WO2018072064A1 - 一种基于单分子测序技术及dna条形码分子鉴定技术联用的生物物种组成的监测方法 - Google Patents

一种基于单分子测序技术及dna条形码分子鉴定技术联用的生物物种组成的监测方法 Download PDF

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WO2018072064A1
WO2018072064A1 PCT/CN2016/102293 CN2016102293W WO2018072064A1 WO 2018072064 A1 WO2018072064 A1 WO 2018072064A1 CN 2016102293 W CN2016102293 W CN 2016102293W WO 2018072064 A1 WO2018072064 A1 WO 2018072064A1
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sequence
dna
monitoring
biological species
single molecule
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PCT/CN2016/102293
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English (en)
French (fr)
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宋经元
辛天怡
徐志超
贾静
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中国医学科学院药用植物研究所
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Priority to PCT/CN2016/102293 priority Critical patent/WO2018072064A1/zh
Publication of WO2018072064A1 publication Critical patent/WO2018072064A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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

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  • the invention belongs to the technical field of quality control of foods and medicines, and particularly relates to a monitoring method for biological species composition based on single molecule sequencing technology and DNA barcode molecular identification technology.
  • the current version of the Chinese Pharmacopoeia's methods for quality control of proprietary Chinese medicines mainly include microscopic identification, TLC identification, content determination, and fingerprints.
  • TLC identification and content determination can only qualitatively or quantitatively monitor whether a proprietary chemical component is contained in a proprietary Chinese medicine, and it cannot be determined whether the target chemical component is derived from the corresponding medicinal material in the prescription of the proprietary Chinese medicine or artificially added, resulting in The monitoring of species composition has limitations.
  • DNA barcode molecular identification is a molecular biology technique that uses a recognized relatively short DNA sequence in the genome for species identification, and is an effective complement to traditional identification methods.
  • the 2015 edition of the Chinese Pharmacopoeia contains the guiding principles for DNA barcode molecular identification of Chinese herbal medicines, and it has been widely used as one of the quality control methods for Chinese herbal medicines.
  • Single-molecule sequencing technology also known as third-generation sequencing technology, includes single-molecule fluorescence sequencing and nanopore sequencing. It has the characteristics of long read speed, high speed and high precision.
  • the technology is mainly applied to genome sequencing, DNA methylation research, and SNP detection. Therefore, how to use the above technology to develop a method for effective monitoring of biological species composition to overcome the limitations of current monitoring of biological species composition, thereby effectively controlling the quality of biological species composition to maintain clinical drug safety. It is an important direction of research in this field.
  • the invention aims to provide a monitoring method for the biological species composition based on the single molecule sequencing technology and the DNA barcode molecular identification technology, which can effectively supplement the quality control method of the existing biological species composition and maintain the safety of clinical medication.
  • An aspect of the present invention provides a monitoring method for a biological species composition based on a single molecule sequencing technology and a DNA barcode molecular identification technology, comprising the following steps:
  • step 2) using the genomic DNA in step 1) as a template, and amplifying the target DNA or cDNA sequence by PCR to obtain a PCR product;
  • step 2) based on a single molecule sequencing platform, and according to the standard operating procedure of the sequencing platform, the library product obtained in step 2) is subjected to library construction and single molecule sequencing;
  • step 4 applying the data processing software of the sequencing platform to perform data processing on the single molecule sequencing result measured in step 3), and obtain a DNA or cDNA sequence of interest which can be used for subsequent analysis;
  • step 4) The DNA or cDNA sequence of interest in step 4) is placed in a DNA barcode database, and BLAST alignment is performed to obtain a species identification result.
  • the invention provides a monitoring method for biological species composition based on single molecule sequencing technology and DNA barcode molecular identification technology, which combines single molecule sequencing technology and DNA barcode molecular identification technology to detect and can be amplified.
  • the biological species component of the target Chinese patent medicine, herbal product or dietary supplement sample of the target DNA or cDNA sequence has wide applicability, and can effectively supplement the quality control method of the existing biological species composition, thereby achieving the safety of clinical medication. purpose.
  • An embodiment of the present invention provides a monitoring method for a biological species composition based on a single molecule sequencing technology and a DNA barcode molecular identification technology, comprising the following steps:
  • S1 extracting genomic DNA of the sample to be tested
  • the excipients contained in the sample to be inspected should be removed as much as possible, and the sample amount can be increased for samples with a higher degree of processing.
  • S2 using the genomic DNA in step 1) as a template, and amplifying the target DNA or cDNA sequence by PCR to obtain a PCR product;
  • the obtained PCR product needs to be purified before being used in the sequencing of the next step.
  • the PCR product obtained by step 2) is used for library construction. If only one PCR purification product is obtained in step 2), the library is directly constructed and sequenced on the machine; if step 2) two or more PCR purifications are obtained.
  • the products can be combined and built, or the libraries can be separately constructed, and each library is separately sequenced.
  • DNA or cDNA sequence of interest obtained in this step can be obtained by steps 1) to 3), but can be obtained by other suitable means in other embodiments, and is not limited thereto.
  • S5 The target DNA or cDNA sequence in step 4) is placed in a DNA barcode database, and BLAST alignment is performed to obtain a species identification result.
  • the DNA or cDNA sequence of interest amplified in step 2) is selected from one or more of the ITS2 sequence, the psbA-trnH sequence, the ITS sequence, the rbcL sequence, the matK sequence, and the COI sequence.
