WO2022246783A1 - 鉴别或辅助鉴别哺乳动物物种的探针组合物及其试剂盒与应用 - Google Patents

鉴别或辅助鉴别哺乳动物物种的探针组合物及其试剂盒与应用 Download PDF

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WO2022246783A1
WO2022246783A1 PCT/CN2021/096624 CN2021096624W WO2022246783A1 WO 2022246783 A1 WO2022246783 A1 WO 2022246783A1 CN 2021096624 W CN2021096624 W CN 2021096624W WO 2022246783 A1 WO2022246783 A1 WO 2022246783A1
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identifying
sequence
mammalian species
probe
assisting
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由玉岩
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北京动物园管理处
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

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  • the invention relates to a probe composition for identifying or assisting in identifying mammalian species in the field of biotechnology, as well as a kit and application thereof.
  • Cytochrome C oxidase I gene is one of the three cytochrome oxidase subunits encoded by mitochondrial genes. It is the gene with the largest molecular weight and the most conserved functional structure. The COI gene has the characteristics of multiple variations, easy amplification by universal primers, and few insertions and deletions in the sequence itself. Therefore, the COI gene is selected as a marker gene (DNA barcode) for DNA classification. The length of the coding gene is generally about About 658bp, it can be used not only for DNA classification, but also for the study of phylogenetic relationship and molecular evolution of species.
  • the COI gene sequence was generally obtained by the method of first-generation sequencing. This method has the advantages of flexibility, convenience, and quick results; but at the same time, there are also cumbersome batch sample operations, and the unstable results of the first-generation sequencing make it impossible to obtain accurate COI gene sequences. risk.
  • a technical problem to be solved by the present invention is how to identify or assist in the identification of mammalian species in batches.
  • the present invention provides a probe composition for identifying or assisting in identifying mammalian species.
  • the probe composition for identifying or assisting in identifying mammalian species provided by the present invention is to perform sequence clustering on mammalian COI gene sequences to obtain representative sequences, and to design probe coverage for each SNP site for representative sequences, and obtain Probe composition.
  • the sequence clustering is performed using the Angiosperms353 method, the genetic distance is set to 0.05 (ie, the sequence similarity is 95%), and the coverage depth is 2X.
  • the probe design is carried out according to the standard of GC content>30%.
  • the probe coverage is designed for each SNP site, and 2 probe coverages are designed for each SNP site.
  • the probe composition for identifying or assisting in identifying mammalian species provided by the present invention is specifically a combination of 3590 single-stranded DNAs shown in Sequence 1-Sequence 3590 of the Sequence Listing.
  • the present invention also provides a method for using the above-mentioned probe composition to identify or assist in the identification of mammalian species: comprising using the above-mentioned probe composition to capture the COI gene of the mammal to be tested, building a library, and obtaining it through high-throughput next-generation sequencing The DNA sequence of the COI gene is used to determine the mammalian species to be tested according to the obtained COI gene sequence.
  • the determination of the mammalian species to be tested according to the obtained COI gene sequence is comparison with the COI gene of known species, for example, comparison with the COI gene in the mitochondrial whole gene data.
  • mammalian species can be identified in batches.
  • the present invention also protects reagents or kits for identifying or assisting in the identification of mammalian species, said reagents or kits comprising said probe composition.
  • the invention applies targeted sequencing genotyping technology, designs and synthesizes liquid-phase probes according to the sequence selected by evaluation and analysis, and tests the capture efficiency of the probes, finally forms a COI gene capture kit, and realizes the purpose of identifying mammals in batches .
  • FIG. 1 is a flow chart of the development of probe combinations in Example 1 of the present invention.
  • the inventors designed capture probes based on the polymorphism of COI sequences among different species to capture the COI gene of the target species, and then built a library to obtain the DNA sequence of the COI gene by the method of high-throughput detection of next-generation sequencing.
  • This solution can take into account the advantages of flexibility, convenience, and quick results of next-generation sequencing, and at the same time solve the problem of cumbersome batch sample operations in first-generation sequencing, and avoid the risk of inability to obtain accurate COI gene sequences due to instability of partial results of first-generation sequencing;
  • the capture probe was developed into a mammalian COI gene capture kit for the study of DNA barcoding of a large number of mammalian species, which can realize the identification of mammalian species. The specific development process is carried out according to the flow chart in Figure 1:
  • the inventor applied the genotyping by target sequencing (Genotyping By Target Sequencing, GBTS, also known as GenoBaits) technology, designed and synthesized liquid phase probes according to the sequence selected by the evaluation analysis, and tested the capture efficiency of the probes, and finally formed the COI gene
  • GBTS Genotyping By Target Sequencing
  • the capture kit realizes the purpose of identifying mammals in batches.
