RESUMO
Objective To establish an accurate TaqMan probe real-time quantitative PCR (qPCR)method for detection of Theiler''s-like virus of rats (TLV).Methods Primers and TaqMan probes specific to 3622~3729 nt region were designed according to the whole genomic sequence of TLV representative strain.Using a synthesized plasmid as DNA standard template, the stability, specificity, and sensitivity of the qPCR method were determined.Results In the standard curve, R2 value was 0.99 with a high specificity.The sensitivity of the real-time PCR was less than 10 copies/μL, which was 100 times higher than the ordinary PCR method.No cross reactions appeared to the other rat viruses.Conclusions The TaqMan probe qPCR method established in this study has advantages such as simple to use, high sensitivity and specificity.
RESUMO
Objective To establish an optimized method to determine the entrapment efficiencies(EE)of cinobufagin(CBG) and resibufogenin(RBG)in the toad skin extract loaded with solid lipid nanoparticles. Methods Dialysis,high-speed centrifugation and low-speed centrifugation were selected to determine the entrapment efficiencies of toad skin extract loaded solid lipid nanoparti?cles. The effects of different dialytic media on the entrapment efficiencies were investigated ,then the EE of every method were com?pared. Results Different methods had different results of EE,the EE of low-speed centrifugation,high-speed centrifugation and dialysis method in CBG and RBG were(74.00±1.69)%and(75.01±2.05)%,(83.60±0.99)%and(82.51±1.56)%,(91.01±0.75)%and(89.22± 0.88)%,respectively. The EE determined by different dialytic media of dialysis were different,but the results did not have the signifi?cant differences. Conclusion Different determination methods of EE have some significant influences on the results of EE,dialysis method is more suitable for the determination of EE for CBG and RBG in the toad skin extract loaded solid lipid nanoparticles.