CN106834497B - Method and kit for identifying cynomorium songaricum of different producing genuine medicinal materials - Google Patents
Method and kit for identifying cynomorium songaricum of different producing genuine medicinal materials Download PDFInfo
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- CN106834497B CN106834497B CN201710124288.7A CN201710124288A CN106834497B CN 106834497 B CN106834497 B CN 106834497B CN 201710124288 A CN201710124288 A CN 201710124288A CN 106834497 B CN106834497 B CN 106834497B
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Abstract
The invention relates to a method for identifying cynomorium songaricum of different producing genuine medicinal materials and a kit thereof, wherein the method comprises the following steps: 1) amplifying a segment containing a sequence shown in SEQ ID NO.1 by using genome DNA of a sample to be detected as a template; 2) sequencing the amplified product, removing a primer region after splicing to obtain a complete amplified fragment SEQ ID No.1, and detecting the 188 th, 304 th or 305 th base from the 5' end in the SEQ ID No.1 sequence; identifying the Tacheng cynomorium songaricum if the 188 th position from the 5' end of the SEQ ID NO.1 sequence is T, the 304 th position is A, or the 305 th position is C; if the 188 th position or the 304 th position or the 305 th position from the 5' end of the SEQ ID NO.1 sequence is C or T, then the other producing area is identified as cynomorium. The method has the advantages of wider adaptability, high primer specificity and high amplification efficiency, and can detect any cynomorium songaricum sample from which DNA can be extracted.
Description
Technical Field
The invention belongs to the technical field of identification of Chinese medicinal material source varieties, and particularly relates to an identification method of different producing areas of genuine medicinal materials cynomorium songaricum by using specific SNP markers.
Background
Cynomorium songaricum is dry fleshy stem of Cynomorium songaricum (Cynomorium songaricum Rupr.) of Cynomoriaceae, also known as Bulao Yao, Wulan high waist, herba Elephantopi scaberis, herba Cynomorii, etc. The fleshy stem of the inflorescence is removed for medicinal use. The fleshy stem is rich in tannin, can be used for extracting tannin extract, contains starch, and can be used for brewing wine, feed and food substitute. Has effects in invigorating kidney, replenishing vital essence, and moistening dryness, and can be used for treating sexual impotence, spermatorrhea, soreness of waist and knees, constipation due to intestinal dryness, menoxenia, infertility, and deficiency of qi and yin. Cynomorium songaricum is mainly produced in provinces such as Xinjiang, Qinghai, Gansu, Ningxia and inner Mongolia in China. Due to the difference of long-term growing environment and cultivation mode, the cynomorium songaricum forms certain intraspecific variation, thereby showing the difference in quality. The Xinjiang Tacheng area has mild climate, abundant rainfall and good quality of the produced cynomorium songaricum. The cynomorium songaricum in different producing areas has similar appearance characters, and is difficult to distinguish by traditional morphological identification, particularly difficult to identify medicinal material powder, decoction pieces, Chinese patent medicine and the like. The identification method utilizing the SNP marker can solve the problem of difficult identification of the genuine cynomorium songaricum, and has the advantages of accuracy, high efficiency, good repeatability and the like.
Disclosure of Invention
The invention aims to provide an SNP marker capable of accurately and quickly identifying cynomorium songaricum of different production areas, a primer pair, a kit and a detection method thereof, which comprise key reagents, primer pairs, templates and detection steps of an SNP detection method.
The SNP marker for identifying cynomorium songaricum in different producing areas has a nucleotide sequence shown as SEQ ID No.1, wherein only three SNPs exist in the sequence shown as SEQ ID No. 1: the 188 th site of the cynomorium songaricum from the 5' end of the sequence of SEQ ID NO.1 is T, and the cynomorium songaricum of other producing areas (the cynomorium songaricum of non-Tacheng producing areas is referred to herein, the same applies below) is C; the 304 th tower city cynomorium songaricum is A, and other producing areas cynomorium songaricum are T; the 305 th site of the Cynomorium songaricum is C, and the other producing sites of the Cynomorium songaricum are T.
