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...
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| Veröffentlicht in: | Virology journal 2014-01, Vol.11 (1), p.16-16, Article 16 |
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| creator | de Oliveira dos Santos, Alcione Souza, Luan Felipo Botelho Borzacov, Lourdes Maria Villalobos-Salcedo, Juan Miguel Vieira, Deusilene Souza |
| 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 × 10³ – 2 × 10⁹ copies/ml. The threshold was adjusted automatically during analysis and the data collected were analyzed by linear regression (r² = 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 (r² = 0.9965 and p |
| doi_str_mv | 10.1186/1743-422X-11-16 |
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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 × 10³ – 2 × 10⁹ copies/ml. The threshold was adjusted automatically during analysis and the data collected were analyzed by linear regression (r² = 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 (r² = 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.</description><identifier>ISSN: 1743-422X</identifier><identifier>EISSN: 1743-422X</identifier><identifier>DOI: 10.1186/1743-422X-11-16</identifier><identifier>PMID: 24472141</identifier><language>eng</language><publisher>England: Springer-Verlag</publisher><subject>Analysis ; Antiretroviral drugs ; automation ; blood ; Blood banks ; blood transfusion ; Brazil ; cost effectiveness ; data analysis ; Deoxyribonucleic acid ; detection limit ; DNA ; Drug therapy ; equations ; Health aspects ; Hepatitis ; hepatitis B ; Hepatitis B - virology ; Hepatitis B virus ; Hepatitis B virus - genetics ; Hepatitis B virus - isolation & purification ; HIV ; Human immunodeficiency virus ; Humans ; linear models ; Liver diseases ; Methodology ; Occult sciences ; patients ; plasmids ; quantitative polymerase chain reaction ; Real-Time Polymerase Chain Reaction - methods ; risk ; screening ; Sensitivity and Specificity ; strains ; testing ; Viral Load - methods</subject><ispartof>Virology journal, 2014-01, Vol.11 (1), p.16-16, Article 16</ispartof><rights>COPYRIGHT 2014 BioMed Central Ltd.</rights><rights>2014 de Oliveira dos Santos et al.; licensee BioMed Central Ltd. 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.</rights><rights>Copyright © 2014 de Oliveira dos Santos et al.; licensee BioMed Central Ltd. 2014 de Oliveira dos Santos et al.; licensee BioMed Central Ltd.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-b638t-7024d82fae46d4905ce31f6895bfb85313c75bbc52f9b0ced935977f85dfe7393</citedby><cites>FETCH-LOGICAL-b638t-7024d82fae46d4905ce31f6895bfb85313c75bbc52f9b0ced935977f85dfe7393</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3906887/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3906887/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,27924,27925,53791,53793</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/24472141$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>de Oliveira dos Santos, Alcione</creatorcontrib><creatorcontrib>Souza, Luan Felipo Botelho</creatorcontrib><creatorcontrib>Borzacov, Lourdes Maria</creatorcontrib><creatorcontrib>Villalobos-Salcedo, Juan Miguel</creatorcontrib><creatorcontrib>Vieira, Deusilene Souza</creatorcontrib><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><title>Virology journal</title><addtitle>Virol J</addtitle><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 × 10³ – 2 × 10⁹ copies/ml. The threshold was adjusted automatically during analysis and the data collected were analyzed by linear regression (r² = 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 (r² = 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.</description><subject>Analysis</subject><subject>Antiretroviral drugs</subject><subject>automation</subject><subject>blood</subject><subject>Blood banks</subject><subject>blood transfusion</subject><subject>Brazil</subject><subject>cost effectiveness</subject><subject>data analysis</subject><subject>Deoxyribonucleic acid</subject><subject>detection limit</subject><subject>DNA</subject><subject>Drug therapy</subject><subject>equations</subject><subject>Health aspects</subject><subject>Hepatitis</subject><subject>hepatitis B</subject><subject>Hepatitis B - virology</subject><subject>Hepatitis B virus</subject><subject>Hepatitis B virus - genetics</subject><subject>Hepatitis B virus - isolation & purification</subject><subject>HIV</subject><subject>Human immunodeficiency virus</subject><subject>Humans</subject><subject>linear models</subject><subject>Liver diseases</subject><subject>Methodology</subject><subject>Occult sciences</subject><subject>patients</subject><subject>plasmids</subject><subject>quantitative polymerase chain reaction</subject><subject>Real-Time Polymerase Chain Reaction - methods</subject><subject>risk</subject><subject>screening</subject><subject>Sensitivity and Specificity</subject><subject>strains</subject><subject>testing</subject><subject>Viral Load - methods</subject><issn>1743-422X</issn><issn>1743-422X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2014</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><recordid>eNqFkk9v1DAQxSMEomXhzA0scYFDWv9J4qQHpN0t0EoVoJYibpbjjFNXib3E3i30G_CtcdiydFER8sGW5_eeRm8mSZ4SvEdIWewTnrE0o_RLSkhKinvJ7ubn_q33TvLI-0uMGS149TDZoVnGKcnIbvLjEFbQuUUPNiCnkXI-pKA1qGBWgAaQXRpMD-jj_BQF8OEABYcaCBFAEl2ZJkLStjCKj2af0eH7aTSxKvoNMhhnPTIWhQtAV1ENg0Wyl9fORus2VkfZbJDXpnucPNCy8_Dk5p4k52_ffJofpScf3h3PpydpXbAypBzTrCmplpAVTVbhXAEjuiirvNZ1mTPCFM_rWuVUVzVW0FQsrzjXZd5o4Kxik-T12nexrHto1o12YjGYXg7fhZNGbFesuRCtWwlW4aIseTSYrQ1q4_5hsF1RrhfjKMQ4CkGIIEU0eXnTxeC-LmMyojdeQddJC27pBckx5iVmhP8fzSpWxlQiPUle_IVeuuVgY5y_KMxyXmV_qFZ2IIzVLrapRlMxzVlVlBQXo9feHVQ8DfQmThi0if9bgldbgsgE-BZaufReHJ-dbrP7a1YNzvsB9CY-gsW42XcE9uz22Db871WOwPM1oKUTsh2MF-dnFI9BUjLuBfsJ8vL7dQ</recordid><startdate>20140128</startdate><enddate>20140128</enddate><creator>de