CN103093120A - Design method of probe for high throughput testing of vertebrate pathogen gene chips - Google Patents

Design method of probe for high throughput testing of vertebrate pathogen gene chips Download PDF

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CN103093120A
CN103093120A CN 201110348756 CN201110348756A CN103093120A CN 103093120 A CN103093120 A CN 103093120A CN 201110348756 CN201110348756 CN 201110348756 CN 201110348756 A CN201110348756 A CN 201110348756A CN 103093120 A CN103093120 A CN 103093120A
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probe
sequence
species
design
protein
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CN 201110348756
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张鑫磊
蒋小云
肖琛
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北京健数通生物计算技术有限公司
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Abstract

The invention relates to a design method of a probe for high throughput testing of vertebrate pathogen gene chips, in particular to the design method of the probe used for high throughput testing of pathogens, such as viruses, bacteria and fungi, with vertebrates as hosts. The design method includes: (1) a probe design method with a ribosomal robonucleic acid ( rRNA) as the template is adopted for detection objects to be the three pathogens: bacteria, fungi and protozoa; and (2) a robe design method with a structural protein coding sequence as the template is adopted for the detection objects to be viruses.

Description

高通量检测脊椎动物病原体基因芯片的探针设计方法 Probe design high throughput gene detecting chip vertebrate pathogens

技术领域 FIELD

[0001] 本发明涉及生物芯片探针的一种设计方法。 [0001] The present invention relates to a method of one design of biochip probe. 尤其是基于此方法设计得到的探针用来对以脊椎动物为宿主的病毒、细菌、真菌、原生动物进行高通量检测,本发明对不同类型的病原体分别采用了有针对性的设计流程。 Probes based on the particular method used to design obtained as a vertebrate host to a virus, bacteria, fungi, protozoa high-throughput detection, the present invention is different types of pathogens were used to design a targeted process.

背景技术 Background technique

[0002] 传染病是由各种病原体引起的能在人与人、动物与动物或人与动物之间相互传播的一类疾病。 [0002] Infectious diseases are a class of diseases can be mutually spread from person to person, animal or animals and human beings and animals caused by various pathogens. 每种传染病都有其特异的病原体,包括病毒、细菌、真菌、原虫、螺旋体、立克茨体等。 Each has its own specific infectious pathogens, including viruses, bacteria, fungi, protozoa, spirochetes, Li Klotz the like. 传染病与其他种类疾病相比,具有发病强度高、传播速度快、波及范围广、地域性和季节性强等特点,传染病产生的危害性极大,不但患者死亡率高,而且容易导致社会恐慌心理,产生的次级危害往往更大,直接影响社会的经济活动和人的正常生活秩序。 Compared with other types of infectious diseases with high incidence of strength, spread fast, wide geographical coverage, strong regional and seasonal characteristics, generated enormous dangers of infectious diseases, not only high mortality, but also easily lead to social panic, secondary hazards resulting often more direct impact on people's social and economic activities of normal life order.

[0003] 虽然传染病理论上分为人与人、动物与动物或人与动物专有传染病,但是许多传染性疾病,甚至包括流行病,都起于人畜共通的特性,要区分哪些疾病从感染动物逐步演化成可以感染人类并不简单,但有证据显示麻疹、天花、流行性感冒、白喉等皆是如此。 [0003] Although theoretically divided into infectious diseases from person to person, animal to animal or human and animal infectious diseases proprietary, but many infectious diseases, even epidemic, have played in zoonotic characteristics, to distinguish which disease from infection animals evolved into can infect humans is not simple, but there is evidence that measles, smallpox, influenza, diphtheria mused the case. 而艾滋病、感冒和结核也都来自人类以外的物种。 And AIDS, colds and tuberculosis are also from species other than humans. 人畜通病在国际间引起密切关注,因为它们通常是过去未被发现的疾病,或是毒力在演化过程中增强,或偶然传入不具对抗该疾病之免疫力的族群或物种。 Drinking a common problem closely watched internationally, because they are usually the last undiscovered disease, or increased virulence in the process of evolution, or accidentally introduced species or population does not have immunity against the diseases of. 因此,对以脊椎动物为宿主的病原体进行系统地监测,是有效进行传染病防控的一个必要环节。 Therefore, in order for the host vertebrate pathogens monitoring system it is a necessary step for effective prevention and control of infectious diseases.

