海水养殖动物部分病原检测基因芯片的初步设计
摘要
病害严重威胁海水养殖业的可持续发展,如何寻求一种有效的途径来缓解病害给养殖业带来的危害与损失,一直是人们所关注的问题。现有的病原检测技术局限性大、效率低、系统性差,急需一种海水养殖动物疾病高通量检测手段进行策略上的改进,打破技术瓶颈。随着水产动物病原基础信息数据的日渐丰富,水产动物疾病诊断技术的发展趋势不可避免地向着高通量技术方向跨越。
本论文根据Lightner蓝皮书、国际兽疫局(OIE)水生动物疾病名录、亚太水产养殖中心网络(NACA)水生动物疾病名录等,选择海水养殖鲽形目鱼类、鲈形目鱼类、对虾类和双壳贝类等宿主的主要病毒、真菌、原生动物等几十种病原微生物作为研究对象,包括14类鱼病毒、14类对虾病毒、鱼立克次氏体、3类真菌以及8类原生动物。针对这些研究对象,在Genbank中共检索收集了27969条相关基因序列,包括海水养殖动物的病原序列26861条,质控系统所需序列1108条。
把在Genbank中分别检索到病原微生物的核酸序列,进行BLAST搜索比对,Omiga软件Alignment多重比对,找出各种病原核酸序列的保守区和特异区序列,将筛选确定的各条序列按照宿主的不同,分3类同时导入到微阵列专业的引物和探针设计软件AlleleID 6.0中,根据引物和探针设计原则,在同一个任务下批量设计出退火温度等各项参数十分相似,扩增片段长度为200~500bp的引物。再根据扩增片段序列,设计长度为57bp的寡核苷酸探针。将设计好的探针再全部导入Omiga软件进行比对,确定探针之间最多不超过四个连续碱基相同,而且位置处于引物扩增区,与引物没有结合位点,然后交由上海生工合成。
本研究中针对选择的研究对象,共设计了检测鱼类病原包括备选引物和探针在内的31对引物和31条相对应的寡核苷酸探针,检测对虾类病原的37对引物和37条相对应的寡核苷酸探针,检测双壳贝类病原的8对引物和8条相对应的寡核苷酸探针。另外还有以宿主序列为模板设计的引物和探针;用来组成质控体系中全程监控的阳性内对照,以及由非同源性宿主序列为模板设计的引物和探针,用来组成质控体系中的阳性外对照。
根据引物和探针的设计结果,按照病原微生物感染宿主的不同,采用基因芯片的原理,将设计合成后的探针按照预先设计的点阵分布用手动点样仪在尼龙膜上点样,经紫外交联后固定在膜上制备成寡核苷酸膜芯片。病原检测膜芯片分为鱼类病原检测芯片、对虾类病原检测芯片和双壳贝类病原检测芯片。
根据实验室保存和课题合作单位提供的鱼类和对虾类的阳性病料,本文先针对部分引物进行了特异性验证实验。在鱼类芯片中,所设计的9对特异性引物,分别以含有EHNV、LCDV、TRBIV、LYCIV、ISKNV、RGNNV、IHNV、VHSV、IPNV和YAV的DNA或cDNA为模板,均能特异性的PCR扩增出与实验设计相符的产物;在对虾类芯片中,所设计的8对特异性引物,分别以含有WSSV、IHHNV和TSV的DNA或cDNA为模板,均能特异性的PCR扩增出与实验设计相符的产物;同时验证了质控体系中的阳性内对照及阳性外对照引物对。之后,采用同步PCR方法和两步法RT-PCR方法,用基因特异性引物加入地高辛标记的dUTP扩增并标记各个目标序列,标记后的PCR产物用1%的琼脂糖凝胶电泳检查后测定标记产量,并测定标记浓度为10 ng/μl—40 ng/μl之间。将DIG标记的扩增产物分别与鱼类病原检测膜芯片和对虾类病原检测膜芯片进行杂交实验,杂交结束后的反应信号用NBT-BCIP液显色后进行分析,验证了鱼类病原检测膜芯片上11条寡核苷酸探针的杂交特异性,包括3条质控系统的探针和8条分别检测EHNV、LCDV、TRBIV、LYCIV、ISKNV、RGNNV、IHNV、VHSV、IPNV和YAV的探针;对虾类病原检测膜芯片上12条寡核苷酸探针,包括4条质控系统的探针和8条分别检测WSSV、IHHNV、TSV病原的探针。之后,采用膜芯片检测人工合成的ISAV和MBV基因以及从河北唐山取样的发病对虾样品,又成功验证了鱼类病原检测芯片上ISAV探针和对虾病原检测芯片上MBV和HPV探针。
本文最后对基因芯片技术应用于海水养殖动物病原检测领域进行了分析和展望,海水养殖动物病原检测芯片必将能快速的、高通量的检测和诊断海水养殖动物疾病,系统的、全面的反映海水养殖动物病害发生的原因和状况,确定有效的预警和防治措施,保障海水养殖业的健康持续发展。
Disease is a serious threat to the sustainable development of mariculture industry, so how to find an effective way to alleviate the disease have been the people's concerns. The existing detection technology of pathogen has great limitation of efficiency and systematic. It is in urgent need of some high-throughput means to improve the strategy in order to break the technical bottleneck. With the growing wealth of basis data of fish pathogen, the diagnostic technology of aquatic animal disease is inevitable trend toward high-throughput techniques.
