CN106047993A - 五种重要病原体分子标记及其应用 - Google Patents

五种重要病原体分子标记及其应用 Download PDF

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CN106047993A
CN106047993A CN201610245537.3A CN201610245537A CN106047993A CN 106047993 A CN106047993 A CN 106047993A CN 201610245537 A CN201610245537 A CN 201610245537A CN 106047993 A CN106047993 A CN 106047993A
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dna
pathogen
kinds
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刘骁
胡薇
杨忠
殷明波
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GENE FOR HEALTH BIOTECHNOLOGY (SHANGHAI) Co Ltd
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Abstract

本发明公开了一种利用核苷酸DNA片段作为分子标记鉴别五种重要病原体：鼠疫耶尔森菌（Yersinia pestis)，恶性疟原虫（Plasmodium falciparum)，霍乱弧菌(Vibrio cholerae)，杜氏利氏曼原虫（Leishmania donovani)和布氏锥虫（Trypanosoma brucei)的方法。这些序列可作为快速、准确鉴定病原体的有效遗传标记，有助于鉴定准确鉴定从原始感染地迁徙人群的快速甄别，并为研究病原体的分子流行病学学提供了依据。

Description

五种重要病原体分子标记及其应用
技术领域
本发明属于生物技术和遗传学领域，具体地说，本发明涉及一种利用核苷酸DNA片段作为分子标记鉴别五种重要病原体的分子标记在分子流行病学中的应用。
背景技术
鼠疫，霍乱，黑热病，疟疾和昏睡病是著名的流行病，常见于非洲。昏睡病在非洲36个国家，7千万人面临感染风险；在近代，就已经爆发过三次。疟疾是在2012年，估计发生了2.07亿例疟疾病例，造成大约62.7万例疟疾死亡。全球估计仍有34亿人有感染疟疾的风险，多数人在非洲和东南亚。大约80%的疟疾病例发生在非洲。鼠疫是烈性传染病，历史上爆发过多次，夺取了几千万人的生命， 所以一直是国家五年规划中的重点。霍乱传播很快，历史上有过6次世界性的大流行。目前主要集中在非洲和南美，每年有550多万人感染，十余万人失去生命。黑热病在我国流行于长江以北17个省市自治区。要实现普遍获得预防和治疗，还有漫长的路要走。随着生活水平的提高，我国每年有超过1亿人出国旅游，国内旅游超过30亿人次，南美，东南亚，非洲等地区也日渐热门，这给病原菌传播提供了便利的条件。这几种流行病的病原菌为鼠疫耶尔森菌（Yersinia pestis)，恶性疟原虫（Plasmodium falciparum)，霍乱弧菌( Vibrio cholerae)，杜氏利氏曼原虫（Leishmania donovani)和布氏锥虫（Trypanosoma brucei)。目前临床上对这些病原菌的检测手段各不相同；一般检查程序包括显微镜检查、培养、菌体裂解试验和动物实验等四步试验，操作繁琐且耗时，对病情的早期发现不利。近年来，随着生物技术的普及和基础研究的深入，我们已经掌握了这些病原菌的基因组信息，通过生物信息学手段，从遗传进化和基因组突变重组的角度，对这些序列进行同源和特异性分析，可以获取病原菌中特异的核酸序列片段，配合聚合酶链反应及DNA测序技术检测具有敏感性、特异性高的特点，而且可以在数小时内获取结果，便于广泛推广使用。
发明内容
本发明的目的在于提供一种、准确鉴定病原体的有效遗传标记的方法，该方法分辨率高，可靠性强。
本发明的另一目的在于提供所述的有效遗传标记作为分子标记应用于流行病学的研究。
本发明在另一方面提供了用于扩增所述五段序列(SEQ NO 11-15)的特异引物对(SEQ NO 1-10)。
本发明的技术方案是：利用所选的五个基因（作为分子标记快速鉴别对应的病原体，方法步骤如下：
(1)收集样本血清，逐个提取基因组DNA；
(2)合成上述五个基因的特异性扩增引物（对）；
(3)在适当条件下对每个个体的每个基因分别进行PCR扩增；
(4)对PCR产物进行纯化并测序；
(5)用生物信息学软件将测序结果与病原数据库进行blast比对和分析。
在本发明的一优选实施例中，步骤(2)中所用特异性扩增引物为：
1F CATAATCAACTACCATATCCCAAAC
1R CGGCTCCTGATACAATAATAAATAC
2F CCCTTGGTTTTATTATATCTTCG
2R AATAATGATAAACCAAACGATAAAC
3F TATTTACCTTCATGCAGAGCGTTGG
3R CCTAGCCTTGGCATCAATCGAATAC
4F TGAAGATTTGCTCGATTAAGATGCC
4R ACGGCTGCCATCACCGACAT
5F TTCTGGCGACTATCCTTAGGTAA
5R ATAGGGAGAAGGATGTTGAAGAAG
步骤(3)所述的PCR扩增程序为：
① 94°C预变性10min；
② 94°C变性30s；
③ 退火30s （1和5的退火温度为57°C，2为55°C， 3和4则为65°C）；
④ 72°C延伸90s；
⑤ 步骤②—④重复35个循环后，72°C延伸10min。
附图说明
图1是判断样品中是否携带巴贝虫（Babesia）的结果比对图。
图2是判断样品中是否携带利什曼虫（Leshimania）的结果比对图。
图3是判断样品中是否携带恶性疟原虫（Plasmodium falciparum)的结果比对图。