[0004] 在传染病爆发过程中,人类由以前被动承受,到治疗控制,再到提前预防控制,积累了大量和传染病做斗争的技术经验。 [0004] In the course of the outbreak of infectious diseases, human bear from the previous passive, to control treatment, to advance prevention and control, and accumulated a great deal of technical experience to fight infectious diseases. 特别是随着现代医学和分子生物学技术发展,人类已经建立起了多种具体传染病检测方法,I)微生物培养法;2)血清学标记物检测法;3)血液或分泌物中所含病毒和病原相关蛋白质检测,其中包括ELISA、胶体金等方法;4)通过传染源核酸序列,进行特异性的荧光定量PCR检测法;5)快速发展的生物芯片微阵列高通量方法。 Especially with the development of modern medical technology and molecular biology, humans have established a variety of detection methods specific diseases, I) a microorganism culture; 2) serological detection marker; 3) contained in the blood or secretions virus and pathogen-related protein detection, including ELISA, colloidal gold or the like; 4), by infection nucleic acid sequence, to specifically quantitative PCR assays; 5) biochip microarrays for high-throughput method of rapid development.

[0005] 微生物培养法由于直观方便等特点,还是最主要的传染病诊断工具,但有些传染原如病毒和钩端螺旋体无法进行人工培养,就只能借助其他诊断工具;血清学标记物检测法,也是通过病原体感染机体后产生的特异性抗体进行检测,通行的抗体检测由于存在“血清学窗口期”,只能在感染2-4周后才能确诊,而且该方法还需要和微生物培养法相互引证;基于血液或分泌物的病原蛋白质检测,如ELISA和胶体金法也是对血清学检测方法的改进,也同样存在以上弊端;新近涌现的针对病原体的RNA或DNA的荧光定量PCR检测方法,有灵敏性高,准确率高,能够有效缩短“病原窗口期”等特点。 [0005] microbial culture and convenient since visualized, or the most important tool for diagnosis of infectious diseases, but some infectious agents such as viruses and Leptospira can not be cultured, it is only by means of other diagnostic tools; serological marker assay , is detected by a specific antibody produced after infection the pathogen organism, due to passage of antibody detection "serological window period", it can only be confirmed 2-4 weeks after infection, but this method also requires another and microbial culture Citation; pathogen protein detection based on blood or secretions, such as ELISA and colloidal gold are improved serological detection methods, the above drawbacks also exist; newly emerging method for quantitative PCR RNA or DNA of the pathogen, there high sensitivity, high accuracy, can effectively shorten the "window period pathogen" and so on. 但该检测方法也只能针对已知病原体设计特异的PCR引物和探针,不能实现高通量检验检测,不能满足新发与突发性传染病的快速、准确、灵敏的诊断需求,是重大传染性疾病的防疫防控与及时救治的主要技术瓶颈之一。 However, the detection method can only design specific PCR primers and probes for known pathogens, high throughput inspection can not meet the emerging infectious diseases and sudden rapid, accurate, sensitive diagnostic needs, is a major one of the main technical bottlenecks epidemic prevention and control of infectious diseases and timely treatment of.

[0006] 生物芯片方法,在考虑了传统和现有传染病检测方法的局限性基础之上,结合现代分子生物学高通量技术优势,而建立起来的传染病病原体诊断检测方法。 [0006] The bio-chip method, in consideration of the conventional method for detecting infectious diseases and limitations of the prior basis, combined with modern high-throughput molecular biology technology, and set up diagnostic test pathogens infectious diseases. 该方法主要技术优点包括:1)高通量。 The main technical advantages of this method include: 1) high throughput. 一张芯片上的一个点阵可以对一份样本同时分析成千上万种的病原体,而一张芯片上有可以同时分析数十个临床样本;2)快速、准确和灵敏。 A dot on a chip can simultaneously analyze thousands of samples of a pathogen, but there are dozens of clinical samples can be analyzed simultaneously on a single chip; 2) rapid, accurate and sensitive. 单次检测I天即可完成,加之高通量特异性,检测效力明显优于现有的其他方法;由于检测过程中采用全封闭的荧光自动化检测系统,集合特异性探针,检测准确度高、灵敏度好;3)可检测未知病原体。 Single detected I days to complete, combined with high-throughput specificity, efficacy is superior to other conventional detection methods; since the detection process is fully enclosed automated fluorescence detection system, a set of specific probes, detection of high accuracy , good sensitivity; 3) can detect unknown pathogens. 现有病原体检测方法,只能对已知病原体进行确认,对于未知病原体检测则无能为力,例如荧光定量PCR方法,有很多技术优势,但前提必须知道被检病原体核酸序列,否则将无法检测。 Existing methods for pathogen detection, only confirmation of known pathogens, for detecting unknown pathogens is powerless, for example, quantitative PCR methods, there are many technical advantages, but only need to know the subject nucleic acid sequences of pathogens, otherwise it will not be detected. 而生物芯片检测系统,由于探针设计本身就具有兼容性,检测序列发生突变将不会影响杂交检测。 And biochip detection system, since the design of the probe itself has compatibility, the detection sequence mutation will not affect the hybridization. 大部分病原体新品种其实都是已知病原体在药物和环境压力下的突变体,序列具有很高同源性。 Most pathogens of new varieties are actually known pathogens mutants in drug and ambient pressure, having a high sequence homology.

