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Article(id=1208491447133782952, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1208491433888166474, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2021-0230, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1613232000000, receivedDateStr=2021-02-14, revisedDate=1615737600000, revisedDateStr=2021-03-15, acceptedDate=null, acceptedDateStr=null, onlineDate=1766056413681, onlineDateStr=2025-12-18, pubDate=1631376000000, pubDateStr=2021-09-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1766056413681, onlineIssueDateStr=2025-12-18, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1766056413681, creator=13701087609, updateTime=1766056413681, updator=13701087609, issue=Issue{id=1208491433888166474, tenantId=1146029695717560320, journalId=1189982191388893191, year='2021', volume='56', issue='9', pageStart='2325', pageEnd='2596', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1766056410524, creator=13701087609, updateTime=1766137069648, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1208829742833332989, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1208491433888166474, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1208829742833332990, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1208491433888166474, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=2577, endPage=2583, ext={EN=ArticleExt(id=1208491447565796309, articleId=1208491447133782952, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Identification of Fritillaria taibaiensis and its relatives by real-time PCR with a TaqMan-MGB probe, columnId=1190335348761793317, journalTitle=Acta Pharmaceutica Sinica, columnName=Original Articles, runingTitle=null, highlight=null, articleAbstract=

The molecular identification of Fritillaria taibaiensis and its relatives was studied by real-time PCR with a TaqMan-MGB probe. DNA was extracted from F. taibaiensis and its relatives. According to the sequence of ITS1 region, the mutation sites of F. taipaiensis and its related species were identified by MEGA7.0 software. The specific primers (a pair) and a TaqMan-MGB probe were designed by Primer Premier 6.0 software. In the Roche LightCycler 96 system, the lowest limit of detection for F. taipaiensis DNA template was 0.002 39 ng·μL-1, and the optimal Tm value range was 60 and 61℃. Specificity identification showed that the method had good specificity for F. taipaiensis, as it could be distinguished from other 13 different Fritillaria species including F. unibracteata. Since this method could accurately identify F. taipaiensis and its related species, it provides technical support for rational development of F. taipaiensis resources, management of Chinese medicinal market and supervision of raw materials in Chinese medicine manufacturing enterprises.

, correspAuthors=Jia-yu ZHOU, Hai LIAO, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2021 Acta Pharmaceutica Sinica. All rights reserved., copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=Tian ZHANG, Jiao CHEN, Rui-ping JIANG, Meng ZOU, Tie-chui YANG, Shao-bing FU, Jia-yu ZHOU, Hai LIAO), CN=ArticleExt(id=1208491449906217158, articleId=1208491447133782952, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=TaqMan-MGB实时荧光PCR法检测太白贝母及其近缘种的研究, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

采用TaqMan-MGB探针实时荧光PCR方法对太白贝母及其近缘种进行分子鉴定研究。提取太白贝母及其近缘种的DNA，依据ITS1区序列，利用MEGA7.0软件进行比较分析，找出太白贝母及其近缘种的变异位点，通过Primer Premier 6.0软件设计一对特异性引物和TaqMan-MGB探针。在Roche LightCycler 96系统中，该方法对太白贝母DNA模板的检测下限为0.002 39 ng·μL-1，最适Tm值区间为60与61℃。特异性鉴定研究表明，该方法对太白贝母有良好的特异性，能与暗紫贝母等其余13种不同贝母基原植物明显区分。TaqMan-MGB实时荧光PCR方法可实现对太白贝母及其近缘种的准确鉴定，为太白贝母资源的合理开发、中药材市场的管理和中药生产企业的原料监管提供技术支撑。