According to the Blue Lightner, the Office International Des Epizooties (OIE) and the Network of Aquaculture Centers in Asia-Pacific (NACA), and other aquatic animals diseases directory, select dozens of pathogenic micro-organisms such as viruses, fungi and protozoa as research subjects which pick mariculture Pleuronectiformes fish, Perciformes fish, shrimp and Shellfish as their main host, including 14 species of fish virus, 14 species of shrimp virus, piscirickettsia, 3 species of fungi and 8 species of protozoa. There are 27969 related gene sequences were collected in GenBank, including aquaculture animal pathogens sequence 26861, quality control system sequence 1108.
First, there are pathogenic microorganisms were retrieved nucleic acid sequence in GenBank carryed out BLAST search against, Omiga Multiple Alignment software than to find a variety of pathogen nucleic acid sequences conserved region and sequence specific respectively. Then,the screening of various different sequences in accordance with the host is divided into 3 categories at the same time import AlleleID 6.0 software, in accordance with primers and probe design principles, in the same project to design the parameters such as annealing temperature is very similar to amplified fragment length of 200 ~ 500 bp primers. Then according to amplified fragment sequence, to design length of 57 bp oligonucleotide probe. Finally, put the design of the probes into Omiga software again, to determine between the probes up to a maximum of more than four consecutive identical base pairs and the location of primers in the amplified region with the primer binding sites do not. Sequence information submitted to the Shanghai Sangon Biological Engineering Technology Services Co., Ltd. to synthesis of primers and probes
In this study, 31 pairs of primers and 31 corresponding oligonucleotide probes were designed in detect fish pathogen; 37 pairs of primers and 37 corresponding oligonucleotide probes were designed in detect shrimp pathogen; 8 pairs of primers and 8 corresponding oligonucleotide probes were designed in detect Bivalve molluscs pathogen. There is also the host sequence as a template to design primers and probes, composed of quality control system used to monitor the whole process of the positive internal control; as well as by the unusual host-derived sequence as a template design primers and probes used to group quality control system into a positive external control.
According to primers and probes design results, in accordance with the pathogenic microorganism infections in different hosts, using the principle of gene chip, the synthesized probes in accordance with the pre-distribution, was pointed into positively charged nylon membrane by Manual Glass Slide Arrayer Replicator, after UV cross-linked the oligonucleotide membrane-array was constructed.
According to laboratory and subject project cooperation unit provided infected fish and shrimp materials, some of the primers specific validation experiments have been developed in this paper. In fish chips, the specific PCR product that consistent with the experimental design were amplified by nine pairs of primers against EHNV, LCDV, TRBIV, LYCIV, ISKNV, RGNNV, IHNV, VHSV, IPNV and YAV DNA or cDNA as templates respectively. Also in shrimp chips, the specific PCR product that consistent with the experimental design were amplified by nine pairs of primers against WSSV, IHHNV, TSV DNA or cDNA as templates respectively. At the same time the inner and outer positive control in quality control system were also verified. Through the synchronization PCR and two-step RT-PCR method, Digoxigenin-labeled target genes are amplified by gene specific primers and DIG-labeled dUTP, then the amplified fragments are hybridized to the membrane-array, and measured the concentration is 10 ng/μl~40 ng/μl. In DIG labeled amplification products and membrane array hybridization experiments, verify the detection of fish pathogens membrane chip 11 oligonucleotide probe hybridization specificity, including 3 oligonucleotide probe in quality control system, 8 oligonucleotide probe were used to detected EHNV, LCDV, TRBIV, LYCIV, ISKNV, RGNNV, IHNV , VHSV, IPNV and YAV pathogen respectively shrimp pathogen detection chip 12 oligonucleotide probes, including four oligonucleotide probe in quality control system, 8 oligonucleotide probe were used to detect WSSV, IHHNV, TSV pathogen respectively. Then,the membrane-chip detected ISAV and MBV synthetic gene, as well as sampling from Hebei Tangshan infected shrimp samples, has also successfully tested fish pathogen detection chip ISAV probe and shrimp pathogen detection chip MBV and HPV probe.
