Optimization and Developmental Validation of 38-plex InDel Panel for Ancestry Inference / 法医学杂志

OBJECTIVES@#The previously established 38-plex InDel system was optimized and its performance was validated according to the Scientific Working Group on DNA Analysis Method (SWGDAM) application guidelines. The ancestry inference accuracy of individuals from East Asian, European, African and mixed populations was verified.@*METHODS@#DNA standard sample 9947A was used as the template to establish the optimal amplification conditions by adjusting primer balance, Mg2+ final concentration and optimizing PCR thermal cycle parameters and amplification volume. The allelic dropout, nonspecific amplification and whether the origin of the inferred samples matched the known information were compared to evaluate the performance of this system.@*RESULTS@#The optimal dosage of this system was 0.125-2 ng DNA template. The results of InDel typing were accurate, the amplification equilibrium was good, and the species specificity was good. This system showed certain tolerance to DNA samples including the inhibitor such as hemoglobin (≤80 μmol/L), indigo (≤40 mmol/L), calcium ion (≤1.0 mmol/L), and humic acid (≤90 ng/μL). The system enabled the direct amplification of DNA from saliva and blood on filter paper, and the results of ethnic inference were accurate. The system successfully detected the mixed DNA sample from two individuals. The test results of the system for common biological materials in practical cases were accurate.@*CONCLUSIONS@#The results of the 38-plex InDel system are accurate and reliable, and the performance of the system meets the requirement of the SWGDAM guidelines. This system can accurately differentiate the ancestry origins of individuals from African, European, East Asian, and Eurasian populations and can be implemented in forensic practice.

Effect of iguratimod on diclofenac metabolism by CYP2C9 in rats and human recombinant CYP2C9 yeast cells

Iguratimod (IGU, also known as T-614), a novel disease modifying antirheumatic drug intended to cure patients with rheumatoid arthritis (RA). The purpose of this study is to evaluate the effect of IGU on the pharmacokinetics of CYP2C9 probe drug diclofenac and its metabolite 4′-hydroxy diclofenac in vivo and in vitro. In in vivo experiments, 24 rats were randomly assigned to three groups consisting of the control group (Normal saline), low dose IGU group (10 mg/kg) and high dose IGU group (30 mg/kg). Blood samples were collected from orbital sinuses vein before 1 hour and serial times of giving diclofenac (15 mg/kg) to all the rats. Plasma concentration of diclofenac and its metabolite 4´-hydroxy diclofenac were assayed by high performance liquid chromatography. Pharmacokinetic parameters were assessed by Winnonlin 6.4 pharmacokinetic software. Moreover, in vitro studies were performed in recombinant human CYP2C9 yeast cell system. IGU at low dose showed no significant differences in the pharmacokinetic parameters of diclofenac and 4-hydroxy diclofenac in vivo when compared with control group (p>0.005). However, at the high dose of IGU, the pharmacokinetic parameters of 4´-hydroxy metabolite of diclofenac increase in half-life (T1/2) and mean area under the curve (AUC0→24), while a decrease in mean clearance (CL, mL/h/kg) and volume of distribution Vz (mL/kg). In addition, in in vitro study, high doses of IGU reduces the metabolism rate of diclofenac. IGU at high dose significantly increase the pharmacokinetics parameters of 4´-hydroxy diclofenac in rats. Additionally, it also showed the potent inhibitory effect on diclofenac metabolism in recombinant human CYP2C9 yeast cells.

Construction of a 15-plex Rapid STR Multiplex Amplification System / 法医学杂志

OBJECTIVE@#To establish a 15-plex rapid STR multiplex amplification system.@*METHODS@#Fourteen auto-chromosome loci and one sex-chromosome were selected to compare the situations of allelic losses and nonspecific amplication under different conditions. FastStart Taq DNA polymerase and DNA standard sample 9947A were used during amplification and optimization process.15-plex rapid STR amplification system was achieved by performing various experiments including selection of amplification conditions and the volume of DNA polymerase, adjustment of inter-locus balance, optimization of rapid amplification, screening of reaction buffers, selection of reaction volume, and a variety of additives.@*RESULTS@#Using 10 μL rapid PCR system, including 1 ng DNA templates, 0.4 μL polymerase and 10xFastStart high fidelity reaction buffer, a complete and well-balance DNA profile of 15 STR loci for standard genomic DNA was obtained in 32 minutes, without the allele drop-out and non-specific amplicons. Meanwhile, 5% glycerinum, 0.01% gelatin, 0.05% gelatin and 5 mmol/L ammonium sulfate could be used as the reactive additive during the amplification procedure.@*CONCLUSION@#The 15-plex rapid STR multiplex amplification system can be used to decrease reaction time and enhance sample throughput.

Low volume amplification in single cell separation and inspection / 法医学杂志

OBJECTIVE@#To establish optimal amplification conditions with the application of 0.2 mL test tube in single cell separation and inspection.@*METHODS@#Oral epithelium cell suspension was prepared. Five or ten cells were collected with 0.2 mL test tube. Then DNA were amplified with Identifiler Plus kit in three different conditions in which the proteinase K addition, gold enzyme concentration in PCR reaction, and PCR reaction cycles were adjusted separately. Finally the detection rate, allelic dropout rate and artificial alleles were compared among the groups.@*RESULTS@#In these 3 different conditions: addition of proteinase K, addition of 0.4 microL gold enzyme in PCR reaction, and use of 32 cycles, the detection rate was higher and allelic dropout rate was lower than the other conditions.@*CONCLUSION@#In single cell separation and inspection, the usage of 0.2 mL test tube could be an effective supplement to chip-low volume amplification.