Tianyi Xin1, Chang Su2, Yulin Lin1, Shuhong Wang2, Zhichao Xu3, Jingyuan Song4. 1. Key Lab of Chinese Medicine Resources Conservation, State Administration of Traditional Chinese Medicine of the People's Republic of China, Institute of Medicinal Plant Development, Peking Union Medical College & Chinese Academy of Medical Sciences, No.151, Malianwa North Road, Haidian District, Beijing 100193, PR China. 2. Shenzhen Institute for Drug Control, Shenzhen 518057, PR China. 3. Key Lab of Chinese Medicine Resources Conservation, State Administration of Traditional Chinese Medicine of the People's Republic of China, Institute of Medicinal Plant Development, Peking Union Medical College & Chinese Academy of Medical Sciences, No.151, Malianwa North Road, Haidian District, Beijing 100193, PR China. Electronic address: zcxu@implad.ac.cn. 4. Key Lab of Chinese Medicine Resources Conservation, State Administration of Traditional Chinese Medicine of the People's Republic of China, Institute of Medicinal Plant Development, Peking Union Medical College & Chinese Academy of Medical Sciences, No.151, Malianwa North Road, Haidian District, Beijing 100193, PR China. Electronic address: jysong@implad.ac.cn.
Abstract
BACKGROUND: Current quality control methods for traditional Chinese patent medicines (TCPMs), e.g., microscopy, thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC), cannot detect herbal species composition with adequate precision. To address this issue, more effective detection methods should be explored. HYPOTHESIS/ PURPOSE: We hypothesized that shotgun metagenomic sequencing can fulfill the requirements for the species detection of multi-ingredient TCPMs. METHODS: Longdan Xiegan Wan (LDXGW), once thought to be the chief culprit in aristolochic acid nephropathy (AAN), was selected to establish the method. It was used for both reference and commercial LDXGW samples. The precision authentication of herbal species contained in multi-ingredient TCPM is based on the shotgun metagenomic sequencing of genomic DNA without PCR amplification. Chemical analyses were also conducted as a contrast test. RESULTS: Over 100 G of raw data was obtained, and this value represented more than 0.75 billion reads. After assembling and filtering all the reads, a total of 261 contigs were obtained, which belonged to the ITS2, psbA-trnH, and matK regions of the reference and commercial samples. Because the homology of the rbcL region was high, it was not analyzed in the HTS data. Bioinformatics analysis indicated that the ITS2 region, as a DNA barcode, showed the highest identification efficiency. It could successfully detect all prescribed species, including four processed herbal ingredients, in the lab-made reference samples. The commercial samples all met the requirements of the Chinese Pharmacopoeia according to the TLC and HPLC tests. However, the shotgun metagenomic sequencing detected the substitution of Akebiae Caulis (Mutong) in the commercial samples, while the chemical analyses could not distinguish. CONCLUSION: The results highlight that shotgun metagenomic sequencing is a complementary method for the precise species detection of TCPMs.
BACKGROUND: Current quality control methods for traditional Chinese patent medicines (TCPMs), e.g., microscopy, thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC), cannot detect herbal species composition with adequate precision. To address this issue, more effective detection methods should be explored. HYPOTHESIS/ PURPOSE: We hypothesized that shotgun metagenomic sequencing can fulfill the requirements for the species detection of multi-ingredient TCPMs. METHODS: Longdan Xiegan Wan (LDXGW), once thought to be the chief culprit in aristolochic acid nephropathy (AAN), was selected to establish the method. It was used for both reference and commercial LDXGW samples. The precision authentication of herbal species contained in multi-ingredient TCPM is based on the shotgun metagenomic sequencing of genomic DNA without PCR amplification. Chemical analyses were also conducted as a contrast test. RESULTS: Over 100 G of raw data was obtained, and this value represented more than 0.75 billion reads. After assembling and filtering all the reads, a total of 261 contigs were obtained, which belonged to the ITS2, psbA-trnH, and matK regions of the reference and commercial samples. Because the homology of the rbcL region was high, it was not analyzed in the HTS data. Bioinformatics analysis indicated that the ITS2 region, as a DNA barcode, showed the highest identification efficiency. It could successfully detect all prescribed species, including four processed herbal ingredients, in the lab-made reference samples. The commercial samples all met the requirements of the Chinese Pharmacopoeia according to the TLC and HPLC tests. However, the shotgun metagenomic sequencing detected the substitution of Akebiae Caulis (Mutong) in the commercial samples, while the chemical analyses could not distinguish. CONCLUSION: The results highlight that shotgun metagenomic sequencing is a complementary method for the precise species detection of TCPMs.
Authors: Niina Haiminen; Stefan Edlund; David Chambliss; Mark Kunitomi; Bart C Weimer; Balasubramanian Ganesan; Robert Baker; Peter Markwell; Matthew Davis; B Carol Huang; Nguyet Kong; Robert J Prill; Carl H Marlowe; André Quintanar; Sophie Pierre; Geraud Dubois; James H Kaufman; Laxmi Parida; Kristen L Beck Journal: NPJ Sci Food Date: 2019-11-19