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Development of cost-effective real-time PCR test: to detect a wide range of HBV DNA concentrations in the western amazon region of Brazil
BACKGROUND: Currently there is a significant risk of infection with hepatitis B virus (HBV) during blood transfusion in high epidemic area. This is due to the pre-seroconversion window period, immunovariant viral strains and the presence of occult HBV infection (OBI). The aim of this study was to de...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3906887/ https://www.ncbi.nlm.nih.gov/pubmed/24472141 http://dx.doi.org/10.1186/1743-422X-11-16 |
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author | de Oliveira dos Santos, Alcione Souza, Luan Felipo Botelho Borzacov, Lourdes Maria Villalobos-Salcedo, Juan Miguel Vieira, Deusilene Souza |
author_facet | de Oliveira dos Santos, Alcione Souza, Luan Felipo Botelho Borzacov, Lourdes Maria Villalobos-Salcedo, Juan Miguel Vieira, Deusilene Souza |
author_sort | de Oliveira dos Santos, Alcione |
collection | PubMed |
description | BACKGROUND: Currently there is a significant risk of infection with hepatitis B virus (HBV) during blood transfusion in high epidemic area. This is due to the pre-seroconversion window period, immunovariant viral strains and the presence of occult HBV infection (OBI). The aim of this study was to develop an in-house real-time PCR-based method, which was both ultra-sensitive and efficient offering an alternative method for nucleic acid testing (NAT). METHODS: A precore fragment with 109 bp was cloned and serial diluted to standard curve construction. The calibration of the HBV - DNA values was performed against OptiQuant® HBV-DNA Quantification Panel, Acrometrix Europe B.V.). RESULTS: From our in-house plasmid we prepared serial dilutions ranging from 2 × 10(3) – 2 × 10(9) copies/ml. The threshold was adjusted automatically during analysis and the data collected were analyzed by linear regression (r(2) = 0.99). The limit of detection for the assay with pHBVRO standards was 2000/ml in a total reaction volume of 30 μl. We found a strong correlation between the two methods (r(2) = 0.9965 and p < 0.0001). The regression line give us the following equation: Log 10 (IU/mL) = 0.9038Log 10 (copies/mL) − 1.0643, suggesting that 1 IU/mL = 15 copies/mL. CONCLUSIONS: Therefore, we can affirm that the qHBVRO PCR can detect HBV DNA in individuals with hepatitis B at any stage of the disease showing high capacity for NAT screening in hepatitis b donors. This results of sensitivity could provide an advance for automation in blood banks and increasing safety of patients who receive blood transfusions. |
format | Online Article Text |
id | pubmed-3906887 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-39068872014-01-31 Development of cost-effective real-time PCR test: to detect a wide range of HBV DNA concentrations in the western amazon region of Brazil de Oliveira dos Santos, Alcione Souza, Luan Felipo Botelho Borzacov, Lourdes Maria Villalobos-Salcedo, Juan Miguel Vieira, Deusilene Souza Virol J Methodology BACKGROUND: Currently there is a significant risk of infection with hepatitis B virus (HBV) during blood transfusion in high epidemic area. This is due to the pre-seroconversion window period, immunovariant viral strains and the presence of occult HBV infection (OBI). The aim of this study was to develop an in-house real-time PCR-based method, which was both ultra-sensitive and efficient offering an alternative method for nucleic acid testing (NAT). METHODS: A precore fragment with 109 bp was cloned and serial diluted to standard curve construction. The calibration of the HBV - DNA values was performed against OptiQuant® HBV-DNA Quantification Panel, Acrometrix Europe B.V.). RESULTS: From our in-house plasmid we prepared serial dilutions ranging from 2 × 10(3) – 2 × 10(9) copies/ml. The threshold was adjusted automatically during analysis and the data collected were analyzed by linear regression (r(2) = 0.99). The limit of detection for the assay with pHBVRO standards was 2000/ml in a total reaction volume of 30 μl. We found a strong correlation between the two methods (r(2) = 0.9965 and p < 0.0001). The regression line give us the following equation: Log 10 (IU/mL) = 0.9038Log 10 (copies/mL) − 1.0643, suggesting that 1 IU/mL = 15 copies/mL. CONCLUSIONS: Therefore, we can affirm that the qHBVRO PCR can detect HBV DNA in individuals with hepatitis B at any stage of the disease showing high capacity for NAT screening in hepatitis b donors. This results of sensitivity could provide an advance for automation in blood banks and increasing safety of patients who receive blood transfusions. BioMed Central 2014-01-28 /pmc/articles/PMC3906887/ /pubmed/24472141 http://dx.doi.org/10.1186/1743-422X-11-16 Text en Copyright © 2014 de Oliveira dos Santos et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. |
spellingShingle | Methodology de Oliveira dos Santos, Alcione Souza, Luan Felipo Botelho Borzacov, Lourdes Maria Villalobos-Salcedo, Juan Miguel Vieira, Deusilene Souza Development of cost-effective real-time PCR test: to detect a wide range of HBV DNA concentrations in the western amazon region of Brazil |
title | Development of cost-effective real-time PCR test: to detect a wide range of HBV DNA concentrations in the western amazon region of Brazil |
title_full | Development of cost-effective real-time PCR test: to detect a wide range of HBV DNA concentrations in the western amazon region of Brazil |
title_fullStr | Development of cost-effective real-time PCR test: to detect a wide range of HBV DNA concentrations in the western amazon region of Brazil |
title_full_unstemmed | Development of cost-effective real-time PCR test: to detect a wide range of HBV DNA concentrations in the western amazon region of Brazil |
title_short | Development of cost-effective real-time PCR test: to detect a wide range of HBV DNA concentrations in the western amazon region of Brazil |
title_sort | development of cost-effective real-time pcr test: to detect a wide range of hbv dna concentrations in the western amazon region of brazil |
topic | Methodology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3906887/ https://www.ncbi.nlm.nih.gov/pubmed/24472141 http://dx.doi.org/10.1186/1743-422X-11-16 |
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