Identification of three Bupleurum species through a rapid detection method using the sequence-specific oligonucleotide within ribosomal DNA internal transcribed spacer as probe

• Main Authors: Wen-Yu Lin, 林文煜
• Other Authors: Tsai-Yun Lin
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/9939xm

碩士 === 國立清華大學 === 生物技術研究所 === 92 === Authentication of Chinese medicinal herbs gains more difficulties with the limitation of few unique morphological characters, and the process after harvesting. This study aims to develop an efficient method for rapid identification of Chinese medicinal herbs based on molecular biotechnology. Bupleuri Radix is a valuable and well known crude drug of Chinese medicine that is widely used in health care and remedy of liver disease. This herb is also one of the commonly misused crude drugs in Taiwan. The first part of this thesis is to analyze the genetic divergence among Bupleurum samples using the ribosomal DNA internal transcribed spacer (rDNA ITS) as marker. Our results showed that the ITS sequences of the three examined Bupleurum species share high identity with only few variations. In the second part, a rapid detection method was developed for identification of B. kaoi Liu Chao et Chuang, B. falcatum and B. chinense DC. based on oligonucleotide array. Oligomers of 15-20 bases within ITS containing one to three variation sites among different species were bound to nylon membrane. The ITS fragments were amplified with PCR using Dig-11-dUTP labeling and hybridized to a membrane holding the designed probes. The intensity of fluorescence signal was used to distinguish the binding strength between the amplified target sequence and the probe on membrane. Subsequently the suspected Bupleurum can be precisely identified through evaluation of the hybridization result in a short time and low-price. The appropriate hybridization temperature was measured below the Tm value of probe about 10-13ºC in our experiments. The signals of eight SSOP sets can be acquired after single reaction when we set hybridization temperature at 40ºC. The hybridization results of signals of forty-eight SSOPs with six dried Bupleurum samples displayed high accuracy (more than 90%). This method is reliable for authentication on condition that the factors of detection are optimized and sufficient SSOPs from the adulterate Chinese medicinal herbs are designed.