[0007]由于生物芯片检测技术本身技术优点和临床应用的潜在价值,使得国内外众多科技专家专注于生物芯片检测技术在传染病学中的研究。 [0007] Due to the potential value of their technical merit and clinical application of biochip technology, making a number of domestic and international technology experts biochip technology in the epidemiology of focus on. 例如,美国加州大学旧金山分校DeRisi实验室研发的能检测多种病毒的Virochip芯片,美国哥伦比亚大学Lipkin实验室研发的能同时检测多种病毒、细菌、真菌和寄生虫的GreeneChip芯片等。 For example, the University of California, San Francisco DeRisi laboratory developed can detect a variety of viruses Virochip chip, Lipkin of Columbia University Laboratory developed to simultaneously detect multiple viruses, bacteria, fungi and parasites GreeneChip chips.

[0008] 生物芯片探针设计的目的在于:经过计算方法优化后的探针能够在检测到更多的生物分子的同时,保证有较高的检测可靠性,即同时兼顾覆盖率和准确率两个方面,对于高通量的病原体检测这一点是至关重要的。 [0008] The object of the design is that a biochip probe: a probe through the optimization calculation method capable of simultaneously detect more biomolecules, ensure high detection reliability, i.e. taking into account the coverage and accuracy of two aspect for high-throughput pathogen detection this point is crucial. 通常的做法是首先查询如EMBL和GenBank等国际公共数据库,取得相应的DNA序列数据作为生物芯片探针设计的参照目标序列,然后从中选择特异性很高的核苷酸片段来设计探针。 The usual practice is as EMBL and GenBank first query public databases, etc., to obtain the corresponding DNA sequence data as a reference target sequence biochip probe design, high specific nucleotide fragment was then choose to design a probe. 特异性是指目标物种和非目标物种间的存在的差异,是检测型生物芯片鉴别物种的核心依据。 Specificity refers to differences between the target species and non-target species, based on the core detection type biochip to identify species. 特异性探针的选择是探针设计过程中的关键环节,探针优化设计算法研究已成为检测型基因芯片信息处理中一个急需解决的问题。 Specific probe selection is the key to the probe design process, research and design optimization algorithm probe has become a problem detection gene chip information processing in a need to be resolved. 对于小规模物种的鉴别,主要是通过序列比对的结果依靠人工分析选择,但是随着对单个芯片检测物种数量需求的快速增加,待分析的序列越来越多,再加上探针设计还要考虑很多其他方面的复杂因素,人工设计不仅费时费力,而且质量难以保证,因此计算方法在探针设计方面得到了广泛的应用。 For the identification of small species, mainly through the sequence than to rely on the results of the analysis of artificial selection, but with the rapid increase in demand for the number of individual species microarray sequence to be analyzed more and more, plus the probe design also other factors to consider many complex aspects of artificial design is not only time-consuming, and difficult to guarantee quality, the calculation method has been widely used in the design of the probe. Waibhav提出了一套从病原体全基因组序列出发的探针设计流程,Satya在此流程基础之上又进行了改进,除了有效地减少了计算时间以外,还使用了多套度量探针专一性的判据对探针质量进行了理论评估。 Waibhav proposed probe design process starting from a pathogen genome sequence, Satya on the basis of this process has been improved, in addition to effectively reduce the computation time, it also uses a plurality of sets of measurement probes specific probe quality criterion for theoretical assessment. Jabado等人进行了针对于病毒检测芯片的探针设计工作,他们认为在序列保守性分析方面,使用蛋白质-蛋白质比对相较于核酸序列之间的比对更有优势,因此他们提出了基于一套从病毒蛋白质序列出发的探针设计流程。 Jabado, who conducted the probe is designed to work against the virus detection chip, they believe in conservative sequence analysis using protein - protein alignment compared to the ratio between the nucleic acid sequence of an advantage, so they made based on a probe design process starting from viral protein sequences. 为了兼顾对探针高覆盖率的要求,还补充了一些以非编码区域为模板设计出来的探针。 In order to take into account the requirements of the high coverage of the probe, also added some non-coding regions of a template designed probes. 综上所述,目前的生物芯片探针设计方法,更加科学、合理,所设计出的探针有着比较好的覆盖率和准确率,能够满足高通量检测的需求。 In summary, the current biochip probe design methods, more scientific and rational, the design of the probe has better coverage and accuracy to meet the demand for high-throughput detection. 但是这些设计方法也存在着两方面的主要不足:1)计算耗时,设计效率较低。 But these design methods there are two main problems: 1) to calculate time-consuming, low efficiency of the design. 以Satya等人的TOF1-beta流程为例,在74个CPU上设计一个物种Brucella melitensis的检测探针,就需要21个小时;2)很多的探针设计流程,由于序列资源的限制,只能在属的层次上得到满足条件检测探针,难以做到更加精细的检测。 In Satya TOF1-beta et al process, for example, to design a detector probe species Brucella melitensis in the CPU 74, it requires 21 hours; 2) designed to process many probes, the sequence due to the limitation of resources, only conditions are satisfied at the detection probe genus level, it is difficult to achieve a finer detection. 随着序列资源的不断丰富,检测种或者亚种层次上的病原体都将成为可能,而现有的设计流程都缺少一个动态的数据管理更新系统,不能做到与快速增长的序列数据库做到同步更新。 With rich resources of the sequence, the detection of pathogens on species or subspecies level will become possible, and existing design processes are the lack of a dynamic data management system update, you can not do with the rapid growth of sequence databases be synchronized update.