, correspAuthors=周嘉裕, 廖海, authorNote=null, correspAuthorsNote=
*周嘉裕, E-mail: ;
廖海, E-mail:
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M: DNA Maker (DL2000), lane 1 to 14 indicated <i>F. unibracteata</i>, <i>F. delavayi</i>, <i>F. cirrhosa</i>, <i>F</i>. <i>taipaiensis</i>, <i>F. przewalskii</i>, <i>F. unibracteata</i> Hsiaoet K. C. Hsia var. <i>wabuensis</i>, <i>F. sinica</i>, <i>F. cirrhosa</i> var. <i>ecirrhosa</i> Franch, <i>F. pallidiflora</i> Schrenk, <i>F. mellea</i>, <i>F. thunbergii</i>, <i>F. ussuriensis</i>, <i>F. walujewii</i> and <i>F. hupehensis</i>, respectively , figureFileSmall=1ur7xmsyRMlPR6CiO6yWKw==, figureFileBig=539G9UxyBMILvnbgu+gSTw==, tableContent=null), ArticleFig(id=1208491458735227732, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1208491447133782952, language=EN, label=null, caption=null, figureFileSmall=HqJMxUXhFAdenr6H0TncYg==, figureFileBig=NK43MAVoanjMskamE4q5Kg==, tableContent=null), ArticleFig(id=1208491458873639780, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1208491447133782952, language=CN, label=Figure 4, caption= Real time PCR performed at various <i>T</i><sub>m</sub> , figureFileSmall=HqJMxUXhFAdenr6H0TncYg==, figureFileBig=NK43MAVoanjMskamE4q5Kg==, tableContent=null), ArticleFig(id=1208491459020440438, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1208491447133782952, language=EN, label=null, caption=null, figureFileSmall=kW5hr00xbkQVoq0KsVTjIA==, figureFileBig=oz4cjenFUgN438cxnwCHlw==, tableContent=null), ArticleFig(id=1208491460199039873, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1208491447133782952, language=CN, label=Figure 5, caption= Limit of detection based on TaqMan-MGB real time PCR. A: The probe sensitivity of the 238.54 ng·μL<sup>-1</sup> group; B: There were two replicates in 238.54 ng·μL<sup>-1</sup> group; C: Sensitivity of different DNA templates , figureFileSmall=kW5hr00xbkQVoq0KsVTjIA==, figureFileBig=oz4cjenFUgN438cxnwCHlw==, tableContent=null), ArticleFig(id=1208491460350034831, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1208491447133782952, language=EN, label=null, caption=null, figureFileSmall=s/yjzkHWLzis8CqmPXPlpg==, figureFileBig=cHBbyYYdMH30q9K3wxnvjQ==, tableContent=null), ArticleFig(id=1208491460463281050, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1208491447133782952, language=CN, label=Figure 6, caption= Taq-Man MGB real time PCR of 14 various <i>Fritillaria</i> species , figureFileSmall=s/yjzkHWLzis8CqmPXPlpg==, figureFileBig=cHBbyYYdMH30q9K3wxnvjQ==, tableContent=null), ArticleFig(id=1208491460610081704, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1208491447133782952, language=EN, label=null, caption=null, figureFileSmall=X2yvEBc9y+GmkgvevMXUDA==, figureFileBig=GwBl1vqjQmEE0+vQD2VsbA==, tableContent=null), ArticleFig(id=1208491460731716537, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1208491447133782952, language=CN, label=Figure 7, caption= Taq-Man MGB real time PCR of seven Bei-mu medicines and <i>F. taipaiensis</i> , figureFileSmall=X2yvEBc9y+GmkgvevMXUDA==, figureFileBig=GwBl1vqjQmEE0+vQD2VsbA==, tableContent=null), ArticleFig(id=1208491460849157064, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1208491447133782952, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Latin name Accession number
F. unibracteata KT861541.1
F. delavayi KP711999.1
F. cirrhosa KT861548.1
F. taipaiensis KT861551.1
F. przewalskii KT861534.1
F. unibracteata var. wabuensis KX669649.1
F. sinica KF906211.1
F. mellea KF906209.1
F. pallidiflora KY884644.1
F. thunbergii DQ191622.1
F. ussuriensis MG946146.1
F. walujewii Regel MG946163.1
F. hupehensis KF906203.1
), ArticleFig(id=1208491461008540640, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1208491447133782952, language=CN, label=Table 1, caption=

