaaaggcatg accaatcgtt cttttatgtt tgaatgtaaa gacaaaagtt 16800
acattatgag aattcctggg gagggaaccg ataaattaat taatcgagaa caggaagctg 16860
aagtttacag agtaattgct ggggaaagta ttagtgatga attgatttat attagccctg 16920
aaaaaggata taaaattact tcttttattg atggtgcaag aaactgtgat tcaaataata 16980
aaagcgatgt ttctttatgt atgaaaaaat taagaagttt tcatgaaagg gaattaacta 17040
cttctcatga gtttgattta tttggagaga tagaatttta tgaaagctta agaggaaata 17100
gggagtctat atacgaagac tatcaatctg ttaaaaatag agtacttact cttaagagtt 17160
atattcagtt gaatatagaa aaaaaggttc tttgtcacat tgatgcaaat cccgataatt 17220
tcttaatctt tgagaaaaat aatcaaacag aagttcgttt aattgattgg gagtatgcgg 17280
ggatgcagga tcctgattta gacattgcga tgtttgctat ttattctcaa tacaataggg 17340
agcaaataga ttttttgata ggcgcatact ttgaagaagg gtgtgaagaa agaataagaa 17400
tgaaaattta tgcatatgtt gctactgctg gtttgttatg gagcaattgg tgtgaatata 17460
aacagcaact aggggttgag tttggcgact atgctcagtc ccagtatgac tatgcaaaag 17520
agtattcttt tattgtcagt gagtatctat caactttcga agataaggat aattaggaag 17580
ggcgaatatt tttattttct tgtttgaatt atgaatgcaa atataatcca gagatgtttg 17640
ctttataaaa tagataggga ggaagtatat tgaaattaaa tgggaacaga attattttaa 17700
tcaccggctc aggtggtttc atcggcagca acctagtatt agaactgtta aggagtgacg 17760
aagtatatca tattatcggt cttgataata tgaacgatta ctacgatgtg tcgctcaagc 17820
agtatcgtct tgaacagatt gagcaacagt tgactcatag tataagcaag tggactttca 17880
tagagggaga tttggcagac aaggatctgg tcacttcaat ttttgaagat taccatccga 17940
atattgttgt caacctagct gcacaggctg gtgttcgcta ttccattacc aatccagatg 18000
tttatataga gagcaatatt attggttttc agaatatcct agaagcctgt cgtcactacc 18060
cagttgagca tttagtatat gcttcatcat cttcggttta tggtggaaat gagaaggtac 18120
cgttttctac ggatgataag gtagataatc cagtatcgct ctatgcagca actaaaaagt 18180
caaatgaact gtttgcacat gcctacagta agctctatga tattccaaca acgggactac 18240
gtttttttac ggtctacggt ccagcaggtc gtccagatat ggcttatttt ggattcacaa 18300
ataaactgat aaaaggtgaa acgattgaaa ttttcaacta tggaaattgc aagcgtgatt 18360
ttacctatgt cgatgatatt gtggaaggtg tcaagcgtgt aatgtttggc gcaccgcaga 18420
agagaagcgg aagtgatggt ctcccaattc cgccgtatgc aatctacaat attggcaata 18480
gcaatcctga aaacttattg gattttgttc acatattgag tgaggaacta gttttggcag 18540
gagtattgcc agcagatttt gatattgaag cacataagaa acttgtgcct atgcaagcag 18600
gagatgtacc agtgacctat gctgatacct ctgatttgga gagagatttt ggttttagcc 18660
cctctaccac actcagagaa ggcttacgcc aatttgctca atggtataaa gagtattaca 18720
aatagtttta ttttgctatt ggggcgatag ccttttggac aaatgctata atttgtgtca 18780
ggattagtaa gaaaggacta ataaataaag ttcactgcta ataaggaggt tttatgtata 18840
gtaagtatga aattgctcac gtagcatgtg atagaaaaca ggatgtaaag acagtttata 18900
atgagtacaa ggctaataag caacaattca taaacaaaat tgcaaattgt gttaaggatg 18960
atggattaaa gacttatttt aatcggattg ttcatgatta tgatgagaag atgtatattg 19020