发明内容[0009] 生物芯片是在现代分子生物学高通量技术的基础之上,建立起来的可用于病原体诊断检测方法。 SUMMARY OF THE INVENTION [0009] The biochip is based on techniques of modern molecular biology over the high-throughput, can be used to set up a pathogen diagnostic test. 随着序列资源的不断丰富,检测属、种甚至于亚种层次上的病原体都将成为可能,各大医疗和公共卫生机构对单个芯片检测物种数量需求也相应地在快速增加。 With rich resources of the sequence to detect the genus, species and even species on subspecies level will become possible, major medical and public health agencies on the number of individual species microarray demand correspondingly increasing rapidly. 传统的探针设计方法主要集中于对小规模物种的鉴别,主要是通过序列比对的结果依靠人工分析选择,设计效率较低,且质量不高。 The traditional method of probe design focuses on the identification of small species, mainly through the sequence than to rely on the results of the analysis of artificial selection, design efficiency is low, and the quality is not high.

[0010] 本发明在整合了国际上最先进的探针设计方法的基础之上,进行了有针对性的改进。 [0010] The present invention incorporates the most advanced method for probe design basis, were targeted improved. 对于细菌、病毒、真菌等不同类型的病原体,采用了不同的序列模板进行探针设计。 For different types of pathogenic bacteria, viruses, fungi and the like, using different sequences for probe design templates. 在设计流程中,充分考虑了病原体序列的情况,尽量在从属到种再到亚种,越来越精细的层次上设计检测探针。 In the design process, the full consideration of the case where the sequence of the pathogen, and then as far as possible in the dependent sub-species to species, the more sophisticated design level detection probe. 同时兼顾了探针的覆盖率和准确率,这对于高通量的病原体检测是非常重要的。 Taking into account the coverage and accuracy of the probe, which for high-throughput pathogen detection is very important.

附图说明 BRIEF DESCRIPTION

[0011] 图1是针对检测对象为细菌、真菌以及原生动物三类病原体的,以rRNA为模板的探针设计流程。 [0011] FIG. 1 is a target for the detection of bacteria, fungi, and protozoan pathogens three to rRNA probes were designed for the process template.

[0012] 图2是针对检测对象为病毒,以结构蛋白编码序列为模板的探针设计流程。 [0012] FIG. 2 is for the detection target is a virus, the structural protein coding sequence as the probe template design process.