ITS1 accession numbers of 13 various Fritillaria species

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Latin name Accession number
F. unibracteata KT861541.1
F. delavayi KP711999.1
F. cirrhosa KT861548.1
F. taipaiensis KT861551.1
F. przewalskii KT861534.1
F. unibracteata var. wabuensis KX669649.1
F. sinica KF906211.1
F. mellea KF906209.1
F. pallidiflora KY884644.1
F. thunbergii DQ191622.1
F. ussuriensis MG946146.1
F. walujewii Regel MG946163.1
F. hupehensis KF906203.1
), ArticleFig(id=1208491461260198901, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1208491447133782952, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Designation Sequences Tm/℃
Forward primer 5′-CCCGTAAACGGATGACAC-3′ 54
Reverse primer 5′-ATATGTTCCTTGGCGCAG-3′ 53
TaqMan-MGB 5′-FAM-TCTCTCATAGCACGAT-MGB-3′ 48
), ArticleFig(id=1208491461495078925, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1208491447133782952, language=CN, label=Table 2, caption=

Sequences of primers and probe

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Designation Sequences Tm/℃
Forward primer 5′-CCCGTAAACGGATGACAC-3′ 54
Reverse primer 5′-ATATGTTCCTTGGCGCAG-3′ 53
TaqMan-MGB 5′-FAM-TCTCTCATAGCACGAT-MGB-3′ 48
), ArticleFig(id=1208491461625102364, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1208491447133782952, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Tm/℃ Cq*
56 12.83, 13.07, 12.37, 26.06 (F. ussuriensis),
26.96 (F. hupehensis)
57 14.58, 13.48, 14.67
58 13.29, 13.48, 13.68
59 12.93, 14.22, 14.01, 24.13 (F. hupehensis)
60 14.20, 14.03, 14.07
61 12.63, 12.50, 12.56
), ArticleFig(id=1208491461750931502, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1208491447133782952, language=CN, label=Table 3, caption=

Cq at various Tm.*The former Cq in each line were three replicates of F. taipaiensis, respectively. Cq: Cycle quantity

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Tm/℃ Cq*
56 12.83, 13.07, 12.37, 26.06 (F. ussuriensis),
26.96 (F. hupehensis)
57 14.58, 13.48, 14.67
58 13.29, 13.48, 13.68
59 12.93, 14.22, 14.01, 24.13 (F. hupehensis)
60 14.20, 14.03, 14.07
61 12.63, 12.50, 12.56
), ArticleFig(id=1208491461843206202, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1208491447133782952, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Concentration of DNA templates/ng·μL-1 Cq
238.54 11.91
23.854 16.09
2.385 4 19.67
0.238 54 23.46
0.023 85 25.19
0.002 39 25.76
0.000 24 26.29
0.000 02 26.60
), ArticleFig(id=1208491462044532811, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1208491447133782952, language=CN, label=Table 4, caption=

Cq under various concentration of DNA templates

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Concentration of DNA templates/ng·μL-1 Cq
238.54 11.91
23.854 16.09
2.385 4 19.67
0.238 54 23.46
0.023 85 25.19
0.002 39 25.76
0.000 24 26.29
0.000 02 26.60
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TaqMan-MGB实时荧光PCR法检测太白贝母及其近缘种的研究
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张田 1 , 陈娇 1 , 蒋瑞平 1 , 邹萌 1 , 仰铁锤 2 , 付绍兵 2 , 周嘉裕 1, * , 廖海 1, *
药学学报 | 研究论文 2021,56(9): 2577-2583
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药学学报 | 研究论文 2021, 56(9): 2577-2583
TaqMan-MGB实时荧光PCR法检测太白贝母及其近缘种的研究
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张田1, 陈娇1, 蒋瑞平1, 邹萌1, 仰铁锤2, 付绍兵2, 周嘉裕1, * , 廖海1, *
作者信息
  • 1.西南交通大学生命科学与工程学院, 四川 成都 610031
  • 2.青海绿康生物开发有限公司, 青海 西宁 810003

通讯作者:

