CN106987658A - 用于检测竹鼠细小病毒的pcr引物及其试剂盒 - Google Patents
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Abstract
本发明公开了用于检测竹鼠细小病毒的PCR引物,包括引物P1‑F和引物P1‑R,它们分别具有序列表中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PCR试剂盒并建立相应的PCR检测方法,该试剂盒含有引物P1‑F和引物P1‑R、DNA提取试剂、PCR扩增试剂、竹鼠细小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
本发明属于细小病毒检测技术领域,尤其涉及用于检测竹鼠细小病毒的PCR引物及其试剂盒。
背景技术
细小病毒属于无囊膜单链DNA病毒,病毒基因组大约5kb。其主要成员包括猪细小病毒、犬细小病毒、阿留申病毒、貂肠炎病毒、猫泛白细胞减少症病毒等。细小病毒是广泛的病原体,在动物中会引起一系列的疾病。
申请人课题组前期的竹鼠病毒宏基因组学研究发现竹鼠细小病毒有很高的感染率,并首次用细胞成功分离到三株竹鼠细小病毒,首次完成其全基因测序。目前申请人分离到的竹鼠细小病毒是一种完全没有报道的新的细小病毒。该病毒粒子无囊膜,不含脂质和糖类,结构坚实致密,对外界环境有强大的抵抗力。60℃加热30min不能使病毒完全灭活,在50%的甘油生理盐水中能在普通冰箱4℃中最长可保存4个月,在25℃保存5d的含毒脏器,病毒滴度不降低;在粪便和固体污染物上的病毒可存活数月至数年。因此,动物养殖场一但发生本病毒感染很难通过消毒彻底杀灭。细小病毒对乙醚、氯仿和丙酮等脂溶性溶剂和酸、碱、酚(0.5%)及胰蛋白酶有抵抗力,但0.5%福尔马林和0.175%次氯酸钠能有效地杀灭病毒,可用作细小病毒的消毒剂。
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细小病毒性疾病领域的研究和报道还处于空白状态,临床上针对竹鼠细小病毒病及由此引起的继发性疾病的防治也处于被动甚至空白状态。迄今,国内外没有任何关于该病毒的基因参考序列。因此,急需一种快速检测竹鼠细小病毒病的方法,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细小病毒的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:
用于检测竹鼠细小病毒的PCR引物,包括引物P1-F和引物P1-R,它们分别具有以下碱基序列:
引物P1-F 5′-GGCAGTCACACGTCACTAGC-3′,
引物P1-R 5′-TTACTCTTTTATGCACCAACTC-3′。
用于检测竹鼠细小病毒的PCR试剂盒,该试剂盒含有引物P1-F和引物P1-R,它们分别具有以下碱基序列:
引物P1-F 5′-GGCAGTCACACGTCACTAGC-3′,
引物P1-R 5′-TTACTCTTTTATGCACCAACTC-3′。
引物P1-F和引物P1-R的摩尔比为1∶1。
引物P1-F和引物P1-R的浓度均为20μM。
该试剂盒还含有以下试剂:DNA提取试剂、PCR扩增试剂、竹鼠细小病毒阳性对照和阴性对照。
DNA提取试剂为AXYGEN体液病毒DNA/RNA小量试剂盒,PCR扩增试剂为2×Es TaqMasterMix。
竹鼠细小病毒阳性对照为竹鼠细小病毒NS1蛋白基因片段cDNA的质粒,阴性对照为竹鼠细小病毒阴性的昆明小鼠血清。
针对竹鼠细小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细小病毒的PCR引物,包括引物P1-F和引物P1-R,它们分别具有序列表中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PCR试剂盒并建立相应的PCR检测方法,该试剂盒含有引物P1-F和引物P1-R、DNA提取试剂、PCR扩增试剂、竹鼠细小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.阴性样品;3.53℃;4. 56℃;5. 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.竹鼠细小病毒;2.ddH2O;3.竹鼠内源性反转录病毒;4.竹鼠圆环病毒;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.DNA浓度为125ng/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.阳性对照;2.阴性对照;3-4.可疑样品;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细小病毒基因序列(其全长序列见SEQ.ID.No.4)的保守区域设计引物,得到一对位于NS1蛋白基因的引物P1-F和P1-R(见表1),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
实施例2 PCR反应体系与反应程序的建立
1、DNA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DNA,产物直接用于PCR反应或置于-20℃保存。
2、PCR扩增反应
按PCR反应体系(见表2)配制反应混合物放入TaKaRa PCR仪,PCR反应程序:预变性:95℃5min;扩增32个循环(包括:变性:94℃30sec;退火:56℃45sec;延伸:72℃90sec);最后延伸:72℃7min。反应结束后,取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1086bp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细小病毒。
表2 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DNA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PCR,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,以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),最终确定检测方法的退火温度设为56℃。
实施例4 特异性实验
