CN106978512B - 用于检测竹鼠源阿卡斑病毒的rt-pcr引物及其试剂盒 - Google Patents
用于检测竹鼠源阿卡斑病毒的rt-pcr引物及其试剂盒 Download PDFInfo
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Abstract
本发明公开了用于检测竹鼠源阿卡斑病毒的PCR引物，包括引物AKVM1和引物AKVM2，它们分别为序列表中SEQ.ID.No.1和SEQ.ID.No.2的碱基序列。据此，发明人还制备了相应的RT‑PCR试剂盒并建立相应的PCR检测方法，该试剂盒含有引物AKVM1和引物AKVM2、RNA提取试剂、PCR扩增试剂、竹鼠源阿卡斑病毒阳性对照和阴性对照。实验表明，本发明具有特异、敏感、快速、简便的特点，弥补了目前临床上检测该病毒的技术空缺，可作为养殖场、基层实验室等场所开展快速检测的良好方法。
Description
技术领域
本发明属于阿卡斑病毒检测领域，尤其涉及用于检测竹鼠源阿卡斑病毒的RT-PCR引物及其试剂盒。
背景技术
阿卡斑病又名赤羽病，主要引起牛、羊等流产、早产、死胎、新生胎儿关节弯曲及积水性无脑症等。阿卡病给畜牧业造成严重的经济损失，世界动物卫生组织(OIE)将其列为法定报告动物疫病，我国《进境动物一、二类传染病、寄生虫病名录》将其列为二类动物疫病。
竹鼠源阿卡斑病是国内外首次报道的在自然环境下引起大规模的成年竹鼠死亡的病例。迄今，国内外没有任何关于该病毒的基因参考序列。2013年11-12月广西柳州三江、2014年4-5月广西桂林及2016年6-8月广西柳城3个不同地区县市先后分别在人工养殖的竹鼠群中暴发一种怪病。据不完全统计，有11个竹鼠养殖场(三江3个场，桂林5个场，柳城3个场)共计3万多只竹鼠受影响。该病临床特征是在单侧或双侧后腿麻痹、拖行，颤抖、转圈、幼年竹鼠出现神经症状，抽搐、四肢僵直，口鼻、耳朵、眼眶、前后肢等少毛或无毛的部位皮肤出现红疹，部分足掌龟裂。发病竹鼠，食欲下降或不食，出现症状后1-3天内发生死亡。因其都表现出后腿拖行现象，且为烈性发病，在广西俗称为“拖垃圾病”。剖检可见大脑充血、出血，肺有出血点、出血斑，肝质脆、色浅，脾肿大、部分梗死，肾有出血点。幼年和成年竹鼠均发病，发病率20-30％，但死亡率几乎100％。流行病学调查发现，竹鼠养殖场之间存在相互引种外，大多数发病场之间无明显其他联系。细菌分离结果显示除大肠杆菌外，未见其他有意义的致病菌。RT-PCR或PCR检测痘病毒、疱疹病毒、腺病毒、淋巴细胞脉络丛脑膜炎病毒、狂犬病病毒、仙台病毒常见大鼠病毒均为阴性。经申请人课题组诊断排除了其他细菌和病毒病，通过病毒宏基因组学、RT-PCR扩增和细胞分离培养证实该病为阿卡斑病毒感染。研究结果发现该毒株对竹鼠属于强毒力。
随着我国特种动物养殖业的迅速发展，竹鼠养殖业得以不断壮大，而目前国内外在竹鼠源阿卡斑病毒性疾病领域的研究和报道还处于空白状态，没有任何参考文献和序列报道，临床上针对竹鼠源阿卡斑病毒病的防治也处于空白状态。因此，急需一种快速检测竹鼠源阿卡斑病毒病的方法，来对发病竹鼠进行及时诊治。
发明内容
本发明要解决的技术问题是提供一种特异、敏感、快速、简便的用于检测竹鼠源阿卡斑病毒的RT-PCR引物及其试剂盒。
为解决上述技术问题，本发明采用以下技术方案：
用于检测竹鼠源阿卡斑病毒的RT-PCR引物，包括引物AKVM1和引物AKVM2，它们分别为以下碱基序列：
引物AKVM1 5′-ATGATTATTACAATTCTCAACAT-3′，
引物AKVM2 5′-GCTAAAAGGATGAATTGGT-3′。
用于检测竹鼠源阿卡斑病毒的RT-PCR试剂盒，该试剂盒含有引物AKVM1和引物AKVM2，它们分别为以下碱基序列：
引物AKVM1 5′-ATGATTATTACAATTCTCAACAT-3′，
引物AKVM2 5′-GCTAAAAGGATGAATTGGT-3′。
引物AKVM1和引物AKVM2的摩尔比为1:1。
引物AKVM1和引物AKVM2的浓度均为10μM。
该试剂盒还含有以下试剂：RNA提取试剂、PCR扩增试剂、竹鼠源阿卡斑病毒阳性对照和阴性对照。
RNA提取试剂为AXYGEN体液病毒DNA/RNA小量试剂盒，PCR扩增试剂为一步法RT-PCR试剂盒。
竹鼠源阿卡斑病毒阳性对照为竹鼠源阿卡斑病毒糖蛋白M基因片段cDNA的质粒，阴性对照为竹鼠源阿卡斑病毒阴性的昆明小鼠血清。
针对竹鼠源阿卡斑病毒检测和诊断的缺乏有效可靠技术的问题，发明人设计并制备了用于检测竹鼠源阿卡斑病毒的PCR引物，包括引物AKVM1和引物AKVM2，它们分别为序列表中SEQ.ID.No.1和SEQ.ID.No.2的碱基序列。据此，发明人还制备了相应的RT-PCR试剂盒并建立相应的PCR检测方法，该试剂盒含有引物AKVM1和引物AKVM2、RNA提取试剂、PCR扩增试剂、竹鼠源阿卡斑病毒阳性对照和阴性对照。实验表明，本发明具有特异、敏感、快速、简便的特点，弥补了目前临床上检测该病毒的技术空缺，可作为养殖场、基层实验室等场所开展快速检测的良好方法。
附图说明
图1是本发明PCR检测方法退火温度检测结果图，图中：1.ddH2O；2.53℃；3.57℃；4.60℃；M：DL 2000DNA分子质量标准。
图2是本发明PCR检测方法特异性试验结果图，图中：1.竹鼠源阿卡斑病毒；2.ddH2O；3.竹鼠内源性反转录病毒；4.竹鼠源阿卡斑病毒；5.竹鼠细小病毒；6.竹鼠乳酸脱氢酶升高病毒；M：DL 2000DNA分子质量标准。
图3是本发明PCR检测方法敏感性试验结果图，图中：1.cDNA浓度为23ng/μL；2-5.10-1～10-4倍比稀释模板；M：DL 2000DNA分子质量标准。
图4是本发明PCR检测方法的临床检测结果图，图中：1.正常竹鼠脑组织对照；2.ddH2O；3-8.分别为发病竹鼠的脑、心、肝、脾、肺、肾；M：DL 2000DNA分子质量标准。
具体实施方式
实施例1引物设计
根据前期病毒宏基因组学测序结果得到的竹鼠源阿卡斑病毒全基因序列(该病毒包含小(S)基因片段(编码核蛋白)、中(M)基因片段(编码糖蛋白)、大(L)基因片段(编码转录酶和复制酶)，其序列分别为SEQ.ID.No.7、SEQ.ID.No.8、SEQ.ID.No.9)的保守区域设计引物，得到一对位于核蛋白基因的引物AKVS1和AKVS2以及一对位于糖蛋白基因的引物AKVM1和AKVM2，送深圳华大基因合成，用DEPC处理水溶解，-20℃保存。
