CN111057793B - 一种鲤冠状病毒hl39的rt-lamp检测引物及应用 - Google Patents

一种鲤冠状病毒hl39的rt-lamp检测引物及应用 Download PDF

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CN111057793B
CN111057793B CN202010001470.5A CN202010001470A CN111057793B CN 111057793 B CN111057793 B CN 111057793B CN 202010001470 A CN202010001470 A CN 202010001470A CN 111057793 B CN111057793 B CN 111057793B
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陈孝宇
郑剑
王振华
曾宪东
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Abstract

本发明属于病毒分子检测技术领域，具体涉及一种鲤冠状病毒HL39的RT‑LAMP检测引物及应用。本发明是针对保藏编号为CCTCC NO：V201988的鲤冠状病毒HL39设计的RT‑LAMP检测引物，包括外引物F3和B3、内引物FIP和BIP。利用该引物制备的试剂盒，在检测鲤冠状病毒时具有简便、快速、高特异性和灵敏性的特点，仅需一个水浴锅或金属浴，2小时内即可准确检测出样品中的鲤冠状病毒HL39（CyNV‑HL39），非常适用于CyNV‑HL39的现场快速检测。

Description

一种鲤冠状病毒HL39的RT-LAMP检测引物及应用
技术领域
本发明属于病毒分子检测技术领域，具体涉及一种鲤冠状病毒HL39的RT-LAMP检测引物及应用。
背景技术
套式病毒目(Nidovirales)于1996年首次由国际病毒分类委员会(InternationalCommitt ee on Taxonomy of Viruses,ICTV)提出，该目病毒宿主广泛，可引起哺乳动物、鸟类、爬行动物、鱼虾等水生动物多种重大疾病。套式病毒目冠状病毒科是一类线形单股正链RNA病毒，最早于1937年从鸡身上分离出来，Almeida等对这些病毒进行了形态学研究，电子显微镜观察发现这些病毒的包膜上有形似日冕的棘突，故提出命名这类病毒为冠状病毒[1]。1975年，国际病毒命名和分类委员会正式命名了冠状病毒科。由冠状病毒科的严重急性呼吸综合征病毒(SARS-CoV)引起的非典型性肺炎，世界上有30多个国家和地区相继报道出现病例，病死率超过10％。2012年，中东地区开始出现新型冠状病毒，病死率超过50％，引起了世界范围的广泛关注，造成社会对冠状病毒的恐慌。因此，对冠状病毒进行深入研究具有重要意义。
2018年ICTV调整了套式病毒目的分类，新增了Abyssoviridae、Medioniviridae、Mono niviridae、Euroniviridae和Tobaniviridae等5个病毒科，其中在Tobaniviridae科下新增Onc otshavirus属。2017年，申请人发表了鲫冠状病毒的相关研究成果，目前鲤冠状病毒(Cypr inid Nidovirus,CyNV)尚无公开报道，更也无有效的检测方法。
申请人首次从鲤鱼病变组织中分离出一株鲤冠状病毒，针对该病毒，设计了RT-LAMP引物，从而实现鲤冠状病毒的快速、精确检测。
发明内容
本发明的目的在于提供了一种鲤冠状病毒HL39的RT-LAMP检测引物，所述的引物为引物组：F3：5’-TGCCCACCAAGACGACTA-3’；B3：5’-TTTGCGCTAGCGCGATTC-3’；FIP：5’-CGTTCCAAACCGTCTTGGGTCAAGAGTGACGTAACTGCTACTG-3’和BIP：5’-ACTCACTTCTGCTCCTTGCGACGCAGAGTGTAAGAGGTTTTCCA-3’，所述病毒的保藏编号为CCTCC NO：V201988。
本发明的另一个目的在于提供了一种鲤冠状病毒HL39的RT-LAMP检测引物的应用，利用该引物，可制备成鲤冠状病毒HL39的检测试剂盒。
为了达到上述目的，本发明采取以下技术措施：
一种鲤冠状病毒HL39的RT-LAMP检测引物，所述的引物为引物组：F3：5’-TGCCCACCAAGACGACTA-3’；B3：5’-TTTGCGCTAGCGCGATTC-3’；FIP：5’-CGTTCCAA ACCGTCTTGGGTCAAGAGTGACGTAACTGCTACTG-3’和BIP：5’-ACTCACTTCTGCTCCTTGCGACGCAGAGTGTAAGAGGTTTTCCA-3’，所述病毒的保藏编号为CCTCC NO：V201988。
鲤冠状病毒HL39的RT-LAMP检测引物在制备成鲤冠状病毒HL39的检测试剂盒中的应用；
以上所述的应用中，优选的，所述的鲤冠状病毒HL39的检测试剂盒包括：
FIP、BIP、F3和B3，dNTPs，Betaine，MgSO4，Bst DNA聚合酶，AMV逆转录酶，模板RNA，10×ThermoPol Reaction Buffer。
与现有技术相比，本发明具有以下优点：
本发明公开了一种鲤冠状病毒HL39的RT-LAMP检测引物，利用该引物制备的试剂盒，特异性好，能有效检测出CyNV病毒；灵敏性好，最低检测限可达12.27pg/μL的RNA；快速高效，检测时间约1小时，非常适用于CyNV-HL39的现场快速检测。
利用本发明的引物制备的RT-LAMP检测试剂盒不需要特殊试剂与设备，检测成本低。结果判定简单，通过反应过程中大量产生的焦磷酸根和体系中的镁离子形成白色稳定的焦磷酸镁沉淀，经肉眼就可判断结果，或通过加入1000×SYBR Green I，可提高结果判定的灵敏度。
具体实施方式
本发明所述技术方案，如未特别说明，均为本领域的常规方案，所用试剂或原料，如未特别说明，均购自商业渠道或是已公开。
实施例1：
一种鲤冠状病毒HL39，其分离过程如下：
1)将携带病毒的鲤鱼肝、脑、脾、肾等组织取出并碾磨后，培养基稀释100倍，2000rpm离心后取上清过滤，接种到草鱼性腺细胞(Grass carp ovary cell line,GCO)单层，25℃培养7天，产生细胞病变效应(CPE)，可见GCO细胞收缩变圆、折光度增加，部分细胞开始脱落，细胞单层开始破裂，培养基：M199，10％胎牛血清，pH 7.0～7.2；
2)收集病变细胞，利用透射电镜进行形态学鉴定：鲤冠状病毒HL39的形态特征：病毒呈棒状，四周有明显的日冕状棘突结构，与典型的冠状病毒形态相符。
3)收集病毒，对其进行全基因组测序，序列为SEQ ID NO.1所示。
鲤冠状病毒的形态特征：病毒呈棒状，四周有明显的日冕状棘突结构，与典型的冠状病毒形态相符，该病毒已于2019年11月26日送至中国典型培养物保藏中心进行保藏，分类命名：鲤冠状病毒(Cyprinid Nidovirus)HL39，保藏编号：CCTCC NO：V201988，地址：中国武汉武汉大学，本发明或称该病毒为CyNV-HL39。
实施例2：
基于鲤冠状病毒HL39的全序列(SEQ ID NO.1所示)与其他病毒的全序列的差异设计的RT-LAMP检测引物为：
F3：5’-TGCCCACCAAGACGACTA-3’；
B3：5’-TTTGCGCTAGCGCGATTC-3’；
FIP：5’-CGTTCCAAACCGTCTTGGGTCAAGAGTGACGTAACTGCTACTG-3’；
BIP：5’-ACTCACTTCTGCTCCTTGCGACGCAGAGTGTAAGAGGTTTTCCA-3’。
实施例3：
利用鲤冠状病毒HL39的RT-LAMP引物对CyNV-HL39进行检测，方法步骤如下：
1)、病毒RNA的获取：
采用
Figure BDA0002353652880000031
或
Figure BDA0002353652880000032
LS试剂或柱吸附洗脱法提取样本总RNA。
2)、RT-LAMP扩增：
采用25μL反应体系：内引物FIP和BIP各0.8μM，外引物F3和B3各0.1μM，dNTPs1mM，Betaine0.5M，MgSO4 5mM，Bst DNA聚合酶8U，AMV逆转录酶5U，模板RNA5μL，10×ThermoPolReaction Buffer 2.5μL，选择64℃和60min进行RT-LAMP反应之后，80℃灭活2min。
3)、检测结果判定，采用下述三种方式之一：
A、发生扩增反应时，从dNTPs析出的焦磷酸根离子和反应液中的镁离子形成白色焦磷酸镁沉淀，通过肉眼判断：检测管出现明显浑浊为阳性，未见浑浊为阴性。
B、每反应管加1000×SYBR Green I 2μL，轻轻混匀，1-5min后在黑色背景下观察结果，反应液变绿为阳性，保持橙色为阴性。
C、取扩增产物，用2％w/v的琼脂糖凝胶电泳后，置于凝胶成像系统中成像，电泳图片显示LAMP特征性梯状条带，则结果为阳性；如无任何条带，则结果为阴性。
实施例4：
鲤冠状病毒HL39RT-LAMP检测试剂盒特异性试验
利用实施例3所述的方法，对表1中的待检病毒样品进行检测，同时设置阴性对照组(双蒸水)和阳性对照组(CyNV-HL39)，特异性试验组具体为：鲤春病毒血症病毒(SVCV)，草鱼呼肠孤病毒873(GCRV-873)，草鱼呼肠孤病毒873(GCRV-9014)，传染性造血器官坏死病毒(IHNV)。
表1鲤冠状病毒RT-LAMP检测试剂盒特异性试验
试验编号 名称 扩增结果
1 CyNV-HL39 +
2 SVCV －
3 GCRV-873 －
4 GCRV-9014 －
5 IHNV －
结果如表1所示，仅阳性对照组(CyNV-HL39)显示LAMP特征性梯状条带，其余组均无特征条带，说明鲫鱼冠状病毒HL39RT-LAMP检测引物具备很强的特异性。
实施例5：
鲤冠状病毒HL39RT-LAMP检测试剂盒的灵敏度
将1227ng/μL的CyNV-HL39 RNA模板进行10倍梯度稀释，利用实施例2所述的方法进行检测，检测本发明提供的试剂盒的检测限，结果显示105倍稀释样品还可观察到特征性梯状条带，本发明提供的试剂盒最低可检测12.27pg/μL的病毒RNA模板。
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<110> 武汉海关技术中心
<120> 一种鲤冠状病毒HL39的RT-LAMP检测引物及应用
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<210> 1
