基于便携式和CFX96实时荧光PCR仪的冬虫夏草及其混伪品鉴定研究
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摘要
采用TaqMan探针实时荧光PCR方法对冬虫夏草及其混伪品进行分子鉴定研究。从100份冬虫夏草及其混伪品中提取总DNA,依据核糖体(rDNA)内部转录间隔区(internal transcribed spacer, ITS)序列,利用MEGA7.0软件进行比较分析,找出冬虫夏草及其混伪品的变异位点,通过Primer Premier 6.0软件设计一对特异性引物和TaqMan探针,分别在两种实时荧光PCR仪(Genesig q16和Bio-Rad CFX96)上进行灵敏度和特异性鉴定研究。灵敏度研究表明,在Bio-Rad CFX96系统中,该方法对冬虫夏草DNA模版的检测下限为0.016 ng·μL~(-1);在Genesig q16系统中,该方法对冬虫夏草DNA模版的检测下限为15.527 ng·μL~(-1)。特异性鉴定研究表明,在Genesig q16和Bio-Rad CFX96系统上,该方法对冬虫夏草均具有良好的特异性,能与混伪品下垂虫草、古尼虫草、蛹虫草、蝉花、凉山虫草、新疆虫草明显区分。TaqMan探针实时荧光PCR方法可实现对冬虫夏草及其混伪品的准确鉴定,为药材市场的管理提供技术支撑,对名贵中药材的鉴别具有较好应用前景。
The molecular identification of Ophiocordyceps sinensis and its adulterants was carried out by real-time fluorescent PCR with TaqMan probe. Genomic DNA was extracted from 100 samples of Ophiocordyceps sinensis and its adulterants. MEGA 7.0 software was used for comparative analysis to define the variable sites between Ophiocordyceps sinensis and its adulterants according to the internal transcribed spacer(ITS) region of ribosomal DNA(rDNA). A set of specific primers and TaqMan probe were designed using Primer Premier 6.0 software, and sensitivity and specificity studies were performed on two different real-time fluorescent PCR systems(Genesig q16 and Bio-Rad CFX96). The sensitivity study showed that the detectable DNA template concentra‐tion of Ophiocordyceps sinensis for the real-time fluorescent PCR was 0.016 ng · μL~(-1) in the Bio-Rad CFX96 system and 15.527 ng · μL~(-1) in the Genesig q16 system, respectively. Meanwhile, this method had good specificity for Ophiocordyceps sinensis on Genesig q16 and Bio-Rad CFX96 systems, so Ophiocordyceps sinensis could be clearly distinguished from Ophiocordyceps nutans, Cordyceps gunnii, Cordyceps militaris, Cordyceps cicadae,Cordyceps liangshanensis, Cordyceps gracilis. Our results indicate that real-time fluorescent PCR with TaqMan probe can be used to accurately identify Ophiocordyceps sinensis from its adulterants. This provides a technical method that has wide applications for market management and quality control of Chinese materia medica.
The molecular identification of Ophiocordyceps sinensis and its adulterants was carried out by real-time fluorescent PCR with TaqMan probe. Genomic DNA was extracted from 100 samples of Ophiocordyceps sinensis and its adulterants. MEGA 7.0 software was used for comparative analysis to define the variable sites between Ophiocordyceps sinensis and its adulterants according to the internal transcribed spacer(ITS) region of ribosomal DNA(rDNA). A set of specific primers and TaqMan probe were designed using Primer Premier 6.0 software, and sensitivity and specificity studies were performed on two different real-time fluorescent PCR systems(Genesig q16 and Bio-Rad CFX96). The sensitivity study showed that the detectable DNA template concentra‐tion of Ophiocordyceps sinensis for the real-time fluorescent PCR was 0.016 ng · μL~(-1) in the Bio-Rad CFX96 system and 15.527 ng · μL~(-1) in the Genesig q16 system, respectively. Meanwhile, this method had good specificity for Ophiocordyceps sinensis on Genesig q16 and Bio-Rad CFX96 systems, so Ophiocordyceps sinensis could be clearly distinguished from Ophiocordyceps nutans, Cordyceps gunnii, Cordyceps militaris, Cordyceps cicadae,Cordyceps liangshanensis, Cordyceps gracilis. Our results indicate that real-time fluorescent PCR with TaqMan probe can be used to accurately identify Ophiocordyceps sinensis from its adulterants. This provides a technical method that has wide applications for market management and quality control of Chinese materia medica.