表1引物序列
分别用引物AKVS1和AKVS2及AKVM1和AKVM2扩增竹鼠源阿卡斑病毒阳性样品，初步筛选引物的特异性及稳定性，结果发现：引物AKVS1和AKVS2能扩增出非特异目的条带，且非特异条带非常明显，已影响结果判定。故被淘汰。
实施例2 RT-PCR反应体系与反应程序的建立
1、RNA模板制备
均匀剪取发病竹鼠的心、肝、脾、肺、肾等组织与抽滤除菌DMEM按体积比1:5的比例研磨制成病料悬液，分装到1.5ml EP管内，经3次冻融后，将病料悬液8000rpm 4℃离心5min，取上清200μL，同时等量吸取阳性对照与阴性对照(见实施例6)并编号。按照AXYGEN体液病毒DNA/RNA小量试剂盒提取模板RNA，产物直接用于RT-PCR反应或置于-80℃保存。
2、RT-PCR扩增反应
按PCR反应体系(见表2)配制反应混合物放入TaKaRa RT-PCR仪，RT-PCR反应程序：反转录反应：50℃30min；预变性：94℃4min；变性：94℃30sec；退火：57℃45sec；延伸：72℃1min；共进行35个循环；延伸：72℃5min。反应结束后，取RT-PCR产物5μL点样于1％琼脂糖凝胶加样孔内(含0.5μg/ml溴化乙锭)电泳进行鉴定，如电泳结果在816bp处出现扩增条带，同时阴、阳性成立，即表明待检样品中有竹鼠源阿卡斑病毒。
表2 RT-PCR反应液组份
实施例3 PCR检测方法退火温度确定
用提取好的样品RNA做模板，设置不同的退火温度，然后其他条件均设置成相同进行RT-PCR，RT-PCR完成后取产物5μL点样于1％琼脂糖凝胶加样孔内(含0.5μg/ml溴化乙锭)。电泳后的凝胶于紫外分析仪下观察结果，已确定其最佳退火温度，结果显示退火温度从53℃-60℃，PCR产物结果均很特异(图1)，最终确定本检测方法的退火温度设为57℃。
实施例4特异性实验
用前述已建立的PCR方法，分别提取竹鼠源阿卡斑病毒、竹鼠圆环病毒、竹鼠细小病毒、竹鼠内源性反转录病毒、竹鼠乳酸脱氢酶升高病毒核酸。按表2的体系配制反应液，按照实施例2的方法进行特异性试验。结果显示只有竹鼠源阿卡斑病毒能进行特异性扩增，其它病毒不能扩增出目的条带(图2)。结果表明，本发明检测方法具有很高的特异性。
实施例5灵敏度实验
用蛋白质核酸测定仪测定了模板cDNA浓度为23ng/μL，对模板cDNA进行10倍倍比稀释然后进行PCR，35个PCR循环后，检测该方法的敏感性，最终测定该方法的最低检测量为23pg(图3)。
实施例6试剂盒组装
根据以上实施例的研究结果，组装检测试剂盒以方便使用。
1、引物为AKVM1和AKVM2，浓度均为10μM。
2、RNA提取试剂：使用总RNA纯化试剂为AXYGEN体液病毒DNA/RNA小量试剂盒。
3、PCR扩增试剂：天根生物公司生产的病毒检测用一步法RT-PCR试剂盒(目录号：SD102)。
4、竹鼠源阿卡斑病毒阳性对照和阴性对照
阳性对照：将需扩增的糖蛋白基因的目的序列(SEQ.ID.No.5)经RT-PCR扩增并克隆进pMD-18T，构成并测序验证100％同源，最后制备目的片段重组质粒pMD-M1，在含氨苄青霉素的LB细菌培养液中进行培养，收获菌液提取质粒作为阳性对照。
阴性对照：无菌采集的阿卡斑病毒阴性的昆明小鼠血清。
实施例7临床应用
对临床上疑似竹鼠源阿卡斑病毒的病鼠进行解剖采样检测，使用前述试剂盒按照本发明的PCR检测方法进行。结果显示，临床检测样品扩增出816bp的特异性带，为竹鼠源阿卡斑病毒阳性(图4)。
SEQUENCE LISTING
<110> 广西壮族自治区兽医研究所
<120> 用于检测竹鼠源阿卡斑病毒的引物及其试剂盒
<130> 用于检测竹鼠源阿卡斑病毒的引物及其试剂盒
<160> 9
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<222> (1)..(22)
<400> 1
tgcaatggca aatcaattta tt 22
<210> 2
<211> 22
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<222> (1)..(22)
<400> 2
cgagcagctg aacaaaggtg tg 22
<210> 3
<211> 23
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<222> (1)..(23)
<400> 3
atgattatta caattctcaa cat 23
<210> 4
<211> 19
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<222> (2)..(19)
<400> 4
gctaaaagga tgaattggt 19
<210> 5
<211> 816
<212> DNA
<213> 人工序列
<400> 5
atgattatta caattctcaa cataattaca ttagcagtca ttgtgactgc aatgcctcct 60
cgtaatacga atgggggtcg atgcttttat ggcggcaata tgtttcgtca gatcaactca 120
acatcgccaa tgagtgaaat ctgcgtcaga gacgacatat ccctggttaa atctattggt 180
tatcacaaac tggcagcaaa tagggaagtt atcgaatcaa gtatgtcata ctacaggttg 240
tattatgtca aaaattggtt cgaatgcaat cctgttcaag atattcttgg aacttttata 300
gtatttgatg taaatcatga gggcatctta gcacctaaga catatgcttg tagagctacg 360
tgttcaataa gcttagaacg agacactggc aatgttgtgt tggaatcccc agcattaaat 420
cattacacca tacatgggac gactatcaag aatggctggt ttaagacaaa ggtctccata 480