Oliveira dos Santos, Alcione</creator><creator>Souza, Luan Felipo Botelho</creator><creator>Borzacov, Lourdes Maria</creator><creator>Villalobos-Salcedo, Juan Miguel</creator><creator>Vieira, Deusilene Souza</creator><general>Springer-Verlag</general><general>BioMed Central Ltd</general><general>BioMed Central</general><scope>FBQ</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>ISR</scope><scope>3V.</scope><scope>7U9</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BENPR</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>H94</scope><scope>K9.</scope><scope>M0S</scope><scope>M1P</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>7X8</scope><scope>7TM</scope><scope>5PM</scope></search><sort><creationdate>20140128</creationdate><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><author>de Oliveira dos Santos, Alcione ; Souza, Luan Felipo Botelho ; Borzacov, Lourdes Maria ; Villalobos-Salcedo, Juan Miguel ; Vieira, Deusilene Souza</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-b638t-7024d82fae46d4905ce31f6895bfb85313c75bbc52f9b0ced935977f85dfe7393</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2014</creationdate><topic>Analysis</topic><topic>Antiretroviral drugs</topic><topic>automation</topic><topic>blood</topic><topic>Blood banks</topic><topic>blood transfusion</topic><topic>Brazil</topic><topic>cost effectiveness</topic><topic>data analysis</topic><topic>Deoxyribonucleic acid</topic><topic>detection limit</topic><topic>DNA</topic><topic>Drug therapy</topic><topic>equations</topic><topic>Health aspects</topic><topic>Hepatitis</topic><topic>hepatitis B</topic><topic>Hepatitis B - virology</topic><topic>Hepatitis B virus</topic><topic>Hepatitis B virus - genetics</topic><topic>Hepatitis B virus - isolation & purification</topic><topic>HIV</topic><topic>Human immunodeficiency virus</topic><topic>Humans</topic><topic>linear models</topic><topic>Liver diseases</topic><topic>Methodology</topic><topic>Occult sciences</topic><topic>patients</topic><topic>plasmids</topic><topic>quantitative polymerase chain reaction</topic><topic>Real-Time Polymerase Chain Reaction - methods</topic><topic>risk</topic><topic>screening</topic><topic>Sensitivity and Specificity</topic><topic>strains</topic><topic>testing</topic><topic>Viral Load - methods</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>de Oliveira dos Santos, Alcione</creatorcontrib><creatorcontrib>Souza, Luan Felipo Botelho</creatorcontrib><creatorcontrib>Borzacov, Lourdes Maria</creatorcontrib><creatorcontrib>Villalobos-Salcedo, Juan Miguel</creatorcontrib><creatorcontrib>Vieira, Deusilene Souza</creatorcontrib><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Gale In Context: Science</collection><collection>ProQuest Central (Corporate)</collection><collection>Virology and AIDS Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>ProQuest Central</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Access via ProQuest (Open Access)</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>MEDLINE - Academic</collection><collection>Nucleic Acids Abstracts</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Virology journal</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>de Oliveira dos Santos, Alcione</au><au>Souza, Luan Felipo Botelho</au><au>Borzacov, Lourdes Maria</au><au>Villalobos-Salcedo, Juan Miguel</au><au>Vieira, Deusilene Souza</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Development of cost-effective real-time PCR test: to detect a wide range of HBV DNA concentrations in the western amazon region of Brazil</atitle><jtitle>Virology journal</jtitle><addtitle>Virol J</addtitle><date>2014-01-28</date><risdate>2014</risdate><volume>11</volume><issue>1</issue><spage>16</spage><epage>16</epage><pages>16-16</pages><artnum>16</artnum><issn>1743-422X</issn><eissn>1743-422X</eissn><abstract>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 × 10³ – 2 × 10⁹ copies/ml. The threshold was adjusted automatically during analysis and the data collected were analyzed by linear regression (r² = 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 (r² = 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.</abstract><cop>England</cop><pub>Springer-Verlag</pub><pmid>24472141</pmid><doi>10.1186/1743-422X-11-16</doi><tpages>1</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Analysis Antiretroviral drugs automation blood Blood banks blood transfusion Brazil cost effectiveness data analysis Deoxyribonucleic acid detection limit DNA Drug therapy equations Health aspects Hepatitis hepatitis B Hepatitis B - virology Hepatitis B virus Hepatitis B virus - genetics Hepatitis B virus - isolation & purification HIV Human immunodeficiency virus Humans linear models Liver diseases Methodology Occult sciences patients plasmids quantitative polymerase chain reaction Real-Time Polymerase Chain Reaction - methods risk screening Sensitivity and Specificity strains testing Viral Load - methods |
| 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 |
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