attctgtgga gagagtttta aagaatcgag aattagtatc tcaaaataaa gataaaattt 19080
taaaaacatt gaaattttat gataaaagaa atcggtctga ttgtgagttt ttaaatgaga 19140
gtaatgtaga agtatatcta aactcccttc atgttaacaa tgacaggctt gctttctact 19200
taaaacagtt aaatggtgat tataatgatg cttaagttgt atatttgatt ttactttctt 19260
tttgataaca attattttga ttttttttgt aacaaatggt atactttgta tatcattttt 19320
ttgatgaggt gaaattatga gtaacaaaga tttttctcaa gaatttctta aaactattat 19380
tttttctaaa agaaaagagt tagggtattc tcagcaggaa ctttctgatt tgacaggaat 19440
aaatagggct atgattagca agttggagca aggggagtat cttccgtcaa ttccgcaatt 19500
actttctctg agcaatgtgc ttgattttga aataaacagt ttatttgttt ctgattttcc 19560
gaagtcaatc ggcactcttc cggcaaaaaa aatcgctgtc gcaggaacag gctatgttgg 19620
tctatctatc gctgttttac tagcccagca tcatcaactc acagctgtgg atattatcca 19680
agaaaaagtt gatttaatca atgcaaagaa gtctcctatt caagatgatt atatagagaa 19740
atatctagca gagaaggatc tcaatttagt agcaacactt gatgcagaaa cagcctacag 19800
agaggcagaa attgttgtga tcgctgctcc aacgaactat gatagtgcta agaattactt 19860
cgatacaagt catgtggaag aagttattca aacagtatta tcggtcaacc ctcaagcttt 19920
aatggttatc aaatcaacga ttcctgttgg ctttacccaa tctatgcgtg agaaatttgc 19980
tacggacaat attattttta gcccggagtt tcttagagag tcaaaagcac tttatgataa 20040
tctttaccct tcacgaatta ttgtgtcttg tgaagaaggg actccaactt cggtagtaga 20100
aggttcaaga ttatttgcct cacttctaca agagggagcc ttgaaggaag atgttgatgt 20160
actctatatg gggtcgacag aagcagaagc agtcaagctc tttgctaata cttacttagc 20220
cctgcgtgtt tcttacttca atgaattgga tacctatgca gagttgaaag ggctggatac 20280
cgaatccatt atcaagggtg ttggcttaga tccacgtatt ggtgaccact acaataaccc 20340
ttcttttggc tatggtggtt actgtctacc aaaagatacc aagcagttat tggctaatta 20400
caatgatgtt ccgcagaata tgatgtctgc tattgttgag gcgaataaga caaggaaaga 20460
tttcattgcc gatcaagtat tgaagatggc tggctattat gattactctg agcatgatag 20520
ctattctcgc cagcaagaga aagaaatcac tgtcggtgta taccgattga ctatgaagag 20580
taactctgac aacttcagac agagttctat tcaaggcgtt atgaagcgat tgaaagccaa 20640
aggagtaaag gtcattattt ttgaaccaac tttagaggac ggctcaactt tctttagttc 20700
taaaattgtc aatgatttga agaagttcaa gtctcaatgt caagccattg tagccaatcg 20760
ttatgatagt tctttagacg atgtgcagga taaggtttat acacgagata tttttaggag 20820
agactgaaga atgtatcgac tttttatata tatgcttatt cctaaaagga gcaattatga 20880
agcttagttt aagaggaaat ttacagggct tattatcagg catgttttgg gggttagaca 20940
catccttgaa tagcttaata ttattaatgg ctcctttcat tgttgaggat agtaaactat 21000
tatcttctac tttactattg gcattttttc atgatttctt ttctgcgatt tttttaacaa 21060
ttgacttatc tatcagaggt tgttttaaaa atacgataca aattatgaaa actagaagtg 21120
cttattttgt aatgatagca gcagtattcg ctggtccttt agggatgaga gcctatcttt 21180
ttgcggttga aaagttgggt gctggtttaa cagcaacaat atctgctata tatcctgcga 21240
tagctgcttt attaggaatg atttttttaa aagattattt gacaagaaag ggatggtttg 21300
