Chinese patent medicine biological species constituent component monitoring method based on SMRT (single molecule real-time) sequencing technique

A technology for composition and Chinese patent medicine, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problem of inability to determine the source of target chemical components, and achieve the effect of wide applicability and maintenance of safety

Inactive Publication Date: 2017-03-15
INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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  • Abstract
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  • Claims
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Problems solved by technology

However, thin-layer chromatography identification and content determination can only be used for qualitative or quantitative monitoring of the target chemical components in Chinese patent medicines, and it is impossible to determine whether the target chemical components are derived from the corresponding medicinal materials in the prescription of Chinese patent medicines or artificially added.

Method used

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  • Chinese patent medicine biological species constituent component monitoring method based on SMRT (single molecule real-time) sequencing technique

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] 1. Materials:

[0023] The test material is the Chinese patent medicine Jiuweiqianghuo Pills. The materials used in this example are self-made in the laboratory according to the prescribed dosage and preparation method of the prescription of this Chinese patent medicine in the "Chinese Pharmacopoeia". In addition, in order to verify the sensitivity of this method, an equal amount of ginseng was added as a positive control according to the dosage of the herb Asarum with the smallest dose in the prescription.

[0024] 2. Experimental steps:

[0025] 1) The prescription of Jiuwei Qianghuo Pills, a Chinese patent medicine, consists of 9 traditional Chinese medicines including Notopterygium, Fangfeng, Scutellaria, Asarum, Dihuang, Chuanxiong, Cangzhu, Angelica dahurica and Licorice. Each medicinal material used in this example has been identified by morphology and DNA barcode to ensure the accuracy of its species. Since the DNA barcode sequence of Atractylodes Rhizome and A...

Embodiment 2

[0044] 1. Materials:

[0045] Basically the same as Example 1, the difference is that the positive control drug ginseng in Example 1 is not added.

[0046] 2. Experimental steps:

[0047] With embodiment 1.

[0048] After comparing the ITS2 sequence obtained in this example with the psbA-trnH sequence by BLAST, the results are as follows:

[0049] ITS2 sequence can detect Notopterygium, Fangfeng, Asarum, Chuanxiong, Rehmannia, Licorice.

[0050] The psbA-trnH sequence can detect notopterygium, chuanxiong, scutellaria baicalensis and licorice.

[0051] The combination of ITS2 sequence and psbA-trnH sequence failed to detect Atractylodes atractylodes and Angelica dahurica. Consistent with Example 1.

[0052] The results show that the method for monitoring biological species composition of Chinese patent medicines based on SMRT sequencing technology disclosed in the present invention can be used to monitor the species composition of Chinese patent medicine Jiuweiqianghuo Pil...

Embodiment 3

[0054] 1. Materials:

[0055] The test material is Jiuwei Qianghuo Pills (batch number: 20141101), a Chinese patent medicine with drug approval number, produced by a pharmaceutical factory and purchased from a pharmacy.

[0056] 2. Experimental steps:

[0057] Basically the same as Example 1, the difference is that this example starts from the DNA extraction step, and gradually completes the experimental operation and data processing until species identification.

[0058] After comparing the ITS2 sequence obtained in this example with the psbA-trnH sequence by BLAST, the results are as follows:

[0059] ITS2 sequence can detect Notopterygium, Atractylodes Rhizome, Asarum, Chuanxiong and Licorice.

[0060] psbA-trnH sequence can detect Notopterygium, Fangfeng, Cangzhu, Chuanxiong, Scutellaria baicalensis and Licorice.

[0061] The combination of ITS2 sequence and psbA-trnH sequence failed to detect rehmannia root and Angelica dahurica. The reason may be that the DNA of the ab...

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Abstract

The invention discloses a Chinese patent medicine biological species constituent component monitoring method based on an SMRT (single molecule real-time) sequencing technique. The Chinese patent medicine biological species constituent component monitoring method comprises the following steps: 1) extracting genome DNA of a sample to be detected; 2) by taking the DNA as a template, amplifying an ITS2 sequence and a psbA-trnH sequence of the DNA; 3) based on a Pacbio RSII sequencing platform, performing library construction on a PCR (polymerase chain reaction) product according to standard operation procedures of the platform, and performing SMRT sequencing; 4) filtering original data by using SMRT Analysis Server 2.3.0 software, obtaining circular-consensus sequencing (CCS) on the basis of the RS_ReadsOfInsert.1 operation procedures, and clustering by using the CCS sequence by using CD-HIT (cluster database at high identity with tolerance) software, so as to obtain an ITS2 sequence and a psbA-trnH sequence for later analysis finally; 5) putting the sequences into a traditional Chinese medicinal material DNA bar code identification system (http: / / www.tcmbarcode.cn / china / ), performing BLAST(basic linear algebra subprograms) comparison, thereby obtaining species identification results. By adopting the method, monitoring on Chinese patent medicine biological species constituent components can be achieved.

Description

technical field [0001] The invention belongs to the technical field of quality control of Chinese patent medicines, and in particular relates to a method for monitoring biological species composition of Chinese patent medicines by using SMRT sequencing technology and DNA barcode molecular identification technology. Background technique [0002] The current version of "Chinese Pharmacopoeia" mainly includes microscopic identification, thin-layer chromatography identification, content determination, fingerprinting, etc. for the quality control methods of Chinese patent medicines. However, thin-layer chromatography identification and content determination can only be used to qualitatively or quantitatively monitor whether the target chemical component is contained in the Chinese patent medicine, and cannot determine whether the target chemical component is derived from the corresponding medicinal material in the Chinese patent medicine prescription or artificially added. [000...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6869C12Q2531/113C12Q2563/185
Inventor 宋经元辛天怡徐志超贾静
Owner INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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