  • the DNA or cDNA sequence of interest amplified in step 2) is selected from the group consisting of an ITS2 sequence and/or a psbA-trnH sequence.
  • the primers used in amplifying each DNA or cDNA sequence of interest and the corresponding primer sequences are listed in Table 1 below, as follows:
  • the present embodiment is not limited to the above-exemplified sequences, and may be other sequences known in the art and suitable for use in the above methods, as long as the biological species capable of amplifying the DNA or cDNA sequence of interest Can be applied to this method.
  • the single molecule sequencing platform is selected from any one of a PacBio RSII sequencing platform, a Heliscope single molecule sequencing platform, and a MinION sequencing platform. It will be appreciated that the present embodiment is not limited to the sequencing platforms listed above, but may be other sequencing platforms known in the art that are suitable for use in the above methods.
  • the data processing steps are: applying the SMRT Analysis Server software to filter the original data, and obtaining a circular consistency sequence based on the RS_ReadsOfInsert.1 software, using CD-HIT
  • the software clusters the circular consensus sequences to obtain a DNA or cDNA sequence of interest that can be used for subsequent analysis.
  • specific processing steps are given when the PacBio RSII sequencing platform is selected.
  • corresponding data processing and subsequent related operations can be performed according to the corresponding selected sequencing platform.
  • DNA extraction and PCR amplification are performed in accordance with the "Guidelines for Molecular Identification of DNA Barcodes of Chinese Medicinal Materials".
  • the principle of operation of the DNA extraction and PCR amplification steps is given, but it can be understood that the present embodiment is not limited to the above-mentioned principles, and may be other known in the art. Suitable guidelines or methods.
  • the monitoring method is suitable for monitoring the composition of biological species in any of a proprietary Chinese medicine, herbal product or dietary supplement.
  • An embodiment of another aspect of the present invention provides a monitoring method for biological species composition based on single molecule sequencing technology and DNA barcode molecular identification technology according to any of the above embodiments.
  • the specific embodiments will be combined with the ITS2 sequence and the psbA-trnH sequence.
  • the 1-5 samples of the traditional Chinese medicine Jiuweijing live pill were used for multi-angle analysis, and the examples 6-10 were analyzed by using the Chinese patent medicine Yimu pill as an example.
  • the test material is a Chinese patent medicine Jiuweizhi live pill.
  • the materials used in this example are prepared in the laboratory according to the prescribed dosage and preparation method of the Chinese patent medicine prescription in the Chinese Pharmacopoeia.
  • an equivalent amount of ginseng medicinal material was added as a positive control according to the dosage of the lowest dose of the medicinal material Asarum in the prescription.
  • the prescription of Chinese traditional medicine Jiuweizhi live pill consists of 9 kinds of traditional Chinese medicines, such as scorpion, wind, jaundice, asarum, rehmannia, chuanxiong, atractylodes, white peony and licorice.
  • the sputum, wind, scutellaria, asarum, rehmannia, and chuanxiong used in this example have been identified by morphology and DNA barcode, while Atractylodes chinensis and Radix Paeoniae Alba have not been able to obtain DNA barcode sequences, and their species have been identified by morphology to ensure each The accuracy of the species.
  • the above-mentioned medicinal materials and the ginseng of the positive control medicinal materials are mixed and pulverized and sieved to prepare a pill.
  • the ITS2 sequence and the psbA-trnH sequence were amplified, respectively.
  • primer sequences used to amplify the ITS2 sequence are:
  • Reverse primer GACGCTTCTCCAGACTACAAT
  • the primer sequences used to amplify the psbA-trnH sequence are:
  • Reverse primer CGCGCATGGTGGATTCACAATCC, synthesized by Shenggong Bioengineering Co., Ltd. (Beijing).
  • reaction system 2 ⁇ PCR MasterMix 12.5 ⁇ L, 1.0 ⁇ L (2.5 ⁇ mol/L) of each of the forward and reverse primers, 20 to 100 ng of template DNA, and sterilized double distilled water to 25 ⁇ L for PCR amplification.
  • ITS2 sequence and psbA-trnH sequence amplification reactions were performed on a PCR machine according to the following procedure:
  • the PCR product was subjected to gel electrophoresis, and subjected to gelatinization recovery using a QIAquick Gel Extraction Kit (QIAGEN N.V., Germany) to obtain a corresponding PCR purified product.
  • the original data can be filtered by using SMRT Analysis Server 2.3.0 software, and circular-consensus sequencing reads (CCS reads) are obtained based on the RS_ReadsOfInsert.1 operation flow, and ITS2 sequences usable for subsequent analysis are obtained. And the psbA-trnH sequence.
  • CCS reads circular-consensus sequencing reads
  • the ITS2 sequence and the psbA-trnH sequence obtained above were placed in a Chinese herbal medicine DNA barcode identification system (http://www.tcmbarcode.cn/china/), and BLAST alignment was performed to obtain a species identification result.
  • BLAST alignment was performed to obtain a species identification result.
  • the knot is The result is as follows:
  • the ITS2 sequence can detect scorpion, wind, asarum, Chuanxiong, Dihuang, and licorice.
  • the psbA-trnH sequence was able to detect ginseng, chuanxiong, scutellaria, licorice and positive control ginseng.
  • the ITS2 sequence was combined with the psbA-trnH sequence, and no atractylodes and white peony were detected, which was consistent with the DNA barcode identification results in the experimental step 1).