gacctggata acacatgtga ggacctacat attacatgcg gtcacaaaac ccttaatgtt 540
catgcatgct ttaggcaaca taaatcatgt attcgttact tcaagggatc aatcttacct 600
gaaatcatga tcgagtcaat ctgtacaaat gcagagctga tattactatg ctgcttctct 660
gcaatctcct gctttgtggc cattatgttg acaaagacat atttggtgta tctgcttata 720
ccaatttttt acccgtttgt caagctttat ggcttcttct tgctgagatt ctgtaagcaa 780
tgtaagaact gcttattacc aattcatcct tttagc 816
<210> 6
<211> 788
<212> DNA
<213> 人工序列
<400> 6
tgcaatggca aatcaattta ttttcaacga tgttccacaa cggaatgcag ctacatttaa 60
tccggatgca gggtatgtgg catttatcag taagtatggg cagcagctca actttactgt 120
tgctagagtc ttcttcctca accagaagaa ggccaagatg gtcttacata agacgccaca 180
accaagtgtc gatcttactt ttgcaggggt caaatttaca gtggttaata accattttcc 240
ccagtacact gcaaacccag tgtcagacac tgcctttacg ctccatcgca tttcgggcta 300
cctagctcgc tgggttgctg agcagtgcaa ggctaatcag atcaaacttg cagaggcagc 360
tgctacaatt gtgatgccac tggctgaagt gaagggttgc acctggagtg atgggtatgc 420
aatgtactta gggttcgctc ctggtgccga gatgtttctg gaaacttttg agttttaccc 480
actggttatc gacatgcacc gtgtgataaa ggacgggatg gatgttaact tcatgaggaa 540
agtcttacgc cagaggtatg ggcagctgac tgcagaggaa tggatgacat ctaaattgga 600
cgcagtcaag gctgcattta actcagttgc ccagatatcc tgggccaaat ctggcttctc 660
acctgcagct agagctttct tggctcaatt tggtattcag atctaatctc tgctgattct 720
ccagttttct tacttctatt tctaagtgca ccatgtctat gtggtgcaca cctttgttca 780
gctgctcg 788
<210> 7
<211> 788
<212> DNA
<213> Rhizomys pruinosus Akabane virus
<400> 7
tgcaatggca aatcaattta ttttcaacga tgttccacaa cggaatgcag ctacatttaa 60
tccggatgca gggtatgtgg catttatcag taagtatggg cagcagctca actttactgt 120
tgctagagtc ttcttcctca accagaagaa ggccaagatg gtcttacata agacgccaca 180
accaagtgtc gatcttactt ttgcaggggt caaatttaca gtggttaata accattttcc 240
ccagtacact gcaaacccag tgtcagacac tgcctttacg ctccatcgca tttcgggcta 300
cctagctcgc tgggttgctg agcagtgcaa ggctaatcag atcaaacttg cagaggcagc 360
tgctacaatt gtgatgccac tggctgaagt gaagggttgc acctggagtg atgggtatgc 420
aatgtactta gggttcgctc ctggtgccga gatgtttctg gaaacttttg agttttaccc 480
actggttatc gacatgcacc gtgtgataaa ggacgggatg gatgttaact tcatgaggaa 540
agtcttacgc cagaggtatg ggcagctgac tgcagaggaa tggatgacat ctaaattgga 600
cgcagtcaag gctgcattta actcagttgc ccagatatcc tgggccaaat ctggcttctc 660
acctgcagct agagctttct tggctcaatt tggtattcag atctaatctc tgctgattct 720
ccagttttct tacttctatt tctaagtgca ccatgtctat gtggtgcaca cctttgttca 780
gctgctcg 788
<210> 8
<211> 4236
<212> DNA
<213> Rhizomys pruinosus Akabane virus
<400> 8
atggttatta caattctcaa cataattaca ttagcagtca ttgtgactgc aatgcctcct 60
cgtaatacga atgggggtcg atgcttttat ggcggcaata tgtttcgtca gatcaactca 120
acatcgccaa tgagtgaaat ctgcgtcaga gacgacatat ccctggttaa atctattggt 180
tatcacaaac tggcagcaaa tagggaagtt atcgaatcaa gtatgtcata ctacaggttg 240
tattatgtca aaaattggtt cgaatgcaat cctgttcaag atattcttgg aacttttata 300