  • Option 1 Use the Angiosperms353 method for sequence clustering, set the genetic distance to 0.1, that is, the sequence similarity is 90%, and the coverage depth is 1X; clustering with this parameter, the final representative sequence is 378, and the development and detection costs are relatively low. Low, there is a risk that when the target sequence mutates again, that is, when there is a difference between the sequence captured during the detection and the provided sequence, the corresponding sequence may not be captured, thereby affecting the species identification results;
  • Scheme 2 Use the Angiosperms353 method for sequence clustering, set the genetic distance to 0.05, that is, the sequence similarity is 95%, and the coverage depth is 2X; clustering with this parameter, the final representative sequence is more, 479, development and The detection cost is high, the probability of capturing the target sequence will be greatly improved, and the accuracy of the final identification result will be higher.
  • Probe Designer software to design liquid-phase capture probes for the 479 representative sequences obtained in Scheme 2.
  • the probe length is set to 110bp, and the GC content is >30%.
  • Each SNP site is designed to be covered by 2 capture probes .
  • the evaluation result is the result of probe design, not as long as the probe is designed, it will be able to capture the target site.
  • a total of 3590 probes targeting mammals were selected, each with a length of 110bp.
  • the nucleotide probe sequence modified with biotin (B) was synthesized by chip in situ synthesis technology (biotin is located at the 5' end of the probe).
  • the 3590 probes selected in step 2 were synthesized, and a total of 10 samples of 7 test species were tested.
  • the seven tested species are yak (sample number qh421, qh565), argali (sample number M-15, M-11), ibex (sample number 661, 660), white-cheeked gibbon (sample number B12), barking deer (sample number 62), Guizhou golden monkey (sample number 125), black deer (sample number 188), the samples used are blood samples. All samples were obtained during their physical examination, and the sample acquisition was reviewed and approved by the Academic Committee of Beijing Zoo.
  • the DNA concentration of each test sample was determined by Qubit Fluorometric Quantitation (Thermo Fisher Company), and the integrity of the DNA was detected by 1% agarose gel electrophoresis. Qualified samples were placed in a 4°C refrigerator for storage and subsequent use.
  • GenoBaits DNA Probe Beads (Boruidi Company) to the reaction system completed by hybridization in the previous step, pipette up and down 10 times, put it on an ABI 9700 PCR instrument and incubate at 65°C for 45 minutes to bind the magnetic beads to the probe.
  • GenoBaits Wash Buffer II (Boruidi Company)
  • 150 ⁇ L GenoBaits Wash Buffer III (Boruidi Company) were used to wash the magnetic beads at room temperature respectively.
  • the magnetic beads after washing were resuspended in 20 ⁇ L Nuclease-Free Water.
  • Thermo Fisher Company Use Qubit Fluorometric Quantitation (Thermo Fisher Company) to measure the DNA concentration of the library, and then use agarose gel electrophoresis to detect whether the fragment size of the library DNA is between 300-400bp.
  • the constructed DNA library was sequenced with an Illumina Hiseq X ten sequencer.
  • the original sequenced reads (Sequenced Reads) or raw reads obtained by sequencing contain low-quality reads with adapters.
  • the distribution of the sequencing error rate has the following two main reasons: 1) Due to the consumption of chemical reagents in the sequencing process, the sequencing error rate will increase with the increase of the length of the sequencing sequence (Sequenced Reads). 2) The incomplete combination of random primers and DNA templates during PCR may lead to a higher error rate in the first few bases.
  • the sequencing results are assembled in full length, and then analyzed again, either by clustering or by Blast, to finally find the sequence closest to the sequencing results and determine the species of the target sample.
  • test results are A coverage rate of 1 (i.e. 100%) indicates that the sample to be tested is the corresponding species, and a coverage rate of not 1 indicates that the sample to be tested is not the corresponding species.
  • the test results are as follows in Table 1:
  • the COI gene capture kit is produced with the probe composition composed of single-stranded DNA from sequence 1 to sequence 3590, which can be used for the detection of batch samples.
  • the invention discloses a probe composition for identifying or assisting in identifying mammalian species, a kit and application thereof.
  • the probe is the nucleotide probe shown in sequence 1-sequence 3590 of the sequence listing.
