A specific SNP-based multiplex PCR assay for the simultaneous identification of two biological ingredients for the Chinese patent medicine, Danggui Buxue pill.

Background: An increasing number of Chinese patent medicines (CPM) have been widely used in East Asian and North American countries, and the safety and efficacy of CPM have highly attracted public attention. However, it is difficult to supervise the authenticity of multiple biological ingredients within CPM based on microscopic inspection and physical and chemical detection. The raw materials may have similar characteristics of tissue structures and ergastic substances or similar chemical composition and contents when substitutes and/or adulterants are added. DNA molecular markers have been used to distinguish the biological ingredients within CPM based on conventional PCR assay. However, it was proved to be time- and labor-consuming and reagent-wasting, as multiple PCR amplification strategies were required for identifying the complex species composition within CPM. Here, we took the CPM (Danggui Buxue pill) as an example and aimed to establish a specific SNP-based multiplex PCR assay and simultaneously determine the authenticity of the two biological ingredients (Angelicae Sinensis Radix and Astragali Radix) within this CPM. Methods: We, respectively, designed the species-specific primers based on highly variable nrITS for discriminating Angelicae Sinensis Radix and Astragali Radix from their common substitutes and adulterants. The specificity of the primers was checked through conventional PCR assay and multiplex PCR assay. Furthermore, we used a handcrafted Danggui Buxue pill sample (DGBXP) to optimize annealing temperatures for the primers with multiplex PCR, and the sensitivity was also assessed. Finally, fourteen batches of commercial Danggui Buxue pills were used to verify the stability and practicability of the established multiplex PCR assay. Results: Two pairs of highly species-specific primers for amplifying Angelicae Sinensis Radix and Astragali Radix were screened, and our established multiplex PCR assay showed high specificity and sensitivity (lowest detection concentration: 4.0 × 10-3 ng/µL) at an optimal annealing temperature of 65°C. The method could simultaneously identify both biological ingredients within the Danggui Buxue pill. Conclusion: The specific SNP-based multiplex PCR provided a simple, time-, and labor-saving method for the simultaneous identification of the two biological ingredients within Danggui Buxue pills. This study was expected to provide a novel qualitative quality control strategy for CPM.

Limited genetic diversity and high differentiation in Angelica dahurica resulted from domestication: insights to breeding and conservation.

BACKGROUND: Angelica dahurica belongs to the Apiaceae family, whose dry root is a famous traditional Chinese medicine named as "Bai zhi". There are two cultivars (A. dahurica cv. 'Hangbaizhi' and A. dahurica cv. 'Qibaizhi'), which have been domesticated for thousands of years. Long term artificial selection has led to great changes in root phenotypes of the two cultivars, and also decreased their adaptability to environment. We proposed hypothesis that the cultivars may have lost some of the genetic diversity found in the wild species and may be highly differentiated from the latter during the domestication process. However, few studies have been carried out on how domestication affected the genetic variation of this species. Here, we accessed the levels of genetic variation and differentiation within and between wild A. dahurica populations and two cultivars using 12 microsatellite markers. RESULTS: The results revealed that the genetic diversity of the cultivars was much lower than that of wild A. dahurica, and A. dahurica cv. 'Qibaizhi' had lower genetic diversity compared to A. dahurica cv. 'Hangbaizhi'. AMOVA analysis showed significant genetic differentiation between the wild and cultivated A. dahurica populations, and between A. dahurica cv. 'Hangbaizhi' and A. dahurica cv. 'Qibaizhi'. Results from Bayesian, UPGMA, NJ and PcoA clustering analysis indicated that all 15 populations were assigned to two genetic clusters corresponding to the wild and cultivated populations. Bayesian clustering analysis further divided the cultivated populations into two sub-clusters corresponding to the two cultivars. CONCLUSIONS: Our study suggests that the domestication process is likely the major factor resulting in the loss of genetic diversity in cultivated A. dahurica populations and in significant genetic differentiation from the wild populations due to founder effect and/or artificially directional selections. This large-scale analysis of population genetics could provide valuable information for genetic resources conservation and breeding programs of Angelica dahurica.

The complete chloroplast genome sequence of Ficus hirta (Moraceae).

The dry root (Radix Fici Hirtae) of Ficus hirta has been used as a traditional herbal medicine in Ling nan regions of China for a long time. As its large market demand, the wild resources of F. hirta have sharply reduced. It is necessary to conduct the study of conservation genetics. However, there is still lack of complete genome information for the research on evolutionary biology, population genetics and phylogeography of this species. Here, we sequenced the complete chloroplast (CP) genome of F. hirta using Next Generation Sequencing technology (NGS). The CP genome of F. hirta is 160,374 bp in length, which contains a large single-copy (LSC) region of 88,446 bp, a small sing-copy (SSC) region of 18,134 bp, and two inverted repeat (IRa and IRb) regions of 26,897 bp. A total of 130 genes were successfully annotated containing 85 protein-coding genes, 37 tRNA genes and 8 rRNA genes. Phylogenetic analysis support genus Ficus is monophyletic and F. hirta is closely related to F. carica within this genus.

The -137G>C polymorphism in interleukin-18 promoter region and cancer risk: evidence from a meta-analysis of 21 studies.

Interleukin-18 (IL-18) is a key cytokine responsible for immune response and involved in the process of cancer development. The association of -137G>C polymorphism in the promoter region of IL-18 with cancer risk is still elusive based on current genetic association studies. We performed this meta-analysis to determine whether the -137G>C polymorphism is associated with cancer risk. A comprehensive search was conducted for databases of PubMed, EMBASE, and China National Knowledge Infrastructure. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to estimate the association strength. Publication bias was detected by Egger's and Begg's test. Twenty-one eligible studies including 3,498 cancer patients and 5,222 controls were identified and analyzed. In the overall analysis, no significant association between -137G>C polymorphism and cancer risk was observed. In the sub-group analyses of ethnicities, the -137G>C polymorphism significantly increased cancer risk in Asian population (GC/CC vs. GG: OR = 1.313, 95% CI = 1.053-1.638, heterogeneity P < 0.001) but not in Caucasian population. Further stratified analyses showed that the variant -137C allele was significantly associated with increased risk of nasopharyngeal carcinoma (C vs. G: OR = 1.484, 95% CI = 1.193-1.847, heterogeneity P = 0.213). No publication bias was detected. We provide evidence that the -137G>C polymorphism in IL-18 promoter region significantly increases cancer risk in Asian population but not in Caucasian population, and the variant -137C allele is associated with increased risk of nasopharyngeal carcinoma.