用前述已建立的PCR方法,分别以竹鼠细小病毒、竹鼠圆环病毒、竹鼠内源性反转录病毒、ddH2O分别为病毒核酸模板进行PCR试验,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。结果显示只有竹鼠细小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DNA浓度为125ng/μL,对模板DNA进行10倍倍比稀释然后进行PCR,32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125pg(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P1-F和P1-R,浓度均为20μM。
2、DNA提取试剂:使用总DNA纯化试剂为AXYGEN体液病毒DNA/RNA小量试剂盒。
3、PCR扩增试剂:普通的2×Es Taq MasterMix(天根生物公司生产)
4、竹鼠细小病毒阳性对照和阴性对照
阳性对照:将需扩增的NS1蛋白基因的目的序列(SEQ.ID.No.3)经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-NS1,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:无菌采集的竹鼠细小病毒阴性的昆明小鼠血清。
实施例7 临床应用
对临床上疑似患竹鼠细小病毒病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1086bp的特异性带,为竹鼠细小病毒阳性(图4)。
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<110> 广西壮族自治区兽医研究所
<120> 用于检测竹鼠细小病毒的引物及其试剂盒
<130> 用于检测竹鼠细小病毒的引物及其试剂盒
<160> 4
<170> PatentIn version 3.3
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<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<222> (1)..(20)
<400> 1
ggcagtcaca cgtcactagc 20
<210> 2
<211> 22
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<222> (1)..(22)
<400> 2
ttactctttt atgcaccaac tc 22
<210> 3
<211> 1086
<212> DNA
<213> 人工序列
<400> 3
ggcagtcaca cgtcactagc gtttcacatg gttggtcagt tctaaaaatg ataagcggtt 60
cagggagttt aaaaccaagg cgggaaaagg aagtgggcgt ggctaactgt atataagcag 120
tcactctggt cggttattca ctctgctttt atttctgagt ctgtgagaga cacaggagcg 180
agactaacca actaaccatg gctggaaatg cttactccga tgaggttttg ggagtaacca 240
actggctaaa ggacaaaagt agccaggagg tattctcatt tgtttttaaa aatgagaacg 300
tccaactaaa tgggaaggac atcggttgga atagttacag aaaggagcta caagatgacg 360
agctgaagtc tctacaacga ggagcggaaa ccacttggga ccaaagcgag gacatggaat 420
gggagagcgc agtagatgac ttgaccaaaa agcaagtatt catttttgat tctttggtta 480
agaagtgttt gtttgaagtg ctcagcacaa agaacatagc tcctagtaat gttacttggt 540
tcgtgcagca tgaatgggga aaggaccaag gctggcactg tcatgtgttg attggaggca 600
aggactttag tcaagcgcaa ggaaaatggt ggagaaggca actaaatgtg tactggagta 660
gatggttggt gactgcctgt aatgttcaac taacaccagc tgaaagaatt aaactaagag 720
aaatagtaga ggacagtgaa tgggtcactt tgcttaccta taagcataag cacaccaaaa 780
aggactatac caagtgtgtt ctttttggaa acatgattgc ttattacttt ttaagcaaaa 840
agaaaatatg taccagtcca ccaagggacg gaggctattt tcttagcagt gactctggct 900
ggaaaactaa ctttttgaaa gagggcgagc gccatctagt aagcaaacta tatactgatg 960
aaatgaaacc agaaacggtt gagaccacag tgaccacagc acaggaagca aagcgcggca 1020
gaattcaaac tagaaaggag gtctctatta aaaccacact caaagagttg gtgcataaaa 1080
gagtaa 1086
<210> 4
<211> 4758
<212> DNA
<213> Rhizomys pruinosus Parvovirus
<400> 4
ggcagtcaca cgtcactagc gtttcacatg gttggtcagt tctaaaaatg ataagcggtt 60
cagggagttt aaaaccaagg cgggaaaagg aagtgggcgt ggctaactgt atataagcag 120
tcactctggt cggttattca ctctgctttt atttctgagt ctgtgagaga cacaggagcg 180
agactaacca actaaccatg gctggaaatg cttactccga tgaggttttg ggagtaacca 240
actggctaaa ggacaaaagt agccaggagg tattctcatt tgtttttaaa aatgagaacg 300
tccaactaaa tgggaaggac atcggttgga atagttacag aaaggagcta caagatgacg 360
agctgaagtc tctacaacga ggagcggaaa ccacttggga ccaaagcgag gacatggaat 420
