AIM To investigate and compare the analytical and clinical performance of Tian Long automatic hypersensitive hepatitis B virus(HBV) DNA quantification system and Roche CAP/CTM system.METHODS Two hundred blood samples ...AIM To investigate and compare the analytical and clinical performance of Tian Long automatic hypersensitive hepatitis B virus(HBV) DNA quantification system and Roche CAP/CTM system.METHODS Two hundred blood samples for HBV DNA testing, HBV-DNA negative samples and high-titer HBV-DNA mixture samples were collected and prepared. National standard materials for serum HBV and a worldwide HBV DNA panel were employed for performance verification. The analytical performance, such as limit of detection, limit of quantification, accuracy, precision, reproducibility, linearity, genotype coverage and cross-contamination, was determined using the Tian Long automatic hypersensitive HBV DNA quantification system(TL system). Correlation and Bland-Altman plot analyses were carried out to compare the clinical performance of the TL system assay and the CAP/CTM system. RESULTS The detection limit of the TL system was 10 IU/m L, and its limit of quantification was 30 IU/m L. The differences between the expected and tested concentrations of the national standards were less than ± 0.4 Log10 IU/m L, which showed high accuracy of the system. Results of the precision, reproducibility and linearity tests showed that the multiple test coefficient of variation(CV) of the same sample was less than 5% for 102-106 IU/m L; and for 30-108 IU/m L, the linear correlation coefficient r2 = 0.99. The TL system detected HBV DNA(A-H) genotypes and there was no cross-contamination during the "checkerboard" test. When compared with the CAP/CTM assay, the two assays showed 100% consistency in both negative and positive sample results(15 negative samples and 185 positive samples). No statistical differences between the two assays in the HBV DNA quantification values were observed(P > 0.05). Correlation analysis indicated a significant correlation between the two assays, r2 = 0.9774. The Bland-Altman plot analysis showed that 98.9% of the positive data were within the 95% acceptable range, and the maximum difference was-0.49.CONCLUSION The TL system has good analytical performance, and exhibits good agreement with the CAP/CTM system in clinical performance.展开更多
The purpose of this study is to detect the plasmid copies encoding HBsAg in recombinant yeast Saccharomyces cerevisiae by real-time PCR, and provide the data for setting up an optimal process of manufacturing hepatiti...The purpose of this study is to detect the plasmid copies encoding HBsAg in recombinant yeast Saccharomyces cerevisiae by real-time PCR, and provide the data for setting up an optimal process of manufacturing hepatitis B vaccine in future. A single copy URA3 gene in the yeast genome was employed as internal control to test the plasmid copies by real-time PCR. The yeast cells carrying plasmids encoding HBsAg were cultured in regular non-feeding process and fed-batch process in shaking flasks at laboratory level and industrial fermentor. The cells at different stages of cultivation were collected and broken. Then the total DNAs of the cells were extracted and the plasmid copies were detected by real-time PCR with the method of relative quantification. The method of the real-time relative quantitative PCR used in this study was reliable with good accuracy and quickness. The plasmid copies in non-feeding and fed-batch flasks were 39.22±12.00 and 43.06±5.70, while the copies in fermentor were 96.16±21.00 and 82.50±7.78, respectively. The data show that the variation of the result is very small and the data precision is quite good. To prolong the fermentation time may increase the cell density without influence on the stability of the plasmid, and the method of relative quantification can also be applied to test the copy number of interest target gene in any host cell with certain known copy gene.展开更多
文摘AIM To investigate and compare the analytical and clinical performance of Tian Long automatic hypersensitive hepatitis B virus(HBV) DNA quantification system and Roche CAP/CTM system.METHODS Two hundred blood samples for HBV DNA testing, HBV-DNA negative samples and high-titer HBV-DNA mixture samples were collected and prepared. National standard materials for serum HBV and a worldwide HBV DNA panel were employed for performance verification. The analytical performance, such as limit of detection, limit of quantification, accuracy, precision, reproducibility, linearity, genotype coverage and cross-contamination, was determined using the Tian Long automatic hypersensitive HBV DNA quantification system(TL system). Correlation and Bland-Altman plot analyses were carried out to compare the clinical performance of the TL system assay and the CAP/CTM system. RESULTS The detection limit of the TL system was 10 IU/m L, and its limit of quantification was 30 IU/m L. The differences between the expected and tested concentrations of the national standards were less than ± 0.4 Log10 IU/m L, which showed high accuracy of the system. Results of the precision, reproducibility and linearity tests showed that the multiple test coefficient of variation(CV) of the same sample was less than 5% for 102-106 IU/m L; and for 30-108 IU/m L, the linear correlation coefficient r2 = 0.99. The TL system detected HBV DNA(A-H) genotypes and there was no cross-contamination during the "checkerboard" test. When compared with the CAP/CTM assay, the two assays showed 100% consistency in both negative and positive sample results(15 negative samples and 185 positive samples). No statistical differences between the two assays in the HBV DNA quantification values were observed(P > 0.05). Correlation analysis indicated a significant correlation between the two assays, r2 = 0.9774. The Bland-Altman plot analysis showed that 98.9% of the positive data were within the 95% acceptable range, and the maximum difference was-0.49.CONCLUSION The TL system has good analytical performance, and exhibits good agreement with the CAP/CTM system in clinical performance.
文摘The purpose of this study is to detect the plasmid copies encoding HBsAg in recombinant yeast Saccharomyces cerevisiae by real-time PCR, and provide the data for setting up an optimal process of manufacturing hepatitis B vaccine in future. A single copy URA3 gene in the yeast genome was employed as internal control to test the plasmid copies by real-time PCR. The yeast cells carrying plasmids encoding HBsAg were cultured in regular non-feeding process and fed-batch process in shaking flasks at laboratory level and industrial fermentor. The cells at different stages of cultivation were collected and broken. Then the total DNAs of the cells were extracted and the plasmid copies were detected by real-time PCR with the method of relative quantification. The method of the real-time relative quantitative PCR used in this study was reliable with good accuracy and quickness. The plasmid copies in non-feeding and fed-batch flasks were 39.22±12.00 and 43.06±5.70, while the copies in fermentor were 96.16±21.00 and 82.50±7.78, respectively. The data show that the variation of the result is very small and the data precision is quite good. To prolong the fermentation time may increase the cell density without influence on the stability of the plasmid, and the method of relative quantification can also be applied to test the copy number of interest target gene in any host cell with certain known copy gene.