gactagcttt aaccatcttg gcagttattt ttcttggata tagtggcttt tctcaaatag 21360
aagaattatt cgttggctct ttagcagcat tgttatgtgt aattggctgg gcttcagaga 21420
gtgtaattac cgcttatggt atgaaagaag atatcttacc taaacaagct ttatttatca 21480
ggcaatggac ttcaagctta gtctatctgt tgtttatgat agtagagggg gatgttgttt 21540
acagtgtagc aactgtttta aactcacaat caattcttgt tatcgtttgt cttgcattaa 21600
tagggactct ttcctattta ttctattatt ctgctattga taatatagga cctgtaagag 21660
cgacaggttt aaatgttact tattcgattt ggacagtcat tttttcattg ttgttttttg 21720
gaggacaatt agatattaaa ttagttatca gtagtgtagc tatcattatt ggtacgctat 21780
ttgttttaaa aaactaaaag gagaatttat gagagcgatt attttagcag ctggaatggg 21840
aactagatta agacccttga ctttaacaac tcctaaatct ttgattaaag ttggaggtca 21900
aacattaata gaaagacaaa ttttgttttt aaatgaaatt ggtatttcgg aaattattat 21960
tgtgacaggt tatttgtcag aaaagtttga ctttttagag agtaaatatg gggtaaggtt 22020
aattcacaat gataaatatg atacctataa taacttttat agcatgtatc ttgtgaggga 22080
ttttttaagt gatgcttatg tgattgatgc ggacaattat ttgcacacga acttcttaga 22140
ttctgatgta accatttcta catatttttc ggcatataaa gataattttg aggatgagtg 22200
gcttttaaaa tgtgatgata atggtctagt gaaagaaatt gtgatagata gtggcgaggg 22260
ctctattcta tctggggtat catattggga tgcaaaatct ggtcaggttc taaacaaact 22320
tattgaagaa gtgttagaat caaacgattt ttgtaatttg tattgggata atcttgtaaa 22380
agataacttg gataagttga cagtgtataa gcgagatatt ccttcgaacg ttatatttga 22440
gattgataat cttgatgatt taggacagtt acaagaattt ttaagttccc atacctagtc 22500
atagagtagc tggattttcg gaactattcg aattaatgaa cgtattgctg atgtggtaca 22560
cgataatcgt atgaagaaaa cactgatttc agaaaaccgt attttcatat tttatatagt 22620
cttttagcaa aaatttatgc tataattagt ctgaatttga ggaattattc atgactaaac 22680
gtaaaaacca tgaaaataaa ttgaaaaaag catgtaacaa gaagggctct ttatcaactg 22740
tagtgggtag acgaaaagct aacacctaga gaggacgaaa ttcgttctct ctttttgtat 22800
gtacgacggg catgtcgtac atctgaggtg aaagtcctcc gtggacagta accgcccaat 22860
aacaaggtat ctatccactc actcactcct tcctccagta tactgttata aaaaacaaac 22920
tggaggattt tttatgtctc agcccatcgt cccattgact attcccaaat ctcgccgctt 22980
tgaaaagaaa aggtaaatgc ctcataacaa ggtacctatc caatcatctc attctccact 23040
atacttttta tttattgtga tattaatatt aagaataacc agttaaattg aagtgatgca 23100
aagcatttta atggaatagt ataggggtgt tgtttatcgt ttactcatgc tctgtaaaag 23160
tggaaaagat ttggctacca tgctataata tagctagaat attttccgaa taaataggta 23220
gagagaattt ctcaatcact ttatttggag gaaaaaaatg actaaacgtc gtcaggcagt 23280
cagtcccatg gctgatgtta ttgcaaaaaa gattttcaat gaccacgaaa ttaccattga 23340
ctttattgat acctttttag gttttcgtcc caaatctgtt cagatattga atgggactat 23400
cgcggatgta aaaaaagaaa ggactagaca ctttagtaca acggtagata ttttggctcg 23460
gttagaagat gggacacagg tcattatcga gattcaggtt gcctatcaaa atagttttat 23520
taagcgtttg tggacttatt cctgccaaca tttggtgaag gacttgccca atattcgtga 23580
