Sample preparation: a challenge in the development of point-of-care nucleic acid-based assays for resource-limited settings
Identifieur interne : 000619 ( Istex/Corpus ); précédent : 000618; suivant : 000620Sample preparation: a challenge in the development of point-of-care nucleic acid-based assays for resource-limited settings
Auteurs : Magda Anastassova Dineva ; Lourdes Mahilum-Tapaypresent Address Diagnostics For The Real World Ltd. Sunnyvale Ca Usa. ; Helen LeeSource :
- Analyst [ 0003-2654 ] ; 2007.
English descriptors
- KwdEn :
- Acid, Acid testing, Additional reagents, Amplification, Assay, Available technologies, Chaotropic, Chaotropic reagent, Chaotropic salt, Chargeswitch1, Clinical specimens, Complex instrumentation, Detection limit, Detection signals, Detection steps, Electrochemical sensor array, Enigma diagnostics, Enzyme treatment, Expensive instrumentation, Extraction, Extraction efficiency, Extraction procedure, Extraction procedures, Horseradish peroxidase, Human plasma, Infectious disease, Inhibitory substances, Laboratory spaces, Larger sample volumes, Liattm analyzer, Magnetic particles, Microbiol, Microfluidic, Microfuge, Model analyte, Next generation, Nucleic, Nucleic acid, Nucleic acid extraction, Oligonucleotide probes, Organic solvents, Other systems, Reagent, Room temperature, Royal society, Salt extraction microfuge, Sample preparation, Sample preparation step, Solidphase extraction, Such assays, Test site, Verigene1, Viral, Viral load, Whole blood.
- Teeft :
- Acid, Acid testing, Additional reagents, Amplification, Assay, Available technologies, Chaotropic, Chaotropic reagent, Chaotropic salt, Chargeswitch1, Clinical specimens, Complex instrumentation, Detection limit, Detection signals, Detection steps, Electrochemical sensor array, Enigma diagnostics, Enzyme treatment, Expensive instrumentation, Extraction, Extraction efficiency, Extraction procedure, Extraction procedures, Horseradish peroxidase, Human plasma, Infectious disease, Inhibitory substances, Laboratory spaces, Larger sample volumes, Liattm analyzer, Magnetic particles, Microbiol, Microfluidic, Microfuge, Model analyte, Next generation, Nucleic, Nucleic acid, Nucleic acid extraction, Oligonucleotide probes, Organic solvents, Other systems, Reagent, Room temperature, Royal society, Salt extraction microfuge, Sample preparation, Sample preparation step, Solidphase extraction, Such assays, Test site, Verigene1, Viral, Viral load, Whole blood.
Abstract
Currently available nucleic acid testing (NAT)-based assays are complex and time-consuming, and they require expensive instrumentation and dedicated laboratory spaces for sample preparation as well as for amplification and detection of the nucleic acid target. Reagents required for these tests are also expensive and must be transported and stored refrigerated or frozen. These characteristics have limited the use of such assays for point-of-care (POC) testing, especially in resource-poor settings. Efforts to develop simple and rapid NAT-based assays have focused predominantly on the amplification and detection steps, with sample preparation and nucleic acid extraction remaining the bottleneck in the development of NAT systems suitable for POC applications or resource-limited settings. A review of NAT platforms and technologies currently under development and validation for rapid field testing revealed that, in addition to requiring expensive and complex instrumentation, many of these systems also require off-line sample preparation and reagent handling. In their current format, they are therefore not appropriate for POC testing in resource-limited settings. We evaluated several commercially available technologies and procedures for the isolation of nucleic acid with the extraction of HIV-1 RNA from human plasma as a model system. Our results indicate that solid-phase extraction with silica or glass in the presence of a chaotropic salt provides the highest extraction efficiency. However, none of the existing methods and technologies is readily adaptable to a POC system. The integration of sample preparation procedures well suited to NAT-based assays in resource-limited settings therefore remains a challenge.