  • the probe of the present invention is used to capture the COI gene of the target species, and the DNA sequence of the COI gene is obtained by using the high-throughput detection method of next-generation sequencing to build a library, which can take into account the advantages of flexibility, convenience, and quick results of the first-generation sequencing. At the same time, it solves the problem of cumbersome batch sample operation in the first-generation sequencing, and avoids the risk of not being able to obtain accurate COI gene sequences due to unstable partial results of the first-generation sequencing.
  • the probe is developed into a mammalian COI gene capture kit for DNA barcoding research on a large number of mammalian species, which can realize rapid and batch identification of unknown mammalian species.

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Abstract

提供了鉴别或辅助鉴别哺乳动物物种的探针组合物及试剂盒。探针组合物为序列表序列1-序列3590所示的核苷酸探针。利用探针捕获目标物种的COI基因，以此建库用二代测序高通量检测的方法获得COI基因的DNA序列，可以同时兼顾一代测序灵活、方便、可以快速得到结果的优点，同时解决一代测序中存在的批量样本操作繁琐的问题，以及避免一代测序部分结果不稳定造成无法得到准确COI基因序列的风险。将探针开发成哺乳动物COI基因捕获试剂盒，可实现对哺乳动物物种的快速、批量鉴别。

Description

鉴别或辅助鉴别哺乳动物物种的探针组合物及其试剂盒与应用 技术领域
本发明涉及生物技术领域中鉴别或辅助鉴别哺乳动物物种的探针组合物及其试剂盒与应用。
背景技术
细胞色素C氧化酶I基因(COI基因)是线粒体基因编码的3种细胞色素氧化酶亚基之一，它是其中分子量最大、功能结构最为保守的基因。COI基因具有多变异、易被通用引物扩增、序列本身又很少存在插入和缺失等特点，因此COI基因被选为DNA分类的标记基因(DNA条形码)，该段编码基因的长度一般大约在658bp左右，其除可以用于DNA分类外，还可以用于物种的系统发育关系和分子进化的研究。
以往获得COI基因序列，一般采用一代测序的方法，该方法有灵活、方便、可以快速得到结果的优点；但同时也存在批量样本操作繁琐，一代测序部分结果不稳定造成无法得到准确COI基因序列的风险。
发明公开
本发明所要解决的一个技术问题是如何批量鉴别或辅助鉴别哺乳动物物种。
为了解决以上技术问题，本发明提供了鉴别或辅助鉴别哺乳动物物种的探针组合物。
本发明所提供的鉴别或辅助鉴别哺乳动物物种的探针组合物为对哺乳动物COI基因序列进行序列聚类得到代表性序列，针对代表性序列，每个SNP位点设计探针覆盖，得到的探针组合物。
上述探针组合物，所述序列聚类为使用Angiosperms353方法进行序列聚类，设置遗传距离为0.05(即序列相似度为95％)，覆盖深度2X。
上述探针组合物中，所述设计探针，按照GC含量＞30％的标准进行。
上述探针组合物中，所述每个SNP位点设计探针覆盖，为每个SNP位点设计2条探针覆盖。
本发明所提供的鉴别或辅助鉴别哺乳动物物种的探针组合物具体为 由序列表序列1-序列3590所示的3590个单链DNA组成的组合。
本发明还提供一种利用上述探针组合物鉴别或辅助鉴别哺乳动物物种的方法：包括以上述探针组合物捕获待测哺乳动物的COI基因，建库，通过二代测序高通量检测获得COI基因的DNA序列，根据得到的COI基因序列确定待测哺乳动物物种。
上述方法中，所述根据得到的COI基因序列确定待测哺乳动物物种，为与已知物种COI基因比对，例如与线粒体全基因数据中的COI基因比对。