  • Example 1 Basically the same as in Example 1, except that the positive control medicinal ginseng in Example 1 was not added.
  • the ITS2 sequence can detect scorpion, wind, asarum, Chuanxiong, Dihuang, and licorice.
  • the psbA-trnH sequence was able to detect sputum, Chuanxiong, Astragalus and licorice.
  • the ITS2 sequence was combined with the psbA-trnH sequence, and no atractylodes and white peony were detected, which was consistent with Example 1.
  • the test material is a Chinese patent medicine Jiuweiyu live pill (batch number: 20141101) produced by a pharmaceutical factory with the drug approval number, purchased from a pharmacy.
  • Embodiment 1 Basically the same as Embodiment 1, except that this embodiment starts from the DNA extraction step and is completed step by step. Experimental manipulation and data processing until species identification.
  • the ITS2 sequence was able to detect scorpion, atractylodes, asarum, Chuanxiong, and licorice.
  • the psbA-trnH sequence is able to detect sputum, wind, atractylodes, Chuanxiong, Astragalus and licorice.
  • the ITS2 sequence was combined with the psbA-trnH sequence, and no rehmannia or white peony was detected.
  • the reason may be that the DNA of the above two species is seriously degraded in the test sample of the Chinese patent medicine used in the present embodiment, or the powder of the above two Chinese herbal medicines is not added in accordance with the provisions of the Chinese Pharmacopoeia in the preparation process.
  • the test material is a Chinese patent medicine Jiuweiyu live pill (batch number: 20140501) produced by a pharmaceutical factory with the drug approval number, purchased from a pharmacy.
  • Example 2 Basically the same as Example 1, except that this example starts with the DNA extraction step, and the experimental operation and data processing are gradually completed until species identification.
  • the ITS2 sequence can detect scorpion, wind, atractylodes, asarum, Chuanxiong, and licorice.
  • the psbA-trnH sequence can detect Chuanxiong and Licorice.
  • the ITS2 sequence was combined with the psbA-trnH sequence, and no jaundice, rehmannia, or white peony was detected.
  • the reason may be that the DNA of the above three species is seriously degraded in the test sample of the Chinese patent medicine used in the present embodiment, or the powder of the above three Chinese herbal medicines is not added in accordance with the provisions of the Chinese Pharmacopoeia in the preparation process.
  • the test material is a Chinese patent medicine Jiuweiyu live pill (batch number: 20140501) produced by a pharmaceutical factory with the drug approval number, purchased from a pharmacy.
  • Example 2 Basically the same as Example 1, except that this example starts with the DNA extraction step, and the experimental operation and data processing are gradually completed until species identification.
  • the ITS2 sequence can detect scorpion, wind, atractylodes, asarum, Chuanxiong, and licorice.
  • the psbA-trnH sequence was able to detect scorpion, atractylodes, chuanxiong, scutellaria, and licorice.
  • the ITS2 sequence was combined with the psbA-trnH sequence, and no rehmannia or white peony was detected.
  • the reason may be that the DNA of the above two species is seriously degraded in the test sample of the Chinese patent medicine used in the present embodiment, or the powder of the above two Chinese herbal medicines is not added in accordance with the provisions of the Chinese Pharmacopoeia in the preparation process.
  • test material is a proprietary Chinese medicine Yimu Pill.
  • the materials used in this example are prepared in the laboratory according to the prescribed dosage and preparation method of the Chinese patent medicine prescription in the Chinese Pharmacopoeia.
  • an equivalent amount of ginseng medicinal material was added as a positive control according to the dosage of the lowest dose of the medicinal wood scent in the prescription.
  • the prescription of Chinese medicine Yimu Pill consists of 4 Chinese herbal medicines including Motherwort, Chuanxiong, Angelica and Muxiang.
  • Each of the medicinal materials used in this example has been identified by morphology and DNA barcode to ensure the accuracy of its species.
  • the dosage and preparation method prescribed in the Chinese Pharmacopoeia the above-mentioned medicinal materials and the ginseng of the positive control medicinal materials are mixed and pulverized and sieved to prepare a pill.
  • the ITS2 sequence and the psbA-trnH sequence were amplified, respectively.
  • primer sequences used to amplify the ITS2 sequence are:
  • Reverse primer GACGCTTCTCCAGACTACAAT
  • the primer sequences used to amplify the psbA-trnH sequence are:
  • Reverse primer CGCGCATGGTGGATTCACAATCC, synthesized by Shenggong Bioengineering Co., Ltd. (Beijing).
  • reaction system 2 ⁇ PCR MasterMix 12.5 ⁇ L, 1.0 ⁇ L (2.5 ⁇ mol/L) of each of the forward and reverse primers, 20 to 100 ng of template DNA, and sterilized double distilled water to 25 ⁇ L for PCR amplification.
  • ITS2 sequence and psbA-trnH sequence amplification reactions were performed on a PCR machine according to the following procedure:
  • the PCR product was subjected to gel electrophoresis, and subjected to gelatinization recovery using a QIAquick Gel Extraction Kit (QIAGEN N.V., Germany) to obtain a purified product.
  • the original data can be filtered by using SMRT Analysis Server 2.3.0 software.
  • the circular-consensus sequencing reads (CCS reads) are obtained based on the RS_ReadsOfInsert.1 operational flow, and the ITS2 sequence and the psbA-trnH sequence available for subsequent analysis are obtained.
  • the ITS2 sequence was able to detect motherwort, Chuanxiong, Angelica, Muxiang and positive control ginseng.