Url:
DOI: 10.1039/b705672a
Links to Exploration step
ISTEX:EE568A42B9A541FB73C5D09C26A9EFFA741CC966Le document en format XML
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Sunnyvale Ca Usa.">Lourdes Mahilum-Tapaypresent Address Diagnostics For The Real World Ltd. Sunnyvale Ca Usa.</name><affiliation><mods:affiliation>Department of Haematology, University of Cambridge, CB2 2PT, Cambridge, UK</mods:affiliation></affiliation></author><author wicri:is="90%"><name sortKey="Lee, Helen" sort="Lee, Helen" uniqKey="Lee H" first="Helen" last="Lee">Helen Lee</name><affiliation><mods:affiliation>Department of Haematology, University of Cambridge, CB2 2PT, Cambridge, UK</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Analyst</title><title level="j" type="abbrev">Analyst</title><idno type="ISSN">0003-2654</idno><idno type="eISSN">1364-5528</idno><imprint><publisher>The Royal Society of Chemistry.</publisher><date type="published" when="2007">2007</date><biblScope unit="volume">132</biblScope><biblScope unit="issue">12</biblScope><biblScope unit="page" from="1193">1193</biblScope><biblScope unit="page" to="1199">1199</biblScope></imprint><idno type="ISSN">0003-2654</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0003-2654</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Acid</term><term>Acid testing</term><term>Additional reagents</term><term>Amplification</term><term>Assay</term><term>Available technologies</term><term>Chaotropic</term><term>Chaotropic reagent</term><term>Chaotropic salt</term><term>Chargeswitch1</term><term>Clinical specimens</term><term>Complex instrumentation</term><term>Detection limit</term><term>Detection signals</term><term>Detection steps</term><term>Electrochemical sensor array</term><term>Enigma diagnostics</term><term>Enzyme treatment</term><term>Expensive instrumentation</term><term>Extraction</term><term>Extraction efficiency</term><term>Extraction procedure</term><term>Extraction procedures</term><term>Horseradish peroxidase</term><term>Human plasma</term><term>Infectious disease</term><term>Inhibitory substances</term><term>Laboratory spaces</term><term>Larger sample volumes</term><term>Liattm analyzer</term><term>Magnetic particles</term><term>Microbiol</term><term>Microfluidic</term><term>Microfuge</term><term>Model analyte</term><term>Next generation</term><term>Nucleic</term><term>Nucleic acid</term><term>Nucleic acid extraction</term><term>Oligonucleotide probes</term><term>Organic solvents</term><term>Other systems</term><term>Reagent</term><term>Room temperature</term><term>Royal society</term><term>Salt extraction microfuge</term><term>Sample preparation</term><term>Sample preparation step</term><term>Solidphase extraction</term><term>Such assays</term><term>Test site</term><term>Verigene1</term><term>Viral</term><term>Viral load</term><term>Whole blood</term></keywords><keywords scheme="Teeft" xml:lang="en"><term>Acid</term><term>Acid testing</term><term>Additional reagents</term><term>Amplification</term><term>Assay</term><term>Available technologies</term><term>Chaotropic</term><term>Chaotropic reagent</term><term>Chaotropic salt</term><term>Chargeswitch1</term><term>Clinical specimens</term><term>Complex instrumentation</term><term>Detection limit</term><term>Detection signals</term><term>Detection steps</term><term>Electrochemical sensor array</term><term>Enigma diagnostics</term><term>Enzyme treatment</term><term>Expensive instrumentation</term><term>Extraction</term><term>Extraction efficiency</term><term>Extraction procedure</term><term>Extraction procedures</term><term>Horseradish peroxidase</term><term>Human plasma</term><term>Infectious disease</term><term>Inhibitory substances</term><term>Laboratory spaces</term><term>Larger sample volumes</term><term>Liattm analyzer</term><term>Magnetic particles</term><term>Microbiol</term><term>Microfluidic</term><term>Microfuge</term><term>Model analyte</term><term>Next generation</term><term>Nucleic</term><term>Nucleic acid</term><term>Nucleic acid extraction</term><term>Oligonucleotide probes</term><term>Organic solvents</term><term>Other systems</term><term>Reagent</term><term>Room temperature</term><term>Royal society</term><term>Salt extraction microfuge</term><term>Sample preparation</term><term>Sample preparation step</term><term>Solidphase extraction</term><term>Such assays</term><term>Test site</term><term>Verigene1</term><term>Viral</term><term>Viral load</term><term>Whole blood</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract">Currently available nucleic acid testing (NAT)-based assays are complex and time-consuming, and they require expensive instrumentation and dedicated laboratory spaces for sample preparation as well as for amplification and detection of the nucleic acid target. Reagents required for these tests are also expensive and must be transported and stored refrigerated or frozen. These characteristics have limited the use of such assays for point-of-care (POC) testing, especially in resource-poor settings. Efforts