图4是判断样品中是否携带锥虫（Trypanosoma brucei)的结果比对图。
图5是判断样品中是否携带弓形虫（Toxbplasma）的结果比对图。
具体实施方式
结合实例进一步说明本发明
实例：利用本发明的方法，快速鉴定目标个体是否携带有病原体
(1)收集来自从非洲回国务工人员血清样本，提取个体的基因组DNA。用于提取高质量基因组DNA的试剂为QIAGEN 公司(德国)生产的DNeasy Blood & Tissue Kit试剂盒。具体方法如下：
将个体血清放入1.5ml EP管中，加入1.0 mM EDTA 和10mM Tris缓冲液500 µl。振荡后吸尽液体，重复三次。加入试剂盒中提供的ATL缓冲液 180µl；加入20µl蛋白酶K，振荡混匀。在56°C水浴中孵育4h，中间振荡数次。从水浴中取出后振荡15s，加入200µl缓冲液AL，振荡混匀。再加入200µl 95%以上的乙醇，混匀。将所有液体移入DNeasy抽提柱里，8000g离心1min，弃滤液。向抽提柱中加入500µl缓冲液AW1，8000g离心1min，弃滤液。再向抽提柱中加入500µl缓冲液AW2，12000g离心3min，弃滤液。保留的抽提柱移入新的EP管中，加入150µl缓冲液AE(或ddH2O)。室温下静置1min，12000g离心1min。滤液中即含有基因组DNA，-20°C保存。
步骤(2)合成特异性扩增引物的方法为固相亚磷酰胺三酯法，由生工生物工程（上海）股份有限公司合成，用HAP法纯化。
步骤(3)所用PCR反应体系为20µl：包括2µl 10×Buffer，dNTP 0.25mM，Taq DNA聚合酶1.5U，上、下游引物各0.5 µmol，模板DNA 20—50ng。
扩增反应在LifePro PCR仪上进行，反应条件为：
① 94°C预变性10min；
② 94°C变性30s；
③ 退火30s （1和5的退火温度为57°C，2为55°C， 3和4则为65°C）
④ 72°C延伸90s；
⑤ 步骤②—④重复35个循环后，72°C延伸10min。
扩增产物送至华大基因上海分公司进行测序。采用ABI3730进行双向测序，测序引物即扩增所用引物。如果有测序结果，经拼接和人工校对后，与人病原菌库进行blast比对，并分别与图1到图5标准结果进行比较，如果比对结果中最佳匹配（第一条）结果与图中所示物种名一致，检测结果为阳性，反之检测结果为阴性。
SEQUENCE LISTING
<110>金弗康生物科技（上海）有限公司
<120>五种重要病原体分子标记及其应用
<160> 10
<210> 1
<211> 25
<212> DNA
<213> 人工序列
<400> 1
CATAATCAACTACCATATCCCAAAC 25
<210> 2
<211> 25
<212> DNA
<213> 人工序列
<400> 2
CGGCTCCTGATACAATAATAAATAC 25
<210> 3
<211> 23
<212> DNA
<213> 人工序列
<400> 3
CCCTTGGTTTTATTATATCTTCG 23
<210> 4
<211> 25
<212> DNA
<213> 人工序列
<400> 4
AATAATGATAAACCAAACGATAAAC 25
<210> 5
<211> 25
<212> DNA
<213> 人工序列
<400> 5
TATTTACCTTCATGCAGAGCGTTGG 25
<210> 6
<211> 25
<212> DNA
<213> 人工序列
<400> 6
CCTAGCCTTGGCATCAATCGAATAC 25
<210> 7
<211> 25
<212> DNA
<213> 人工序列
<400> 7
TGAAGATTTGCTCGATTAAGATGCC 25
<210> 8
<211> 20
<212> DNA
<213> 人工序列
<400> 8
ACGGCTGCCATCACCGACAT 20
<210> 9
<211> 23
<212> DNA
<213> 人工序列
<400> 9
ACGGCTGCCATCACCGACAT 23
<210> 10
<211> 24
<212> DNA
<213> 人工序列
<400> 10
ATAGGGAGAAGGATGTTGAAGAAG 24
<210> 11
<211> 300
<212> DNA
<213> Babesia2035449-2035749
<400> 11
GCCGTTTATACATACATTAAACAATTTAAATATCGAATAGCTAAGATACACAAAAAATC
GTCATATAGCATTAAATGCTACTTGTAATGCCATAAATGTTGATTATGTATTTAATATA
TACGTAATAAATCAGATTGAAAGGGATTAAATATGTGATTAGCATTTTAATATATTATT
GCGACTAATAATGTAACCAATGATATATATGTTCATTTTATTTTATATAGACATACATA
TTTGCTCTATGTGGAATAATAACTATTATTGGCTAAATTAAATTCAAACAAATGTTTTA
GCCAC
<210> 12
<211> 24
<212> DNA
<213> Leishmania236331-236761
<400> 12
GAGTACCAGCGCACCCTGCCTGTGCGCACCGTCGTGCCGGAGCCGCCGAACACGCTGCG
CGACTACACTCGGCATCAGGTGTGGCGCATCGGCCGCAGTTGCTTCCTCTTTTTCTTCT
TCCTGTTCACGTGGCCGGTCTGGCTCACGCTGCACCTACTCCTGAAGCTGCTCGATGTC
CATCAGCGGCGGGTGCGGCTGCGGTACCACCGCAGTTGGGCGCACGCGAGCGTGCCGCA
CATCCGCCCTAACGTGCACAAGCCGTCGAGTGAGTTCCCGCTGAGCGCGTGGCACCTAC