gtatttgatg taaatcatga gggcatctta gcacctaaga catatgcttg tagagctacg 360
tgttcaataa gcttagaacg agacactggc aatgttgtgt tggaatcccc agcattaaat 420
cattacacca tacatgggac gactatcaag aatggctggt ttaagacaaa ggtctccata 480
gacctggata acacatgtga ggacctacat attacatgcg gtcacaaaac ccttaatgtt 540
catgcatgct ttaggcaaca taaatcatgt attcgttact tcaagggatc aatcttacct 600
gaaatcatga tcgagtcaat ctgtacaaat gcagagctga tattactatg ctgcttctct 660
gcaatctcct gctttgtggc cattatgttg acaaagacat atttggtgta tctgcttata 720
ccaatttttt acccgtttgt caagctttat ggcttcttct tgctgagatt ctgtaagcaa 780
tgtaagaact gcttattacc aattcatcct tttagcccat gtccaacaat gtgtatctgc 840
gggatggttt ataattctag tgaagcctta aaagttcaca gaaaatgctt aaactgcact 900
ggctacaaga ctctaactaa aacaaggtat ttgtgtaaaa agaaactccc taatgtggtg 960
ttggccacta tcagcacagt gctattcttt acttttataa cccctataac agccgaatgc 1020
tacaattaca caagcttgcc agcagatttc caggctgcta tagataaaaa taattcttgc 1080
acacgtgagc aactaataat gatcatatta acaaccctat gtattgcctc aatcattgtg 1140
gttatatcag cagttcacct gtacatacgg atatgctatg tattttgtgc ttattgcggg 1200
atggtacacg aaaagaaggg cctggtttta ctagataatt tcacgtctca ttgccttaca 1260
tgcatttgtc gtgataaaag catacacaga gccaacaata attgtgttgc tccaatgaaa 1320
tacaaaactg ccaagtatac taatctagtg tgcattgtct tcctgattat cattgctgtt 1380
acccctgctt ttagtacttg catacaagag aaggaaatag aaacaataga tgatgctgca 1440
atgtgcattg ctctatatca aaatataacc cagccaaagg aatacaatgc ctttgtgaaa 1500
gaactttcat caacattaag cagccatgaa attgaattcc tcctgccagc tgttaagcca 1560
tcattcgact atctgacaac taaatcatct acatctaaat atctccacac tgccactatt 1620
catgaattta tagctgcaaa tctctatcct aacaacttca aaaaacacct gacaccagca 1680
gggccagaca gcatacaatg gagaacatat attcataata acaacttgca tttatgcaac 1740
gaccatgttg tcaaaatgat atgtcgctgt gtaatcaagc aagaggaatg cagctctaca 1800
acagtagatg atggtgaaca gattgtgcat tactataaga ggaacaaaga attttataaa 1860
gctgatctag agatactgta cacagttata tcaagggcaa tcccaggatt ggtcggtaat 1920
ttactgcgac aagttctaaa gtcgcaaaag tatgatgagt cattgcatgt cctaaataag 1980
attaagaaag atgtttctaa aattaaccaa ctgaatggca tagtagaatt cttaatacat 2040
atcaattcaa agaacataac agaagaggtc agggagttaa gaattaagcc tgatcttagt 2100
atcagggggt ccaaatttac aaacaagaac tcagggactc ctaacatcaa agaatgccaa 2160
actccattgt tcatcacatg cactgggaag agattccgat cactaacgaa acaatatatt 2220
gcgtgctcca atggaggtgt taaattgtac cagagaccaa acaaacccct aacacttgta 2280
ggcgataagc tctgcattgg tgacaaacac tgtatgatag catttgaccc agcagtagta 2340
gatgaaaata tacaaaaact ggattgctat agtctggctg ccacagacca atcaaatggg 2400
atgttgaaac ctgaaaggtc tattcgactc ctcaaaacag gcgaatgtaa aatagcaggg 2460
gtattatcta gaattgcagt gtcaataaat cagaaaaatt ataaatacac cactatagtt 2520
cataagaaaa gtgatttagt cgatgagtat tgtctaagcc caaattgcga tgtagattgc 2580