to develop simple and rapid NAT-based assays have focused predominantly on the amplification and detection steps, with sample preparation and nucleic acid extraction remaining the bottleneck in the development of NAT systems suitable for POC applications or resource-limited settings. A review of NAT platforms and technologies currently under development and validation for rapid field testing revealed that, in addition to requiring expensive and complex instrumentation, many of these systems also require off-line sample preparation and reagent handling. In their current format, they are therefore not appropriate for POC testing in resource-limited settings. We evaluated several commercially available technologies and procedures for the isolation of nucleic acid with the extraction of HIV-1 RNA from human plasma as a model system. Our results indicate that solid-phase extraction with silica or glass in the presence of a chaotropic salt provides the highest extraction efficiency. However, none of the existing methods and technologies is readily adaptable to a POC system. 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Reagents required for these tests are also expensive and must be transported and stored refrigerated or frozen. These characteristics have limited the use of such assays for point-of-care (POC) testing, especially in resource-poor settings. Efforts to develop simple and rapid NAT-based assays have focused predominantly on the amplification and detection steps, with sample preparation and nucleic acid extraction remaining the bottleneck in the development of NAT systems suitable for POC applications or resource-limited settings. A review of NAT platforms and technologies currently under development and validation for rapid field testing revealed that, in addition to requiring expensive and complex instrumentation, many of these systems also require off-line sample preparation and reagent handling. In their current format, they are therefore not appropriate for POC testing in resource-limited settings. We evaluated several commercially available technologies and procedures for the isolation of nucleic acid with the extraction of HIV-1 RNA from human plasma as a model system. Our results indicate that solid-phase extraction with silica or glass in the presence of a chaotropic salt provides the highest extraction efficiency. However, none of the existing methods and technologies is readily adaptable to a POC system. The integration of sample preparation procedures well suited to NAT-based assays in resource-limited settings therefore remains a challenge.</abstract><qualityIndicators><score>9.396</score><pdfVersion>1.2</pdfVersion><pdfPageSize>595.276 x 779.528 pts</pdfPageSize><refBibsNative>true</refBibsNative><keywordCount>0</keywordCount><abstractCharCount>1735</abstractCharCount><pdfWordCount>4468</pdfWordCount><pdfCharCount>31441</pdfCharCount><pdfPageCount>7</pdfPageCount><abstractWordCount>244</abstractWordCount></qualityIndicators><title>Sample preparation: a challenge in the development of point-of-care nucleic acid-based assays for resource-limited settings</title><genre><json:string>review-article</json:string></genre><host><title>Analyst</title><language><json:string>unknown</json:string></language><publicationDate>2007</publicationDate><issn><json:string>0003-2654</json:string></issn><eissn><json:string>1364-5528</json:string></eissn><volume>132</volume><issue>12</issue><pages><first>1193</first><last>1199</last><total>7</total></pages><genre><json:string>journal</json:string></genre></host><categories><wos><json:string>science</json:string><json:string>chemistry, analytical</json:string></wos><scienceMetrix><json:string>natural sciences</json:string><json:string>chemistry</json:string><json:string>analytical chemistry</json:string></scienceMetrix><inist><json:string>sciences appliquees, technologies et medecines</json:string><json:string>sciences biologiques et medicales</json:string><json:string>sciences biologiques fondamentales et appliquees. psychologie</json:string></inist></categories><publicationDate>2007</publicationDate><copyrightDate>2007</copyrightDate><doi><json:string>10.1039/b705672a</json:string></doi><id>EE568A42B9A541FB73C5D09C26A9EFFA741CC966</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/document/EE568A42B9A541FB73C5D09C26A9EFFA741CC966/fulltext/pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/document/EE568A42B9A541FB73C5D09C26A9EFFA741CC966/fulltext/zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/document/EE568A42B9A541FB73C5D09C26A9EFFA741CC966/fulltext/tei"><teiHeader><fileDesc><titleStmt><title level="a">Sample preparation: a challenge in the development of point-of-care nucleic acid-based assays for resource-limited settings</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>The Royal Society of Chemistry.</publisher><availability><p>This journal is © The Royal Society of Chemistry</p></availability><date>2007</date></publicationStmt><notesStmt><note>This paper was published as part of the special issue on the Rapid Diagnostic Testing of Infection and Disease States.