ABSTRACT
OBJECTIVE: To optimize DNA extraction methods and PCR reaction parameters, and develop an excellent and accurate rapid detection reagent for Fetus Cervi. METHODS: The DNA of Fetus Cervi was extracted by the modified salting method, modified SDS method A and modified SDS method B. Four DNA polymerase were chosen from the market and compared with each other. The annealing temperature and annealing time were optimized by classical control variable method and intersected experiment. A rapid detection reagent of Fetus Cervi was developed and then evaluated. RESULTS: The A260 /A280 ratio of the DNA extracted by modified SDS method B was (1.74 ± 0.05), and the mass concentration was (0.250 ± 0.005) μg•L -1. With high fidelity Taq DNA polymerase, the PCR product concentration could reach (0.185 ± 0.005) μg•L-1. Through these experiments, the annealing temperature was set at 58 ℃ and the annealing time was 30 s. The rapid detection reagent course was established to quickly and accurately identify Fetus Cervi and their artefacts, with one clear and bright band at 563 bp. CONCLUSION: The rapid detection reagent of Fetus Cervi combines the optimal DNA extraction method and the optimal PCR reaction parameters, and can accomplish the identification of Fetus Cervi and its pseudo-products with high accuracy and specificity.
ABSTRACT
The objective of this study was to develop stable lyophilized product of Bivalirudin, a direct thrombin inhibitor with good reconsti-tution time, % yield of good vials and cake quality. The % loss of crystallinity was determined by DSC for any crystallographic changes of the bulking agent after freeze drying. The glass transition temperature of 5% w/v of Bivalirudin, 3% w/v of mannitol and 0.03% of sodium acetate was approximately -31°C and the collapse temperature was approximately -28°C. Tubular vials were found to withstand the thermal transition during freeze drying. Water content was inversely proportional to the primary drying set point. Reconstitution time was inversely proportional to annealing temperature and vacuum set point. The % yield of good vials and cake quality was directly proportional to annealing time and primary drying set point. The % loss of crystallinity by DSC was independ-ent of all factors and directly proportional to annealing time. Drying at -10°C results in transition of β form of mannitol, whereas drying at -7.5°C and -5°C results in α and δ form of mannitol respectively. On annealing at -7°C for 54 minutes and drying at -6.5°C at 215mT lyophilized product with less than 2% water content, reconstitution time less than 10 seconds, with high yield of more than 95% yield of good vials with best cake quality was obtained.
ABSTRACT
The objective of this study was to develop stable lyophilized product of Bivalirudin, a direct thrombin inhibitor with good reconsti-tution time, % yield of good vials and cake quality. The % loss of crystallinity was determined by DSC for any crystallographic changes of the bulking agent after freeze drying. The glass transition temperature of 5% w/v of Bivalirudin, 3% w/v of mannitol and 0.03% of sodium acetate was approximately -31°C and the collapse temperature was approximately -28°C. Tubular vials were found to withstand the thermal transition during freeze drying. Water content was inversely proportional to the primary drying set point. Reconstitution time was inversely proportional to annealing temperature and vacuum set point. The % yield of good vials and cake quality was directly proportional to annealing time and primary drying set point. The % loss of crystallinity by DSC was independ-ent of all factors and directly proportional to annealing time. Drying at -10°C results in transition of β form of mannitol, whereas drying at -7.5°C and -5°C results in α and δ form of mannitol respectively. On annealing at -7°C for 54 minutes and drying at -6.5°C at 215mT lyophilized product with less than 2% water content, reconstitution time less than 10 seconds, with high yield of more than 95% yield of good vials with best cake quality was obtained.
ABSTRACT
Objective: To establish a stable, reproducible, and suitable reaction system for ISSR analysis of genetic differences in Illicium difengpi. Methods: The ISSR-PCR amplification system on I. difengpi in five factors (Mg2+, dNTPs, primers, Taq DNA polymerase, and DNA template) was optimized by orthogonal design, and the PCR result was analyzed by SPSS. Then based on the optimal ISSR-PCR amplification system, the annealing temperature and cycle times in PCR were proposed by gradient determenation. Results: Most of the factors in different levels had the significant effects on the result of PCR, and the most remarkable factor was the quantity of Taq DNA polymerase. The optimized ISSR-PCR reaction system (20 μL) for I. difengpi was constructed of Mg2+ (1.60 mmol/L), dNTP (0.22 mmol/L), primer (0.90 μmol/L), Taq polymerase (0.50 U), and DNA template (70.00 ng). The optimized annealing temperature and cycle times were 51.8 °C and 40 cycles, respectively. Thirteen ISSR primers with stable amplification and abundant polymorphism were selected from 62 ISSR primers. Conclusion: The established and optimized ISSR reaction system is stable and credible according to the testing results of 16 samples of I. difengpi, and provides the basis for the genetic analysis of I. difengpi.
ABSTRACT
Objective: To optimize the each factor affecting on ISSR-PCR reaction system of Schisandra henryi and establish the stable ISSR-PCR system. Methods: Based on the analysis of orthogonal design test, an orthogonal design was used to optimize the ISSR-PCR amplification system on S. henryi by five factors (Taq polymerase, Mg2+, DNA template, dNTP, and primer) at four concentration levels, respectively. Results: A suitable ISSR-PCR reaction system was constructed with the 20 μL reaction system containing 1.00 U Taq polymerase, Mg2+1.50 mmol/L, DNA template 40.00 ng, dNTP 0.25 mmol/L, and primer 0.50 μmol/L. Twelve effective ISSR primers were selected and the optional annealing temperature of every one primers was fixed. Conclusion: ISSR-PCR is significantly influenced by the concentration of S. henryi. This ISSR-PCR system could provide clear bands, reliable reaction system, and abundant polymorphisms. It proves a reference for molecular research of S. henryi.