The SNP marker provided by the invention can be used for identifying cynomorium songaricum in different producing areas.
Identifying the Tacheng cynomorium songaricum if the 188 th position from the 5' end of the SEQ ID NO.1 sequence is T, the 304 th position is A or the 305 th position is C;
identifying other producing areas as cynomorium songaricum if the 188 th position or the 304 th position or the 305 th position from the 5' end of the SEQ ID NO.1 sequence is C or T;
identifying the sample as not being cynomorium if the 188 th position from the 5' end of the sequence of SEQ ID No.1 is neither T nor C, and the 304 th position is neither A nor T, and the 305 th position is neither C nor T.
The invention also provides a cynomorium songaricum identification method in different producing areas, which comprises the following steps:
1) amplifying a segment containing a sequence shown in SEQ ID NO.1 by using genome DNA of a sample to be detected as a template;
2) sequencing the amplified product, removing a primer region after splicing to obtain a complete ITS fragment, and determining the 188 th, 304 th or 305 th base from the 5' end in the SEQ ID NO.1 sequence by analyzing the sequence shown in SEQ ID NO. 1.
Identifying the Tacheng cynomorium songaricum if the 188 th position from the 5' end of the SEQ ID NO.1 sequence is T, the 304 th position is A, or the 305 th position is C;
identifying other producing areas as cynomorium songaricum if the 188 th position or the 304 th position or the 305 th position from the 5' end of the SEQ ID NO.1 sequence is C or T;
identifying the sample as not being cynomorium if the 188 th position from the 5' end of the sequence of SEQ ID No.1 is neither T nor C, and the 304 th position is neither A nor T, and the 305 th position is neither C nor T.
The primers used for amplifying the sequence segment shown in SEQ ID NO.1 are as follows:
SY1F 5’-AAGGAAGCAGCAGCACATTGAGT-3’
SY1R 5’-AACCTGCGGAAGGATCATTGTTG-3’
the primers can also be used for identifying cynomorium songaricum in different producing areas.
The invention also provides a kit containing the primer.
The invention provides application of the primer or the kit in cynomorium songaricum identification.
The SNP provided by the invention can accurately distinguish the cynomorium songaricum from other cynomorium songaricum producing areas, the method provided by the invention has wider adaptability, the primer has high specificity and high amplification efficiency, and any sample of the cynomorium songaricum from which DNA can be extracted can be detected. Can realize the rapid identification of the original plants, medicinal materials, seeds, powder, decoction pieces and Chinese patent medicines of the Tacheng cynomorium songaricum.
Drawings
FIG. 1 is a diagram of interspecific variation of Cynomorium songaricum from different producing areas.
FIG. 2 is a gel diagram of the amplification of primers SY1F-SY 1R.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.
Example 1 identification method of Cynomorium songaricum raw materials in different producing areas
1) 46 parts of cynomorium songaricum samples from different production places (see table 1) are collected, 25mg of the medicinal materials are respectively ground on an MM400 ball mill (Retsch, Germany), and total DNA is extracted by using a plant genome DNA kit of Tiangen Biochemical technology (Beijing) Co., Ltd.
TABLE 1 Suo-yang samples from different producing areas
2) PCR amplification
The primer sequence is SY1F 5'-AAGGAAGCAGCAGCACATTGAGT-3',
SY1R 5’-AACCTGCGGAAGGATCATTGTTG-3’，
synthesized by Shanghai Meiji biological medicine science and technology Co., Ltd. (Beijing).
The PCR amplification procedure was:
the PCR reaction system is as follows:
PCR Buffer(10×)2.5u L，Mg2+2U L (25mmol/L), dNTPs mixture 2U L (2.5mmol/L), primers 1U L (2.5umol/L), template DNA 2U L (about 30ng), Taq DNA polymerase 1.0U, sterile double distilled water to 25U L, PCR amplification.
