Most of Chinese medicinal herbs are subjected to traditional processing procedures, including stir-frying, charring, steaming, boiling, and calcining before they are released into dispensaries. The marketing and ident...Most of Chinese medicinal herbs are subjected to traditional processing procedures, including stir-frying, charring, steaming, boiling, and calcining before they are released into dispensaries. The marketing and identification of processed medicinal materials is a growing issue in the marketplace. However, conventional methods of identification have limitations, while DNA mini-barcoding, based on the sequencing of a short-standardized region, has received considerable attention as a new potential means to identify processed medicinal materials. In the present study, six DNA barcode loci including ITS2, psb A-trn H, rbc L, mat K, trnL(UAA) intron and its P6 loop, were employed for the authentication of 45 processed samples belonging to 15 species. We evaluated the amplification efficiency of each locus. We also examined the identification accuracy of the potential mini-barcode locus, of trnL(UAA) intron P6 loop. Our results showed that the five primary barcode loci were successfully amplified in only 8.89%——20% of the processed samples, while the amplification rates of the trnL(UAA) intron P6 loop were higher, at 75.56% successful amplification. We compared the mini-barcode sequences with Genbank using the Blast program. The analysis showed that 45.23% samples could be identified to genus level, while only one sample could be identified to the species level. We conclude that trnL(UAA) p6 loop is a candidate mini-barcode that has shown its potential and may become a universal mini-barcode as complementary barcode for authenticity testing and will play an important role in medicinal materials control.展开更多
目的建立一种高效、精准的DNA条形码技术准确快速鉴定鱼胶物种。方法以细胞色素C氧化酶I(cytochrome C oxidase subunit I,COI)基因为靶标,筛选并优化DNA条形码引物,通过聚合酶链式反应(polymerase chain reaction,PCR)扩增和Sanger测...目的建立一种高效、精准的DNA条形码技术准确快速鉴定鱼胶物种。方法以细胞色素C氧化酶I(cytochrome C oxidase subunit I,COI)基因为靶标,筛选并优化DNA条形码引物,通过聚合酶链式反应(polymerase chain reaction,PCR)扩增和Sanger测序评估其鉴定效果。对市售鱼胶样品进行特异性扩增,结合生命条形码数据系统(barcode of life data systems,BOLD)和美国国家生物技术信息中心(national center for biotechnology information,NCBI)数据库进行物种比对,并采用邻接法(neighbor-joining,NJ)和系统发育树分析验证鉴定结果。结果通过筛选8对COI基因微型DNA条形码引物,最终确定了一组扩增目标片段约190 bp的引物组合,其PCR测序成功率达100%,物种鉴定准确率达98.15%。基于该引物对16份鱼胶样品的鉴定结果显示,仅56.25%的样本与标注物种一致,其中不符样品主要是鳘鱼胶和白花胶,鉴定阈值设为95%。进一步通过NJ分析和系统发育树构建,证实鱼胶样品的系统发育关系与BOLD/NCBI数据库比对结果高度一致,验证了鉴定结果的可靠性。结论本研究成功筛选出适用于鱼胶微型DNA条形码鉴定的引物,为鱼胶产品的真伪鉴别和质量控制提供了高效、可靠的技术手段。展开更多
基金supported by the Major Scientific and Technological Special Project for“Significant New Drugs Creation(No.2014ZX09304307)the Key Projects in he National Science and Technology Pillar Program(No.2011BAI07B08)
文摘Most of Chinese medicinal herbs are subjected to traditional processing procedures, including stir-frying, charring, steaming, boiling, and calcining before they are released into dispensaries. The marketing and identification of processed medicinal materials is a growing issue in the marketplace. However, conventional methods of identification have limitations, while DNA mini-barcoding, based on the sequencing of a short-standardized region, has received considerable attention as a new potential means to identify processed medicinal materials. In the present study, six DNA barcode loci including ITS2, psb A-trn H, rbc L, mat K, trnL(UAA) intron and its P6 loop, were employed for the authentication of 45 processed samples belonging to 15 species. We evaluated the amplification efficiency of each locus. We also examined the identification accuracy of the potential mini-barcode locus, of trnL(UAA) intron P6 loop. Our results showed that the five primary barcode loci were successfully amplified in only 8.89%——20% of the processed samples, while the amplification rates of the trnL(UAA) intron P6 loop were higher, at 75.56% successful amplification. We compared the mini-barcode sequences with Genbank using the Blast program. The analysis showed that 45.23% samples could be identified to genus level, while only one sample could be identified to the species level. We conclude that trnL(UAA) p6 loop is a candidate mini-barcode that has shown its potential and may become a universal mini-barcode as complementary barcode for authenticity testing and will play an important role in medicinal materials control.
文摘目的建立一种高效、精准的DNA条形码技术准确快速鉴定鱼胶物种。方法以细胞色素C氧化酶I(cytochrome C oxidase subunit I,COI)基因为靶标,筛选并优化DNA条形码引物,通过聚合酶链式反应(polymerase chain reaction,PCR)扩增和Sanger测序评估其鉴定效果。对市售鱼胶样品进行特异性扩增,结合生命条形码数据系统(barcode of life data systems,BOLD)和美国国家生物技术信息中心(national center for biotechnology information,NCBI)数据库进行物种比对,并采用邻接法(neighbor-joining,NJ)和系统发育树分析验证鉴定结果。结果通过筛选8对COI基因微型DNA条形码引物,最终确定了一组扩增目标片段约190 bp的引物组合,其PCR测序成功率达100%,物种鉴定准确率达98.15%。基于该引物对16份鱼胶样品的鉴定结果显示,仅56.25%的样本与标注物种一致,其中不符样品主要是鳘鱼胶和白花胶,鉴定阈值设为95%。进一步通过NJ分析和系统发育树构建,证实鱼胶样品的系统发育关系与BOLD/NCBI数据库比对结果高度一致,验证了鉴定结果的可靠性。结论本研究成功筛选出适用于鱼胶微型DNA条形码鉴定的引物,为鱼胶产品的真伪鉴别和质量控制提供了高效、可靠的技术手段。