*周嘉裕, E-mail: ;
廖海, E-mail:
Identification of Fritillaria taibaiensis and its relatives by real-time PCR with a TaqMan-MGB probe
Tian ZHANG1, Jiao CHEN1, Rui-ping JIANG1, Meng ZOU1, Tie-chui YANG2, Shao-bing FU2, Jia-yu ZHOU1, * , Hai LIAO1, *
Affiliations
  • 1. School of Life Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China
  • 2. Qinghai Lvkang Biological Development Co., Ltd., Xining 810003, China
出版时间: 2021-09-12 doi: 10.16438/j.0513-4870.2021-0230
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摘要
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采用TaqMan-MGB探针实时荧光PCR方法对太白贝母及其近缘种进行分子鉴定研究。提取太白贝母及其近缘种的DNA，依据ITS1区序列，利用MEGA7.0软件进行比较分析，找出太白贝母及其近缘种的变异位点，通过Primer Premier 6.0软件设计一对特异性引物和TaqMan-MGB探针。在Roche LightCycler 96系统中，该方法对太白贝母DNA模板的检测下限为0.002 39 ng·μL-1，最适Tm值区间为60与61℃。特异性鉴定研究表明，该方法对太白贝母有良好的特异性，能与暗紫贝母等其余13种不同贝母基原植物明显区分。TaqMan-MGB实时荧光PCR方法可实现对太白贝母及其近缘种的准确鉴定，为太白贝母资源的合理开发、中药材市场的管理和中药生产企业的原料监管提供技术支撑。

TaqMan-MGB探针  /  太白贝母  /  ITS1  /  实时荧光PCR  /  分子鉴定

The molecular identification of Fritillaria taibaiensis and its relatives was studied by real-time PCR with a TaqMan-MGB probe. DNA was extracted from F. taibaiensis and its relatives. According to the sequence of ITS1 region, the mutation sites of F. taipaiensis and its related species were identified by MEGA7.0 software. The specific primers (a pair) and a TaqMan-MGB probe were designed by Primer Premier 6.0 software. In the Roche LightCycler 96 system, the lowest limit of detection for F. taipaiensis DNA template was 0.002 39 ng·μL-1, and the optimal Tm value range was 60 and 61℃. Specificity identification showed that the method had good specificity for F. taipaiensis, as it could be distinguished from other 13 different Fritillaria species including F. unibracteata. Since this method could accurately identify F. taipaiensis and its related species, it provides technical support for rational development of F. taipaiensis resources, management of Chinese medicinal market and supervision of raw materials in Chinese medicine manufacturing enterprises.