GCTGCGAAGACGGGCGCCAGCGGTGGTACTTTGGTGAGCCACTGCGCGAAGGGGAGGCA
TGCAATGAGCTCGCCTTGGCGCAGCTAAGTGGCCTGCACACCGTCAACGACTATCATGA
GCTGCTCCGCCAGCGCG
<210> 13
<211> 643
<212> DNA
<213> Plasmodium143443-144086
<400> 13
TTTAACGAGGATAGTGAAATTCATGAAGTGAAAGATGATGGAGAAATAAAAATCCAGAA
AAAAATCTCGCTAGTCAATTTAAGAGACATAAAAATAGCAACAAAGGAAACCCCATTCA
CAGCAGATTTGATAGGTATAGTTAAGCACATTGGCACCATAAGCAATTTGAAGACAAAG
CAAGGAAATGACATAACGAAGCAAAATATAATCATCGTTGACGACACGAAACACTCCTT
CGAAATAGCCTTCTGGGACAGCAACGTAAATTTAATTAAAGATGAAATAAAGGAAAACG
AAATTTATGTATTTACCAACATAAGTATACGAAATTGGAATGACATGAAAAATGGAACC
TTTGGCGTGACCAGTTCGATCGAGAAAATAGAAAACTTAAATGAAGAATTAAAAGCTAA
ATGTACAATGATATCAGAATGGTATAATACAAATGGAAAATATGAGCAATTCACCAACA
TGAGGAATATCTTATCCAATGATGTCAGCCAAATTCCTGACAAGCATTACGCCTTAAGT
GACGTAAATGATGTTTTAGCAAAAATTTCTGGTACCTACACACTTGTAGGAAGAATTAA
AAGAATATATTGGAAGAGTAAAGAGAATGAGCATCGATTTTACTATCCTGCTT
<210> 14
<211> 743
<212> DNA
<213> Toxoplasma686613-687356
<400> 14
TCATGTATGTACTTGGCTCTCTCTGTCGATATACACATGAGTATTTGTAGGCTCAACTG
TGGCTTTCTCTCTTGAGCTGTGTCGCATGGCGATCTGGCTTTCGTATCTGCATTAGCAG
GCACAATGCGTTGTCTTCCAAGAGTGATGTCTTGATAGTTCTCTTTCAGTTCCTTTTCC
GCTTTGCTGGTCCGACAAGGCATAGGGGGGGGGGTCGCCCTGAAGCCTGTGAGAGCGCG
TGTATTCTGGGTCACTTCTGCGTCGATCTTCTTGCGTCTGCATGTCCTGGACTTCAGAA
GCTCTCTTCGCGTTCGAAACGTCTTTTCCACCAATTCTTTCCTTTCCATTCTGTCTTCT
CTCTTGCGCCTCCAGTTCGCAGAAGACCCGGTTAGAACCTGGTCGCACGCAGATGGCGG
CGGCGATCTCGCGGAAGAAGTTCTTGAGAGAAGCAACGTCGGCATGGGGACATGCATCT
TGAACAAGTCAGAGTTGTCTCCCTCGAAGCCCCGCGTTTCCGGCACCACGCATGCAGCC
GTTGCGCTTTTTTCTTCCGTCGAACCTTTTCAACTCCCCTGTCTCCACCCGCGGCGCCC
TGCGCTCTCAGTCTCGCTCTGGTTGTGTCTCCCAATTTCTGTCGTTGCTCATACGCTTG
CGTCCTGAAAAAGAGCCAGATCTTTGCTATAGAAGCACATAAATATGTACATAAAACTA
CATAGATATGCACATGTATTCTTGTGCGAGTTATG
<210> 15
<211> 824
<212> DNA
<213> Trypanosoma1561994-1562818
<400> 15
GGGAAGAAACCGCATCATTTCCTCCGTCGAGGAATTTTCTTTTCCCATAGGGATTTGAA
CCTTCTATTGGACGTGTACGAGTCCGGTCAACCTTTTTATCTCTACACTGGCAGAGGTC
CAAGCAGCGAGTCCATGCATATGGGTCATTTGATTCCTTTCATGTTTACTAAGTGGCTT
CAGGACTCCTTTAGGGTACCCCTCGTGATACAAATGACAGATGATGAGAAGTTTTATTT
CCGTAATATCCCAATGGAACAAGTCGAGGCCATGACAACAGAAAATATAAAAGATATTA
TCGCTATGGGGTTCGACCCCGAGTTAACTTTCATTTTCCGTGACTTTGATTATATGGGA
TGCATGTACCGTACCGTTGCCAAGATTGAGAGGGCCTTCACTGCGAGTCAAGTACGTGG
TTGCTTTGGTTTTGCAATGGAAGACAACTGTGGCCGCTGGATGTTTCCTGCTATACAGG
CTGCACCATCTTTCTCAGCAGCTTTTCCTCATATCTTTCCACCATCGATGGGAAATGTG
TTTTGTCTCATTCCACAGGCCATTGATCAGGATCCATATTTCCGCCTGACACGTGACAT
CGCCCCACGTCTCGGTTATTTAAAGCCGGCGGTGATTCATTCCAAGTTCTTCCCCGGAT
TAAGTGGCCCAAAAGGAAAGATGAGTTCATCTTCCGGTACTGCAGTGCTGTTGACGGAT
ACAGAAAAGATGGTGAAGGATAAAATTAACAAACATGCCTTTAGCGGCGGTGGGGCAAC
CAAGCAAGAGCACTTTCTTCTTGGCGCCAACGTGGAGGTGGATGTTCCTATACAGTG