</note><note>Point-of-care nucleic acid testing (POC-NAT), a rapidly developing field, can improve healthcare in resource-limited settings. This paper reviews the current status and challenges presented by current POC-NAT systems. [b705672a-ga.tif]</note><note>Reports on the global AIDS epidemic, UNAIDS/WHO, 2006; http://www.unaids.org/en/HIV_data/2006globalreport/default.asp (accessed 1 October 2007)</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a">Sample preparation: a challenge in the development of point-of-care nucleic acid-based assays for resource-limited settings</title><author xml:id="author-0000"><persName><forename type="first">Magda Anastassova</forename><surname>Dineva</surname></persName><affiliation>Department of Haematology, University of Cambridge, CB2 2PT, Cambridge, UK</affiliation></author><author xml:id="author-0001"><persName><forename type="first">Lourdes</forename><surname>Mahilum-TapayPresent address: Diagnostics for the Real World Ltd., Sunnyvale, CA 94085, USA.</surname></persName><affiliation>Department of Haematology, University of Cambridge, CB2 2PT, Cambridge, UK</affiliation></author><author xml:id="author-0002"><persName><forename type="first">Helen</forename><surname>Lee</surname></persName><affiliation>Department of Haematology, University of Cambridge, CB2 2PT, Cambridge, UK</affiliation></author><idno type="istex">EE568A42B9A541FB73C5D09C26A9EFFA741CC966</idno><idno type="DOI">10.1039/b705672a</idno><idno type="ms-id">b705672a</idno></analytic><monogr><title level="j">Analyst</title><title level="j" type="abbrev">Analyst</title><idno type="pISSN">0003-2654</idno><idno type="eISSN">1364-5528</idno><idno type="coden">ANALAO</idno><idno type="RSC-sercode">AN</idno><imprint><publisher>The Royal Society of Chemistry.</publisher><date type="published" when="2007"></date><biblScope unit="volume">132</biblScope><biblScope unit="issue">12</biblScope><biblScope unit="page" from="1193">1193</biblScope><biblScope unit="page" to="1199">1199</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>2007</date></creation><langUsage><language ident="en">en</language></langUsage><abstract><p>Currently available nucleic acid testing (NAT)-based assays are complex and time-consuming, and they require expensive instrumentation and dedicated laboratory spaces for sample preparation as well as for amplification and detection of the nucleic acid target. Reagents required for these tests are also expensive and must be transported and stored refrigerated or frozen. These characteristics have limited the use of such assays for point-of-care (POC) testing, especially in resource-poor settings. Efforts to develop simple and rapid NAT-based assays have focused predominantly on the amplification and detection steps, with sample preparation and nucleic acid extraction remaining the bottleneck in the development of NAT systems suitable for POC applications or resource-limited settings. A review of NAT platforms and technologies currently under development and validation for rapid field testing revealed that, in addition to requiring expensive and complex instrumentation, many of these systems also require off-line sample preparation and reagent handling. In their current format, they are therefore not appropriate for POC testing in resource-limited settings. We evaluated several commercially available technologies and procedures for the isolation of nucleic acid with the extraction of HIV-1 RNA from human plasma as a model system. Our results indicate that solid-phase extraction with silica or glass in the presence of a chaotropic salt provides the highest extraction efficiency. However, none of the existing methods and technologies is readily adaptable to a POC system. The integration of sample preparation procedures well suited to NAT-based assays in resource-limited settings therefore remains a challenge.</p></abstract></profileDesc><revisionDesc><change when="2007">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/document/EE568A42B9A541FB73C5D09C26A9EFFA741CC966/fulltext/txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="corpus rsc-journals not found" wicri:toSee="no header"><istex:xmlDeclaration>version="1.0" encoding="UTF-8"</istex:xmlDeclaration><istex:document><article type="PER" xsi:schemaLocation="http://www.rsc.org/schema/rscart38 http://www.rsc.org/schema/rscart38/rscart38.xsd" dtd="RSCART3.8"><metainfo><articletype code="PER" pubmedForm="other" headingForm="Perspectives" group="Review" fullForm="Perspective"></articletype></metainfo><art-admin><ms-id>b705672a</ms-id><doi>10.1039/b705672a</doi></art-admin><published type="web"><journalref><link>AN</link></journalref><volumeref><link>Unassigned</link></volumeref><issueref><link>Advance Articles</link></issueref><pubfront><fpage></fpage><lpage></lpage><no-of-pages></no-of-pages><date><year>2007</year><month>October</month><day>1</day></date></pubfront></published><published