Finally, this article analyzes the prospects of gene chip technology in the field of aquaculture animal pathogen detection,aquaculture animal pathogen detection chips will be able to fast, high-throughput detection and diagnosis of animal diseases of mariculture, systematic and comprehensive reflection of mariculture cause animal diseases and conditions, to identify effective early-warning and prevention measures to protect the health of marine aquaculture sustainable development.
本论文根据Lightner蓝皮书、国际兽疫局(OIE)水生动物疾病名录、亚太水产养殖中心网络(NACA)水生动物疾病名录等,选择海水养殖鲽形目鱼类、鲈形目鱼类、对虾类和双壳贝类等宿主的主要病毒、真菌、原生动物等几十种病原微生物作为研究对象,包括14类鱼病毒、14类对虾病毒、鱼立克次氏体、3类真菌以及8类原生动物。针对这些研究对象,在Genbank中共检索收集了27969条相关基因序列,包括海水养殖动物的病原序列26861条,质控系统所需序列1108条。
把在Genbank中分别检索到病原微生物的核酸序列,进行BLAST搜索比对,Omiga软件Alignment多重比对,找出各种病原核酸序列的保守区和特异区序列,将筛选确定的各条序列按照宿主的不同,分3类同时导入到微阵列专业的引物和探针设计软件AlleleID 6.0中,根据引物和探针设计原则,在同一个任务下批量设计出退火温度等各项参数十分相似,扩增片段长度为200~500bp的引物。再根据扩增片段序列,设计长度为57bp的寡核苷酸探针。将设计好的探针再全部导入Omiga软件进行比对,确定探针之间最多不超过四个连续碱基相同,而且位置处于引物扩增区,与引物没有结合位点,然后交由上海生工合成。
本研究中针对选择的研究对象,共设计了检测鱼类病原包括备选引物和探针在内的31对引物和31条相对应的寡核苷酸探针,检测对虾类病原的37对引物和37条相对应的寡核苷酸探针,检测双壳贝类病原的8对引物和8条相对应的寡核苷酸探针。另外还有以宿主序列为模板设计的引物和探针;用来组成质控体系中全程监控的阳性内对照,以及由非同源性宿主序列为模板设计的引物和探针,用来组成质控体系中的阳性外对照。
根据引物和探针的设计结果,按照病原微生物感染宿主的不同,采用基因芯片的原理,将设计合成后的探针按照预先设计的点阵分布用手动点样仪在尼龙膜上点样,经紫外交联后固定在膜上制备成寡核苷酸膜芯片。病原检测膜芯片分为鱼类病原检测芯片、对虾类病原检测芯片和双壳贝类病原检测芯片。
根据实验室保存和课题合作单位提供的鱼类和对虾类的阳性病料,本文先针对部分引物进行了特异性验证实验。在鱼类芯片中,所设计的9对特异性引物,分别以含有EHNV、LCDV、TRBIV、LYCIV、ISKNV、RGNNV、IHNV、VHSV、IPNV和YAV的DNA或cDNA为模板,均能特异性的PCR扩增出与实验设计相符的产物;在对虾类芯片中,所设计的8对特异性引物,分别以含有WSSV、IHHNV和TSV的DNA或cDNA为模板,均能特异性的PCR扩增出与实验设计相符的产物;同时验证了质控体系中的阳性内对照及阳性外对照引物对。之后,采用同步PCR方法和两步法RT-PCR方法,用基因特异性引物加入地高辛标记的dUTP扩增并标记各个目标序列,标记后的PCR产物用1%的琼脂糖凝胶电泳检查后测定标记产量,并测定标记浓度为10 ng/μl—40 ng/μl之间。将DIG标记的扩增产物分别与鱼类病原检测膜芯片和对虾类病原检测膜芯片进行杂交实验,杂交结束后的反应信号用NBT-BCIP液显色后进行分析,验证了鱼类病原检测膜芯片上11条寡核苷酸探针的杂交特异性,包括3条质控系统的探针和8条分别检测EHNV、LCDV、TRBIV、LYCIV、ISKNV、RGNNV、IHNV、VHSV、IPNV和YAV的探针;对虾类病原检测膜芯片上12条寡核苷酸探针,包括4条质控系统的探针和8条分别检测WSSV、IHHNV、TSV病原的探针。之后,采用膜芯片检测人工合成的ISAV和MBV基因以及从河北唐山取样的发病对虾样品,又成功验证了鱼类病原检测芯片上ISAV探针和对虾病原检测芯片上MBV和HPV探针。
本文最后对基因芯片技术应用于海水养殖动物病原检测领域进行了分析和展望,海水养殖动物病原检测芯片必将能快速的、高通量的检测和诊断海水养殖动物疾病,系统的、全面的反映海水养殖动物病害发生的原因和状况,确定有效的预警和防治措施,保障海水养殖业的健康持续发展。
Disease is a serious threat to the sustainable development of mariculture industry, so how to find an effective way to alleviate the disease have been the people's concerns. The existing detection technology of pathogen has great limitation of efficiency and systematic. It is in urgent need of some high-throughput means to improve the strategy in order to break the technical bottleneck. With the growing wealth of basis data of fish pathogen, the diagnostic technology of aquatic animal disease is inevitable trend toward high-throughput techniques.