3) Agarose gel electrophoresis
The PCR amplification products of 3u L were each collected, electrophoresed on 1% agarose gel, and the results were examined on a gel imager after electrophoresis (see FIG. 2, in which numbers 1 to 10 are TC1, TC2, HT1, HT2, AL1, AL2, ZY1, ZY2, GR1, and GR2, respectively).
4) Sequencing
The PCR product was directly sent to Meiji Biomedicine science and technology Limited (Beijing) of Shanghai for sequencing. The sequencing primer is the same as the PCR primer. To ensure sequence reliability, square bidirectional sequencing was performed.
5) Sequence splicing
This example applies the software CodonCode Aligner 3.7.1(CodonCode Co., Germany) to perform sequence alignment and proofreading. Firstly, sequence quality evaluation and pretreatment are carried out, namely two parts of addresses at two ends of a sequencing result are removed, and the quality of the rest part is evaluated. Respectively sliding from the 5 'end to the 3' end of the sequence by a 20bp window, deleting one base if the Q value of more than 2 bases in the window is less than 20, continuing sliding the window, and stopping sliding if the number of the Q values of the bases in the window which are less than 20 is less than or equal to 2. This operation was repeated from the 3' end. The cut forward and reverse sequences are more than 100bp, the number of low-quality bases of the forward and reverse sequences is less than 1%, the length of the cut sequence is less than 50% of the original sequence, and the average Q value is more than or equal to 30. And splicing the forward and reverse sequencing results to ensure that the forward and reverse sequences have more than 50% of overlapping regions.
6) Sequence alignment
Comparing the sequenced and spliced samples by MEGA5 software to obtain 3 SNP loci, wherein the 188 th site of the Tacheng cynomorium songaricum from the 5' end of the SEQ ID NO.1 sequence of all the Tacheng cynomorium songaricum is T, and the other producing areas of the Tacheng cynomorium songaricum are C; the 304 th tower city cynomorium songaricum is A, and other producing areas cynomorium songaricum are T; the 305 th site of the cynomorium songaricum is C, other producing areas of the cynomorium songaricum are T, and the sequencing result is shown in figure 1.
Example 2 identification method of cynomorium songaricum decoction pieces
Basically the same as example 1, except that cynomorium songaricum decoction pieces in different producing areas are taken as samples, DNA is extracted, and identification is carried out, so that the 3 SNP sites can be specifically detected, all the cynomorium songaricum 188 th site from the 5' end of the SEQ ID NO.1 sequence of the cynomorium songaricum is T, and the cynomorium songaricum in other producing areas is C; the 304 th tower city cynomorium songaricum is A, and other producing areas cynomorium songaricum are T; the 305 th site of the Cynomorium songaricum is C, and the other producing sites of the Cynomorium songaricum are T. The sequencing results are the same as in FIG. 1.