上述方法中，可批量鉴别哺乳动物物种。
本发明还保护鉴别或辅助鉴别哺乳动物物种的试剂或试剂盒，所述试剂或试剂盒包括所述的探针组合物。
上述探针组合物、上述方法和/或上述试剂或试剂盒在鉴别或辅助鉴别哺乳动物物种中的应用，或在制备鉴别或辅助鉴别哺乳动物物种产品中的应用属于本发明的保护范围。
本发明应用靶向测序基因型分型技术，根据评估分析选择的序列设计合成液相探针，并对探针进行捕获效率测试，最终形成COI基因捕获试剂盒，实现批量进行哺乳动物鉴别的目的。
附图说明
图1为本发明实施例1中探针组合开发的流程图。
实施发明的最佳方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1
发明人根据不同物种间COI序列的多态性，设计捕获探针，用于捕获目标物种的COI基因，然后建库用二代测序高通量检测的方法获得COI基因的DNA序列。该方案可以同时兼顾一代测序灵活、方便、可以快速得到结果的优点，同时解决一代测序中存在的批量样本操作繁琐的问题，以及 避免一代测序部分结果不稳定造成无法得到准确COI基因序列的风险；将捕获探针开发成哺乳动物COI基因捕获试剂盒，用于大量哺乳动物物种DNA条形码研究，可实现对哺乳动物物种的鉴别。具体开发过程按照图1中的流程图进行：
1、技术路线及方案的确定
两种二代测序技术GenoBaits和GenoPlexs的对比见表1:
表1 GenoBaits和GenoPlexs技术的对比
Figure PCTCN2021096624-appb-000001
发明人应用靶向测序基因型分型(Genotyping By Target Sequencing,GBTS，又称GenoBaits)技术，根据评估分析选择的序列设计合成液相探针，并对探针进行捕获效率测试，最终形成COI基因捕获试剂盒，实现批量进 行哺乳动物鉴别的目的。
根据300多种哺乳动物的COI基因序列，以及部分从线粒体全基因数据中提取的COI基因，共413条COI基因进行序列聚类，目的是找到每一个分类中最具有代表性的COI序列，并以最终聚类得到的序列进行探针设计。因设计时使用不同参数有不同效果，给出两套评估方案，具体如下：
方案一：使用Angiosperms353方法进行序列聚类，设置遗传距离为0.1即序列相似度为90％，覆盖深度1X；以此参数进行聚类，最终得到的代表性序列为378条，开发及检测成本较低，存在风险是当目标序列再次发生变异的时候即检测时捕获到的序列和提供的序列都存在差异的时候，可能会出现无法捕获相应序列，从而影响物种鉴结果的情况；
方案二：使用Angiosperms353方法进行序列聚类，设置遗传距离为0.05即序列相似度为95％，覆盖深度2X；以此参数进行聚类，最终得到的代表性序列较多，为479条，开发及检测成本较高，对目标序列捕获的概率会大幅提升，最终鉴定结果的准确性更高。
最终确认以方案二进行试剂盒的开发，进行探针设计。
2、探针设计和挑选原则
2.1探针设计原则：
用GenoBaits Probe Designer软件对方案二中获得的479条代表性序列进行液相捕获探针设计，探针长度设置为110bp，GC含量＞30％，每个SNP位点设计由2条捕获探针覆盖。
2.2探针挑选原则：
1)选取探针含量在30％-80％之间的；
2)选取同源性区域个数＜10的；
3)选取探针区域不包含SSR、N区域的。
探针设计结果见表2:
表2探针设计结果
Figure PCTCN2021096624-appb-000002
Figure PCTCN2021096624-appb-000003
注：该评估结果为探针设计结果，并非只要设计出探针就一定能捕获目标位点。
挑选出针对哺乳动物的探针总数3590个，每个长度110bp。利用芯片原位合成技术合成带有生物素(biotin，B)修饰的核苷酸探针序列(生物素位于探针5’端)。
3、引物合成及开发测试
合成步骤2挑选的3590个探针，对7个测试物种共计10个样品进行测试。7个测试物种分别为牦牛(样本号qh421、qh565)、盘羊(样本号M-15、M-11)、北山羊(样本号661、660)、白颊长臂猿(样本号B12)、赤麂(样本号62)、黔金丝猴(样本号125)、黑麂(样本号188)，所用样本为血液样本。所有样品在其体检时获得，样本获取经北京动物园学术委员会的审核及许可。
3.1样品DNA质检
将每个测试样品DNA用Qubit Fluorometric Quantitation(Thermo Fisher公司)对DNA浓度进行测定，用1％琼脂糖凝胶电泳检测DNA的完整性。检测合格的样品放入4℃冰箱，保存、备用。
3.2样品DNA测序文库构建