<211> 25833
<212> DNA
<213> 人工序列(Artificial Sequence)
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ttgaattacc aataaactac cacccgctat tatagcacta gacactagcg aactgctagc 60
ttcctccaag ctgcacaacg caaactgtct ttttaactct cctaacctcg aacgcagcgc 120
aggtaccctc acccggacca cgcgtgtgcc cttgactgga aaccctacct tcgatcaggc 180
cttcgaacgc agcaaaggta ccctcacccg gaccacttgt gtgcccctac gcctgctctt 240
acgtagcccc atcctaccca cccgccaact cttaacctat cagctttgcc caccgtgacg 300
actagacgtg atctaactgc tactaccaat cctattacaa ccaacttgcc caccaagacg 360
actagagagt gacgtaactg ctactgccta aggcactacc agcgaaatca taatctgctc 420
ctcttgaccc aagacggttt ggaacggtgc tactagtgaa ctcacttctg ctccttgcga 480
cccaaagacg gttattattt ggaaaacctc ttacactctg cccctttacc tactcaatat 540
aacttaaatc tcttttagaa tcgcgctagc gcaaaccttc accggtaacc gccagaacgt 600
cctacaagct ctcaaccacc ggcactttgt tgctaaatcc tacgtgcaag catctttttg 660
ctataacatc cacgttcgaa aatcaacaaa aatgcccaac atgcgaatcc gcaagattgc 720
tcctaaagcc gctgttaaac caacccatag cggcccaaga gtggtctcca atcccgcaag 780
ctgccacatc tacatcggac cccaactgcg tgctcacaac tacccgagcg tgctaaaggg 840
ctcctttaag gctgtcagcg cggaaaccat aacagctacc aacctaattg ccaccgtccg 900
caaggatgct ggtatttcag gtcaatatgc tgggaagatg ggttatgacc ccgagatggg 960
acttttcgtc tccgtcgaag gtgttcgtgc tctccaagct tccaaactcc aaactgccct 1020
taaggcaatt aaaaagggta ctagaccctt tggccttttc aggcctaaac agagtgtgac 1080
taacacccac gctcaaaatt tgctagccct ccaccgtgct agctccaccg gcccttggac 1140
caaacactta ccatctcaag gtcccatagg taactgcctc gcagaattca tgcaggagta 1200
cactaccaca attccaccat ttgatagtgt cctcactgaa aacggttggc aatacgctgt 1260
cacgattgac ggtgtgacta aatctcgcct cccgtacacc gaaattcgcc gcggtggtta 1320
tgaatttaaa acaactaatt accacatcgg tgcaatccag gttactgcca accacattgg 1380
caaagtaaac cctggagata ctaaattgga ctgtcaatat ttcatgttgg cacgtaatca 1440
aaagccccaa aacttcactg ctgagcaact cgaaggaaac atccataaca tcatcttcga 1500
cctcagtcat gaaactggca aacccaaact ctatggaatg gaagaaacac gacaatgggc 1560
tgggacaaca ccaaaaccac ctaagaaaaa gcttagactt gtagctatga catcaactct 1620
cagtgccact ccatcaacca cacacccagc gcccctaaca accacacgcg caccacttaa 1680
aacacacatt tcaattggcc cgcacaactg caccatcata gatattaata aagcccaatt 1740
cgcctttgat aactacactt ctcaactccc taactcttgt gttcaccgtg ccaacaaagc 1800
tgtttgcgcc ggtttgaatg ctgatcttct ttctacccac tttaactacc tctctaaagt 1860
agaaacatct aaaggaactt atcaatgggt acattttagc cccaaaacta cggcccctgc 1920
tatccaattc tacgtcttgc aatttcagaa ccacgtctac ctacgtcttc tccccatgcc 1980
tatgaacctt tctttccgca ctatctgcga cattgaaacc gcccacggtc aaccacaccc 2040
aacttacaca agcccagact ggcgcgtgta ttacctcgca aagtacgatc ccaccaaagt 2100
actcagagtt caagttgttg attgggcagg ttcagatacc attactgaag atcatgtcac 2160
cctcttccca cgccgacaca ttatgactaa agttttgaaa aacgaccaaa ctaaagttaa 2220
ctacatcgac ccaactaaag atgcagcaac aacacccttc acaattgaaa cagttgaagg 2280
aggcaaagtt gaagtactct ccgccaacgt catccgccaa attaacattg aaactggtcg 2340
cggcaagaaa cgtaaaacag ctaaattaga tgtaaaaatt tcaattagag ttaaagctac 2400
taaaacccgc acaacaattc acatcttcat aacccaagca aaagatgaaa gcaaaatcgt 2460
cactccaacc ttagaaatgc agggagtagt cgacgtttta atttccagag catgtgtcct 2520
ctcacaacac gcaggacctg acatgactta tgaactgcac acacctgacg gttcttgcac 2580
taaccccctc gttcaattct ttgaagaaga aggcgaggtt cctgaagatg tttacgatct 2640
cttcccatct gtagaacgta acgctgaagg tttagttatg tgcatgccag aaggttttgc 2700
cctcatttct ggttgcattc ctcatccact tgcttactat gctgacaagt acggtcaaac 2760
actcgtacct gaattcaaag tgacaagaac tcacgttgaa cgcggcccca ttccaccatc 2820
acggtccatt tactacacat tcatcccaat taatgtagac tccatcccac ccttcggaca 2880
cacctacaaa tccctcgcca aacttaacac atttaacctg ggctgggaag atcaagacat 2940
gttagataac gctgacaccc tccttccatg tctcttagaa atgaaaacat gaaacttcac 3000
ctaacatgga aaatcatcgc ccgtgacttc tcagataaca ccactgaaac agcccttacc 3060
atctacactg attttacaac tagtgatttc accccagcca accaaacagg atccagctct 3120
caatccctta gctctccacc tctttcccaa ctctcttccc acaaatctct tgctaacctc 3180
tcagaaactg gctcaataga taacatcccg gaaaacaccc ctgaaacaat ccaagaagaa 3240
gaagatatcg aagaggaagt aacatctcct gttcaaccca cactcaacac actccaagaa 3300
caagctactc tcaacgaatc cattcaatca ctcgttgaat cagttgattc tgatgtccct 3360
tacacctggg aacaacgtgt tgaagaaagt gaggttgcga ctattgattt acaagctgta 3420
gaattagatc aaacacccac tcccaccact ctctcaacag aagcagtgca agaatgggtc 3480
aacgcaacat cacctgacct tgctacagta gactggaaca gtgcaataac tgaacaggaa 3540
accacagtaa cttcactaat agaagaaatc tccctacgcc ccaacaccaa caacaccggc 3600
tataacttgt ttaaacctaa ccaacgtaac aacaacagcc catcagacta cctatcacga 3660
acaacatcag caaccaaaca ctgcttaatt aacggtctta cttcaggttc caccatcctt 3720
