Euroasian journal of hepato-gastroenterology

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VOLUME 6 , ISSUE 1 ( January-June, 2016 ) > List of Articles


High Functional Stability of a Low-cost HBV DNA qPCR Primer Pair and Plasmid Standard

Gerardo García, Yamila León, Eduardo Canales, José Angel Silva

Citation Information : García G, León Y, Canales E, Angel Silva J. High Functional Stability of a Low-cost HBV DNA qPCR Primer Pair and Plasmid Standard. Euroasian J Hepatogastroenterol 2016; 6 (1):19-24.

DOI: 10.5005/jp-journals-10018-1160

License: CC BY-NC 4.0

Published Online: 01-12-2017

Copyright Statement:  Copyright © 2016; The Author(s).


Aims: We studied the functional stability of a primer pair and the standard curve based on a plasmid carrying full-length HBV genome, from a novel low-cost real-time quantitative polymerase chain reaction (qPCR) assay. The assay was developed at the Center for Genetic Engineering and Biotechnology (CIGB) in Havana, to quantify the serum hepatitis B virus (HBV) DNA from chronic HBV-infected (CHB) patients. Materials and methods: In-house generated oligonucleotides and plasmids were incubated at 37°C during 1 month and compared with the same materials incubated at –20, 4, and 25°C during the same time in qPCR experiments. Results: This work shows that the oligonucleotide pair and the plasmid for the quantitative standard curve are functionally stable in severe temperature conditions during 1 month. Polymerase chain reaction amplification with both materials after its incubation 30 days at 37°C produced similar cycle threshold (CT) values and similar degree of sample quantifications compared with the same materials preserved using the conventional storage conditions at –20°C. Conclusion: These results are indicative of the robustness of this low-cost qPCR system for HBV DNA quantification. These results also support that this qPCR assay can be used as a low-cost technology in clinical studies to monitor the viral load changes of serum HBV DNA of CHB patients, which could be used by poor people of third world countries, where there are frequent blackouts and temperature changes that can hinder the primer and plasmid stability.

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  1. Lavanchy D. Hepatitis B virus epidemiology, disease burden, treatment, and current and emerging prevention and control measures. J Viral Hepat 2004 Mar;11(2):97-107
  2. Long-term outcomes in hepatitis B: the REVEAL-HBV study. Clin Liver Dis 2007 Nov;11(4):797- 816
  3. Natural history of chronic hepatitis B REVEALed. J Gastroenterol Hepatol 2011 Apr;26(4):628-638
  4. Natural history of chronic hepatitis B: what exactly has REVEAL revealed? Liver Int 2012 Oct;32(9):1333-1341
  5. Serum HBV DNA as a marker of efficacy during therapy for chronic HBV infection: analysis and review of the literature. Hepatology 2003 Jun;37(6):1309-1319
  6. Hepatitis B virus DNA quantification using a cost effective and simple real time PCR in Cuban carriers. J Infect Dis Ther 2014;(2): 49-58
  7. Realtime PCR quantification of hepatitis B virus DNA using automated sample preparation and murine cytomegalovirus internal control. J Virol Methods 2005 Jun;126(1-2):207-213
  8. Application of real-time PCR to quantify hepatitis B virus DNA in chronic carriers in The Gambia. Virology J 2006 Apr 4; (3):23-30
  9. Real-t ime PCR for mRNA quantitation. Biotechniques 2005 Jul;39(1):75-85
  10. Serious Overestimation in Quantitative PCR by Circular (Supercoiled) Plasmid Standard: Microalgal pcna as the Model Gene. PLoS One 2010 Mar 5;5(3):e9545
  11. The hepatitis B virus (HBV) precore protein inhibits HBV replication in transgenic mice. J Virol 1996 Oct;70(10): 7056-7061
  12. Characterization of the intracellular deproteinized relaxed circular DNA of hepatitis B Virus: an intermediate of covalently closed circular DNA formation. J Virol 2007 Nov;81(22):12472-12484
  13. Quantitation of covalently closed circular hepatitis B virus DNA in chronic hepatitis B patients. Hepatology 2004 Sep;40(3):727-737
  14. Development of costeffective real-time PCR test: to detect a wide range of HBV DNA concentrations in the western amazon region of Brazil. Virology J 2014 Jan 28;(11):16.
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