[0013] 图3为细菌Brevibacterium epidermidis中四条序列进行多序列比对后的片段 [0013] FIG. 3 is a sequence of four bacteria Brevibacterium epidermidis multiple sequence fragment in the ratio

[0014] 图4是从进化树的分支上寻找细菌Brevibacterium epidermidis最近邻菌种的示意图 [0014] FIG. 4 is a schematic diagram of a bacterial strain Brevibacterium epidermidis nearest neighbor search from the branch of the phylogenetic tree

具体实施方式 detailed description

[0015] 下面结合具体的实例及附图对本方法作进一步说明。 [0015] The following specific examples and in conjunction with the accompanying drawings of the present process is further illustrated.

[0016] 一、针对细菌、真菌以及原生动物的以rRNA为模板的设计流程 [0016] First, the design process for bacteria, fungi and protozoa to rRNA as a template

[0017] 我们将细菌Brevibacterium epidermidis作为目标物种,并以它为例介绍图1所示的探针设计流程。 [0017] We Brevibacterium epidermidis bacteria as the target species, and probes were designed to introduce it as an example of a flow shown in FIG. 首先,从Ribosomal Database Project (RDP)数据库里得到目标物种的16S rRNA序列。 First, get 16S rRNA sequence of the target species from Ribosomal Database Project (RDP) database. 根据这些16S rRNA序列,进行序列比对,从GenBank中抽提出该物种更多的16S rRNA序列,同时对序列的种属描述信息进行校正,确保为目标物种的16S rRNA序列。 According to the 16S rRNA sequences, sequence alignments, The extracted from GenBank more species of the 16S rRNA sequence, while the description of the sequence is corrected species, 16S rRNA sequence to ensure target species. 对目标物种的多条16SrRNA进行多序列比对,抽提出种内保守的序列区域。 16SrRNA objective about many species of multiple sequence alignment, pumping propose conserved sequence regions within species. 图3所示为其中的一段保守序列片段。 Figure 3 is a section of the conserved sequence fragment.

[0018] 通过系统发生分析,对所研究额全部细菌菌种的代表性序列构建进化树,从进化树的分支中可以找到目标物种的最近邻物种,如图4所示,细菌Brevibacteriumepidermidis的最近邻菌种为Kineosporia aurantiaca。 Analysis [0018] The phylogenetic, phylogenetic tree was constructed to study the amount of the entire sequence of a representative bacterial species, from the branch of the phylogenetic tree species can be found in the nearest neighbor of the target species, 4, nearest neighbor bacteria Brevibacteriumepidermidis species is Kineosporia aurantiaca. 将两个菌种进行序列比对,得到种间的保守区。 The two species sequence alignment to give conserved regions between species. 从Brevibacterium epidermidis的种内保守区域中去除这部分种间保守区,即得到了目标菌种的特异性区域,作为备选序列进行下一步的探针设计。 Removing portions of the conserved between these regions from the conserved region of Brevibacterium epidermidis, i.e., to obtain a specific region of the target species, as an alternative to the next sequence of probe design.

[0019] 根据如下的几类实验条件,包括探针长度为60mer,所有探针的理论融解温度在2度内波动,GC含量在30% -70%的范围内等等,从备选序列中抽提出满足条件的备选探针集合。 [0019] The following types of experimental conditions, including the length of the probe 60mer, all probes theoretical melting temperature fluctuations within 2 degrees, the GC content in the range of 30% -70%, etc., from the candidate sequence the extracted candidate probe sets satisfied the condition. 构建将脊椎动物序列和相应的病原体序列整合到一起的非目标物种序列库,通过Blastn对备选探针进行同源性检测。 Construction of the integration vertebrate sequence and the corresponding sequence of pathogens to non-target species with the library of sequences, homology detection probe alternatively by Blastn. 我们设置的特异性标准是备选探针对于非目标物种基因的连续互补片段长度小于15bp,总的互补长度应小于75%。 We set the standard specific probe is an alternative for the continuous non-target species is complementary to the gene fragment length less than 15bp, the total length should be less than 75% complementarity. 通过筛选,去除掉可能与非目标物种序列产生交叉杂交的结果,得到高专一性的探针。 By screening, to remove possible cross-hybridization with the results of non-target species sequence, to obtain a high specific probes. [0020] 二、针对病毒以蛋白质编码序列为模板的设计流程 [0020] Second, in order for the virus protein coding sequence as a template of the design process

