浙龟甲的PCR鉴别研究
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摘要
目的建立聚合酶链式反应(PCR)技术鉴别浙龟甲[来源为龟黄喉水龟Clemmysmutica(Cantor)的背甲及腹甲]真伪的方法。方法提取浙龟甲及其混淆品的基因组DNA,从NCBI网站上检索黄喉水龟及其混淆品的线粒体基因,进行分析比对,根据黄喉水龟细胞色素b(cytochromebgene,Cytb)基因上的特异位点应用Oligo软件设计引物,对浙龟甲及其混淆品样本进行PCR扩增。结果所设计的引物能特异性扩增黄喉水龟,扩增片段长度为357 bp,能有效区分浙龟甲及其混淆品种。结论建立的PCR技术鉴别浙龟甲方法,具有特异性高、方法简便快捷、实用性较强的特点,在快速准确鉴别浙龟甲方面具有良好的应用前景。
OBJECTIVE To develop a new polymerase chain reaction(PCR) technology for identifying the authenticity of Zheguijia[the dorsal and abdominal carapaces of Clemmys mutica(Cantor)]. METHODS Extract genomic DNA from Zheguijia and its adulterants was extracted. A series of sequences from mtDNA of Zheguijia and its adulterants were downloaded from the GenBank for analysis and comparison. And the Oligo software was used to design a set of primers, according to the specific sites on Cytb genes of Zheguijia. PCR technology was performed out to amplify the specific fragment of Zheguijia and its adulterants. RESULTS The designed specific primers were only used to amplify the genuine products of Zheguijia and the amplified fragments were 357 bp, which could effectively distinguished Zheguijia and its adulterants. CONCLUSION The PCR assay proposed in this study can be used for the quality control of Zheguijia, which is specificity, sensitivity and applicability.
OBJECTIVE To develop a new polymerase chain reaction(PCR) technology for identifying the authenticity of Zheguijia[the dorsal and abdominal carapaces of Clemmys mutica(Cantor)]. METHODS Extract genomic DNA from Zheguijia and its adulterants was extracted. A series of sequences from mtDNA of Zheguijia and its adulterants were downloaded from the GenBank for analysis and comparison. And the Oligo software was used to design a set of primers, according to the specific sites on Cytb genes of Zheguijia. PCR technology was performed out to amplify the specific fragment of Zheguijia and its adulterants. RESULTS The designed specific primers were only used to amplify the genuine products of Zheguijia and the amplified fragments were 357 bp, which could effectively distinguished Zheguijia and its adulterants. CONCLUSION The PCR assay proposed in this study can be used for the quality control of Zheguijia, which is specificity, sensitivity and applicability.
引文
[1]浙江省食品药品监督管理局.浙江省中药炮制规范2015年版[M].北京:中国医药科技出版社,2016.
[2]LI G H.Advances in the study of animal drug identification[J].Drug Eval Res(药物评价研究),2010,33(3):241.
[3]LI N,YUE B B,ZHANG J H,et al.Molecular identification of processed medicinal insects Chinese Polyphaga based on Cytb gene[J].China Pharm(中国药房),2015,26(31):4354-4356.
[4]WANG M,LI X H,ZHOU Y Q,et al.Identification and characteristics of Carapax et plustrum Testudinis based on PCR technology and modified extraction of genomic DNA[J].J Beihua Univ Nat Sci(北华大学学报:自然科学版),2016,17(6):777-780.
[5]LUO Y M,ZHANG W M,DING X Y,et al.SNP marker and allele-specific diagnostic PCR for authenticating herbs of Perilla[J].Acta Pharm Sin(药学学报),2006,41(9):840-845.
[6]ZHENG C J,ZHAO S J,ZHAO Z H,et al.Molecular identification of Fallopia multiflora by PCR-RFLP based on r DNA ITS sequence[J].J South China Univ Technol(Nat Sci Ed)(华南理工大学学报),2009,37(6):74-78,83.
[7]YUAN T Y,YAN Y,FANG L H,et al.Establishment of DNApolymorphism graphs for standard Chinese medical herbs by RAPD[J].Chin J New Drugs(中国新药杂志),2013,22(19):2250-2255.
[8]GUO H,WANG Q B,JIA L W,et al.Study progress in DNAbarcode of traditional Chinese medicine[J].China Pharm(中国药师),2016,19(3):566-570.
[9]CUI Z H,YUAN Y,HU J.Authentication of Mutong,Chuan-mutong and Guan-mutong by rapid PCR[J].Mod Chin Med(中国现代中药),2016,18(12):1560-1565,1577.
[2]LI G H.Advances in the study of animal drug identification[J].Drug Eval Res(药物评价研究),2010,33(3):241.
[3]LI N,YUE B B,ZHANG J H,et al.Molecular identification of processed medicinal insects Chinese Polyphaga based on Cytb gene[J].China Pharm(中国药房),2015,26(31):4354-4356.
[4]WANG M,LI X H,ZHOU Y Q,et al.Identification and characteristics of Carapax et plustrum Testudinis based on PCR technology and modified extraction of genomic DNA[J].J Beihua Univ Nat Sci(北华大学学报:自然科学版),2016,17(6):777-780.
[5]LUO Y M,ZHANG W M,DING X Y,et al.SNP marker and allele-specific diagnostic PCR for authenticating herbs of Perilla[J].Acta Pharm Sin(药学学报),2006,41(9):840-845.
[6]ZHENG C J,ZHAO S J,ZHAO Z H,et al.Molecular identification of Fallopia multiflora by PCR-RFLP based on r DNA ITS sequence[J].J South China Univ Technol(Nat Sci Ed)(华南理工大学学报),2009,37(6):74-78,83.
[7]YUAN T Y,YAN Y,FANG L H,et al.Establishment of DNApolymorphism graphs for standard Chinese medical herbs by RAPD[J].Chin J New Drugs(中国新药杂志),2013,22(19):2250-2255.
[8]GUO H,WANG Q B,JIA L W,et al.Study progress in DNAbarcode of traditional Chinese medicine[J].China Pharm(中国药师),2016,19(3):566-570.
[9]CUI Z H,YUAN Y,HU J.Authentication of Mutong,Chuan-mutong and Guan-mutong by rapid PCR[J].Mod Chin Med(中国现代中药),2016,18(12):1560-1565,1577.