  • the psbA-trnH sequence was able to detect Motherwort, Chuanxiong and Angelica.
  • the combination of the ITS2 sequence and the psbA-trnH sequence can detect the four herbs in the prescription, which is consistent with the DNA barcode identification results in the experimental step 1).
  • Example 6 Basically the same as in Example 6, except that the positive control medicinal ginseng in Example 6 was not added.
  • the ITS2 sequence can detect Motherwort, Chuanxiong, Angelica, and Woody.
  • the psbA-trnH sequence was able to detect Motherwort, Chuanxiong and Angelica.
  • the ITS2 sequence binds to the psbA-trnH sequence and can detect four herbs in the prescription. Consistent with Example 6.
  • test material is a proprietary Chinese medicine Yimu Pill (batch number: 3015152) produced by a pharmaceutical factory with a drug approval number, purchased from a pharmacy.
  • Example 6 Basically the same as Example 6, except that this embodiment starts with the DNA extraction step, and gradually completes the experimental operation and data processing until species identification.
  • the ITS2 sequence can detect Motherwort, Chuanxiong, Angelica, and Woody.
  • the psbA-trnH sequence was able to detect Motherwort, Chuanxiong and Angelica.
  • the ITS2 sequence binds to the psbA-trnH sequence and can detect four herbs in the prescription.
  • test material is a proprietary Chinese medicine Yimu Pill (batch number: 2015395) produced by a pharmaceutical factory with the drug approval number, purchased from a pharmacy.
  • Example 6 Basically the same as Example 6, except that this embodiment starts with the DNA extraction step, and gradually completes the experimental operation and data processing until species identification.
  • the ITS2 sequence can detect Motherwort, Chuanxiong and Angelica.
  • the psbA-trnH sequence was able to detect Motherwort, Chuanxiong and Angelica.
  • the ITS2 sequence was combined with the psbA-trnH sequence and no woody notes were detected.
  • the reason may be that the DNA of the above-mentioned species is seriously degraded in the test sample of the Chinese patent medicine used in the present embodiment, or the powder of the Chinese herbal medicine is not added in accordance with the provisions of the Chinese Pharmacopoeia in the preparation process.
  • test material is a proprietary Chinese medicine Yimu pill with a drug approval number produced by a pharmaceutical factory (batch number: 2015616), purchased from a pharmacy.
  • Example 6 Basically the same as Example 6, except that this embodiment starts with the DNA extraction step, and gradually completes the experimental operation and data processing until species identification.