引文
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[25]Chen SL,Yao H,Han JP,et al.Principles for molecular identification of traditional Chinese materia medica using DNAbarcoding[J].China J Chin Mater Med(中国中药杂志),2013,38:141-148.
[26]Jiang WC,Yue SW,Jiang H,et al.Application and research progress of TaqMan probe real time PCR[J].Clin Lab J(临床检验杂志),2015,4:797-805.
[27]He GL,Chen N,Liu P,et al.Identification of Bird's Nest using TaqMan real-time quantitative PCR[J].Lett Biotechnol(生物技术通讯),2015,26:111-115.
[28]Wu D,Guo Q.Detection of other animal species in Placenta Hominis by using TaqMan real time PCR[J].J Chin Med Mater(中药材),2017,40:38-41.
[29]Chen SL,Pang XH,Song JY,et al.A renaissance in herbal medicine identification:from morphology to DNA[J].Biotechnol Adv,2014,32:1237-1244.
[30]Li XW,Yang Y,Henry RJ,et al.Plant DNA barcoding:from gene to genome[J].Biol Rev,2015,90:157-166.
[31]Chen XC,Liao BS,Song JY,et al.A fast SNP identification and analysis of intraspecific variation in the medicinal Panax species based on DNA barcoding[J].Gene,2013,530:39-43.
[32]Hou DY,Song JY,Yao H,et al.Molecular identification of Corni Fructus and its adulterants by ITS/ITS2 sequences[J].Chin JNat Med,2013,11:121-127.
[33]Ma HM,Yu J,Dai M,et al.Study on DNA barcoding technology of Qinghai Cordyceps sinensis and Xinjiang Cordyceps grsacilis for identification[J].Mod Tradit Chin Med Mater Med-World Sci Technol(世界科学技术-中医药现代化),2016,18:826-832.
[34]Zhang XK,Liu TH.Studies on identification method by PCRof specificity of aweto[J].Chin Med Pharm(中国医药科学),2015,5:55-57.
[35]Liang HX,Wang W,Duan QZ,et al.A new method based on PCR for identification of Cordyceps sinensis[J].J Chin Med Mater(中药材),2018,41:554-558.
[2]Zhang WJ,Wei F,Ma SC,et al.Application of ITS1 barcode sequence for the identification of Cordyceps sinensis from its counterfeit species[J].Chin J Pharm Anal(药物分析杂志),2015,35:1716-1720.
[3]Winkler D.Yartsa Gunbu(Cordyceps sinensis)and the fungal commodification of Tibet's rural economy[J].Econ Bot,2008,62:291-305.
[4]Xia EH,Yang DR,Jiang JJ,et al.The caterpillar fungus,Ophiocordyceps sinensis,genome provides insights into highland adaptation of fungal pathogenicity[J].Sci Rep,2017,7:1806.
[5]Zhang S,Zhang YJ.Molecular evolution of three protein-coding genes in the Chinese caterpillar fungus Ophiocordyceps sinensis[J].Microbiol China(微生物学通报),2015,42:1549-1560.
[6]Liu Y,Wang XY,Gao ZT,et al.Detection of Ophiocordyceps sinensis and its common adulterates using species-specific primers[J].Front Microbiol,2017,8:1179.
[7]Xu H,Dong TX,Zhao KJ,et al.Study on identification of Cordyceps-a Chinese traditional medicine[J].Chin Pharm J(中国药学杂志),2014,49:283-286.
[8]Yang YQ,Duan JH.Discrimination of Cordyceps Sinensis from the counterfeit[J].World J Integr Tradit West Med(世界中西医结合杂志),2012,7:31-33.
[9]Chen XQ,Liu BL,Zhao ZZ,et al.Studies on macroscopic and microscopic identification of Cordyceps sinensis and its counterfeits[J].China J Chin Mater Med(中国中药杂志),2011,36:1141-1144.
[10]Kang S,Zhang J,Lin RC.Macroscopic and microscopic characteristics of Chinese Caterpillar Fungus[J].Acta Pharm Sin(药学学报),2013,48:428-434.
[11]Liu HJ,Hu HB,Chu C,et al.Morphological and microscopic identification studies of Cordyceps and its counterfeits[J].Acta Pharm Sin(药学学报),2011,46:189-195.
[12]Hu HK,Xiao L,Zheng BG,et al.Identification of chemical markers in Cordyceps sinensis by HPLC-MS/MS[J].Anal Bioanal Chem,2015,407:8059-8066.
[13]Zhang JK,Wang P,Wei X,et al.A metabolomics approach for authentication of Ophiocordyceps sinensis by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry[J].Food Res Int,2015,76:489-497.