gaaggtcacg cagacgcatg atatgtataa taaaatttct ccgatttatt cgattgcttt 23640
agtagcgagt aagtatttcg atgatgaaaa acctatacac agttttgtat tgactgaaaa 23700
agatagcggt caaatcttag aattaccatt tggtgaccag gaagaattaa agagggagtg 23760
ggaaagaact cgaccatcca tagaagagtt cgttttccca cccccgcaca gttgattagg 23820
tcagatttgg agtgtaaaac acgaacaaat ctgccaatca accactgcgc tgagatgttg 23880
acacaaaccc tgagaagtgc gctggacttt ttgcccagcc ttatcattga gctgaaaaag 23940
tttcgtaagg gtcgacttgg gaaaaatcaa cgccaatgga ttgaattttt tgcaaataga 24000
gaatttagtc aatcgacatc agatattatt gaaaaggcag agaatctgct aaacagaaat 24060
acatggacag aggaggaaat agaaatggta gatcaatggt tgcgtaatgc ttccaaccac 24120
tttggagaat tggaaagttc ttttatacgt ggtagaaatc gtggaaagga agaaggaaga 24180
gctgaaggtc tagagaaagg tctagaagaa ggaattctac aaaaaagttt agatgttgca 24240
caaaaattat tagctagagg tcttgacata gaggatgtgt tagaaataac aggactaact 24300
tcggaacagt tgactcgatc ttcgcaagaa catcagttct aggaggaaaa tacaaaatat 24360
atgaaacatt acgactactt agttgttggt gcagggctgt tcggtgcggt ctttgctcat 24420
gaggcagcta aaaagggtaa aaaaataaag gttatcgaaa aacgagacca cattgcaggt 24480
aacatctata caaaagaagt agaaggtatt caagtccatg aatatggcgc gcatattttc 24540
catacttctg aaaaagaaat ttgggattat gtgaatcagt ttgcggagtt caaccgctat 24600
acaaacacac cagtcgctaa ctacaagggt gagatttaca atcttccctt caacatgaat 24660
acctttaaca agctttgggg tgtggtgacc cctgctgaag cagcagcgaa gattgcagag 24720
caacgagctg tattgggagg taaaacacct gagaacttgg aagagcaagc catctcacta 24780
gttggtacag acatctatga aaaattgatc aagtcctaca ccgagaaaca atgggaaaag 24840
ccgtgtacgg agttacctgc cttcatcatc cgtcgcttgc ccgtgcgttt gacttatgat 24900
aataactact ttaacgatac ttatcaaggt attccaatcg gcggttacac gcagattgta 24960
gaaaaaatgt tggatcatga aaatatcgat gtggagacaa atgttgattt ctttgctaat 25020
aaggaagaat acttggcaaa ctatccaaaa gttgtcttta caggtatgat tgatgagttc 25080
tttgattatg agcttggtga attggaatat cgtagccttc gctttgaaac agaagttttg 25140
gatatggaaa actatcaagg gaatgcagtt gtcaactaca cagatagcga gacaccgttt 25200
actcgaatca tcgaacacaa acatttcgaa tttggtacac aagaaaaaac cattattacc 25260
cgtgagtatt cgaagacctg gaaacgaggt gatgagccat attatccagt caacagtgat 25320
cgcaacaaca aactctatac gacctacaag cgccttgcag agcagcaaga aaatgttatt 25380
tttggtgggc gtcttggtca ttatcgttac tacgatatgc accaggttat cggtgcggcc 25440
ttgcaatgtg tgagaaatga agttggggaa tgattttgag aagtcatcaa aaaaaccaga 25500
aattctggtt tttttgatta ttgaattgct gccaacaggt cagctgcttg ttgctcgagt 25560
tttgatagag tttcgtcaga taactccaat aaacctgttc cccatgcttc tggattgatg 25620
gttgaacctg taaattcacc agcaatctga gtacggataa atggaagaag gtgttggaag 25680
tctttgaata ggtggtcgtg accactgttt gcgactgcag agacagttgt gattttatct 25740
tgtaggatag acggtccttt tggattggat aaatcgagcg aacgactgag ccaatcgaga 25800
gcatttttta cgatgcctgg gattgcatag ttatatactg gtgagaaaat ccagatagca 25860