  • the ITS2 sequence can detect Motherwort, Chuanxiong and Angelica.
  • the psbA-trnH sequence was able to detect Motherwort, Chuanxiong and Angelica.
  • the ITS2 sequence was combined with the psbA-trnH sequence and no woody notes were detected.
  • the reason may be that the DNA of the above-mentioned species is seriously degraded in the test sample of the Chinese patent medicine used in the present embodiment, or the powder of the Chinese herbal medicine is not added in accordance with the provisions of the Chinese Pharmacopoeia in the preparation process.

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Abstract

提供了一种基于单分子测序技术及DNA条形码分子鉴定技术联用的生物物种组成的监测方法。

Description

一种基于单分子测序技术及DNA条形码分子鉴定技术联用的生物物种组成的监测方法 技术领域
本发明属于食品、药品等质量控制技术领域，具体涉及一种基于单分子测序技术及DNA条形码分子鉴定技术联用的生物物种组成的监测方法。
背景技术
现行版《中国药典》对中成药质量控制的方法主要包括显微鉴别、薄层色谱鉴别、含量测定、指纹图谱等。然而，薄层色谱鉴别和含量测定仅可对中成药中是否含有目标化学成分进行定性或定量监测，并无法判定该目标化学成分是来源于中成药处方中的相应药材还是人为添加，致使对于生物物种组成的监测具有局限性。
DNA条形码分子鉴定法是利用基因组中一段公认的相对较短的DNA序列进行物种鉴定的一种分子生物学技术，是传统鉴定方法的有效补充。2015年版《中国药典》收载中药材DNA条形码分子鉴定法指导原则，将其作为中药材质量控制方法之一，现已获得广泛应用。单分子测序技术，又称第三代测序技术，包括单分子荧光测序和纳米孔测序两大类，具有读长长、速度快、精度高等特点。目前该技术主要应用于基因组测序、DNA甲基化研究、SNP检测等方面。因此，如何利用上述技术开发出一种能够对生物物种组成进行有效监测的方法，以克服目前对生物物种组成监测的局限性，从而对生物物种组成的质量进行有效控制，以维护临床用药安全将是本领域研究的重要方向。
发明内容
本发明旨在提供一种基于单分子测序技术及DNA条形码分子鉴定技术联用的生物物种组成的监测方法，能够对现有生物物种组成的质量控制方法进行有效补充，维护临床用药安全。
本发明的一方面提供了一种基于单分子测序技术及DNA条形码分子鉴定技术联用的生物物种组成的监测方法，包括如下步骤：
1)提取待检样品基因组DNA；
2)以步骤1)中的基因组DNA为模板，采用PCR方法扩增其目的DNA或cDNA序列，获得PCR产物；
3)基于单分子测序平台，并依照测序平台的标准操作流程，对步骤2)中获得的PCR产物进行文库构建以及单分子测序；
4)应用测序平台的数据处理软件对步骤3)中测得的单分子测序结果进行数据处理，获得可用于后续分析的目的DNA或cDNA序列；
5)将步骤4)中的目的DNA或cDNA序列放入DNA条形码数据库，进行BLAST比对，获得物种鉴定结果。
本发明的另一方面提供了在如上述技术方案所述的基于单分子测序技术及DNA条形码分子鉴定技术联用的生物物种组成的监测方法中使用的试剂盒，包括用于DNA提取、PCR扩增、PCR产物纯化、文库构建和测序步骤的相关试剂盒。
本发明提供了一种基于单分子测序技术及DNA条形码分子鉴定技术联用的生物物种组成的监测方法，该方法将单分子测序技术及DNA条形码分子鉴定技术联用，可检测出能够扩增出目的DNA或cDNA序列的中成药、草药制品或膳食补充剂样品中的生物物种组成组分，适用性广，可对现有生物物种组成的质量控制方法进行有效补充，从而达到维护临床用药安全的目的。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明一方面的实施例提供了一种基于单分子测序技术及DNA条形码分子鉴定技术联用的生物物种组成的监测方法，包括如下步骤：
S1：提取待检样品基因组DNA；
在本步骤中，应尽量去除待检样品中所含有的辅料，对于加工程度较高的样品，可增大取样量。
S2：以步骤1)中的基因组DNA为模板，采用PCR方法扩增其目的DNA或cDNA序列，获得PCR产物；
在本步骤中，需对所获得的PCR产物进行纯化后才可用于下一步骤的测序中。
S3：基于单分子测序平台，并依照测序平台的标准操作流程，对步骤2)中获得的PCR产物进行文库构建以及单分子测序；
在本步骤中，使用步骤2)纯化获得的PCR产物进行文库构建，若步骤2)仅获得1个PCR纯化产物，则直接构建文库并上机测序；若步骤2)获得两个及以上PCR纯化产物，可将各个产物合并后建库，亦可分别建库，每个文库分别上机测序。
S4：应用测序平台的数据处理软件对步骤3)中测得的单分子测序结果进行数据处理，获得可用于后续分析的目的DNA或cDNA序列；
在本步骤中，若获得两个及以上文库的测序结果，需分别进行数据处理。
可以理解的是，在本步骤中所得到的目的DNA或cDNA序列可通过步骤1)-3)获得，但在其它实施例中也可通过其它适合的方式获得，而不仅仅局限于此。
S5：将步骤4)中的目的DNA或cDNA序列放入DNA条形码数据库，进行BLAST比对，获得物种鉴定结果。