Claims (5)

1.一种利用核苷酸DNA片段作为分子标记鉴别五种重要病原体的方法，其特征在于，基于PCR扩增和DNA测序技术获得五种重要病原体的核苷酸序列，用生物信息学软件进行blast比对，根据结果来确定是否感染上述病原体。
2.此方法主要包括以下步骤：
(1)采集病人血清，逐个提取基因组DNA；
(2)合成上述核苷酸DNA片段的特异性扩增引物（对）；
(3)在适当条件下对每条个体的每个基因分别进行PCR扩增；
(4)对PCR产物进行纯化并测序；
(5)用生物信息学软件将测序结果进行blast比对和分析。
3.根据权利要求1所述的方法，其特征在于，步骤(2)所述的引物为：
1F CATAATCAACTACCATATCCCAAAC
1R CGGCTCCTGATACAATAATAAATAC
2F CCCTTGGTTTTATTATATCTTCG
2R AATAATGATAAACCAAACGATAAAC
3F TATTTACCTTCATGCAGAGCGTTGG
3R CCTAGCCTTGGCATCAATCGAATAC
4F TGAAGATTTGCTCGATTAAGATGCC
4R ACGGCTGCCATCACCGACAT
5F TTCTGGCGACTATCCTTAGGTAA
5R ATAGGGAGAAGGATGTTGAAGAAG
如权利要求1所述的方法，其特征在于，步骤(3)所述的PCR扩增程序为：① 94°C预变性10min；
② 94°C变性30s；
③ 退火30s （1和5的退火温度为57°C，2为55°C， 3和4则为65°C）
④ 72°C延伸90s
⑤ 步骤②—④重复35个循环后，72°C延伸10min。
4.根据权利要求1所述的方法，其特征在于，步骤(5)将测序所得的核苷酸序列对已知病原菌数据库进行blast比对，通过与标准病原结果图进行比较判断样本是否感染5种病原菌。
5.该方法在对鼠疫，霍乱，黑热病，疟疾和昏睡病的检测中的应用。
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