gaagtcggat gcggatatgg taaagaattc cacaaataca agaactctaa catcaaagtc 3780
gttgctttgg aacctcacca tgaacgctac accgaagccg caaagaaact accaccaaat 3840
tctaccattt acaacaagac cctagaagaa tacaaccaaa ttgtcattaa agaacaaatt 3900
gacgccctag tttcctttaa cgccattcat tacagtacag ttactgaagc tcttaaagca 3960
tgtaaccttg ccaaagatca agttgtcatt ttaacaccaa atcatgataa acttaaagct 4020
cttcacgata gtaacacaac catcacaaat tacaaaattg aacttgctaa taacaaattt 4080
acattcacaa tcggccaaaa cacctacagc gaaaactact tctcacttca ggatttcatg 4140
attggaatgc atgccagcaa cttcatgctt tcttcccgct cttgtactct gcttgaacat 4200
gttaacacct ttaatgccca cctcttaatt gacgccacaa cagaaacaac caccctgcta 4260
gacgcctatg aagtactcgt cttcataaaa tttagaccac tgctcctcac ctactccaac 4320
accgctcttg caactaacaa ctcaagaaca atccgcagtg cactctcacg tcttattaac 4380
ttccgtgtcg caccttctaa aacaatatca caggtacatg ttcacatcct cacccgcaac 4440
gaccacgcag ccacagaaca agaacatcta gacttagcaa atattgaaat tgcccacatg 4500
aacccaaaac cagcaattat ctttgtaact tcttcccgat ccaaacataa ctacaccacc 4560
ccaactgtct tcttcgactt tgctaaacta tcagccccca ccctcccaga acagcttaaa 4620
ccactcactg atctagccct cgcaatccaa cacactgcta ccaaaactcc aacaacagta 4680
cttacaactg ccccagagga cccaaaaacc ctcccaacag cacccacctc ctttacaaaa 4740
ccttcccgta aacgctacca ctgcttccac gatttaacca ccctaccagt taacaaaaat 4800
ttcatcttag ttaacgcagg taacccccac ttaaaatcag gtggtggaat aactaaagcc 4860
gtctataatc gctacggcca aggattcatg caaactaaca acagcatgca tgctacacaa 4920
ttacctcttg aagttggtga cgtcttggaa tacccctctg accgtaacaa cctgattaac 4980
gtgacagccc cctgcatcaa catcaaacac gcagattaca aaggcccaca accagcccac 5040
gcactactag caacatataa cgcaattatc aactacgttg aacgtaagta cgataatggc 5100
gacgagaaac acgtcttcat tcctctactt ggtgctggat actacggttg gtcacagatc 5160
caaagccttc aagcttttct atccgcgact gataatagtt tccgcaacac atatgttcta 5220
ctcctacacc catcatctgt agatgttgaa acagctgaag tatttgcaaa aatgctcgcc 5280
actactaacc cacactacta ccatgttgag ggcatagcta aagttaatgc caactcttgc 5340
gttctgcgtg cccacattga ccttgcccgc aaaacatctt accctcaacc tagcttagga 5400
ttaatcggcg cttactggat gtacacctgc cccaccctgg ccgccccatt gatacaagct 5460
ggcatgcacg gtcccaacac ctggactcct ggcagcttta ttgacactag tattgctgaa 5520
atgactgctc gcgcttacaa aataaatctt gtttgcgacg gttgcttcac tgatatcatc 5580
aatttcaatc ccgaccaagg tttaatctgc aatacctgca agacacctgt cactctccgg 5640
acaaactatt atcgcccacg cctcccacac atgattaaaa atagtcacgg acaacccatc 5700
cctaacatta ttctggatgg aatctaccac atggccacgc accaattgtt cctaaattcg 5760
gtactcttta aaaattcaca tgctttcaca attaacaacc taagtgaatg tgatctccca 5820
actactatag cagtagcaac ctacttttac aaaccttcag atgctttaaa cttcttcctg 5880
aactttaact ctgctatttc tgctaacgta acttatacat cagaacagat aacacaagct 5940
caaaccaccc ctcaactcac cactgatgat gactactcgg acttggacat tgacacaatt 6000
aaccctacac ctactacttt accacgcgcc ccaacaccgc tttcaattag tgatagtagc 6060
gaagatattt cactctccga cgacacccac accgtagcat cccatcacgc ttcacaaatg 6120
tccataacag ctgacacaac tagtgataga tatagctcta caaactctct agctatctca 6180
gaagatataa catttcaaac aggtgacggt gaaacaacaa cttcacttct gactgattcg 6240
gtcctatact actctgtcgt tacagtctta caactacgta aagcagagga ttgtgtagaa 6300
cctgctgtag aaactattaa cctaagaccc aactcatcac ttcatacctt gacacatgat 6360
acaaacgcca ccgaccaact ttacatccaa gtggcttctg atgcttcaaa tcacactgta 6420
atcaataaca ccacacttga atctatttct aaaacgatgt gtgtgaaaaa ctccttgtac 6480
accctactta ccaactcaaa gtgtataagt caccacatca tagttacagg atatgaccaa 6540
catgctcatg aaacacatac tatcaaaagc gtcacgcgcc aagccatgct cgccatccaa 6600
cccctttcac aacttacgcc cacacacgtt tacagcacac ttcaagaaga tatttccacc 6660
atcttccaaa ctgtcgtcct agaacacgtc aaccacacca agctttccac ttacaacacc 6720
aagcttcact acgcaaccac agtaccaaca aacctactca tcgatggaac aatcgaagat 6780
gttcaaggta agctgcgcga agttgatctt ggcattgttg ttccttacca gtgtgaagtt 6840
gacgagcaaa ttgaatacac aaatgcaatt ggcgacgttc tctaccaaaa cacaaatcac 6900
cactcaccac tagtgacctt tgtttctatc gacaaatctt ttactaaacg tatttttaat 6960
tctttaaagt ttcaaactct acagaaccta tggctctacc ttattaccct acaagtattt 7020
ttcacagtta aaggaattgg cttcggcttc agctcaacgg aaccaaacac acaactcatg 7080
aacggtctct ggatacaata cggcatcttc tacttccaag tacacctcca aaccctcatc 7140
acccacccag tgaaaaatgg ctacagacta ctttcattac caatcatgct atacaacaaa 7200
ggcaaaagat tcaccgaacg ttcagtccaa acaatgacgg gagaaatcgt taacacacga 7260
aaaccctcgt tatcaaacgc tgtacaaaaa tacggcatta acactcgact tgaatacgtt 7320
gatatcttcg atctcctcca ctatacacct ggatacatcg cgtttaacat cgcctttttc 7380
tatctctttg ctggtaatgg ctatataaca ttcttagcct acatcatcca atttttcacc 7440
gctcgcgata tctacagact ctctaaacac ctctacacag tctcaaaact caccaccttc 7500
ctcgcgaaat ttaaaacagc aaccacccgt cacgtcttgt acgccaccaa ccaagtaaac 7560
aaccgttccc ttcatctcac cctcggatcc acgacaattg acttactcag ctttacaagc 7620