在一可选实施例中，步骤2)中扩增的目的DNA或cDNA序列选自ITS2序列、psbA-trnH序列、ITS序列、rbcL序列、matK序列和COI序列中的一种或多种。在一优选实施例中，步骤2)中扩增的目的DNA或cDNA序列选自ITS2序列和/或psbA-trnH序列。在下述表1中列出了扩增各目的DNA或cDNA序列时所使用的引物及相应的引物序列，如下：
表1扩增各目的DNA或cDNA序列时所使用的引物及相应的引物序列
Figure PCTCN2016102293-appb-000001
Figure PCTCN2016102293-appb-000002
可以理解的是，本实施例并不局限于上述所列举的序列，还可以是本领域内已知的、适合用于上述方法的其它序列，只要能够扩增出目的DNA或cDNA序列的生物物种均可适用于本方法。
在一可选实施例中，所述单分子测序平台选自PacBio RSII测序平台、Heliscope单分子测序平台和MinION测序平台中的任意一种。可以理解的是，本实施例并不局限于上述所列举的测序平台，还可以是本领域内已知的、适合用于上述方法的其它测序平台。
在一可选实施例中，在选用PacBio RSII测序平台时，其数据处理步骤为：应用SMRT Analysis Server软件对原始数据进行过滤，并基于RS_ReadsOfInsert.1软件获得环状一致性序列，用CD-HIT软件将所述环状一致性序列进行聚类，获得可用于后续分析的目的DNA或cDNA序列。在本实施例中，给出在选用PacBio RSII测序平台时的具体处理步骤，同理，在选用其它测序平台时，可根据相应选择的测序平台进行相应的数据处理以及后续相关操作。
在一可选实施例中，DNA提取和PCR扩增依照“中药材DNA条形码分子鉴定指导原则”进行。在本实施例中，给出了DNA提取和PCR扩增步骤的操作原则，但可以理解的是，本实施例中并不局限于上述所依据的原则，还可以是本领域内已知的其它适合的指导原则或方法。
在一可选实施例中，所述监测方法适用于中成药、草药制品或膳食补充剂中任意一种中的生物物种组成的监测。
本发明另一方面的实施例提供了一种在如上述任一项实施例所述的基于单分子测序技术及DNA条形码分子鉴定技术联用的生物物种组成的监测方法 中使用的试剂盒，包括用于DNA提取、PCR扩增、PCR产物纯化、文库构建和测序步骤的相关试剂盒。
为了能够更详细地描述本发明实施例所提供的基于单分子测序技术及DNA条形码分子鉴定技术联用的生物物种组成的监测方法，下面将结合具体实施例，并以ITS2序列和psbA-trnH序列为例进行示例性说明，具体的，实施例1-5以中成药九味羌活丸为例进行多角度分析，实施例6-10以中成药益母丸为例进行多角度分析。
实施例1
1.材料：
试验材料为中成药九味羌活丸，本实施例所用材料为依照《中国药典》中该中成药处方的规定剂量及制法，在实验室自制。此外，为验证本方法的灵敏度，根据该处方中剂量最小的一味药材细辛的用量，加入等量人参药材作为阳性对照。
2.实验步骤：
1)中成药九味羌活丸处方由羌活、防风、黄芩、细辛、地黄、川芎、苍术、白芷和甘草共9味中药组成。本实施例所用到的羌活、防风、黄芩、细辛、地黄、川芎均已经过形态学及DNA条形码鉴定，而苍术和白芷由于未能获得DNA条形码序列，由形态学鉴定其物种，以确保各物种的准确性。依照《中国药典》规定剂量及制法，将上述药材及阳性对照药材人参混合粉碎并过筛，制成丸剂。
2)DNA提取
取120mg上述样品，用MM400高通量组织研磨仪(德国Retsch)研磨，用核分离液漂洗至上清液无色，用植物基因组DNA提取试剂盒(天根生化科技(北京)有限公司)提取基因组DNA。
3)PCR扩增及产物回收
以所提取的DNA为模板，分别扩增其ITS2序列和psbA-trnH序列。
具体的，扩增ITS2序列所使用的引物序列为：
正向引物：ATGCGATACTTGGTGTGAAT；
反向引物：GACGCTTCTCCAGACTACAAT；
扩增psbA-trnH序列所使用的引物序列为：
正向引物：GTTATGCATGAACGTAATGCTC；
反向引物：CGCGCATGGTGGATTCACAATCC，由生工生物工程有限公司(北京)合成。
引物用无菌去离子溶解并稀释至2.5μmol/μL。
25μL反应体系：2×PCR MasterMix 12.5μL，正反向引物各1.0μL(2.5μmol/L)，模板DNA 20～100ng，加灭菌双蒸水至25μL，进行PCR扩增。
PCR反应程序：在PCR仪上按照以下程序进行ITS2序列和psbA-trnH序列扩增反应：
Figure PCTCN2016102293-appb-000003
PCR产物经凝胶电泳后，用QIAquick Gel Extraction Kit(QIAGEN N.V.,德国)进行切胶回收，获得相应的PCR纯化产物。
5)文库构建、SMRT测序及数据处理
本实施例基于PacBio RSII测序平台，依照该平台标准操作流程，对上述PCR产物进行文库构建，并进行SMRT测序。
具体的，可应用SMRT Analysis Server 2.3.0软件对原始数据进行过滤，并基于RS_ReadsOfInsert.1操作流程获得环状一致性序列(circular-consensus sequencing reads,CCS reads)，获得可用于后续分析的ITS2序列和psbA-trnH序列。
6)物种鉴定
将上述得到的ITS2序列和psbA-trnH序列放入中药材DNA条形码鉴定系统(http://www.tcmbarcode.cn/china/)，进行BLAST比对，获得物种鉴定结果。在本实施例中，将所得ITS2序列和psbA-trnH序列经BLAST比对后，结 果如下：
ITS2序列能够检出羌活、防风、细辛、川芎、地黄、甘草。
psbA-trnH序列能够检出羌活、川芎、黄芩、甘草以及阳性对照药材人参。
将ITS2序列与psbA-trnH序列结合，未检出苍术、白芷，这与实验步骤1)中DNA条形码鉴定结果一致。
该结果显示本发明所公开的基于SMRT测序技术的中成药生物物种组成监测方法能够用于实验室自制中成药九味羌活丸的物种组成监测，具有良好的可行性。
实施例2
1.材料：
基本同实施例1，所不同的是没有加入实施例1中的阳性对照药材人参。