TaqMan-MGB probe  /  Fritillaria taipaiensis  /  ITS1  /  real-time PCR  /  molecular identification
张田, 陈娇, 蒋瑞平, 邹萌, 仰铁锤, 付绍兵, 周嘉裕, 廖海. TaqMan-MGB实时荧光PCR法检测太白贝母及其近缘种的研究. 药学学报, 2021 , 56 (9) : 2577 -2583 . DOI: 10.16438/j.0513-4870.2021-0230
Tian ZHANG, Jiao CHEN, Rui-ping JIANG, Meng ZOU, Tie-chui YANG, Shao-bing FU, Jia-yu ZHOU, Hai LIAO. Identification of Fritillaria taibaiensis and its relatives by real-time PCR with a TaqMan-MGB probe[J]. Acta Pharmaceutica Sinica, 2021 , 56 (9) : 2577 -2583 . DOI: 10.16438/j.0513-4870.2021-0230
正文
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川贝母(Fritillariae) 源于百合科贝母属多种植物的鳞茎, 是中国乃至亚洲地区一种名贵的中药材。其性味苦、甘、微寒, 有清热润肺、化痰止咳之功效[1], 始载于《神农本草经》。相较于平贝母、浙贝母与伊贝母[2]等药材, 川贝母治疗劳虚咳嗽、吐痰咳血等症状效果更为显著, 被誉为“止咳圣药”[3]。由于川贝母多为野生, 长年采挖已致野生资源渐趋枯竭, 市场上已出现大量混伪品。为满足临床需要, 《中国药典》增补了数种不同基原的贝母品种, 以《中国药典》2020年版为例, 川贝母有6种基原植物, 分别为太白贝母(Fritillaria taipaiensis)、卷叶贝母(F. cirrhosa)、暗紫贝母(F. unibracteata P. K. Hsiao & K. C. Hsia)、甘肃贝母(F. przewalskii)、梭砂贝母(F. delavayi) 和瓦布贝母(F. unibracteata Hsiaoet K. C. Hsia var. wabuensis)。尽管多基原贝母品种缓解了贝母药材的市场空缺, 然而不同基原贝母在生物碱及药效上仍有较大差别。有研究发现, 暗紫贝母、梭砂贝母较之甘肃贝母、卷叶贝母在止咳和化痰两个方面可能更能代表川贝母的功效特点。在镇咳方面, 暗紫贝母、梭砂贝母优于卷叶贝母、甘肃贝母; 在祛痰方面, 暗紫贝母优于甘肃贝母、卷叶贝母、梭砂贝母[4]; 而太白贝母祛痰作用优于暗紫贝母, 镇咳无显著差异[5]。因此, 为了避免多基原混用而导致的有效性与安全性问题, 建立适合于单基原的准确、高效鉴定方法已刻不容缓。
不同基原川贝母及其混伪品的鉴定主要依赖于传统的生药学方法, 此法主要从植株茎高、叶片宽度、叶片是否卷曲、鳞茎色泽与鳞茎抱合度等特征进行观察比较, 需要丰富的鉴定经验[6]。近年来, 由于不同贝母品种的混栽现象, 导致不同基原川贝母甚至常见混伪品形态高度相似, 区分难度大, 常需要结合其他分析鉴定技术进行鉴别。化学方法较为常用, 但需使用高效液相色谱以及液质联用技术, 推广难度较大。2020版《中国药典》中“川贝母”项下已有PCR-RFLP分子鉴定方法, 该法能区分川贝母、伊贝母与浙贝母等药材[7], 但不能区分川贝母的6种基原, 并且也不能区分川贝母与浓蜜贝母、中华贝母和康定贝母等川贝母近缘物种[8, 9]。DNA条形码是近年来建立的分子鉴定方法[10, 11], 广泛用于植物物种鉴别, 但其标准操作流程包含DNA提取、PCR扩增、测序、数据分析及结果判定等, 耗时较长[12]。因此, 急需建立更加便捷的检测方法以满足药材市场的监控需求。
TaqMan-MGB标记技术在传统TaqMan探针的基础上, 用MGB基团修饰了3'末端非荧光猝灭基团, 能够在不增加探针碱基数的情况下, 将探针的Tm值提高10 ℃以上, 从而提高特异性并更容易设计[13, 14]。此外, TaqMan-MGB的3'末端标记有非荧光猝灭基团, 可减少荧光背景并提高信噪比。此法的特异性引物对和特异性探针序列可双重保障反应的特异性, 相较常规PCR更具应用与推广价值[15]。笔者分析太白贝母等13种不同贝母的ITS1序列, 发现太白贝母含有专属性的“ATA”序列, 即作为鉴定太白贝母的特异性标记。本研究基于该“ATA”序列, 设计实时荧光PCR引物与TaqMan-MGB探针, 建立准确、快速鉴定太白贝母的实时荧光PCR方法, 为太白贝母资源的合理开发、中药材市场的管理和中药生产企业的原料监管提供技术支撑。