tacccttatt attcaggcaa cctagctgac tgctcttgga gtgaatcaac tcattctact 2640
ctgagccaaa aagtgatatc tcatacagat atagaatcat tcatatctag tattaaactc 2700
tccttgcaca atgacctgat ccagcaccat ttcaggccac taagtaacat gccacatata 2760
aagcctaatt ttaaatcaat aaatgttcaa gggacgattt ctggtagcaa gattcaagac 2820
agctatataa cattctcaat accattgatg actggactat cacagggttt cacattgcaa 2880
gatcacaaag gcaacgccct ctttgacatt atagcatatg tgaaaagtgc gcgtgtagtt 2940
gcgacataca accatgacta taaaactgga cccacagtga gcataaatgt tcaacataat 3000
gaacaatgta caggatcctg ccccagtaat atacctaaaa aagataattg gctcaccttc 3060
tctcgcgagc atacaagcac ctggggctgt gaagaatggg gttgcctagc aataggcaca 3120
gggtgtgtat atggatcctg ccaagatgtc attagagaag aagcaacggt tatttccaga 3180
gtaaataatg agcaattgga agttgaattt tgtgtttcag agcctacagg tacaatgtgt 3240
aatactatca atgtacttga acctgtactg ggagagcaca tgcagtttga agtccacggt 3300
gtacagacta atctattacc tgaagtggct ttgatcaaaa ataggagggt gtacaaaggg 3360
tcaataaaca ggaaaggtgt attcaaccct caatgtggtt cagtccagtc atttgatggt 3420
aaactctatg gtgttgggaa tccaaagttt gactacattt gccacgcatt atcaaggaaa 3480
gatatagttg tgagaaaatg ttatgaaaat cactactatt catgcgctac actcaaggaa 3540
gctgtagaaa tcaaatcaaa tatcacaaac tctaaaacta tgctttataa cgacaatgct 3600
ttactcggta gtgcatcaat aaagataatg ttaggcgatc taatatatca acaagcatca 3660
gtgcaagaaa gagatataag aggtcatgca acttgtggag gttgcacaga ttgttttaac 3720
gatgttgcgt gtaaagtcag catgacttca aatggtgttt accagtgccc aatagtatca 3780
tcctgtgatt cttatatcaa taatgtttat attaacgaag gcacaaatga tgttagcttg 3840
aaatttcgat gtttaaaggc agaaatcaaa ataagtattt gtggtaaaga gatccctgtt 3900
aaatctgaaa taattaagga cacgaagaag ctggatttag ctagtgctga tcaaacatcc 3960
tatattaaag aatttgataa aaagtgtgcc acatggctat gccgagctta taatgaggga 4020
attggattta tcttggaacc attatggaat gagttgagtt tgtgggggaa gtatgttatg 4080
cttgcattag ctataattat tagtatccta atccttataa aagtaataag gccactggca 4140
agatatattg tcacaatact taaagagaat gacaaagtgt acaaactgga aaataaattg 4200
aaataatcta atttaaatag acataactgg gagggt 4236
<210> 9
<211> 6859
<212> DNA
<213> Rhizomys pruinosus Akabane virus
<400> 9
tagtgtaccc ctaaatacaa catacaacat ggacaattac aagatcaatc aatatcgtgc 60
taggattaac gatgcgaatg atccagaaac tgctaaagat atattagctg atctattaat 120
ggatcgacat aactactttg gcagagaact ctgttactat cttgacattg agtataaaaa 180
tgacacgcca attgatgata ttctcctgga tttcttacca ccagggactg atttcaaggc 240
tagatactgt acacctgaca attatataat tcacaacaga aagttatatg tattggatta 300
caaggttgct gtcgataatg aatcttcagc taagactttt gaaaagtatg acaagatatt 360
tggcgatgtt ttggtgccat tgggcctgga ctatgagata gttgttgtta gggcagaccc 420
ggtgcgagat gtaatacatg tcaattcgga agacttcctt aatgagttcg ggccgattaa 480
tatgaactta gattttactt ggttttacaa tcttcgtgca ttaatatatg ataagtttaa 540
agacgatgaa cgctttttag aaatagccag ccagggtgaa tttacaatga ctgggccatg 600
