The misuse of toxic drugsisseriousone of the threats of public health. In this study, toxic Hyoscyami Semen and its adulterants were identified by DNA barcoding. The genomic DNA was extracted from 61 samples including Hyoscyami Semen and its adulterants by reagent kit method. Their ITS2 sequences were amplified, and purified PCR products were sequenced. Sequence assembly and consensus sequence generation were performed using CodonCode Aligner v 4.25. The genetic distances, variable sites and the neighbor-joning (NJ) phylogenetic tree were computed by MEGA 6.0 in accordance with the Kimura 2-parameter(K2P) model. The results showed that the intra-specific genetic distances of Hyoscyamusniger were 0.005 which were smaller than inter -specific ones (0.360) of H. niger and their adulterants. The NJ tree showed that H. niger was clustered into one monophyletic branch, and clearly separated with other species. Therefore, ITS2 sequence was able to identify Hyoscyami Semen and its adulterants to ensure the safty of medicines.

Objective: To establish fluorescence quantitative polymerase chain reaction (PCR) to detect integrated HIV DNA in peripheral blood mononuclear cells. Methods: A total of 30 HIV-seropositve individuals were enrolled in this study, including 10 subjects with a detection limit of 20 copies/ml of plasma, 10 patients with drug resistance and 10 patients with no history of antiretroviral therapy (ART). Cultivated ACH2 cells carried a single copy of the integrated HIV genome. We have built pMD19T-CD3 plasmid and calculated the copy number. We used oligonucleotides ULF1 specific for the long terminal repeats (LTR) regions and two oliligonucleotides specific for human Alu sequences to pre-amplified the integrated HIV DNA. Samples and serial dilutions of ACH2 cells were all pre-amplified, the products of which were used for the second round fluorescence amplifications. The Lambda T primers, UR2 primers and HIV Taqman probes were used for second round amplifications in integrated HIV DNA assay. The CD3IN5 primers, CD3IN3 primers and CD3 Taqman probes were used for CD3 quantification. Results: Serial 5-fold dilutions of the plasmid were used as standards for CD3 gene quantifications. The equation of the linear regression was y=-2.731x+ 43.01(R²=0.953). The cellular input was quantified by number of human genome equivalents (CD3 gene, 2 copies/cell). The copies of ACH2 cells was 4.271×10^4, which was the cellular input of the ACH2 cells. Serial 5-fold dilutions of ACH2 cells were used to generate a standard curve for the integrated HIV DNA assays. The equation of the linear regression was y=-3.146x+ 39.11 (R²=0.968). The ratio between the number of copies of the integrated HIV DNA form and the number of cells (CD3 copies) was calculated to obtain the frequency of cells. Based on the test between different groups, each group had no difference (P> 0.05). Conclusions This method presented advantage in detection of the lower copies of virus. It hinted that the current antiretroviral therapy can not effectively attack against latent viral reservoirs. It also provided a basis for new therapeutic intervention.