aaagcagctt tccttgccct gtgctcctcc ttcatctcat ggcatttcgg attagctctc 7680
gccggtggtc actctggtga agactactct caaccaccct accacgcagt atttcaacat 7740
gcactccgca ctttcggact tattcctcct aaagaacatt actacagcta cccatcccta 7800
gcttcagcct gcgcagactc atcagcccct ctctgcttcc tcggaacacc atttaacttc 7860
cacttcccat catcctacac acaagcagca tccatgacta cagaacacgt agtaccattc 7920
tggatccaac taatcatcct agccccacca tcgctttttt tagttgtcat tccgtggctg 7980
atcacaatgt tcatctcacc agtattaaca tttaaccaac ttttgctccc tgcactcgcc 8040
gtacatttct tagctatcta tgtaatctac ctttactatt tcaagaaatc gtgctgccat 8100
tctcacacat gccttaaaca cgctaaatta gaccgcccca tccaaattaa aggcctcttc 8160
gatttatcca tgtacttcaa tcacaataaa ctttgccccc gccataactt ttactgtaat 8220
aatgctgacg aacctggcca cacacttgga cccgtcctag cagctcacat cactgaattt 8280
tatcatctta aacctggcac tattaagccc gatgtcgccc tagtacacac agaacaccac 8340
gcttccctga gtcgcaaaga attgaagaaa cacgacgacc caaccgattg tgtctggacc 8400
gccccacaag gcgctgctga tactctcgtc tacgaagcct actcccattt gaaaggagtc 8460
caaattcgca ttgatgccaa caccgataca acaactcgta gcatgcctat tcccatgggc 8520
gccaaaacaa catcttctcc agctacaggt gtcatcaaca caaaagttct cggccatctt 8580
aaaagacatt gtaagaaacc agctctccaa tcttacctct caggatacgt cggtgccccc 8640
aacgctaaaa ctactatcat gcccattacg ttttttaaac atctgcctga aggactcaag 8700
aaatacgcca tcaaaaacat caccttctca actaacatgc taactgacaa ctctaactca 8760
ggtttcatcc cagaaggcat ccacaaccca aacaacctcc caaccaagac tttcctggtc 8820
aagtctatcg atcaaacaat tttcgaccaa ctccacccag aaacacaagc tatgctaaaa 8880
gacgaatacc acattaaact tcatgatata acacctcaat ttcacgaagt tattgcttca 8940
gtgatgtcct acgaaagttt catcttcgca ctcatcattg tcttatccgt cttcctcggt 9000
tttatctcct acagctctac tataactgcc accacatccc tacatgccgg cttgaacccc 9060
actatgcgcg gatccaccac aggtaaacca ttcattcaac cagttactta ccccaactcc 9120
attctaatcc ctctgacatc ttcccaatat aatgtattca gactacaaaa cggaaccctc 9180
tacttcactt cccaaataac tgccccaacc tgcaccgtta aactaccaaa cccctacatt 9240
ggcttaacca cagaaaaatt ctatgaatgc tcatctgccc tcctccacat gcccttcatg 9300
gttcatctcg gtggtgtgaa aatcgcttac cttagagccg gtgttgctta caacatgcca 9360
acatactcag agacaccaca aactagcaaa ctctgtgttc tcactgcact ctcaaagacg 9420
aaatgcttta acatcctacc aggtggcttt catactccag tactagccat cctcgtctca 9480
acttgtgcac ttctggcaac ggtccttttc tacctctacc tactccagat ctttaaattc 9540
tacactaact cagtcttgat ggtggttgcc attcaaatcg caactatctt cctcttcatg 9600
ctatccccag ctcttgctct aacacttcaa ctggtctgtc tcatgtacct ttaccataac 9660
tggatggtcg ccatgattaa catcgcaaac ctcgctgttc tagcccacac aacatttggc 9720
atggtactca tcttcatcta cgcgctctac agactctaca aagtctacca agttacatca 9780
accgctgcag ttacaattaa cgcaactggt gaaatgcgct tccacggaac tcttgatgaa 9840
gttgctctct gcacctttcc aattagctct gcaagtgttt accaaatcat gtctgacctc 9900
agcttagatt acattaaact ggccagttac aaaacatctt ataaccgtgc tacccgcaac 9960
ctggttaaca tgatgcaaag atacattctc gacacctcca tcccagctat actctatgac 10020
ggctcagtaa caactggcat catgcaacaa tccctagtta gacttaaatc cctagttaca 10080
acagtttaca aggataacta ctccaacatc tgcaaaattt catcaacaac ccactcacga 10140
tcaacattct gcgtcggaac cttcctcaac ccaaccactg ttatcacttg ctctcacgga 10200
atcttagcaa actctactat caccgtttcc cacaagtcaa ctatctatga agcaacaatt 10260
gaatcacaac gtaatgacat tacaatgctt aaacttacaa caccaaactc cgcaatcact 10320
cacaccatcg agtgccactc accaactgcc actgacctcg aaagcatgca agttaattac 10380
acccaatttg ttactttcgc tgataaagat cactcagatg ccatagccat gaataacaca 10440
acctacctac catctggtca cttctttgct atcggaaccg aatctggcga atctggcagc 10500
ccctacttct ttaacaacaa acttgtagga attcactacg gagttaacac ctgtgatcaa 10560
tttatgctcg cctctaaacc tgatggcagt tactacatcc cagaaatcca acacaacatc 10620
tccacacaac catcttttag ccttagttgc atagcccatg gtcgaaaatc ggtaaccgaa 10680
aaaaacaccc ccacacttca cgatgctatc accaaagccc ttgcccaaac aacacgctca 10740
gcttacacta acgatttaac aacagaacac acacctgaac aactccaggc tgtccttgat 10800
tttctgacag accctctgaa taaatctggc cctaagacac tccataacta catggcaaca 10860
tccatccaag ttcaatccac caccacagcc caatccgccc acattgacgt ccacaaacca 10920
attagacaag cagcaactct ctactaccta atggttatca tctttgacat catcgcattc 10980
ttcttaaccc ccggtgctga cttttccaac caacttgtat ccatgactgc ctacctagct 11040
gcctggttcc ttaataaaac atgcttctac aacacccatg tcctctacac agtaaccaca 11100
tgcttccaac tttgcaaata cttcaacatt gtctacctct tcacaatcat tcaatcctac 11160
aaaccaattg aaatgtcaat gatggataaa caatatgact tcctctgcgc catcattgta 11220
gccctcctac tctctccaat cctggtcctc atctggaaac gcctctgcaa cttcacctgg 11280
cattacctcg ttgctcacct agttgttggc tgtgtgcttt gcatccacgt cttcttcagc 11340
tacagatcct tcacatcttc aacaggcggt ctcttaacat tctcagaatt cgcctttaac 11400