[14]Zhang WW,Gong T,Han DH.Comparative research on HPLCcharacteristic fingerprint of cultured Cordyceps Militarise and Cordyceps Sinensis[J].Chin J Inf Tradit Chin Med(中国中医药信息杂志),2015,22:92-96.
[15]Qian ZM,Sun PP,Li WQ,et al.HPLC characteristics of Ophiocordyceps sinensis[J].Mod Tradit Chin Med Mater MedWorld Sci Technol(世界科学技术-中医药现代化),2014,16:279-283.
[16]Hebert PDN,Cywinska A,Ball SL,et al.Biological identifications through DNA barcodes[J].Proc R Soc Lond B,2003,270:313-321.
[17]Wang W,Wang LL,Xiong LL,et al.ITS sequences as a DNAbarcode in the identification and phylogenetics of Cordyceps[J].Jiangsu J Agric Sci(江苏农业学报),2012,28:680-682.
[18]Xiang L,Song JY,Xin TY,et al.DNA barcoding the commercial Chinese caterpillar fungus[J].FEMS Microbiol Lett,2013,347:156-162.
[19]Wang YQ,Xue XH.Progress and application of real-time fluorescent quantitative polymerase chain reaction[J].Bull Biol(生物学通报),2016,51:1-6.
[20]An GL.The principle and application of real-time fluorescent quantitative PCR[J].Chin Mod Educ Equip(中国现代教育装备),2018,301:19-21.
[21]Liu ZY,Yao YJ,Liang ZQ,et al.Molecular evidence for the anamorph-teleomorph connection in Cordyceps sinensis[J].Mycol Res,2001,105:827-832.
[22]Chen YQ,Ning W,Qu LH,et al.Determination of the anamorph of Cordyceps sinensis inferred from the analysis of the ribosomal DNA internal transcribed spacers and 5.8S rDNA[J].Biochem Syst Ecol,2001,29:597-607.
[23]Zhang YJ,Xu LL,Zhang S,et al.Genetic diversity of Ophiocordyceps sinensis,a medicinal fungus endemic to the Tibetan Plateau:implications for its evolution and conservation[J].BMC Evol Biol,2009,9:290.
[24]Chen YQ,Hu B,Xu F,et al.Genetic variation of Cordyceps sinensis,a fruit-body-producing entomopathogenic species from different geographical regions in China[J].FEMS Microbiol Lett,2004,230:153-158.
[25]Chen SL,Yao H,Han JP,et al.Principles for molecular identification of traditional Chinese materia medica using DNAbarcoding[J].China J Chin Mater Med(中国中药杂志),2013,38:141-148.
[26]Jiang WC,Yue SW,Jiang H,et al.Application and research progress of TaqMan probe real time PCR[J].Clin Lab J(临床检验杂志),2015,4:797-805.
[27]He GL,Chen N,Liu P,et al.Identification of Bird's Nest using TaqMan real-time quantitative PCR[J].Lett Biotechnol(生物技术通讯),2015,26:111-115.
[28]Wu D,Guo Q.Detection of other animal species in Placenta Hominis by using TaqMan real time PCR[J].J Chin Med Mater(中药材),2017,40:38-41.
[29]Chen SL,Pang XH,Song JY,et al.A renaissance in herbal medicine identification:from morphology to DNA[J].Biotechnol Adv,2014,32:1237-1244.
[30]Li XW,Yang Y,Henry RJ,et al.Plant DNA barcoding:from gene to genome[J].Biol Rev,2015,90:157-166.
[31]Chen XC,Liao BS,Song JY,et al.A fast SNP identification and analysis of intraspecific variation in the medicinal Panax species based on DNA barcoding[J].Gene,2013,530:39-43.
[32]Hou DY,Song JY,Yao H,et al.Molecular identification of Corni Fructus and its adulterants by ITS/ITS2 sequences[J].Chin JNat Med,2013,11:121-127.
[33]Ma HM,Yu J,Dai M,et al.Study on DNA barcoding technology of Qinghai Cordyceps sinensis and Xinjiang Cordyceps grsacilis for identification[J].Mod Tradit Chin Med Mater Med-World Sci Technol(世界科学技术-中医药现代化),2016,18:826-832.
[34]Zhang XK,Liu TH.Studies on identification method by PCRof specificity of aweto[J].Chin Med Pharm(中国医药科学),2015,5:55-57.
[35]Liang HX,Wang W,Duan QZ,et al.A new method based on PCR for identification of Cordyceps sinensis[J].J Chin Med Mater(中药材),2018,41:554-558.