gcttgatgac gaaacaccag aattatatga tcatcctata ttcaaagagt tttatgattc 660
tctaccaaat gagtcaaaaa ttacctttca gcggtcaatg aacttcgatg caacaaaagg 720
tgacaaatgg aatcaaaacc tggttgatac aattcatgaa tatactccaa gttataatct 780
ctttataaaa aatgcctcat ctggcatttt taaatgtcat ggagattacc cgaaacctag 840
caattctgaa atcacacaag gttggcaatt aatgacagag agaataacta gtgaaagaga 900
actaactaat gacataaata aacaaaagcc ttctgtccac ttcctctggt gtccaccaag 960
tgaagagtcc aatgaaaata tcaaaaagtt gttgcgatta tccaaaatgc ttcaaaagtt 1020
ggatggcacc agcacatatt tagacgcatt taaagccatt ggtatcctga tggatttctc 1080
atcaaatgtt ggattgtatg aatcacatac aagcaagcta aagaatatgt caaggcaaac 1140
atcaaaaaag atagacaaga agattgaagc tataaaaatc ggtacatcaa ctgtaatgtg 1200
ggagcagcaa tttgtcttcg atacaaacat cattgatcac cagtcgaagg ggaaactctt 1260
caaagaattc atgggaattg gcaaccataa gcaattttca aagaagacaa ttgaagacat 1320
tgacatctca aagccaacta tattagattt caataacaaa aacataattg acaaatgtat 1380
attccaatac aaaaatgttg acaagatatt atcaaagcca aatatgctaa acaagatggg 1440
atgttatctt gaagaatatg gcccacaaat ttgcgcagca tcagaagaaa catgggaaac 1500
acttaagctt atatgcaaca tgtcatattg gtcagcaata aaagatttct cgacattaat 1560
gaaaaacatg ctggcagtgt cgcagtacaa taggcacaac acattccggg tagtaacttg 1620
tgcaaataac aatttatttg ccattgttat gccatcttca gatattaaga caaagagggc 1680
aacgttggca tactttataa tatgtattca cgatcataaa gatgacatca tgcatcatgg 1740
agctttacat gcaacattta gaagtgaagg gaaatatgta agtgtctcaa aagggatcag 1800
attggataaa gagagatgcc aaaggatagt ttcttcccct ggactattct tattaacaac 1860
actgctgatg tataacaata acccaacgat taagattgag gatgttgcaa attttgcttt 1920
ccatacatct ctatctatta cgaaagctat gttatcctta actgagccat ctagatatat 1980
gataatgaat tcacttgcta tctctagcca tgtcaaagat tacatagcag agaaattctc 2040
accatataca aaaacaagct tctcagtgat tatggctaat ctgataaaaa aaggctgtta 2100
caacgcatat aagcagagat caaaagttga tctcagaagc atacatctaa cagactacga 2160
aataactcag aaaggtgtca acaataaccg tgatttatca tccatttggt ttgagggtaa 2220
agtgtcattg aaagaatata taaaccagat ttacatgccc ttttacttta attcaaaagg 2280
attgcatgaa aagcaccatg ttatgattga tcttgcaaaa acagtactag agatagagca 2340
tgatcaacgc ctcaatatac ctggtatatg gtctaatact cctcagaaac aaacagtgaa 2400
tctgcctgtt ttgatatatg ctatttcaaa aaacctctta atggatacct ctagacataa 2460
ttatattcgg tctagaatag aaaactcaaa caatttaaag cgatctatca ctactatcag 2520
tacttttact agttcaaaat catgcattaa ggtcggtgac tttacagatt tcaagtcaag 2580
agaatcaagg attgtcaatg ctaaactaga gaaagatatc aggaagtaca caatagcaaa 2640
cccagaattc attgaagacc tgactgaaaa ggccactata aggcatgcta tttatgacga 2700
cttaaaaaaa gctataccag attatattga tgtaatgtct actaaagtat ttgacgcatt 2760
gtattataag atcaagcatg gtgaaataac agacagacca gccgttgaac atatactcca 2820
agtcatgaaa gatcataaga gatttgtctt tacatatttc aataaaggac agaaaacagc 2880
aaaagacaga gaaatatttg tgggagagtt tgaagccaaa atgtgcttat acttagtaga 2940