gggagagcgc agtagatgac ttgaccaaaa agcaagtatt catttttgat tctttggtta 480
agaagtgttt gtttgaagtg ctcagcacaa agaacatagc tcctagtaat gttacttggt 540
tcgtgcagca tgaatgggga aaggaccaag gctggcactg tcatgtgttg attggaggca 600
aggactttag tcaagcgcaa ggaaaatggt ggagaaggca actaaatgtg tactggagta 660
gatggttggt gactgcctgt aatgttcaac taacaccagc tgaaagaatt aaactaagag 720
aaatagtaga ggacagtgaa tgggtcactt tgcttaccta taagcataag cacaccaaaa 780
aggactatac caagtgtgtt ctttttggaa acatgattgc ttattacttt ttaagcaaaa 840
agaaaatatg taccagtcca ccaagggacg gaggctattt tcttagcagt gactctggct 900
ggaaaactaa ctttttgaaa gagggcgagc gccatctagt aagcaaacta tatactgatg 960
aaatgaaacc agaaacggtt gagaccacag tgaccacagc acaggaagca aagcgcggca 1020
gaattcaaac tagaaaggag gtctctatta aaaccacact caaagagttg gtgcataaaa 1080
gagtaacctc accagaagac tggatgatga tgcagccaga cagttacatt gaaatgatgg 1140
ctcaaccagg tggagaaaac ttgcttaaaa atacactaga gatctgtaca ctgactctag 1200
caagaaccaa aacagcattt gacttgattc tggaaaaagc tgaaaccagc aaactagcca 1260
acttttccat ggctagcacc agaacctgta gaatctttgc tgaacatggc tggaactata 1320
ttaaagtctg tcatgccatt tgttgtgtgc taaatagaca aggaggcaaa agaaacactg 1380
tgctctttca cggaccagcc agcacaggca aatctattat tgcacaagcc atagcacaag 1440
cagttggtaa tgttggttgt tacaatgctg caaatgtgaa ctttccattt aatgactgta 1500
ccaacaaaaa cttgatttgg gtggaagaag ctggtaactt tggccagcaa gtaaaccaat 1560
tcaaagctat ttgttctggc caaaccatac gcattgatca aaaaggaaaa ggcagcaaac 1620
agattgaacc aacaccagtt atcatgacca ccaacgagaa cattaccgtg gtcagaatag 1680
gctgtgagga aagaccagaa cacactcaac caatcagaga cagaatgctc aacattcacc 1740
tgacacgtac actgcctggt gactttggtc tggtagataa gcacgaatgg cctctgatct 1800
gtgcttggtt ggtgaagaat ggttaccaat ctaccatggc ttgttactgt gctaaatggg 1860
gcaaagttcc tgattggtca gaagactggg cggagccgaa gctagagact cctataaatt 1920
cgctaggttc aatgcgctca ccatctctga ctccgagaag tacgcctctc agccagaact 1980
acgctcttac tccacttgca tcggaccttg cggacctagc tttagagcct tggagcacac 2040
caaatactcc tgttgcgggc actgcagcaa gccagaacac tggggaggct ggttccacag 2100
cctgccaagg tgctcaacgg agcccaacct ggtccgagat cgaggcggat ctaagagctt 2160
gcttcagcca ggaacagctg gagaaagact tcaacgattc actgaccttg gactaaggta 2220
cgatggcgcc tccagctaaa agagctaaaa gaggtaaggg gctaagggat ggttggttgg 2280
tggggtacta atatgtaact acatatgttt tacaggcctg aaatcacttg gttctaggtt 2340
gggtgcctcc tggctataag tacctgggac cagggaacag ccttgaccaa ggagaaccaa 2400
ccaacccatc tgacgccgct gctaaagagc acgacgaggc ctacgaccaa tacatcaaat 2460
ctggaaagaa tccttaccta tacttctctc ctgctgatca acgcttcatt gaccaaacca 2520
aggacgccaa ggactggggc ggcaaggttg gtcactactt cttcagaacc aaaagagctt 2580
ttgcacctaa gctttctact gactctgaac ctggcacttc tggtgtgagc agacctggta 2640
aaagaactaa accacctgct cacatttttg taaatcaagc cagagctaaa aaaaaacgag 2700
cttctcttgc tgctcagcag aggactctga ctatgagtga tggaggggca caacaagaca 2760
acgctacaca gtcagctgct agagttgagc gagcagctga cggcagtgga ggccctggtg 2820
gtagtggtgg tggtggtggt ggtggtgttg gggtttctac ggggagcttt gacaaccaaa 2880