tccacagcgt ctacttgtgc acgtagctct gcaatttcag gaaggatagg tgtctcgatg 25920
tcttggttga ataggggaat ttttgcaaga tcaatgtaag atacttgagc cttatcagcc 25980
aagaacgctt cagctttttt ggccatttga tggttaaaag aaccgtcacg tagtgaacca 26040
actaaaaaca atacgtttgc catgatggtc tccttttcat aaaaacataa atttagtata 26100
tcgtatttgt cttttgttgg caatcctagg tctgctggtt cttgaatttt ttaagtatct 26160
cggctaattg gtcttgttct tctgaactaa ggacagaaaa aatctctcca acgtgatcat 26220
aatgctctgg aaggattttt tcaatggttt cgcgtccaag tggggtgaga gcaatcaagg 26280
aagcacgacg gtcatttgta tcaatagtct tgtagatata accatctcga atcatgtttt 26340
ttataacgac agtcatgttg ccagaagtac ttaataattt atcaattaag tcttgaatcc 26400
gcagatttcc tttgttgtag agtgcctcca tcactccaaa ctgacaaact gtcagtccgt 26460
attttttgat tgtttctaat tctgattttg tgatagtatt agctgctctg cgaaagacga 26520
ctagactgtg aagtgcatgc ttgttctcgt ttaaagattg ttcaacagtt ctcatgagaa 26580
tattttagca ataaatagtt cggttatcaa gcaaaaaaag ttaaatccta tattcatagg 26640
taaagtactt tatatagcat agaggagctg aactgtgggt cccagttttt atattcgttc 26700
tctgtctgaa ggagaatgaa agtttattgg taaattctca aagtaagttc tagttcccgt 26760
ccttccagaa gttactttga gaaaactgca cagcatcaac agaatttagt ctcagactaa 26820
gttctgtttt tgactaaaaa tgttgatagt agtgagtttt agggttgaaa atgttg 26876
Claims (10)
1.用于液相芯片多重PCR鉴别猪链球菌17种新cps型的引物组合,其特征在于,所述引物组合以wzy基因为检测靶标,包括含有以下核苷酸序列的引物对:SEQ ID NO.1-2,SEQ IDNO.3-4,SEQ ID NO.5-6,SEQ ID NO.7-8,SEQ ID NO.9-10,SEQ ID NO11-12,SEQ IDNO.13-14,SEQ ID NO.15-16,SEQ ID NO.17-18,SEQ ID NO.19-20,SEQ ID NO.21-22,SEQID NO.23-24,SEQ ID NO.25-26,SEQ ID NO.27-28,SEQ ID NO.29-30,SEQ ID NO.31-32,SEQ ID NO.33-34。
2.根据权利要求1所述的引物组合,其特征在于,SEQ ID NO.1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33所示核苷酸序列的5’端还含有与对应编号液相芯片微球的anti-TAG序列反向互补的TAG序列,所述TAG序列与SEQ ID NO.1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33所示核苷酸序列之间插入一个12碳胺基基团。
3.根据权利要求1或2所述的引物组合,其特征在于,其包括以下引物对:SEQ IDNO.35-2,SEQ ID NO.36-4,SEQ ID NO.37-6,SEQ ID NO.38-8,SEQ ID NO.39-10,SEQ IDNO.40-12,SEQ ID NO.41-14,SEQ ID NO.42-16,SEQ ID NO.43-18,SEQ ID NO.44-20,SEQID NO.45-22,SEQ ID NO.46-24,SEQ ID NO.47-26,SEQ ID NO.48-28,SEQ ID NO.49-30,SEQ ID NO.50-32,SEQ ID NO.51-34。
4.根据权利要求1~3任一项所述的引物组合,其特征在于,SEQ ID NO.2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34所示引物的5’端含有生物素标记。
5.权利要求1~4任一项所述的引物组合在制备用于液相芯片多重PCR鉴别猪链球菌的试剂盒中的应用。
6.用于液相芯片多重PCR鉴别猪链球菌的试剂盒,其特征在于,其含有权利要求1~4任一项所述的引物组合。
7.根据权利要求6所述的试剂盒,其特征在于,所述试剂盒还含有管家基因thrA的扩增引物,其核苷酸序列如SEQ ID NO.52-53所示。
8.根据权利要求6或7所述的试剂盒,其特征在于,所述试剂盒中各引物的在多重PCR反应体系中的终浓度为0.2-0.3μM。
9.根据权利要求6或7所述的试剂盒,其特征在于,所述引物组合进行多重PCR的反应条件为:94℃预变性5min;94℃变性30s,58℃退火40s,72℃延伸50s,30个循环;最后延伸72℃,10min。
10.一种液相芯片多重PCR鉴别猪链球菌的方法,其特征在于,以待测样品的DNA为模板,利用权利要求1~4任一项所述的引物组合进行多重PCR,将PCR扩增产物与液相芯片微球结合形成PCR扩增产物-液相芯片微球-SAPE复合物,检测所述复合物的荧光强度,根据荧光强度判断待测样品的猪链球菌的cps分型情况。
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