[0021] 图2所示的探针设计流程为针对病毒的,并以蛋白质编码序列为模板的设计流程。 Probe design flow shown in [0021] Figure 2 is against the virus and a protein coding sequence is a template of the design process. 首先,从European Molecular Biology Laboratory(EMBL)数据库中下载病毒序列标准文件。 First, download the file from the viral sequences standard European Molecular Biology Laboratory (EMBL) database. 从中抽提整理属于目标病毒的序列,根据序列文件提供的信息,进一步抽提出编码结构蛋白的核酸序列以及所编码的蛋白质序列。 Target sequence from a virus belonging to the extracted finishing, according to the information sequence files, and further in the extracted nucleic acid sequence encoding structural proteins and the encoded protein sequence. 将这些蛋白质序列与Pfam proteinfamilies database中的种子序列进行比对,得到保守的序列区域,将其对应的核酸编码区作为下一步设计的备选序列。 These protein sequences are aligned with the seed in the Pfam proteinfamilies database sequence, conserved sequence regions obtained, corresponding to a nucleic acid sequence of the coding region as the next alternative design. 对于那些不能够通过与Pfam数据库比对得到保守区的序列,直接将它们的核酸编码区进行序列比对、聚类,得到保守区域,作为备选序列的另一个来源。 For those who can not conserved regions, clusters, obtained by aligning the conserved region sequences obtained directly their nucleic acid coding region sequence alignments with the Pfam database, another source as an alternative sequence. 从备选序列出发设计探针的步骤与以rRNA为模板的设计流程中后面的步骤是是一致的。 The step of designing the probe sequence from alternative step in the design process for the rRNA template is consistent later.

Claims (2)

  1. 1.一种针对细菌、真菌以及原生动物三类病原体的探针,其特征是一种基于16SrRNA或18S rRNA序列模板的设计方法为基础的探针,包括:从RibosomalDatabaseProject(RDP)数据库里得到目标物种的rRNA序列,抽提出物种内部的保守的序列区域; (1)通过系统发生分析,对多个物种的代表性序列构建进化树,从进化树的分支中找到目标物种的最近邻物种; (2)将两个物种进行序列比对得到种间的保守区; (3)从目标物种的种内保守区域中去除这部分种间保守区,得到目标物种的特异性区域,作为备选序列; (4)针对该备选序列进行探针设计,对得到的备选探针进行特异性评估,去除那些可能产生交叉杂交的低质量探针。 A against bacteria, fungi, and protozoan pathogens, three types of probes, wherein a design method of 16SrRNA or 18S rRNA sequence of the template based on a probe, comprising: a target obtained from RibosomalDatabaseProject (RDP) database rRNA of species, conserved sequence regions in the extracted within species; (1) the phylogenetic analysis, phylogenetic tree of various species representative sequence, find the nearest neighbor species target species from the branch of the phylogenetic tree; ( 2) the ratio of the two species obtained sequences conserved regions among species; (3) removing the conserved region between this part from the conserved region of the target species, to obtain a specific region of the target species, as an alternative sequence; (4) the candidate sequences for probe design, the probe was subjected to an alternative specific evaluation, those with low mass remove possible cross-hybridizing probe.
  2. 2.一种针对病毒的探针,其特征是一种基于结构蛋白编码序列的设计方法为基础的探针,通过蛋白-蛋白比对获取保守区域的信息,包括: (1)从EMBL数据库中下载病毒序列标准文件,从中抽提出编码结构蛋白的核酸序列以及相应的蛋白质序列; (2)将这些蛋白质序列与Pfam数据库中的种子序列进行比对,得到保守的序列区域,将与其对应的核酸编码区作为下一步设计的备选序列; (3)按照权利要求1中所述的步骤(4),进行后续的探针设计。 A probe against the virus, which is a structure-based design method based on protein coding sequence probe by Protein - Protein alignment of conserved region acquiring information, comprising: (1) from the EMBL database Download viral sequences standard documents, which in the extracted nucleic acid sequence and corresponding protein sequence encoding a structural protein; (2) the seed sequences of these protein sequences to the Pfam database is to compare, obtained conserved sequence regions, the corresponding nucleic acid as an alternative to coding region sequences of the next design; (3) in the step 1 according to claim (4), for subsequent probe design.
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