Abstract:

Objective To establish a national grade Ⅱ reference material for hepatitis B virus（HBV） DNA. Methods The HBV positive serum was diluted with HBV negative serum. The content was 1.00×10⁶ IU/mL,packed to 1.5 mL each bottle. Roche COBAS AmpliPrep/COBAS TaqMan 48 testing system was used. Matrix interoperability was evaluated. For homogeneity evaluation,30 bottles were randomly selected,and each bottle was determined for 2 times. The prepared materials were placed at room temperature for 14 d,37 ℃ for 7 d,45 ℃ for 3 d,2-8 ℃ for 6 months and -20 ℃ for 12 months,and the stabilities were evaluated. The stabilities of the prepared materials which were repeated freezing and thawing for 15 times,7 d after the opening of bottles and simulated transportation to 2 laboratories were evaluated. The quantity of prepared materials was traced to the national standard material（GBW09150）. The uncertainties included the introduction of heterogeneity,the long-term stability and the quantity. Results Matrix interoperability test showed that the prepared materials' mean value was within the 95% confidence interval of clinical fresh samples. The between-run imprecision was 2.04%,and the within-run imprecision was 1.98%（F=1.052 4,P>0.05）. The stability test indicated the prepared materials were stable at room temperature for 14 d,37 ℃ for 7 d,45 ℃ for 3 d,2-8 ℃ for 6 months and -20 ℃ for 12 months,and the slope rates had no statistical significance compared with the 0th day/month by linear fitting test（P>0.05）. The stability was observed in prepared materials which were repeated freezing and thawing for 15 times,7 d after the opening of bottles and simulated transportation to 2 laboratories with no statistical significance compared with prepared materials which stored at -20 ℃（t=0.46,-0.35,1.53 and 0.75,P>0.05）. The quantity was（2.5±0.9）×10⁶ IU/mL. Conclusions The prepared materials have reached the requirements of national grade Ⅱ reference material,which can be used as a reference material for nucleic acid amplification for HBV DNA.

Key words: Reference materials, Hepatitis B virus, DNA