tcaccattaa cagcagtcat aacagctaca gttgatgaaa gtcctgtttc tgttattgtc 11460
tacccactct tgcagcatgt catcagcgca accctgatct tagcaatcgt gccatctccc 11520
ctcctcgcag caacagtcta ctctttctat ctcattgacc aagacgtcct ctgcctccta 11580
atcttcctcc tgcttacatc caaactgaca ccacttttcc tccgcccact cacaacattc 11640
gtttcctacg atgttgtcat cctctcaccc tctgcttacg ctcaatggct catggcaaaa 11700
tccgaaggta aagaatacgg tctctttttc gctgtcattc aatcagtctt tgactttaca 11760
tctcaacacg aagttgaatt gacacttgaa gcacaagaga ttacacagca agtccgtgtt 11820
gaagttcata acaccgaacc taaagatact aaggatgaca cctgctacat cacacccact 11880
actattatta acccaactct aattagaacc gacaattact tctacaccaa caccaagaaa 11940
gtaatctgcg aagctcccat tgaactatca gataaggcta aagaaatcgt cgctaaacaa 12000
ctcgacgcta ttccacaccc tgatgcagaa acagctgatc tagaagttgc cacacttatt 12060
gactgcacaa ctacaaccaa gcaatcagaa gttaaagaaa ttgaagtaga cgatttaaca 12120
catgcctacc tactcgctct tatcaaagca acttgcatag aatctggcct ccccattcca 12180
gtgatgaact acatctcatc ccagacccaa attgactcag tgatgatcca ttactttgtc 12240
aaagctgcca acaaactact gcttaaagat caggaactgc ctcttatcac tgcccaatct 12300
gacacctcca ccattcaaga aaccttggaa gctctcaaat taaaggacac caccctaatg 12360
aacccccaag aacttaaagc tcaccgcaaa cagattaaca ttctgaaagc tgaacaagaa 12420
cgacagctat ccatggaacg gaaactcaac gctttcctag acacaatgca ccaacgtgct 12480
gtggctgaaa agaatagatc agaagttcac agcaaagtgt cagctgcttt acgcatgcac 12540
ctttcaaagg ttgcaaacgc tgcacattgt gacatggaaa caacagacgg tgttatcaca 12600
ctatgctctg ctttttctat ccgctcagtc tgcattactc aaaatactgc agatgtctct 12660
atctactact cacaacccac agatacatac tcaatcacaa ctaccaccac agtcttcaca 12720
gcttataacc ctatgaatgc caacggacaa cccatcattc caaccactga cccttacaat 12780
cctatcattt cattcccggt tatcttctca ctaaaacacc gtgaggaaaa atcggagata 12840
gaacaacaag caaacgtggg atactccgaa cgtgccataa aaataacaac gactgaacac 12900
gccactggta cagttctcat gattgacaac attacatctg tcatccacgc cactaaaacc 12960
gatgctcgct tcaccgtaaa cggtaaaggc tacaagatcg tcgacaatgc cgttgcccac 13020
aaagctcgtg ctaacatgtc tacaattctt gctgtcttgc gcaaatccac catcagcgca 13080
caagcagcag tagttagaat tggcggtgct agataccaaa catctcacca agctatctct 13140
ttcaccccct tccaaacacc aggctacgtt acctacgcag gaatatgcgt ctgccaacgc 13200
tgcacactca gagaacaaca cgagtgccac tacgcaggta actttgtcca gatacctacc 13260
aatatctcaa cacaaggact taacaaaatc tacgaattaa catctatcgg cccatgtaaa 13320
cacaaccaat ttgaatgcac cacatgccaa ccagacttaa ttacaaaagc ccccggacaa 13380
gtactggtat acttcacagc ccacgcccaa acccactaca aacaaatagt tactcaaatc 13440
tgcaaattct accaagttaa acaatgcaac tcccaaaaac atgcagactt tactatccac 13500
ataattccac acggccactc aactgataca actcatccta acaccaccgt tctagttgta 13560
cgaaaacacc tagtcgatcc agatcctgaa cctccacaca tcgggataag ttccacctta 13620
actggcgaag aactgattac tcttgacaga ttactacaac ctgctgtcga gcctcaaact 13680
aagtcgtttt taaacttctg gagcgggtgg taggtgtatc gttaccatcc gctagcgacc 13740
cttatacatc agggagctat aaaccgatac ctcgtatctt ttacattaac gttgcaacac 13800
gtaatgacac aaaattagat aaagtctcca aactcaacac ctggagcatg gaaacaccaa 13860
gcggaattat catggtaaaa aatcgtgacg cctctaaaga atttgatata gctaacaacc 13920
taccaaagtc actaccaatt ttaacacaca aactcatgac ttacaaccgc gacaacctcc 13980
taatcagata ctacacaaca ccttactcac taggagacct tgtctacgct taccaactcg 14040
gcgattttga acacctttta ctcgcggccg gactttcacc aaatcctgcc caattgctcg 14100
accctggtaa ttacacccaa tatcaacaaa caaaagaaca tctcagacaa attatctaca 14160
gatctaatga aaacctccat aaaatcattt attacttcac caccactgat aaatatctgc 14220
cgctaaccct ggacaacatt gatcagaacg gttatttgta tgatttcggt gactacccag 14280
ctaaaagtcc cgtcaataat caccacgtac tagaatcatg gacacaattc tgtgtattct 14340
gcgaagttaa ccttgctgaa ctaccaggtt ttaacctcta cactcaaacc cactcaactc 14400
cacaaattat aaagagccga caagacctag tctctggctc ctacatggat aaactactag 14460
ctcttaacca cctaatagca gcaaatccag aagccccttc caccgcttcc actcccttct 14520
ttaacgcctc cgcagccaca actgacatgg tcggcctaca ctcaccactc tttggctacc 14580
atgaagtcaa caatgatttg attcgctcta aactctcacc aacagaagtt gacttcatca 14640
gatctcaaga ttctgcaaac tatgctaaac ccaacgtcat ctccatcggc aacacaatta 14700
gctactacgg caaaagaata acaactaaag ctgcccaaag aaaacctcaa tacgatctcg 14760
acatgctcaa aagattagaa tcaattggca tcagcacact cgctacaaca tcttaccact 14820
accaagtagc tgacgatgaa caatgtgtgc aagattttaa atactacgat tacaactcgc 14880
ccaaaaccta tgaccctaac ttcctcaagg ccatctacca ctacatgcaa gacatctttt 14940
ccatttacaa aactgatgtt tcccttaacc atgaaacagg acaaccacgc ccctcttcta 15000
tgggtcttgg aatcacaggt tttactcaaa aagctgtttg gcagtctcta ccacctggtt 15060
ttggcccacg catgcttgaa gttgcaaaag aaactctact accatttaca acaaaggtaa 15120
ttgcaaaata cgctcgcaca aagaaagccc gtgcacgtac agtaggtgct tccagcttcg 15180