材料和方法
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植物材料    六种川贝母基原植物、中华贝母(F. sinica)、浓蜜贝母(F. mellea)、伊贝母(F. pallidiflora Schrenk) 与浙贝母(F. thunbergii) 采自青海省西宁市互助县; 康定贝母(F. cirrhosa var. ecirrhosa Franch) 采自四川省康定市; 平贝母(F. ussuriensis) 采自陕西汉中; 新疆贝母(F. walujewii Regel)由中国科学院苏志豪研究员与天津理工大学刘明玉教授馈赠。湖北贝母(F. hupehensis Hsiao et K. C. Hsia) 药材购自中国食品药品检定研究院, 批号: 120962-201005; 川贝母(松贝、青贝与炉贝)、浙贝母与平贝母药材购自成都荷花池中药材市场; 伊贝母药材购自新疆伊犁。所有基原植物及药材由西南交通大学生命科学与工程学院周嘉裕副教授鉴定, 并保存于生命科学与工程学院。
仪器和试剂   LightCycler 96实时荧光定量PCR仪(Roche), DYY-6C型电泳仪(北京六一生物科技有限公司), 多功能酶标仪(美国Bio-Tek), Veriti 96-Well Thermal Cycler PCR仪(Thermo Fisher)。植物DNA提取试剂盒(DP305) 购自北京天根生化有限公司, DL2000 DNA Marker、2×T5 Fast qPCR Mix (Probe)、Agarose、核酸缓冲液(TAE 1×), 2×Master Mix与核酸染料(TS-GelRed)均购自北京擎科生物科技有限公司。
基于ITS1区序列的系统进化树   从NCBI (https://www.ncbi.nlm.nih.gov/) 下载太白贝母等13种不同贝母的ITS1序列(表 1)。采用MEGAX软件(Version 7) 构建NJ系统进化树, 重复次数为1 000。利用DNAMAN软件(Version 6) 进行序列比对, 筛选太白贝母的特异性碱基序列。利用Primer Premier (Version 6), 设计正向、反向引物和TaqMan-MGB探针, 并由北京擎科生物科技有限公司合成, 核酸序列信息见表 2。
基因组DNA提取    分别取太白贝母等样品的干燥叶片与市售药材样品各约50~60 mg, 75%的酒精和无菌水洗净, 晾干, 液氮研磨, 采用植物基因组DNA提取试剂盒(DP305) 提取基因组DNA。Bio-Tek酶标仪检测DNA的浓度。
常规PCR反应体系    常规PCR反应体系: 2×Master Mix 12.5 μL, 正、反向游引物(10 μmol·L-1) 各1.0 μL, DNA模板2.0 μL, 用灭菌水补足至总体积25 μL。
常规PCR反应参数: 95 ℃预变性10 min, 1次循环; 95 ℃变性30 s, 58 ℃退火30 s, 72 ℃延伸50 s, 共计35次循环; 72 ℃延伸7 min。1.5%琼脂糖电泳检测PCR产物。
TaqMan-MGB实时荧光PCR反应体系   实时荧光PCR反应体系为: 2×T5 Fast qPCR Mix (Probe) 为10.0 μL, 正向与反向引物(浓度为10 μmol·L-1) 各1.0 μL, DNA模板2.0 μL, TaqMan-MGB探针1.0 μL, 用灭菌水补足至总体积20 μL。
实时荧光反应条件参数: 95 ℃预变性2 min, 1次循环, (95 ℃变性10 s, 56 ℃退火60 s, 共计40次循环)。
TaqMan-MGB实时荧光PCR最低灵敏度   选取太白贝母基因组DNA样本, 用灭菌水10倍稀释成7个梯度, 每个浓度梯度各取2 μL作为模板, 按照“TaqMan-MGB实时荧光PCR反应体系”方法进行灵敏度检测。
TaqMan-MGB实时荧光PCR的特异性   取太白贝母等不同贝母样品的基因组DNA。按照“TaqMan-MGB实时荧光PCR反应体系”法进行实时荧光PCR扩增, 利用实时荧光PCR仪自带软件进行扩增数据分析。
结果与分析
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1 13种贝母ITS1的系统进化树与序列比对结果