gaggatatcc aaagagaggt gtaaactcaa cccagatgag atgattagcg agcctggaga 3000
tagcaagtta aaaaagcttg aagacatggc tgaatatgag attcggtata ctgctaacac 3060
attaaaatcc atgaaagaca aggcactgca agaatttagt agatttgcag acgacttcaa 3120
ttttaagcca cactcaacaa aaatagagat aaatgcagac atgtcaaaat ggagtgcaca 3180
ggatgttctc ttcaagtatt tttggttatt tgctcttgac cctgcacttt ataaaccgga 3240
gaaagaaagg attttatact tcttatgcaa ctacatggac aaagtactag ttatccctga 3300
tgatgtgatg acgtcgatat tagatcaacg tgtaaaacga gaacaggaca tcatatacga 3360
aatgacaaat gggctaaaac aaaactgggt ttctattaaa cgaaactggc ttcagggcaa 3420
tctaaattac acatctagct acttacattc ctgttgtatg aatgtctaca aagacattat 3480
caagaatgta gctctgctcc tagaaggaga cgttttagta aactcaatgg ttcattcaga 3540
tgataaccac acttcaataa caatgattca agacaaactc tcggacgata tcattattga 3600
atactgcatt aaattatttg aaaagatatg cctatctttt ggaaaccagg ccaacatgaa 3660
gaaaacttat gttacaaatt tcatcaaaga atttgtttct ctcttcaata tctatggaga 3720
accgttctca gtatatggtc ggtttctgct tactgcagta ggtgattgtg cattcctcgg 3780
cccgtatgag gatacagcaa gcaggttatc tgctactcaa actgcaatca aacatggtgc 3840
tcaaccatct gttgcttggg ttgcaattgc acttactcag tggataacac atagtactta 3900
taacatgcta cctggacaaa acaatgaccc actgaatgtt ttaacttcgc aaaacagatt 3960
tgacatacct atagaacttt gtggattatt aaatacagat ttgccgacat tggcaatagc 4020
aggcttagaa gcaggcaatt taacttattt ggtcaatttg tctagaagga tgtccaaagt 4080
ccagctactg cgcgaaagca ttcaatctca atatgcagac atagaatctt gggatttaag 4140
taagttgacg cagatggata aatttaaact taagttgttg aggttcatga cattagattc 4200
agctatgagt agcgacgacg gcatgggaga gactagtgat atgcgttctc ggtctctgtt 4260
aacgccacgg aaatttacaa cacatgcgtc tttagttagg ttagaatctt acaatgattt 4320
ccagaaactg gtacaggacc aaagtcaaat agatgactta ttcgaattct ttattaggat 4380
gccgcaattg ttggttacta aaggggagac aactgaagag tttatgaaat caatcttgtt 4440
caggtacaat agcaggaaat tcaaagaatc cttatcaata caaaacccag cacagctttt 4500
tatagaacaa gtgttatttg caaacaagcc catgattgac tatactagca tacatgacag 4560
actttttggc atacaagatg atcctaatat agacgactgt acgatgataa tagggaagaa 4620
gacctttatt gagacgtatc ggcagataca aatagatctt aataaatttg agatcactaa 4680
tcaagatgtc aagaccattt atgcattctg tctaatgaat gatcccatac tagtagcatg 4740
tgctaacaat attttgctat ctatgcatgg tctgcaaatg aatcgtaacg gaatgacatg 4800
ttgtatgatg ccagaaataa aatctctaaa ggtgatatat cattcgccgg ctctagtact 4860
acgagcatat gtacaagaaa ataataatat caagggagct gaacctgatg aaatgcagcg 4920
tgatctgtac catctagagg agttcattgc caaaactaaa cttcgtgaaa gtatgcaagc 4980
tagaattgca aaaaacgaac ttaaaactat gggtagagac tttaaatttg agatccaaga 5040
attgacaaaa ttttatcaga tatgttatga ttatataaaa tctactgaac ataaaatcaa 5100
aatattcatc ttacctaaac gagcttacac tccaattgac ttctgtggca tgattacagg 5160
taacacatta aaagatcaat gctggtttac tatacattat ttaaagcaga ttacggtacc 5220