tcgcatcaac agttttcaga tttcttcaca aacctgtaac catgcgcatg gtcgaacaag 15240
cccaaaacaa cattggccca ctcctaattg gcgtctctaa atttaacggc ggttttgata 15300
aatacatccg tactcaacat ccaaacggta tagaacatta caacgtcatg ggtgcagatt 15360
acacaaaatg tgatcgtagc ttcccattga tcaaccgcgc cttcgccgca gcccttttct 15420
atgatttagg aggtttacca tctgacaaca tgtggtttat caacgaaata tccgccttca 15480
tgactgactt ctgcaacatc tcaggtcaca catttaataa accaggcggc acaacatcag 15540
gcgactcaac aactgccttt tccaactcca tctacaacta cttcgtacac ctcactgtac 15600
aattcctcac cttcctctca gccgacgtgc cagaaacata caaacatctc caaggttctg 15660
ctcatcgcgc ttactcaact ggaactctcg aagattacaa cacctacttc gaactcatcc 15720
atgatttaaa ctgcactgaa tacttctttc actttctcag tgacgattct tttatcttct 15780
caacccaaga agctttcccc atcttcacaa catctaactt ttcaaccaaa cttcaatcta 15840
tccttggctg cactgttgac gtaactaaag cctgggctgc atctggacac atcgaagaat 15900
tctgcagttc tcacattgaa gaaatcgacg gtgtctatca atacattcct gatcctaaca 15960
acatgatcgc aggcttgtac atgtccggtg acccagtacc agtagataag accatctgga 16020
aactagttgc tacactcagt gaattggcca tttttcaaca caaggaccca tcacttttta 16080
acgcccttta ccaactcctt caaaacatgc acagtgaata tgtcactcaa cataaccacg 16140
ctctcctccc tcttcaattg cttaacaaga aattctacac tgatctactt caaccaggat 16200
ttgacactga aacaattttc ctcactgact tgctaacatc tgtcggtgtg aaactccaag 16260
catccaacga ttcacaatgc tacttctgtg acaaccctac aatttcaaca tgtgctgatt 16320
gcgtagtgcc ataccccatg tgtgcactct gctcttacac tcactgggtt caaacaaacc 16380
atgccccatc acaagtacca ccatgtgtgt cctgtggctg tgtcaatcct ggaaaacttt 16440
accactgctt cactggcagc gttaaaattt gctgtcaaga tcacgtctct ggcctttccc 16500
tacccctagt agaccacgcc aagaaagtaa ttaggatacc cctcttccaa aactgcgaac 16560
gtcaacgatc cttagtccat gcaataacac acacggccct atttgacgac aatgacgaac 16620
cacttaacac tgcaacattt tccaaatatg acccagaaca aactaaagaa gaaaacttca 16680
tacgcatcct aaacgactgc tacatgctcg aagcctacca agcccaccaa aacccagcat 16740
acacctacga agttactgaa cctggcacaa tagttgtatc acaacctcca tcaacagcat 16800
atggtaactg tgtctacgct gaaattctcg atgacaaaaa tcgcgttgtc ttgaaagtca 16860
ctcttgatac aaagtcagta ctaacaccta ctgtctacca tgtaacatct tgtcgtggta 16920
cactctacac tcgctacaac aaaattcgca gagttcagca caaaccttca atagtcccta 16980
gatcaaccct agcctacttt aacaacgcac aattcataat tggaccacca ggaacaggaa 17040
aaactacttt cgtcatcaag aactacattg acagagcaga accaactaac aaaatagcct 17100
acatcgcccc aacacacgca ctcgtacaag ctatggatca agccatcttc gaagctcacg 17160
gatatgcagc tagtgtcaac attgttaaat cagaacttaa caacaagacc tacaactaca 17220
aagcaaacat gcctggatgc gatatcatgc tcggaacacc cggcgctgta tcagcacaca 17280
ctggttgcac catgatcttt gacgagatgt cacttgtaca gttgaatact gctatgactg 17340
caattgctcg agtaaaacca tctaaactca tcctgctcgg tgacccattt caattaggcc 17400
cagtagttca ctttagaaac ttccaataca actacgaaga cttcccctat cttcatttcg 17460
ccccaacaat aacatctctc cgcgtatgct tccgatgccc aacccaaatc tttaacgtat 17520
ggagtcacta ctacaccaaa catgacatcc cactcaatgc ctttaaagcc ggcggctcgt 17580
ttgaaatcat taaacaccac tcatgcgcca gagtttcaga aacagctcac cctctggtca 17640
caagactctc agaagaaaat ccagactcca tgatcctttg taactacaaa gctccagtct 17700
cctgcctcga aaacgctcta acaattgact caagtcaagg cagaacctac cgagacgtca 17760
tagtcgtgtt gctcggaaac acaaatttca cccgtgtacc caaccgattt attgtcgctg 17820
cttcaagagc tacacattct ctcaaagttt acctttctga agagatgtac gaccactact 17880
ccacactatt caattggcca caattagaaa caccacaaat ccagccacaa aacgccttca 17940
caacacctaa caacttagaa gaaacaaatc tcacaatggc cgctaataac atcagctccc 18000
ttgtcatctg tgatatagaa tttttccatc aagttcgcag ccttgaaccc aaagtgtgca 18060
cacttgaagt cggtgacatt accatccaca catcaacaag cgctaaacag attatcgttc 18120
cacgcgtttc atcaacatct agtgaaatct tcccgaaata cttcgctcac aacatgccaa 18180
ccctcactct ccccaaacca acaccaaagg gagattggga ttacatgaaa cctcaccgtg 18240
gaatttctcg ccgcattaac atcgacagaa ctcacaaact cttctcccat atggcttgca 18300
caacaaatgg aactcccatc tatgttcttt acggtgcccg caacgatatc ctagcccttt 18360
ccaaattaaa tatcgttggt gactataaat gtttctgcaa tctaccagct aactacttca 18420
ccctagatgc ccaagataac atcaaatact actgcaccca acacgccaaa ccagcagcac 18480
ctctcgctgg aaccattaat gctaaataca ttgacatcca aacagaaaag atcactcttg 18540
ccaaacgcca tgctgaaatt tgtgatcacc accatggtgc agcacataca tctgatgtcg 18600
acaccatcat gaccggatgc atcttaggca atgaattgac actagcaact caacatctca 18660
gccaacttct cttcacggaa cccttcagcc actactcaag tgtaatttct ccagatatgc 18720
accttacagt ttcaacccgc ccttgcagga cactcggaca acttatcata actaacatca 18780
atggtaagaa ctacagattt tcaatccaac cacacctgga ccttaaaaca cactttttca 18840
acatcgttgc taaacaaggc cactcctctg ttttagtcaa cgttccagaa acagttccga 18900
cctgtactaa aattcgcggt gaaacctgcg ctacatgcta caacacctgg cttatcctcc 18960
aagaacttca cgctgaacta gctaacacag gtctcgccct ggttaaccca ctccaacgcc 19020