针对每个测试样，取12μL步骤3.1质检合格的DNA(200ng)放置于0.2μL PCR管中，将管置于超声波破碎仪中对DNA进行随机物理破碎，片段破碎至200-400bp。然后向管中加入4μL GenoBaits End Repair Buffer(博瑞迪公司)和2.7μL GenoBaits End Repair Enzyme(博瑞迪公司)，补水至20μL，放入ABI 9700PCR仪中37℃温育20分钟，完成破碎片段的末端修复和加A过程。
从PCR仪中取出小管加入2μL GenoBaits Ultra DNA ligase(博瑞迪公司)、8μL GenoBaits Ultra DNA Ligase Buffer(博瑞迪公司)和2μL GenoBaits Adapter(博瑞迪公司)，补水至40μL，然后放置于ABI9700PCR仪上22℃反应30分钟，完成测序接头的连接。向连接产物中加入48μL的Beackman AMPure XP Beads(Beackman公司)对连接产物进行纯化，纯化后按照0.65+0.2倍磁珠进行片段筛选，保留插入片段在200-300bp的连接产物。
向上一步的PCR管中加入5μL带有Barcode序列(所用Barcode序列选自序列表序列3591-序列3686中的Barcode序列，不同的Barcode用于区分不同的样品)的测序接头、1μL P5接头、10μL GenoBaits PCR Master Mix(博瑞迪公司)，并用纯水补至20μL；用ABI 9700 PCR仪进行扩增，扩增程序为：95℃预变性5min，95℃变性30s，60℃退火30s，72℃延伸30s；重复2-4步，共8个循环；72℃延伸5min。
向第二轮PCR产物中加入24μL Beckmen AMPure XP Beads(Beckmen公司)，用移液器上下吸打均匀后，将0.2μL的PCR管置于磁力架上至溶液澄清，弃去上清并用75％乙醇洗涤磁珠一次，用pH值为8.0的Tris-HCl将文库DNA洗脱下来。
3.3样品DNA杂交捕获
取500ng已完成构建的样品DNA测序文库，加入5μL GenoBaits Block I(博瑞迪公司)和2μL GenoBaits Block II(博瑞迪公司)，置于Eppendorf Concentrator plus(Eppendorf公司)真空浓缩仪上，在≤70℃的温度下蒸干至干粉。向干粉管中加入8.5μL GenoBaits 2x Hyb Buffer(博瑞迪公司)、2.7μL GenoBaits Hyb Buffer Enhancer(博瑞迪公司)、2.8μL Nuclease-Free Water，用移液器吸打混匀后放置于ABI 9700 PCR仪上95℃温育10分钟，然后取出PCR管加入3μL已经合成的探针(60ng/ul)，旋涡震荡混匀后放置于ABI 9700PCR仪上65℃温育2小时，完成探针杂交反应。
向上一步杂交完成的反应体系中加入100μL GenoBaits DNA Probe Beads(博瑞迪公司)，上下吸打10次，放入ABI 9700 PCR仪上65℃温育45分钟，使磁珠与探针结合。用100μL GenoBaits Wash Buffer I(博瑞迪公司)、150μL GenoBaits Wash BufferII(博瑞迪公司)分别对结合探针后的磁珠进行65℃热洗，然后再用100μL GenoBaits Wash Buffer I(博瑞迪公司)、150μL GenoBaits Wash Buffer II(博瑞迪公司)和150μL GenoBaits Wash Buffer III(博瑞迪公司)分别对磁珠进行常温洗涤。洗涤完成的磁珠用20μL Nuclease-Free Water进行重悬。
取13μL重悬后的DNA(带磁珠)加入到新的0.2mL PCR管中，然后加入15μL GenoBaits PCR Master Mix(博瑞迪公司)、2μL GenoBaits Primer Mix(博瑞迪公司)配置post-PCR体系，用ABI 9700 PCR仪进行文 库扩增，扩增程序为：95℃预变性5min，95℃变性30s，60℃退火30s，72℃延伸30s；重复2-4步，共15个循环；72℃延伸5min。
向post-PCR产物中加入45μL Beckmen AMPure XP Beads(Beckmen公司)并用移液器上下吸打均匀，然后将0.2mL PCR管置于磁力架上至溶液澄清，弃去上清并用75％乙醇洗涤磁珠两次，用pH为8.0的Tris-HCl将文库DNA洗脱下来。完成测试样品的杂交捕获工作。
3.4样品DNA杂交捕获文库的质检与测序
用Qubit Fluorometric Quantitation(Thermo Fisher公司)对文库的DNA浓度进行测定，然后用琼脂糖凝胶电泳检测文库DNA的片段大小是否在300-400bp之间。构建好的DNA文库用Illumina Hiseq X ten测序仪进行测序。
