ABSTRACT
Global concerns have been paid to the potential hazard of traditional herbal medicinal products (THMPs). Substandard and counterfeit THMPs, including traditional Chinese patent medicine, health foods, dietary supplements, etc. are potential threats to public health. Recent marketplace studies using DNA barcoding have determined that the current quality control methods are not sufficient for ensuring the presence of authentic herbal ingredients and detection of contaminants/adulterants. An efficient biomonitoring method for THMPs is of great needed. Herein, metabarcoding and single-molecule, real-time (SMRT) sequencing were used to detect the multiple ingredients in Jiuwei Qianghuo Wan (JWQHW), a classical herbal prescription widely used in China for the last 800 years. Reference experimental mixtures and commercial JWQHW products from the marketplace were used to confirm the method. Successful SMRT sequencing results recovered 5416 and 4342 circular-consensus sequencing (CCS) reads belonging to the ITS2 and regions. The results suggest that with the combination of metabarcoding and SMRT sequencing, it is repeatable, reliable, and sensitive enough to detect species in the THMPs, and the error in SMRT sequencing did not affect the ability to identify multiple prescribed species and several adulterants/contaminants. It has the potential for becoming a valuable tool for the biomonitoring of multi-ingredient THMPs.
ABSTRACT
Objective: To identify Rehmanniae Radiuxand its closely related species using the ITS2 barcode and to guarantee the quality and clinical curative effect of this medical material. Method: The sequences has been ana-lyzed and assembled using corresponding software. The Kimura 2-Parameter (K2P) distances were calculated and NJ (neighbor-joining) tree was established based on the K2P methods to identify the Rehmanniae Radix. Results:The length of the ITS2 sequence of Rehmanniae Radix was 231 bp. The average intra-specific genetic distances of Rehmanniae Radix were 0.0004. The average inter-specific genetic distances between Rehmanniae Radix and its closely related species were 0.0312. The minimum inter-specific divergence is larger than the maximum intra-specific divergence. The Rehmanniae Radix can be identified using the NJ trees method. Conclusions: The ITS2 sequence can be used as DNA barcode to identify Rehmanniae Radix and its closely related species, which will lay the foundation for the clinical medication safety of Rehmanniae Radix.
ABSTRACT
matK is one of the core DNA barcode markers for plant DNA barcode identification and its universality using single makers has been in controversy. However, the universalities of different matK primer pairs in same seed plant group (order) and same matK primer pairs in different seed plant groups (order) are lack of systematic research. In this study, we collected 14563 full-length matK sequences of 11429 species of 3292 genera in 239 families belonging to 36 orders in seed plants. The universalities of 13 matK primer pairs and its 78 primer com-binations have been assessed using bioinformatics methods. The results indicated that xf/5r, 1F/8R, 390F/1326R and 3F_KIM/1R_KIM were the four most universality primer pairs. The four markers' universalities were 91.18%, 84.65%, 79.81% and 80.94% respectively in all 11429 seed plants. The most universality primer pairs in different orders were different. For each order, the primer pair with maximum universality was different. the xf/5r was the basal primer pair for primer combination and 1F/8R, 1F/1R, M3/M4 and 3F_KIM/1R_KIM could be the complementary primer pairs. This study could be a valuable resource for the primer selection of the research DNA barcoding identification in seed plants.
ABSTRACT
The ITS/ITS2 barcodes were used to simply and effectively identify Codonopsis Radix and its adulter-ants. In this study, ITS (internal transcribed spacer of unclear ribosomal DNA) regions were amplified using PCR (polymerase chain reaction) from thirty-three samples of Codonopsis Radix and ITS2 regions were obtained from the ITS sequences using the hidden Markov model (HMMer)-based annotation methods. The sequences of ITS/ITS2 regions were aligned and the genetic distances were computed by MEGA5.0. Species identification efficiency of ITS/ITS2 sequences were evaluated using BLAST1 and nearest distance methods. The results indicated that The sequences lengths of ITS regions of Codonopsis Radix were 654-655 bp, and the lengths of ITS2 regions were 239 bp. The intraspecific genetic distances among Codonopsis Radix were obviously lower than the interspecific genetic distance between Codonopsis Radix and its adulterants. Therefore, ITS/ITS2 regions can stably and accu-rately distinguish Codonopsis Radix and its adulterants.
ABSTRACT
DNA barcoding is a rapidly developing frontier technology in the world and will be useful in promoting the quality control and standardization of traditional Chinese medicine. Until now, many studies concerning DNA barcoding have focused on leaf samples but rarely on Chinese herbal medicine. There are three issues involved in DNA barcoding for traditional Chinese medicinal materials: (1) the extraction methods for total DNA of the rhizomes of the medicinal materials; (2) intra-specific variation among samples from different places of origin; (3) accuracy and stability of this method. In this study, Gentianae Macrophyllae Radix was used to verify the stability and accuracy of DNA barcoding technology. Five regions (ITS2, psbA-trnH, matK, rbcL, and ITS) were tested for their ability to identify 86 samples of Gentianae Macrophyllae Radix and their adulterants. After improving the DNA extraction method, genomic DNA from all samples was successfully obtained. To evaluate each barcode's utility for species authentication, PCR amplification efficiency, genetic divergence, and species authentication were assessed. Among all tested regions only ITS2 locus showed 100% of PCR amplification and identification efficiencies. Based on the established method, we successfully identified two samples of Gentianae Macrophyllae Radix bought in pharmacy to the original species.
ABSTRACT
ObjectiveTo evaluate the sensitivity and accuracy of an in-house detecting method of HIV-1 genotypic drug resistance system. MethodsTotally 130 serum specimens from Henan and Guangxi province were collected from April 2004 to October 2008 and tested in the Military HIV Testing Center of China. ViroSeqTM v2.0 (Abbott, Switzerland), a US FDA approved HIV genotypic drug resistance detecting system was utilized as the reference method. All the specimens were detected by the novel in-house method and the reference method to validate the difference in amplifying efficiency, drug resistance mutation detection and drug resistance report. ResultsConcerning the 14 850 known drug resistance mutation sites,14 752 (99. 3％ ) mutations can be detected by both of the two methods. Rates of concordance of detection in the regions of protease inhibitors-, reverse transcriptase inhibitors- and both two classes inhibitors-resistance were99.7％ ( Kappa =0. 909 9 , P ＜0. 01 ) , 99. 0％ (Kappa=0.952 1, P＜0. 01) and99.3％ (Kappa=0. 948 8, P ＜ 0. 01 ) respectively. Drug resistance reports from these two systems showed similar results (Kappa = 0. 637 4, P ＜ 0. 01 ). The in-house detecting system identified 34 novel mutations besides the ViroSeqTM drug resistance mutation database ( ViroSeqTM software v2. 7). Two mutations, V179F and K238T,had significant effect on HIV drug resistance. ConclusionsThe in-house genotyping system is an accurate,cost-effective method and has a high concordance with commercial ViroSeqTM genotyping system. Database from the in-house assay was superior to this of the ViroSeqTM assay.