agcaaggaaa gcccagatag caacatcgtt agacttagag atacaaatag cctatgaagc 5280
attgagatta attgcacatt ttgcagacac atttattgct gaaaattcaa gagttccgtt 5340
cctcaagcaa gtcatatcaa atttcttata taaaggtatt caaatgcaag tgctatacaa 5400
taagataaaa gcctcccgac ttaggacaaa aatattacca attctgtttt acatgggcga 5460
tttggaacag cttgatgtag acaggtatga tgcagaaaaa gcagaagagc agataacatg 5520
gaataattgg caagtatcta gagaatttac aacgggacca attgacttgt caataaaagg 5580
atatgggaga tctataagaa ttatagggga agatacacga ctaactgcag ctgagctaca 5640
aattaaccgc atgagaaatg atataatatc aaggcatgga cagtcgctgt tgaacaagcc 5700
acatggtcta agatttgaaa aaatggatga ggtagaattt ttggatgata aactgcatta 5760
tattgtctat cagttaaggg acagaaaacg ctattattat aatatattgt ctacaaatgc 5820
tatatatgaa cataatgaca gattgagcca aacaaggtca agaagaaaca atcgatggat 5880
acctgtctgt ccggtagcaa tatcgaagta tgtcaataca gatagaccta tattagatag 5940
aatagaaatg ctcaatattg ataatactaa gctaactagt ctgcaagtgg ctgttagcga 6000
tactgcaatg gttagaaagg cagcacttgc aaaaatgtct ttttttgaag gtccacctat 6060
acagtgtggg ggtatagatc tttcaaaatt gatgcaaaac cagaccatca taaacttaga 6120
tatcaacaag atttcatcaa taagtctatt agacctttgt agaattttct catgtagagg 6180
ctattcaaat gatcaagatg catttgagtt cttatctgat gaagttatga atgaagacat 6240
atctgaagaa ttagacagtt caccagtttt aaaaattaca tatacaaaaa agtcaaattc 6300
acaaaacaca tttaagaatg ctatagtaaa ggctttagta cgtgaatgtg acagattcga 6360
agagatattt gatctttcag atgacgggtt tacctcagat actaatttgg aactgttaga 6420
gaatctagtc tggatactaa accacttaaa gaccaaccag tggtctacag aattgatgga 6480
atgcattcac atgtgcttat acagaaatga aatggaccat atataccaca acttccaaat 6540
accagatgtg tttgtaacaa acccaattga gctgagtatt aaatggttgg atgtaataga 6600
attcctagaa atgatccttg cccatgactt taagtctgaa ccctggatat caataatgaa 6660
ccactctatc acaaaagcta ttgattactc gcggttagaa cataaaaaga cgagtgcaca 6720
agcaaatata tccaaattca taaaggggaa gaaaatgggt ggtcgatcaa aatttgattt 6780
ttaagtgaat tggtagagat cagtacgatt tttattgaca aagtgtgttt agaataagta 6840
tattattgca tttaggggc 6859
Claims (7)
1.用于检测竹鼠源阿卡斑病毒的RT-PCR引物，其特征在于包括引物AKVM1和引物AKVM2，它们分别为以下碱基序列：
引物AKVM1 5′-ATGATTATTACAATTCTCAACAT-3′，
引物AKVM2 5′-GCTAAAAGGATGAATTGGT-3′；
所述引物AKVM1和引物AKVM2的摩尔比为1:1。
2.一种用于检测竹鼠源阿卡斑病毒的RT-PCR试剂盒，其特征在于该试剂盒含有引物AKVM1和引物AKVM2，它们分别为以下碱基序列：
引物AKVM1 5′-ATGATTATTACAATTCTCAACAT-3′，
引物AKVM2 5′-GCTAAAAGGATGAATTGGT-3′。
3.根据权利要求2所述的用于检测竹鼠源阿卡斑病毒的RT-PCR试剂盒，其特征在于：所述引物AKVM1和引物AKVM2的摩尔比为1:1。
4.根据权利要求3所述的用于检测竹鼠源阿卡斑病毒的RT-PCR试剂盒，其特征在于：所述引物AKVM1和引物AKVM2的浓度均为10μM。
5.根据权利要求2所述的用于检测竹鼠源阿卡斑病毒的RT-PCR试剂盒，其特征在于该试剂盒还含有以下试剂：RNA提取试剂、PCR扩增试剂、竹鼠源阿卡斑病毒阳性对照和阴性对照。
6.根据权利要求5所述的用于检测竹鼠源阿卡斑病毒的RT-PCR试剂盒，其特征在于：所述RNA提取试剂为AXYGEN体液病毒DNA/RNA小量试剂盒，PCR扩增试剂为一步法RT-PCR试剂盒。
7.根据权利要求5所述的用于检测竹鼠源阿卡斑病毒的RT-PCR试剂盒，其特征在于：所述竹鼠源阿卡斑病毒阳性对照为竹鼠源阿卡斑病毒糖蛋白M基因片段cDNA的质粒，阴性对照为竹鼠源阿卡斑病毒阴性的昆明小鼠血清。
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