type="print"><journalref><link>AN</link></journalref><volumeref><link>132</link></volumeref><issueref><link>12</link></issueref><pubfront><fpage>1193</fpage><lpage>1199</lpage><no-of-pages>7</no-of-pages><date><year>2007</year><month>11</month><day>19</day></date></pubfront></published><published type="subsyear"><journalref><title type="abbreviated">Analyst</title><title type="full">Analyst</title><title type="journal">Analyst</title><title type="display">Analyst</title><title type="pubmed">Analyst</title><sercode>AN</sercode><publisher><orgname><nameelt>The Royal Society of Chemistry</nameelt></orgname></publisher><issn type="print">0003-2654</issn><issn type="online">1364-5528</issn><coden>ANALAO</coden><cpyrt>This journal is © The Royal Society of Chemistry</cpyrt></journalref><volumeref><link></link></volumeref><issueref><link>12</link></issueref><pubfront><fpage></fpage><lpage></lpage><no-of-pages></no-of-pages><date><year>2007</year><month>Unassigned</month><day>Unassigned</day></date></pubfront></published><art-front><titlegrp><title>Sample preparation: a challenge in the development of point-of-care nucleic acid-based assays for resource-limited settings<fnoteref idrefs="fn1"></fnoteref><footnote id="fn1">This paper was published as part of the special issue on the Rapid Diagnostic Testing of Infection and Disease States.</footnote></title></titlegrp><authgrp><author aff="affa"><person><persname><fname>Magda Anastassova</fname><surname>Dineva</surname></persname></person></author><author aff="affa"><person><persname><fname>Lourdes</fname><surname>Mahilum-Tapay<fnoteref idrefs="fn2"></fnoteref><footnote id="fn2">Present address: Diagnostics for the Real World Ltd., Sunnyvale, CA 94085, USA.</footnote></surname></persname></person></author><author aff="affa" role="corres"><person><persname><fname>Helen</fname><surname>Lee</surname></persname></person></author><aff id="affa"><org><orgname><nameelt>Department of Haematology</nameelt><nameelt>University of Cambridge</nameelt></orgname></org><address><city>Cambridge</city><country>UK</country><postcode>CB2 2PT</postcode></address></aff></authgrp><art-toc-entry><ictext>Point-of-care nucleic acid testing (POC-NAT), a rapidly developing field, can improve healthcare in resource-limited settings. This paper reviews the current status and challenges presented by current POC-NAT systems.</ictext><icgraphic xsrc="b705672a-ga.tif" id="ga"></icgraphic></art-toc-entry><abstract><p>Currently available nucleic acid testing (NAT)-based assays are complex and time-consuming, and they require expensive instrumentation and dedicated laboratory spaces for sample preparation as well as for amplification and detection of the nucleic acid target. Reagents required for these tests are also expensive and must be transported and stored refrigerated or frozen. These characteristics have limited the use of such assays for point-of-care (POC) testing, especially in resource-poor settings. Efforts to develop simple and rapid NAT-based assays have focused predominantly on the amplification and detection steps, with sample preparation and nucleic acid extraction remaining the bottleneck in the development of NAT systems suitable for POC applications or resource-limited settings. A review of NAT platforms and technologies currently under development and validation for rapid field testing revealed that, in addition to requiring expensive and complex instrumentation, many of these systems also require off-line sample preparation and reagent handling. In their current format, they are therefore not appropriate for POC testing in resource-limited settings. We evaluated several commercially available technologies and procedures for the isolation of nucleic acid with the extraction of HIV-1 RNA from human plasma as a model system. Our results indicate that solid-phase extraction with silica or glass in the presence of a chaotropic salt provides the highest extraction efficiency. However, none of the existing methods and technologies is readily adaptable to a POC system. The integration of sample preparation procedures well suited to NAT-based assays in resource-limited settings therefore remains a challenge.</p></abstract></art-front><art-body><section><title>Introduction</title><p>Nucleic acid testing (NAT) for an infectious agent is based on the direct and highly sequence-specific detection of nucleic acid and has an analytical sensitivity of at least three orders of magnitude higher than that of other tests based on immune reactions, virus isolation, or cell culture. NAT is the only reliable tool available for testing during the window period between infection and seroconversion,<citref idrefs="cit1">1</citref> for the detection of infection with immunovariant viruses,<citref idrefs="cit2">2</citref> for the identification of immunosilent or occult carriage,<citref idrefs="cit1 cit3">1,3</citref> or for the early diagnosis of viral infections in newborns and infants who harbour maternal antibodies