Application of clone sequencing technology and third-generation nanopore sequencing technology in identifying ingredients of Tibetan patent medicine Shiliujianwei powder
10.19778/j.chp.2025.04.008
- VernacularTitle:克隆测序技术与三代纳米孔测序技术在藏成药石榴健胃散组方药味鉴别中的应用
- Author:
Jie LIU
1
;
Wenliang FANG
1
;
Dongxue WU
1
;
Beijie XU
1
;
Jian ZHENG
1
Author Information
1. 中国食品药品检定研究院,北京 100050
- Publication Type:Journal Article
- Keywords:
Tibetan patent medicine;
Shiliujianwei powder;
polymerase chain reaction;
cloning and sequencing;
third generation nanopore sequencing
- From:
Drug Standards of China
2025;26(4):385-392
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To compare the application of cloning sequencing and third-generation nanopore sequencing technology in the identification of raw materials mixed in Tibetan patent medicine Shiliujianwei powder,and to pro-vide a reference for the establishment of the specific identification method for the formulation of medicinal prepara-tions using the raw powder of medicinal herbs or herbal pieces.Methods:By investigating the different concentra-tions of ExTaq enzyme,genomic DNA,upstream and downstream primers in the PCR amplification system,the suit-able PCR amplification system of genomic DNA universal primers for self-made Tibetan medicine Shiliujianwei powder was determined.The amplified products of Shiliujianwei powder were sequenced by two methods,the first was cloned and sequenced by Sanger sequencing technology,and the second was sequenced by third generation nanopore sequen-cing technology.Results:The addition of ExTaq enzyme,upstream and downstream primers and genomic DNA in 20 μL PCR amplification system of Shiliujianwei powder and single medicinal samples were determined to be 0.4,1.5 and 1 μL,respectively.The amplification products of the DNA from self-made Shiliujianwei powder were cloned and sequenced by Sanger sequencing technology,then only the ITS2 sequence of Carthami flos was obtained.The amplified products of self-made pomegranate Jianwei powder DNA were sequenced by three-generation nanopore se-quencing technology,and the ITS2 sequences of Granati semen,Carthami flos,Piperis longi fructus and Amomi fruc-tus rotundus were finally obtained,but the ITS2 sequence of cinnamomi cortex was not obtained.Conclusion:Both cloning sequencing and the third generation nanopore sequencing could solve the problem of overlapping peaks in the direct sanger sequencing for the universal primer amplification products of patent drugs.The third generation nano-pore sequencing was better than cloning sequencing in the sequencing of the universal primer amplification products of patent drugs,but there were still one raw material medicine that have not been detected.It is of certain reference val-ue for the molecular biological identification and DNA barcoding study of different raw materials in the patent medi-cine to establish the specific identification method of Tibetan patent medicine Shiliujianwei powder.