系统进化树显示(图 1) 6种川贝母与浓蜜贝母、中华贝母等形成单系群, 其中太白贝母与其余贝母品种构成姐妹群, 亲缘关系较远, 表明太白贝母是分化较早的贝母品种, 推测太白贝母与其余贝母品种在ITS1区域有较高的变异性。随后, 比对不同贝母的ITS1序列, 发现太白贝母ITS1区域的98到100碱基位置存在特异性的“ATA”碱基序列(图 2), 并以该“ATA”序列为核心, 设计MGB探针、正向与反向实时荧光PCR引物, 探针及引物位置见图 2。
2 常规PCR扩增反应
开展实时荧光PCR实验之前, 采用常规PCR扩增所有贝母样品的ITS1区域, 结果显示所有贝母样品均能扩增出预期分子大小的PCR产物, 不仅表明ITS1序列正确, 并且引物设计合理(图 3)。随后, 对太白贝母的PCR扩增产物进行Sanger测序, 进一步确认太白贝母的ITS1区域在相对应的位置上存在“ATA”碱基序列。
3 实时荧光PCR扩增反应条件的探索
3.1 最适Tm值
当Tm值为56 ℃时, 不仅太白贝母出现了特异性荧光扩增曲线, 平贝母与湖北贝母也出现了低强度的非特异性荧光扩增曲线, 表明需要优化Tm值。随后, 选择56、57、58、59、60、61与62 ℃等系列Tm值开展实验(图 4), 对应的循环定量值(Cq) 值见表 3。比较发现, 当Tm值为60和61 ℃时, 实时荧光PCR结果的特异性最高(61 ℃的荧光强度略高) (图 4), 因此确定最适Tm值区间为60与61 ℃。
3.2 TaqMan-MGB探针的最低灵敏度
太白贝母DNA样本的初始浓度为238.54 ng·μL-1, 随后将其稀释到23.854、2.385 4、0.238 54、0.023 85、0.002 39、0.000 24与0.000 02 ng·μL-1等8个浓度梯度。实时荧光PCR扩增结果显示TaqMan-MGB探针可检测出的最低浓度为0.002 39 ng·μL-1 (图 5), 表明该探针检测的灵敏度较高。不同浓度DNA模板的Cq值见表 4。
4 TaqMan-MGB探针特异性
以14种贝母为试样, 将DNA模板浓度控制在100~150 ng·μL-1区间, 进行Taqman-MGB实时荧光PCR (Tm值为61 ℃), 结果仅有太白贝母出现扩增曲线(图 6), 表明该方法具有较高的特异性。
5 对市售药材的鉴定
使用TaqMan-MGB实时荧光PCR法鉴定购买的药材(松贝、青贝、炉贝、浙贝、平贝、伊贝母、湖北贝母), 并以太白贝母为标准品, 结果显示仅有太白贝母标准品产生特异性荧光曲线, 其余药材均不含有太白贝母(图 7)。
讨论
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筛选特异性分子标记是建立准确、高分辨率分子鉴定方法的首要前提[16]。相较于基因编码区, ITS位于核基因组中编码核糖体rRNA基因的间隔区, 受到的选择压力较小, 进化速率较快, 常用于研究属间、种间甚至居群间等较低分类等级的系统发育关系[17]。ITS包括ITS1与ITS2, 其中ITS2是5.8S rRNA基因与28S rRNA基因间的内转录2区。陈士林等[18]提出以ITS2为主, psbA-trnH序列为辅的药用植物DNA条形码鉴定策略。本课题组分析不同贝母的ITS2序列, 结果显示太白贝母与其他川贝母在ITS2区域只相差1个碱基, 相似度过高, 形成特异性分子鉴定方法有一定的难度[12]。相较于ITS2, ITS1是18S rRNA基因与5.8S rRNA基因间的内转录1区, 其序列长度更长, 产生的变异更多[19]。例如, 贠凯祎等[20]利用冬虫夏草及其混伪品在ITS1区域的差异, 建立了特异鉴定冬虫夏草的Taqman方法。从结果1中发现太白贝母与其他川贝母品种构成姐妹群, 亲缘关系较远, 表明太白贝母可能在较早时间即与其他川贝母品种产生了分化, 这与郑辉等[12]基于ITS2序列的系统分类结果相似。导致这种现象的原因可能与太白贝母独特的生境相关, 太白贝母是川贝母中唯一能在低海拔地区(< 2 000 m) 生长的品种, 环境的差异可能导致遗传物质发生变异。为考察该“ATA”序列的稳定性, 比对了NCBI中已有的10条太白贝母ITS1序列(序列号分别为MH588425.1、MH588424.1、MH588423.1、MH588422、MH588421.1、KT861553.1、KT861552.1、KT861551.1、KP712002.1与HM045470.1), 结果发现所有序列均含有“ATA”碱基序列, 结合进一步的Sanger测序, 表明该碱基序列稳定性比较好, 适合用于下一步的qPCR实验验证。