aatacactca acgagaaaca caaattttaa ccaaggtaat gtctgtcact ccaacatcta 19080
caggcaaaat catggtccat ctcgacaaca cactctcctt ccccttccta atcgatttac 19140
caacaagtat ccaaacatac tgcaacaaaa ttgataaacc aatccctaac cctgcaattt 19200
tcacttccct aaaaattaca cacacctacg gatttggaac accatttgtc cctatgacaa 19260
acaacccact cattccatct atcctcagta accaaaaact tgctgacaac ccggactact 19320
actacatcac acatcaacca tcaaacagcg aactttcttt catgactttt tcgccaactg 19380
ctggcttcta ctgctacgac acccctctga tcaacttctc ggaacgtacc atcaataaac 19440
caccctacta tatcacacga attgtccgtg gccaactaca aaaacctgat attggcctct 19500
tctccactaa tagacttttc accaaccaat acgtaccttt acctaatgaa caacaagcgt 19560
acgaagactt tggtaaacat gtctcacttg gcgaaacaac atcaacatcc tggacaatcg 19620
gtggcatgca tctactccca gccttccaaa ccacaaccaa ttggcagatc acatcagaac 19680
atacaccaat tctctcaatt accatagctc gcgatcgcgg ggtcaaacta ggaacaacag 19740
ccttagatgt acctgctgac caactacttg aattgattga ctataaaaac atcacagtct 19800
caaaagtaat agctattaag atcgacggcc aaaaagtaaa catgatggtt ttcactcaaa 19860
acggtaaaat ccaaacagcc tacccagtac aacaagcaaa gattcacgat gtgacaaaac 19920
accacgtcac tcctgactac ataatctggc ctaaaatttt cagcgactca cctgtcgaaa 19980
aatgggatct tcctaattac aaccaaccac ctaaaggtct cacatcaaac gtcaacatcc 20040
ataaatacga tcaaatgtgt gcattctttg acaaacacct aaccattcca acaaatggcc 20100
acgtacacca tcttggtcat gccggacaag tttacagccc tggtgatata gttttaaaaa 20160
gctacttcac aaaagctcgt ttaacatcat acgacttgcg tgattgcata tcaaccagcg 20220
aaacagctca ccctgatgac aactggcgtg cacacttcat cctgtcagat gcatactgtc 20280
ctaacaccga ttttacatct ctctgcaaag actacatgac taaacaccta cgtcttggcg 20340
gaagcatgct ctggaaaatg acagaaacct gcaacatcga cgctgaatcc attctaccat 20400
acttcggatc tttcaccttt gttactttcg ccgttaacta ctcatcctct gaaacttacg 20460
ctttccttaa ggggtacatg ggtgaaccta attacaatac tacactccca gctaacatca 20520
tgggtcaact cggcgcgtac agacaaaacc tattgtacgt cccctatgct agtgagtata 20580
caggtaccag agtttataaa gattctggtg ctgtgaaagt tattccggcc atgtatgcag 20640
atcgtctgaa tacttcccac tatgctctcg gctgcatcta cagcacttag acatggcaaa 20700
cttgctatgg ctgactcttc tcatcactct cagtctcgtg tccgctcaaa ttaacacgac 20760
acttccagac tgtcctaccc caacctgcac ctacacacca ccaacaacga ccccatttga 20820
ccacacatca ctacactctt ttttcacaca cgacacaaac tatgctttcc ttcttgctct 20880
taagtataat ggtactttca ttcctgccac tatggctatg cctgctaaca acctttcgcg 20940
aacatatata ctcaactata aaaatgtctt tgtcaacgta cctgattctc aactcccccc 21000
tcctcaaaga agtaatactc agttggctgt tgttgcaact acagcttcct gtctcactgc 21060
aacgtatact gcgattcctg gtactattcc tggcctttca accaatcaaa tgtattgtgt 21120
gaaatttact cagtcgtcca actttgtgta cttgtacgat accactgtga acacccctgt 21180
tggcgtactt ttaaaaaacc caactgcccc agtagttaca actatcctat ccactatcat 21240
ggttaaccgc cctacaacag tttcaacctc acagctgcta gctaatatct actcccttat 21300
gagaacgaga agcaacgtga ttcaacaaca attcacttgc acatcttgcc tctttgccat 21360
cgacgcatct acaggaagag cagctacctc accaactact tacctcctaa acaactggtc 21420
cccatccttc tttgaaacta aaacagctat gttggaccac gtctctatga acaccatcta 21480
ccaaacagca cgtggtgtta ctcgtgaaac cttcactggt tacatcctta acgcagctaa 21540
aaccgctgcc tacgctcgtg gttctcccgc ctattacatc ttttcattcg cagccattgc 21600
acgctaccat caagcaatgg ttactcctta taactataac atcggaaaac tttgtgaagc 21660
aacagctgct tgctgcttat caaatggcat tgaacatttt gacatctata cctggctatt 21720
tcactactac ggctttatga aatccaaagt ctacagtgaa acttttacaa caaacccagt 21780
actaacatca gttattaact tagccacatc aattaaaggt aaacattctg tgtactgtga 21840
cctaacacct tacagttcat ttactgccta taatttgatc atgcttcaag atgttgtcga 21900
acaataccaa gggacacgta caacatccac atacgctaac aattttctca acctcactga 21960
tattaacgga tacctaccct attccaccga tacccccctc ccacaaaact accaaaacta 22020
catgcctttt gcaacctacc tacttgcaac attttactct tctttcactt tacaacagaa 22080
atttgacttt cacagattcc tccaatctgt ctgttattat gactctggtt tcatttctcg 22140
taaccaagcc taccattcga tctgtcacat aatcactctg aaaacatcat cttcagtgcc 22200
ttaccccgca gaactccaat ggccctttac tgctctccct tcctctggtt atgactctta 22260
tccagtttct acagatttag gcacagtagc tgttggcaac acaaatccac ttgattaccc 22320
tggaatttgt taccaatcag tttgcttgac tcatcctact attaacttca gacttttgtt 22380
atcagaatta ccaattaaat gggaagtccg acctgaaggt atctatgtaa catctagaaa 22440
cgatcctaca tgcctgaatt tcaaaacatc acagttggga acagttttca ttaccctcta 22500
cgattctacc gctacatgca cactccaaca gactacttac tctcgctctg ctgtcctgcg 22560
ccaagtcaac acgtttacac caaatgctac tgatttagtc gtgccttacg gcaaaacacc 22620
aatatacaat ggagcctata atattaattc cactgcatct ggtgctgtct ctctctctac 22680
caacacacac tacattccac gtctcctcac ttcagtttta acaccgcaaa gtgactacaa 22740
gattacacct acaaccacta atcttgaacc attcgtggaa attaatggca caacctttta 22800