3.5测试数据分析
测序得到的原始测序序列(Sequenced Reads)或者raw reads，里面含有带接头的、低质量的reads。对于二代测序技术，测序错误率分布具有下面两个主要原因：1)由于测序过程中化学试剂的消耗，导致测序错误率会随着测序序列(Sequenced Reads)长度的增加而升高。2)PCR过程中随机引物和DNA模版的不完全结合可能导致前几个碱基测序错误率较高。
为了保证信息分析质量，必须对raw reads过滤，得到clean reads，使用clean reads进行后续分析。使用软件fastp(version 0.20.0,参数：-n 10-q 20-u 40)对raw reads进行过滤，数据处理的步骤如下:
3.5.1去除接头序列(adapter)；
3.5.2当测序read中含有的N的含量超过该条read长度比例的10％时，需要去除此对paired reads；
3.5.3当测序read中含有的低质量(Q≤20)碱基数超过该条read长度比例的40％时，需要去除此对paired reads。
得到检测数据后，将测序结果进行全长组装后，再次进行分析，可以采用聚类的方式，也可采用Blast的方式，最终找到与测序结果亲缘关系最近的序列进而确定目标样本所属物种情况。
上述7个测试物种共计10个样品利用针对哺乳动物的整套探针组合物(共3590条探针，每条长度110bp，具体探针序列见序列表序列1-序列3590) 进行测试，测试结果中覆盖率为1(即100％)的表明待测样品为对应的物种，覆盖率不为1的表明待测样品并不是对应的物种，测试结果具体如下表1：
表1
Figure PCTCN2021096624-appb-000004
Figure PCTCN2021096624-appb-000005
Figure PCTCN2021096624-appb-000006
结果表明，10个样品检测出的物种和其实际所述物种完全相符合，准确率为100％。以序列1-序列3590的单链DNA组成的探针组合物生产COI基因捕获试剂盒，可用于批量样品的检测。
以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。
工业应用
本发明公开了鉴别或辅助鉴别哺乳动物物种的探针组合物及其试剂盒与应用。所述探针为序列表序列1-序列3590所示的核苷酸探针。利用本发明的探针捕获目标物种的COI基因，以此建库用二代测序高通量检测的方法获得COI基因的DNA序列，可以同时兼顾一代测序灵活、方便、可以快速得到结果的优点，同时解决一代测序中存在的批量样本操作繁琐的 问题，以及避免一代测序部分结果不稳定造成无法得到准确COI基因序列的风险。将探针开发成哺乳动物COI基因捕获试剂盒，用于大量哺乳动物物种DNA条形码研究，可实现对未知哺乳动物物种的快速、批量鉴别。

Claims (11)

  1. 鉴别或辅助鉴别哺乳动物物种的探针组合物，其特征在于，所述探针组合物为对哺乳动物COI基因序列进行序列聚类得到代表性序列，针对代表性序列，每个SNP位点设计探针覆盖，得到的探针组合物。
  2. 根据权利要求1所述的鉴别或辅助鉴别哺乳动物物种的探针组合物，其特征在于：所述序列聚类为使用Angiosperms353方法进行序列聚类，设置遗传距离为0.05，覆盖深度2X。
  3. 根据权利要求2所述的鉴别或辅助鉴别哺乳动物物种的探针组合物，其特征在于：所述每个SNP位点设计探针覆盖，为每个SNP位点设计2条探针覆盖。
  4. 根据权利要求1-3任一项所述的鉴别或辅助鉴别哺乳动物物种的探针组合物，其特征在于：所述探针组合物为由序列表序列1-序列3590所示的3590个单链DNA组成的组合。
  5. 利用探针组合物鉴别或辅助鉴别哺乳动物物种的方法，其特征在于：包括以权利要求1-4任一所述的探针组合物捕获待测哺乳动物的COI基因，建库，通过二代测序高通量检测获得COI基因的DNA序列，根据得到的COI基因序列确定待测哺乳动物物种。
  6. 鉴别或辅助鉴别哺乳动物物种的试剂或试剂盒，其特征在于：所述试剂或试剂盒包括权利要求1-4任一所述的探针组合物。
  7. 权利要求1-4任一所述的探针组合物在鉴别或辅助鉴别哺乳动物物种中的应用。
  8. 权利要求1-4任一所述的探针组合物在制备鉴别或辅助鉴别哺乳动物物种产品中的应用。
  9. 权利要求5所述的方法在鉴别或辅助鉴别哺乳动物物种中的应用。
  10. 权利要求5所述的方法在制备鉴别或辅助鉴别哺乳动物物种产品中的应用。
  11. 权利要求6所述的试剂或试剂盒在鉴别或辅助鉴别哺乳动物物种中的应用。
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