2.实验步骤：
同实施例1。
将本实施例所得ITS2序列和psbA-trnH序列经BLAST比对后，结果如下：
ITS2序列能够检出羌活、防风、细辛、川芎、地黄、甘草。
psbA-trnH序列能够检出羌活、川芎、黄芩、甘草。
将ITS2序列与psbA-trnH序列结合，未检出苍术、白芷，与实施例1一致。
该结果显示本发明所公开的基于SMRT测序技术的中成药生物物种组成监测方法能够用于实验室自制中成药九味羌活丸的物种组成监测，具有良好的可行性。
实施例3
1.材料：
试验材料为由某制药厂生产的具备药品批准文号的中成药九味羌活丸(批号：20141101)，购买自某药店。
2.实验步骤：
基本同实施例1，所不同的是本实施例由DNA提取步骤开始，逐步完成 实验操作和数据处理，直至物种鉴定。
将本实施例所得ITS2序列和psbA-trnH序列经BLAST比对后，结果如下：
ITS2序列能够检出羌活、苍术、细辛、川芎、甘草。
psbA-trnH序列能够检出羌活、防风、苍术、川芎、黄芩、甘草。
将ITS2序列与psbA-trnH序列结合，未检出地黄、白芷。其原因可能为，本实施例所用中成药试验样品中上述两个物种DNA降解严重，或该中成药在制剂过程中未按照《中国药典》规定加入上述两种中药材的粉末。
该结果显示本发明所公开的基于SMRT测序技术的中成药生物物种组成监测方法能够用于市售中成药九味羌活丸的物种组成监测，具有良好的可行性。
实施例4
1.材料：
试验材料为由某制药厂生产的具备药品批准文号的中成药九味羌活丸(批号：20140501)，购买自某药店。
2.实验步骤：
基本同实施例1，所不同的是本实施例由DNA提取步骤开始，逐步完成实验操作和数据处理，直至物种鉴定。
将本实施例所得ITS2序列和psbA-trnH序列经BLAST比对后，结果如下：
ITS2序列能够检出羌活、防风、苍术、细辛、川芎、甘草。
psbA-trnH序列能够检出川芎、甘草。
将ITS2序列与psbA-trnH序列结合，未检出黄芩、地黄、白芷。其原因可能为，本实施例所用中成药试验样品中上述三个物种DNA降解严重，或该中成药在制剂过程中未按照《中国药典》规定加入上述三种中药材的粉末。
该结果显示本发明所公开的基于SMRT测序技术的中成药生物物种组成监测方法能够用于市售中成药九味羌活丸的物种组成监测，具有良好的可行性。
实施例5
1.材料：
试验材料为由某制药厂生产的具备药品批准文号的中成药九味羌活丸(批号：20140501)，购买自某药店。
2.实验步骤：
基本同实施例1，所不同的是本实施例由DNA提取步骤开始，逐步完成实验操作和数据处理，直至物种鉴定。
将本实施例所得ITS2序列和psbA-trnH序列经BLAST比对后，结果如下：
ITS2序列能够检出羌活、防风、苍术、细辛、川芎、甘草。
psbA-trnH序列能够检出羌活、苍术、川芎、黄芩、甘草。
将ITS2序列与psbA-trnH序列结合，未检出地黄、白芷。其原因可能为，本实施例所用中成药试验样品中上述两个物种DNA降解严重，或该中成药在制剂过程中未按照《中国药典》规定加入上述两种中药材的粉末。
该结果显示本发明所公开的基于SMRT测序技术的中成药生物物种组成监测方法能够用于市售中成药九味羌活丸的物种组成监测，具有良好的可行性。
实施例6
1.材料：
试验材料为中成药益母丸，本实施例所用材料为依照《中国药典》中该中成药处方的规定剂量及制法，在实验室自制。此外，为验证本方法的灵敏度，根据该处方中剂量最小的一味药材木香的用量，加入等量人参药材作为阳性对照。
2.实验步骤：
1)中成药益母丸处方由益母草、川芎、当归和木香共4味中药组成。本实施例所用到的各个药材均已经过形态学及DNA条形码鉴定，以确保其物种的准确性。依照《中国药典》规定剂量及制法，将上述药材及阳性对照药材人参混合粉碎并过筛，制成丸剂。
2)DNA提取
取120mg上述样品，用MM400高通量组织研磨仪(德国Retsch)研磨， 用核分离液漂洗至上清液无色，用植物基因组DNA提取试剂盒(天根生化科技(北京)有限公司)提取基因组DNA。
3)PCR扩增及产物回收
以所提取的DNA为模板，分别扩增其ITS2序列和psbA-trnH序列。
具体的，扩增ITS2序列所使用的引物序列为：
正向引物：ATGCGATACTTGGTGTGAAT；
反向引物：GACGCTTCTCCAGACTACAAT；
扩增psbA-trnH序列所使用的引物序列为：
正向引物：GTTATGCATGAACGTAATGCTC；
反向引物：CGCGCATGGTGGATTCACAATCC，由生工生物工程有限公司(北京)合成。
引物用无菌去离子溶解并稀释至2.5μmol/μL。
25μL反应体系：2×PCR MasterMix 12.5μL，正反向引物各1.0μL(2.5μmol/L)，模板DNA 20～100ng，加灭菌双蒸水至25μL，进行PCR扩增。
PCR反应程序：在PCR仪上按照以下程序进行ITS2序列和psbA-trnH序列扩增反应：
Figure PCTCN2016102293-appb-000004
PCR产物经凝胶电泳后，用QIAquick Gel Extraction Kit(QIAGEN N.V.,德国)进行切胶回收，获得纯化产物。
5)文库构建、SMRT测序及数据处理
本实施例基于PacBio RSII测序平台，依照该平台标准操作流程，对上述PCR产物进行文库构建，并进行SMRT测序。
具体的，可应用SMRT Analysis Server 2.3.0软件对原始数据进行过滤， 并基于RS_ReadsOfInsert.1操作流程获得环状一致性序列(circular-consensus sequencing reads,CCS reads)，获得可用于后续分析的ITS2序列和psbA-trnH序列。
6)物种鉴定
将上述序列放入中药材DNA条形码鉴定系统(http://www.tcmbarcode.cn/china/)，进行BLAST比对，获得物种鉴定结果。本实施例所得ITS2序列和psbA-trnH序列经BLAST比对后，结果如下：
ITS2序列能够检出益母草、川芎、当归、木香以及阳性对照药材人参。