According to the Blue Lightner, the Office International Des Epizooties (OIE) and the Network of Aquaculture Centers in Asia-Pacific (NACA), and other aquatic animals diseases directory, select dozens of pathogenic micro-organisms such as viruses, fungi and protozoa as research subjects which pick mariculture Pleuronectiformes fish, Perciformes fish, shrimp and Shellfish as their main host, including 14 species of fish virus, 14 species of shrimp virus, piscirickettsia, 3 species of fungi and 8 species of protozoa. There are 27969 related gene sequences were collected in GenBank, including aquaculture animal pathogens sequence 26861, quality control system sequence 1108.
First, there are pathogenic microorganisms were retrieved nucleic acid sequence in GenBank carryed out BLAST search against, Omiga Multiple Alignment software than to find a variety of pathogen nucleic acid sequences conserved region and sequence specific respectively. Then,the screening of various different sequences in accordance with the host is divided into 3 categories at the same time import AlleleID 6.0 software, in accordance with primers and probe design principles, in the same project to design the parameters such as annealing temperature is very similar to amplified fragment length of 200 ~ 500 bp primers. Then according to amplified fragment sequence, to design length of 57 bp oligonucleotide probe. Finally, put the design of the probes into Omiga software again, to determine between the probes up to a maximum of more than four consecutive identical base pairs and the location of primers in the amplified region with the primer binding sites do not. Sequence information submitted to the Shanghai Sangon Biological Engineering Technology Services Co., Ltd. to synthesis of primers and probes
In this study, 31 pairs of primers and 31 corresponding oligonucleotide probes were designed in detect fish pathogen; 37 pairs of primers and 37 corresponding oligonucleotide probes were designed in detect shrimp pathogen; 8 pairs of primers and 8 corresponding oligonucleotide probes were designed in detect Bivalve molluscs pathogen. There is also the host sequence as a template to design primers and probes, composed of quality control system used to monitor the whole process of the positive internal control; as well as by the unusual host-derived sequence as a template design primers and probes used to group quality control system into a positive external control.
According to primers and probes design results, in accordance with the pathogenic microorganism infections in different hosts, using the principle of gene chip, the synthesized probes in accordance with the pre-distribution, was pointed into positively charged nylon membrane by Manual Glass Slide Arrayer Replicator, after UV cross-linked the oligonucleotide membrane-array was constructed.