由于太白贝母ITS1区域中“ATA”碱基序列的上下游含有较多的AT碱基, 导致初始探针的Tm值较低(48 ℃), 但该探针连接MGB基团以后, 其最终Tm值超过实时荧光定量PCR引物的Tm值, 实验能够获得成功。郭丽霞等[21]也利用MGB修饰基团提高探针的Tm值, 建立了小球藻的分子鉴定方法。以上结果表明, MGB适用于AT密集区域的探针设计, 能够提高探针的Tm值, 从而增强了该技术的应用范围。合适的Tm值不仅决定着PCR的扩增效率[22], 也影响MGB探针的特异性杂交[23]。
灵敏度结果显示, 检测太白贝母的DNA浓度最低可达0.002 39 ng·μL-1。贠凯祎等[20]设计Taqman探针, 检测冬虫夏草的最低DNA模板浓度为0.016 ng·μL-1, 与之相比, 本文设计的探针检测的DNA模板浓度更低, 应用前景较好。在对市售药材开展实时荧光PCR检测时, 松贝、青贝与炉贝等川贝母药材都未出现扩增曲线, 表明所购买的川贝母药材中未含有太白贝母。《中国药典》记载, 太白贝母常以“松贝”与“青贝”入药, 其主要栽培区位于陕西、甘肃与重庆等地区, 而本次实验购买的川贝母药材产地为四川阿坝州与甘孜州, 并非太白贝母的主产区, 因此未出现阳性扩增结果, 本文作者将继续收集来自陕西、甘肃与重庆等地区的川贝母药材, 开展进一步的验证。
最后, 采用本文的Taqman-MGB实时荧光PCR方法, 整体仅需1.5 h, 即可完成太白贝母的准确检测。与《中国药典》2020版的PCR-RFLP、DNA条形码等方法[24]比较, 本方法能够实时监测试验结果, 省去电泳及核酸测序等步骤, 操作更加便捷, 大大缩短检测周期[25], 本方法具有特异性高、快速、标准化程度高与高效的特点, 从而为太白贝母资源的合理开发、中药材市场的管理和中药生产企业的原料监管提供技术支撑。
作者贡献: 张田是本研究的实验设计者和实验研究的执行人, 完成数据分析, 论文初稿的写作; 陈娇、蒋瑞平、邹萌、仰铁锤和付绍兵参与样品采集、实验设计, 试验结果分析; 周嘉裕和廖海是项目的构思者及负责人, 指导实验设计、数据分析、论文写作与修改。全体作者都阅读并同意最终的文本。
利益冲突: 本文的研究内容无任何利益冲突。
基金
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  • 国家自然科学基金青年科学基金项目(31500276)
  • 四川省重点研发项目(2018SZ0061)
  • 四川省科技计划项目(2021ZHFP0170)
  • 四川省中医药管理局项目(2020JC0128)
  • 四川省中医药管理局项目(2021MS116)
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2021年第56卷第9期
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doi: 10.16438/j.0513-4870.2021-0230
  • 接收时间：2021-02-14
  • 首发时间：2025-12-18
  • 出版时间：2021-09-12
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  • 收稿日期：2021-02-14
  • 修回日期：2021-03-15
基金
国家自然科学基金青年科学基金项目(31500276)
四川省重点研发项目(2018SZ0061)
四川省科技计划项目(2021ZHFP0170)
四川省中医药管理局项目(2020JC0128)
四川省中医药管理局项目(2021MS116)
作者信息
    1.西南交通大学生命科学与工程学院, 四川 成都 610031
    2.青海绿康生物开发有限公司, 青海 西宁 810003

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*周嘉裕, E-mail: ;
廖海, E-mail:
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2种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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