EXAMPLE 3 identification of Cynomorium songaricum powder
Basically the same as example 1, except that cynomorium songaricum powder in different producing areas is taken as a sample, DNA is extracted, and identification is carried out, so that the 3 SNP sites can be specifically detected, all the talcheng cynomorium songaricum 188 th site from the 5' end of the sequence of SEQ ID NO.1 of the talcheng cynomorium songaricum is T, and other producing areas of cynomorium songaricum are C; the 304 th tower city cynomorium songaricum is A, and other producing areas cynomorium songaricum are A; the 305 th site of the Cynomorium songaricum is C, and the other producing sites of the Cynomorium songaricum are T. The sequencing results are the same as in FIG. 1.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> institute of medicinal plants of academy of Chinese medical science
<120> method for identifying cynomorium songaricum of different producing genuine medicinal materials and kit thereof
<130>KHP171110599.4
<160>3
<170>PatentIn version 3.3
<210>1
<211>410
<212>DNA
<213> cynomorium songaricum
<400>1
gacactcagg cagatgtgcc ctcgacctac aggccttggg cgcaacttgc gttcaaatac 60
tcgatggttc acgggattct gcaattcaca ccaagtatcg catttcgcta cgttcttcat 120
cgatgcaaga gccaagatat ccgttgtcga gagtccttga gattccaaat cgcatcacgc 180
gtgcaactgt ttcgtggcgc acgcgcgagc cacttctagt atgccttgac gctgttcgcg 240
ccggaatttg ttacacgccc aagagaacac acgcatgtct cgaaagtttt agaagcgaga 300
cacacttgga gcttcttcgg cgaaagggca agagtgaggc ccaacaggtt gcccacatct 360
caccccgctt aacgctacag gttaccccag ttgcactgct atgcaagttt 410
<210>2
<211>23
<212>DNA
<213> Artificial sequence
<400>2
aaggaagcag cagcacattg agt 23
<210>3
<211>23
<212>DNA
<213> Artificial sequence
<400>3
aacctgcgga aggatcattg ttg 23
Claims (7)
1. The SNP marker for identifying cynomorium songaricum is characterized in that the nucleotide sequence is shown as SEQ ID No.1, wherein the 188 th site from the 5' end of the SEQ ID No.1 sequence is T or C; position 304 is A or T; position 305 is C or T.
2. The use of SNP marker for identifying Cynomorium songaricum according to claim 1 for identifying Cynomorium songaricum,
identifying the Tacheng cynomorium songaricum if the 188 th position from the 5' end of the SEQ ID NO.1 sequence is T, the 304 th position is A or the 305 th position is C;
if the 188 th position or the 304 th position or the 305 th position from the 5' end of the SEQ ID NO.1 sequence is C or T, then the other producing area is identified as cynomorium.
3. A different-producing-area cynomorium songaricum identification method comprises the following steps:
1) amplifying a segment containing a sequence shown in SEQ ID NO.1 by using genome DNA of a sample to be detected as a template;
2) sequencing the amplified product, removing a primer region after splicing to obtain a complete ITS fragment, and determining the 188 th, 304 th or 305 th base from the 5' end in the sequence of SEQ ID NO.1 by analyzing the sequence shown in SEQ ID NO. 1.
4. The identification method according to claim 3,
identifying the Tacheng cynomorium songaricum if the 188 th position from the 5' end of the SEQ ID NO.1 sequence is T, the 304 th position is A, or the 305 th position is C;
if the 188 th position or the 304 th position or the 305 th position from the 5' end of the SEQ ID NO.1 sequence is C or T, then the other producing area is identified as cynomorium.
5. The method of claim 3 or 4, wherein the primer used for amplification is
SY1F 5’-AAGGAAGCAGCAGCACATTGAGT-3’
SY1R 5’-AACCTGCGGAAGGATCATTGTTG-3’。
6. The method according to claim 3 or 4, wherein the sample to be tested comprises Cynomorium tarum, other original plants of Cynomorium songaricum of origin, herbs, seeds, powder, decoction pieces and Chinese patent drugs.
7. Use of the method of any one of claims 3-6 in the identification of cynomorium.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102181538A (en) * | 2011-03-23 | 2011-09-14 | 内蒙古大学 | Method for assisting in identifying cynomorium plant and special primers thereof |
| CN102888456A (en) * | 2012-09-21 | 2013-01-23 | 中国医学科学院药用植物研究所 | Method for quickly identifying pseudo-ginseng |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102181538A (en) * | 2011-03-23 | 2011-09-14 | 内蒙古大学 | Method for assisting in identifying cynomorium plant and special primers thereof |
| CN102888456A (en) * | 2012-09-21 | 2013-01-23 | 中国医学科学院药用植物研究所 | Method for quickly identifying pseudo-ginseng |
Non-Patent Citations (1)
| Title |
|---|
| 中药材锁阳的ITS2条形码分子鉴定研究;侯典云等;《中国中药杂志》;20131231;第38卷(第23期);4028-4032 * |
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