According to laboratory and subject project cooperation unit provided infected fish and shrimp materials, some of the primers specific validation experiments have been developed in this paper. In fish chips, the specific PCR product that consistent with the experimental design were amplified by nine pairs of primers against EHNV, LCDV, TRBIV, LYCIV, ISKNV, RGNNV, IHNV, VHSV, IPNV and YAV DNA or cDNA as templates respectively. Also in shrimp chips, the specific PCR product that consistent with the experimental design were amplified by nine pairs of primers against WSSV, IHHNV, TSV DNA or cDNA as templates respectively. At the same time the inner and outer positive control in quality control system were also verified. Through the synchronization PCR and two-step RT-PCR method, Digoxigenin-labeled target genes are amplified by gene specific primers and DIG-labeled dUTP, then the amplified fragments are hybridized to the membrane-array, and measured the concentration is 10 ng/μl~40 ng/μl. In DIG labeled amplification products and membrane array hybridization experiments, verify the detection of fish pathogens membrane chip 11 oligonucleotide probe hybridization specificity, including 3 oligonucleotide probe in quality control system, 8 oligonucleotide probe were used to detected EHNV, LCDV, TRBIV, LYCIV, ISKNV, RGNNV, IHNV , VHSV, IPNV and YAV pathogen respectively shrimp pathogen detection chip 12 oligonucleotide probes, including four oligonucleotide probe in quality control system, 8 oligonucleotide probe were used to detect WSSV, IHHNV, TSV pathogen respectively. Then,the membrane-chip detected ISAV and MBV synthetic gene, as well as sampling from Hebei Tangshan infected shrimp samples, has also successfully tested fish pathogen detection chip ISAV probe and shrimp pathogen detection chip MBV and HPV probe.
Finally, this article analyzes the prospects of gene chip technology in the field of aquaculture animal pathogen detection,aquaculture animal pathogen detection chips will be able to fast, high-throughput detection and diagnosis of animal diseases of mariculture, systematic and comprehensive reflection of mariculture cause animal diseases and conditions, to identify effective early-warning and prevention measures to protect the health of marine aquaculture sustainable development.
引文
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[29] Kwok A.Y., Wilson J.T., Coulthart M, Phylogenetic study and identification of human pathogenic Vibrio species based on partial hsp60 gene sequences. Can J Microbiol, 2002, 48(10):903-910.
[30]王馗,史成银,黄倢.用于PCR检测对虾皮下及造血组织坏死杆状病毒的一种快速提取DNA方法.海洋水产研究,2000,21(1):8-12.
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[34]陶保华,胡超群,吴蔚.斑节对虾弧菌病的病原生物学研究.热带海洋学报,2001,20(2):80-86.
[35]李天道,于佳,俞开康.四种弧菌对中国对虾的致病性研究.海洋湖沼通报,1998,1:57-64.
[36] Van Hulten M.C., Witteveldt J., Peter S., et al. The white spot syndrome virus DNA genome sequence. Virology, 2001, 286(1): 7-22.
[37]杨卫帆,易小平.多重PCR检测对虾白斑综合症病毒和传染性皮下组织坏死病毒.水产科学,2006,25(12):649-651.
[38]徐丽美,杨丰.2005.利用多重PCR同时检测WSSV和MBV两种对虾病毒的研究.高技术通讯,15(5):101-104.
[39] Vanderzant C., Nickelson R., Parlier J. C. Isolation of vibrio parahaemolyticus gulf coast shrimp. J. Milk Food Technol., 1970, 33(1): 161-162.
[40] Yang B., Song X.-L., Huang J., Shi C.-Y., Liu Q.-H., Liu L. A single-step multiplex PCR for simultaneous detection of white spot syndrome virus (WSSV) and infectious hypodermal and haematopoietic necrosis virus (IHHNV) in penaeid shrimp. J. Fish Diseases, 2006, 29(5): 301-305.
[41] Yang F., He J., Lin X., et al. Complete genome sequence of the shrimp white spot bacilliform virus. J. Virol., 2001, 75(23): 11811-11820.
[42] H?se C.C., Mekalanos J.J. Effects of changes in membrane sodium flux on virulence gene expression in vibrio cholerae. Microbiology, 1999, 96(6): 3183-3187.
[43] Rodriguez J., Bayot B., Amano Y., et al. White spot syndrome virus infection in cultured Penaeus vannamei (Boone) in Ecuador with emphasis on histopathology and ultrastructure. J Fish Dis., 2003, 26(8): 439-450.
[44] Burton J E, Oshota O J, North E, Hudson M J, Polyanskaya N, Brehm J, Lloyd G, Silman N J. Development of a multipathogen oligonucleotide microarray for detection of Bacillus anthracis. Molecular and Cellular Probes, 2005, (19): 349-357.
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