cgcactatga cttccttggg ggggggtggg tgcgcatcac agcatatgct tcgcgacttg 2940
ttcatataaa catgcctgct tcagaagaat atcatagaat ttttgttaga aatagcagtg 3000
atgttagtca aaaaggaaaa atgtcactag atgatactca cactcaaatc tggactccat 3060
ggagtctagt tgatgcaaat gcatggggct gttggtttca accaagcgat tggcaattca 3120
ttcaaaactc aatggcagaa ctaaatgttg aaacatttga gcaagaaata tttaatgttg 3180
tgctgaaaac tgtaacagaa caaaacagtg gaggagctga agctattaaa gtatacaaca 3240
acgacctaac agcttcactc atggttgctt tggacagtaa caactctttg ccatatacac 3300
cagcagctgc aaccagtgaa acacttggtt tctatccttg gaaaccaacc ataccagctc 3360
catatagata ttacttttat attccaagaa atctttctgt gacatatact gatgacacca 3420
ctgctgtcac agacacagta ggtcacacag atgcctacca ttcaagattt ctcaccattg 3480
aaaacacaac accaatcacc ttgctgcgca caggtgacga atttgccaca ggcatttata 3540
aatttgactg tgaaccaata aaattgactc acctgtggca aaacaacaga gctttgggaa 3600
tgccgccaat ggttggtgcg ctgccatcta atgacactcc aataaacata caaaaaccta 3660
acaccaacag atttggtcaa tcacaaatac acaattcaaa ccttctaaca gaagtaacca 3720
gactaagacc agcacaaatt ggcttcgcac aaccacatga tgtatttgaa gccagtgaca 3780
gtgggccatt caaagtgcct ttgatcagca tgtttccaac agacggtgct gacaatgatg 3840
gcaatggaaa tgtgaggtac atgtacagca aacaacatgg agcatactat gaccaacaag 3900
cagctgacta tgttgaaaga ttcacatatg aaccagcagg cacaggaggc agaaactatc 3960
aaaatggatg gactcaagac gttccatggg aaagcgtagc tacacaagaa aaaatactta 4020
ctagcaaaga tcccattgcc ggtaaaactg gaattcaccc acagaacatc atgaacacct 4080
atggaccact cacagcattt ccacacacca taccaatcta tccacaaggg caaatatggg 4140
acaaagaaat tgatctagaa cacaaaccaa gactgcacgc aaatgcgccg tttgtttgta 4200
aaggcaaccc tccaggacag ctgtttgtta gacttgcacc taacttgact gaccaatttg 4260
acccaaacag ctcaaacctt acacgcatag tgacatatgg aacatttttt tggaaaggca 4320
aactaacatt cattgcaaaa atgagaccaa accagacttg gaacccaata ttccagatca 4380
caacatcaaa tggaggatct ggaagatatg atgacataac caagtggcta ccaacaccaa 4440
caggacacat gaaaacagat ccgctgctta cagcaccaat tgcaagaaac atttactaac 4500
taaccaacca tgattctttg cttgctacac attcttttaa cacatattaa aatacaacat 4560
agaaacataa tattaagtat agactttaat aatagaatta tttggtatta cctaaatgtt 4620
agaaataaca gaacttttgg cttaaatata gttagttggt taatgttaga tagaatataa 4680
gaagactttg tacttgggta aagggaggaa agggtagttg gttggtactc ccatagactg 4740
aatgttaagg accaaaaa 4758
Claims (7)
1.用于检测竹鼠细小病毒的PCR引物,其特征在于包括引物P1-F和引物P1-R,它们分别具有以下碱基序列:
引物P1-F5′-GGCAGTCACACGTCACTAGC-3′,
引物P1-R5′-TTACTCTTTTATGCACCAACTC-3′。
2.一种用于检测竹鼠细小病毒的PCR试剂盒,其特征在于该试剂盒含有引物P1-F和引物P1-R,它们分别具有以下碱基序列:
引物P1-F5′-GGCAGTCACACGTCACTAGC-3′,
引物P1-R5′-TTACTCTTTTATGCACCAACTC-3′。
3.根据权利要求2所述的用于检测竹鼠细小病毒的PCR试剂盒,其特征在于:所述引物P1-F和引物P1-R的摩尔比为1∶1。
4.根据权利要求3所述的用于检测竹鼠细小病毒的PCR试剂盒,其特征在于:所述引物P1-F和引物P1-R的浓度均为20μM。
5.根据权利要求2所述的用于检测竹鼠细小病毒的PCR试剂盒,其特征在于该试剂盒还含有以下试剂:DNA提取试剂、PCR扩增试剂、竹鼠细小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细小病毒的PCR试剂盒,其特征在于:所述DNA提取试剂为AXYGEN体液病毒DNA/RNA小量试剂盒,PCR扩增试剂为2×Es Taq MasterMix。
7.根据权利要求5所述的用于检测竹鼠细小病毒的PCR试剂盒,其特征在于:所述竹鼠细小病毒阳性对照为竹鼠细小病毒NS1蛋白基因片段cDNA的质粒,阴性对照为竹鼠细小病毒阴性的昆明小鼠血清。
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