psbA-trnH序列能够检出益母草、川芎、当归。
ITS2序列与psbA-trnH序列结合，均能检出处方中四种药材，这与实验步骤1)中DNA条形码鉴定结果一致。
该结果显示本发明所公开的基于SMRT测序技术的中成药生物物种组成监测方法能够用于实验室自制中成药益母丸的物种组成监测，具有良好的可行性。
实施例7
1.材料：
基本同实施例6，所不同的是没有加入实施例6中的阳性对照药材人参。
2.实验步骤：
同实施例6。
本实施例所得ITS2序列和psbA-trnH序列经BLAST比对后，结果如下：
ITS2序列能够检出益母草、川芎、当归、木香。
psbA-trnH序列能够检出益母草、川芎、当归。
ITS2序列与psbA-trnH序列结合，均能检出处方中四种药材。与实施例6一致。
该结果显示本发明所公开的基于SMRT测序技术的中成药生物物种组成监测方法能够用于实验室自制中成药益母丸的物种组成监测，具有良好的可行性。
实施例8
1.材料：
试验材料为由某制药厂生产的具备药品批准文号的中成药益母丸(批号：3015152)，购买自某药店。
2.实验步骤：
基本同实施例6，所不同的是本实施例由DNA提取步骤开始，逐步完成实验操作和数据处理，直至物种鉴定。
本实施例所得ITS2序列和psbA-trnH序列经BLAST比对后，结果如下：
ITS2序列能够检出益母草、川芎、当归、木香。
psbA-trnH序列能够检出益母草、川芎、当归。
ITS2序列与psbA-trnH序列结合，均能检出处方中四种药材。
该结果显示本发明所公开的基于SMRT测序技术的中成药生物物种组成监测方法能够用于市售中成药益母丸的物种组成监测，具有良好的可行性。
实施例9
1.材料：
试验材料为由某制药厂生产的具备药品批准文号的中成药益母丸(批号：2015395)，购买自某药店。
2.实验步骤：
基本同实施例6，所不同的是本实施例由DNA提取步骤开始，逐步完成实验操作和数据处理，直至物种鉴定。
本实施例所得ITS2序列和psbA-trnH序列经BLAST比对后，结果如下：
ITS2序列能够检出益母草、川芎、当归。
psbA-trnH序列能够检出益母草、川芎、当归。
将ITS2序列与psbA-trnH序列结合，未检出木香。其原因可能为，本实施例所用中成药试验样品中上述物种DNA降解严重，或该中成药在制剂过程中未按照《中国药典》规定加入上述两种中药材的粉末。
该结果显示本发明所公开的基于SMRT测序技术的中成药生物物种组成监测方法能够用于市售中成药益母丸的物种组成监测，具有良好的可行性。
实施例10
1.材料：
试验材料为由某制药厂生产的具备药品批准文号的中成药益母丸(批号： 2015616)，购买自某药店。
2.实验步骤：
基本同实施例6，所不同的是本实施例由DNA提取步骤开始，逐步完成实验操作和数据处理，直至物种鉴定。
本实施例所得ITS2序列和psbA-trnH序列经BLAST比对后，结果如下：
ITS2序列能够检出益母草、川芎、当归。
psbA-trnH序列能够检出益母草、川芎、当归。
将ITS2序列与psbA-trnH序列结合，未检出木香。其原因可能为，本实施例所用中成药试验样品中上述物种DNA降解严重，或该中成药在制剂过程中未按照《中国药典》规定加入上述两种中药材的粉末。
该结果显示本发明所公开的基于SMRT测序技术的中成药生物物种组成监测方法能够用于市售中成药益母丸的物种组成监测，具有良好的可行性。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (8)

  1. 一种基于单分子测序技术及DNA条形码分子鉴定技术联用的生物物种组成的监测方法，包括如下步骤：
    1)提取待检样品基因组DNA；
    2)以步骤1)中的基因组DNA为模板，采用PCR方法扩增其目的DNA或cDNA序列，获得PCR产物；
    3)基于单分子测序平台，并依照测序平台的标准操作流程，对步骤2)中获得的PCR产物进行文库构建以及单分子测序；
    4)应用测序平台的数据处理软件对步骤3)中测得的单分子测序结果进行数据处理，获得可用于后续分析的目的DNA或cDNA序列；
    5)将步骤4)中的目的DNA或cDNA序列放入DNA条形码数据库，进行BLAST比对，获得物种鉴定结果。
  2. 权利要求1所述的生物物种组成的监测方法，其中，步骤2)中扩增的目的DNA或cDNA序列选自ITS2序列、psbA-trnH序列、ITS序列、rbcL序列、matK序列和COI序列中的一种或多种。
  3. 权利要求2所述的生物物种组成的监测方法，其中，步骤2)中扩增的目的DNA或cDNA序列选自ITS2序列和/或psbA-trnH序列。
  4. 根据权利要求1所述的生物物种组成的监测方法，其中，所述单分子测序平台选自PacBio RSII测序平台、Heliscope单分子测序平台和MinION测序平台中的任意一种。
  5. 根据权利要求4所述的生物物种组成的监测方法，其中，在选用PacBio RSII测序平台时，其数据处理步骤为：应用SMRT Analysis Server软件对原始数据进行过滤，并基于RS_ReadsOfInsert.1软件获得环状一致性序列，用CD-HIT软件将所述环状一致性序列进行聚类，获得可用于后续分析的目的DNA或cDNA序列。
  6. 权利要求1所述的生物物种组成的监测方法，其中，DNA提取和PCR扩增依照“中药材DNA条形码分子鉴定指导原则”进行。
  7. 根据权利要求1所述的生物物种组成的监测方法，其中，所述监测方 法适用于中成药、草药制品或膳食补充剂中任意一种中的生物物种组成的监测。
  8. 一种在如权利要求1-7任一项所述的基于单分子测序技术及DNA条形码分子鉴定技术联用的生物物种组成的监测方法中使用的试剂盒，包括用于DNA提取、PCR扩增、PCR产物纯化、文库构建和测序步骤的相关试剂盒。
PCT/CN2016/102293 2016-10-18 2016-10-18 一种基于单分子测序技术及dna条形码分子鉴定技术联用的生物物种组成的监测方法 WO2018072064A1 (zh)

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