to the virus, which interfere with antibody-based tests and can persist for up to 18 months after birth. In addition to its utility in screening for and diagnosis of infectious diseases, thereby also contributing to the safety of the blood supply, NAT can be applied to monitor treatment efficacy and clinical progression. Outside of the field of infectious disease, NAT can be used to screen for susceptibility to genetic disorders whose causative mutations are known. These many advantages of NAT are offset, however, by the fact that existing, commercially available NAT-based assays are complex (more than 15 steps) and time-consuming (assay times of more than 4 h, sample collection and transportation time excluded) and require expensive instrumentation ($20 000–$120 000) and dedicated laboratory spaces for sample preparation as well as for amplification and detection of the nucleic acid target.<citref idrefs="cit4 cit5">4,5</citref> Reagents required for these tests are also expensive (kit cost $50–$100 per test in the US)<citref idrefs="cit5">5</citref> and must be transported and stored below room temperature. NAT has thus been limited to specialized laboratory settings, with NAT-based assays, in their current forms, being unsuitable for point-of-care (POC) applications. They are also unaffordable in resource-poor regions with a high prevalence of infectious disease (such as Africa, Asia, and Latin America), the very regions where they are needed most.</p><p>The development of new NAT assays that are based on simplified sample preparation as well as amplification and detection chemistry and which can detect multiple nucleic acid analytes in a single sample, without the need for complex instrumentation, is essential if this powerful technology is to move beyond the clinical laboratory environment to resource-limited settings, where the demand for and potential impact of POC testing are greatest. Efforts to develop simple and rapid NAT formats intended for use in POC testing and in resource-limited settings have generally focused on two areas: (1) the development of real-time detection assays, such as those based on real-time polymerase chain reaction (PCR) analysis, and (2) the development of automated miniaturized devices and microarrays for multiplex detection.<citref idrefs="cit6 cit7 cit8 cit9 cit10 cit11 cit12 cit13 cit14">6–14</citref> These approaches apply mainly to the amplification and detection steps of NAT assays and do not relate to the ‘front end’ of such assays – the procedure for sample preparation and nucleic acid extraction, which remains the bottleneck in, and major impediment to, their wider application. Most of the newly emerging NAT systems and platforms intended for POC testing (POC-NAT) require off-line sample extraction, with few assay developers having attempted integration of this step.<citref idrefs="cit11 cit13 cit15">11,13,15</citref> An essential component of the development of the next generation of rapid and simple POC-NAT diagnostic assays must therefore be the development of simple and inexpensive sample processing and nucleic acid extraction protocols.</p><p>We have now surveyed and evaluated existing technologies for nucleic acid extraction with regard to complexity and suitability for integration into POC-NAT systems, with a particular focus on their application to resource-limited settings.</p></section><section><title>Overview of POC-NAT systems and platforms</title><p>One of the main challenges in the development of POC-NAT tests is the development of an efficient, inexpensive, simple, rapid, and, ideally, equipment-free procedure for sample preparation. Few of the newly emerging NAT systems intended for POC testing have an integrated sample extraction step. GeneXpert<sup>®</sup> developed by Cepheid (Sunnyvale, CA) is the first fully integrated NAT system developed for bio-security applications. This system automates nucleic acid extraction and real-time PCR in microfluidic cartridges. Each GeneXpert<sup>®</sup> cartridge incorporates a syringe drive, rotary drive, and sonic horn.<citref idrefs="cit16 cit17">16,17</citref> The test sample is initially filtered to isolate the cells or spores of interest, which are then lysed by ultrasonic energy delivered by the sonic horn, resulting in the release of nucleic acid. The syringe and rotary drives move liquid between the cartridge chambers in order to wash, purify, and concentrate the nucleic acid. After the extraction is complete, the nucleic acid extract is moved into the cartridge reaction chamber and subjected to real-time PCR. The strengths of the Cepheid GeneXpert<sup>®</sup> system related to POC-NAT applications are an integrated sample processing procedure, an enclosed cartridge to prevent contamination, automatic result interpretation, multiplex testing, and the use of stable lyophilized reagents, results within two hours. Among the challenges related to the implementation of the GeneXpert<sup>®</sup> system in resource-limited settings are expensive equipment and consumables, high energy consumption requiring electricity supply, and a sophisticated heating–cooling device that requires specialist repair and maintenance.