Applied Cell Biology
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High Stability of the HBV-DNA in Samples Stored at Different Temperatures or Stressed by Repeated Freeze-Thawing Treatment
Jorge Aguiar, Eduardo Canales, Gerardo García, Jose Angel Silva, Gerardo Guillén and Julio Cesar Aguilar
1Department for the Therapeutic Vaccine against Hepatitis B, Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba
2Department for Plant Genomics, Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba
3Department for Quality Control, Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba
4Department for Oligonucleotide Synthesis, Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba
Abstract
Most of the patients chronically infected with hepatitis B are living in countries with limited resources. The diagnostic systems need to be robust enough to facilitate the assessment of samples under stress due to temperature and power failure. The stability of the HBV-DNA was assessed at different temperatures and under freezethawing conditions in three different kind of HBV containing samples.
HBV positive serum samples and their purified matched HBV-DNA samples were incubated both at 37°C and -20°C and subsequently quantified by HBV-specific qPCR. In addition, the performance in qPCR of the above-mentioned samples and also of an in-house HBV plasmid standard curve based on a plasmid comprising the genome of the hepatitis B virus as a part of a novel qPCR system were assayed under freeze-thawing conditions.
No significant differences were detected between groups of treatment in terms of HBV-DNA levels, both in the case of serum samples or their purified matched counterpart when stored to both 37°C and -20°C or when treated with the freezethawing protocol. Similar results were obtained with the HBV plasmid standards when they were treated with the freeze-thawing procedures.
In conclusion, both serum HBV-DNA samples as well as the purified matched HBVDNA serum samples were stable at 37°C, or after repeated cycles of freeze-thawing like also was the standard curve from the novel qPCR, further demonstrating the robustness of this quantitative system. These results support the use of this novel quantitative system in areas with limitations in cold chain conditions during transportation.