ctacgatgct aatcaagcct acgtcaacac atcggacgtt tatcaagcta acgttgattt 22860
tggactcccc caaacatttt ctcttgatac cagctttctt aattgcacag ctttcctctg 22920
cgcttctgac tacaaatgta tcgccgactt ttcaccaatc tgctcagcta cacaaccact 22980
aatcgacgcc ctactccaaa cctacacact gtacaatcaa caacttacac tgtacaattc 23040
ttccctgctc acaattcaag ccgctgcaaa ctacctctac gacacaagaa cacgtactag 23100
acgatcactc ctatctactg ttaaccttgg ctacggtgac gtttttcaac aaccagcctg 23160
ggtctacgaa ataggtgcaa cagctgcaat tcctatgatc ggccctgcaa tctcagtcgg 23220
tttgttagcc agtagagtag catctattga atcaaccctc taccaaatta cccaaggagt 23280
gaaggagtct attatggcta tgaacaccaa gcttgataag attgccaaca ttttacactc 23340
agacattgaa agattaggat taacagttaa cacattagcc tcttcattca acacattcac 23400
taaacaagta acagataaat ttgcctctgt tgatgttgca tttaacgact tatcaacatc 23460
actgaacaca ctcaccacaa caacaactgt tggtgcctct tatgcagctc aaattaactt 23520
cgcaacaacc acgctaacca atttagaagt tcaaatctct caaacaacag atcacgctct 23580
atcttgcatc gctgcgctta atgaacatgt tctttctcca cactgcattt ccgtttcaca 23640
acttagcgct tacacaccta atgctctctc taacatcggt gaaaaactag tactaactca 23700
ctacatcaac ggatcactca tcacattcta ccatctcatt gctagcaagg gagtttacag 23760
acccctgccc aaaatgtata actctacaca ccatttagtt gcaacaactg gcttttataa 23820
cggccattgt atcttctgtg gtgaacatta ttgtgatatc agcgtttctg taccatgtga 23880
ttatgcgtac gaaccactta aatccactgt actagcagct tacccactaa caactggtgt 23940
agcccttctt actcttgact ctacagctac taacttcgtt attaactata ataatgcccc 24000
tctcatttca tcaagcgttc cacaaccacc tatcatcaat aacaccgatg gtacagttat 24060
tacgctccaa caacaactaa tccagctcca aactcaattc gatcacttta acatctcgtc 24120
gaattttcac gtttcagaaa tccaaccagt catagactac tacaatgcac aaatcaactc 24180
atctctaaac cagatagttg atttcggaaa tggctcaaca tctaatgtag gaacaatcat 24240
cctcattgta actatagttg cctttgctct tgcatcagct attgccatta tctgtggatt 24300
aaagcactaa agaccaacaa gttgcaatgg tttccaccgg tacaaacacc accgcaggag 24360
atcccaacct tgggtttcac ttacaactta atgatgtcct taacaccctg gacatctccg 24420
gcttcagcat ctcaaattct catttgatga gtgcgatgct gaccctcaac cttctctgga 24480
gcgctatctt tctagtctcc ttttatctaa tcttggacat ggttatgtac tacgttcccc 24540
tgctcaataa gattccactg ctgccaacaa tcaaaaacct ttgttggttt atctcgactc 24600
tgcttgttgc tgtttccatt tacgcttcag gaactctgct gatcatcaaa atcttcggag 24660
gcgtagtctt gctccttgct tttctcggca tggtgaagac tttctatatg ctcattgcaa 24720
tggcgctgtc ctactaccga caccgtaaca tcgcagtggc attgaacggc cctaatgtcg 24780
ttatggttaa tggcacagca gtccactctg atttcatccc cacgtctgtg acactggtga 24840
agaaagatgg tgaactctat tttcaaggaa caaaggtccc cggaaacaac ttgattgatg 24900
caaccgtgaa gatcttcact ttcaacgaat cgtttgaatg cctccccatc gcactctcac 24960
gttacactaa aggttcctac gctagatttg agctcaagtc gaccaacacc cactatgaac 25020
tccaaacttt ctatccttcg caaacagaaa ctaaatctgt ttgaaacact agcgaaaaca 25080
aattacaatg gcattctacg gacaaccaat gatgcctatg cctatgcctg cttcctttcc 25140
tcctcgtcgc cgcgccccta aaaagcgccc gccacagaag aaacaagcca agccccaacc 25200
taaacctctt aatgaccaac agaaaacagc tgttgcaaaa gaggctattg ccaaaaccaa 25260
tactgtgcca atgccaccca tggccacagt agtgaagaca aacctgcgcg ttcaggttgt 25320
aaaaagcaaa cgcggtgacc tcctagatgc tcgagggcgt cttacacaat cagagatcaa 25380
ccagctgtat ttccaaatcg ccactgccct caacaacggc ctcggagaaa ttggaataca 25440
caatgcttca acacaggacg gcgttcgtct catcgtgcgc ctcaatttag cccctcctac 25500
ccctattgct gagaaactct acaaatctca cgcggcggct aatctcaact cagaaattct 25560
ggtttctaaa caaaaggaac tggagcgaga ccccattttt catgtttcct cgactgagta 25620
aactgacttc acctcaaaac tgaagtatat ataatcacat ctatcacact atcctttata 25680
aactatatat tggggtggca ttaggccatt ccaaagaatt ctttaagatc acactcctcg 25740
atacctgact agtcatcacg tttcctgggg tctgctcttt aacattctac tatcctataa 25800
gatctaaaac tatccaaaaa aaaaaaaaaa aaa 25833
<210> 2
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
tgcccaccaa gacgacta 18
<210> 3
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
tttgcgctag cgcgattc 18
<210> 4
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
cgttccaaac cgtcttgggt caagagtgac gtaactgcta ctg 43
<210> 5
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
actcacttct gctccttgcg acgcagagtg taagaggttt tcca 44

Claims (3)

1.一种鲤冠状病毒HL39的 RT-LAMP检测引物，所述的引物为引物组：F3：5，-TGCCCACCAAGACGACTA-3，；B3：5，- TTTGCGCTAGCGCGATTC -3，；FIP：5，- CGTTCCAAACCGTCTTGGGTCAAGAGTGACGTAACTGCTACTG -3，和BIP：5，- ACTCACTTCTGCTCCTTGCGACGCAGAGTGTAAGAGGTTTTCCA -3，，所述病毒的保藏编号为CCTCC NO：V201988。
2.权利要求1所述的鲤冠状病毒HL39的 RT-LAMP检测引物在制备鲤冠状病毒HL39的检测试剂盒中的应用。
3.根据权利要求2所述的应用，所述的鲤冠状病毒HL39的检测试剂盒包括：
FIP、BIP、F3和B3，dNTPs，Betaine，MgSO4，Bst DNA聚合酶，AMV逆转录酶，模板RNA，10×ThermoPol Reaction Buffer。
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