</p><p>Other POC-NAT systems that are at the prototype stage of development and under consideration for integration of the sample preparation step include the Liat™ Analyzer (IQuum), the FL rapid PCR system of Enigma Diagnostics, HandyLab's microfluidic system (Ann Arbor, MI), and the Lab-on-a-card POC testing platform of the Program for Appropriate Technology in Health (PATH) and collaborators. These systems remain to be fully validated. The Liat™ (laboratory in a tube) Analyzer [<figref idrefs="fig1">Fig. 1(a)</figref>] integrates sample preparation with real-time PCR detection. Relatively rapid PCR amplification (50 cycles in 1 h) is achieved by ‘flow cycling’, whereby the amplified sample is moved between three different chambers, or ‘segments’, at different temperatures for denaturation, annealing, and elongation.<citref idrefs="cit18">18</citref> Sample preparation is based on conventional solid-phase extraction with magnetic particles. The ‘insert-and-test’ automation system consists of two components: the Liat™ collection and reaction tube, and a portable POC instrument. The flexible sample tube has an area for collection of the raw biological specimen and separate ‘segments’ that contain pre-loaded assay reagents.<citref idrefs="cit13">13</citref> For sample volumes of 10–200 μL, the specimen is collected directly into the tube, a barcode is scanned to identify the test, and the tube is inserted into the analyzer. Larger sample volumes require additional sample processing and concentration.</p><figure xsrc="b705672a-f1.tif" id="fig1" pos="float"><title>Point-of-care systems: (a) IQuum Inc, Liat™ Analyzer; (b) DxBox, A Point-of-Care Diagnostic System for the Developing World (concept design), being developed by UW, PATH, Micronics and Nanogen.</title></figure><p>The FL rapid PCR system of Enigma Diagnostics also integrates real-time PCR detection with sample preparation. Nucleic acid purification and concentration are achieved with an internal ultrasonic disrupter and magnetic beads. Centrifugal force is used to move liquid reagents and the processed sample within the device. Thermal cycling is achieved by passing an electric current through a conducting polymer to heat the reaction mixture directly.<citref idrefs="cit19">19</citref> The instrument contains four-wavelength detection optics that are compatible with multiplex fluorogenic assays and TaqMan probes.</p><p>The sample preparation and testing steps of the microfluidic NAT system from HandyLab are performed in a cartridge, combining sample concentration and enrichment, drop metering, mixing, and detection. Various fluids required for sample washing, analyte release, neutralization, and reagent dissolution are pre-packaged in hermetically sealed barrier domes associated with the microfluidic network. Thermally responsive and expansive materials are used to create valves and a pressure differential within the device for moving liquid through channels and chambers.<citref idrefs="cit20 cit21">20,21</citref> Beads coated with a polycationic agent, such as poly-lysine, together with enzyme treatment with proteases and RNases are used for extraction of DNA. The system relies on PCR amplification and fluorescence detection.</p><p>The Lab-on-a-card DxBox testing system [<figref idrefs="fig1">Fig. 1(b)</figref>] is also under development by collaborators at PATH, Micronics, Nanogen, and the University of Washington (UW). It comprises both a disposable single-use microfluidic card containing dry, heat-stable agents and a handheld instrument for operation of the microfluidic circuits and temperature control. The user inserts a swab containing a stool sample into the card and places the card in the instrument. The card includes four microfluidic sub-circuits for organism capture and lysis, nucleic acid capture, multiplexed PCR amplification, and visual lateral-flow detection of amplified products. A combination of capillary action and positive displacement pumping draws the sample <it>via</it> microfluidic channels through the integrated sub-circuits on the disposable card.<citref idrefs="cit6 cit7">6,7</citref></p><p>Challenges related to the development of these microfluidic NAT platforms and their application in resource-limited settings include the cost associated with both the complexity of microfluidic card production and with the maintenance of the equipment. The ability to work with larger sample volumes in order to improve sensitivity also needs to be addressed. Other PCR-based systems intended for field- and POC-use include R.A.P.I.D.<sup>®</sup> and RAZOR<sup>®</sup> developed by Idaho Technology, HyBeacons<sup>®</sup> probes developed by LGC, and Bio-Seeq™ developed by Smiths Detection.</p><p>Relatively new isothermal amplification methods such as loop-mediated amplification (LAMP)<citref idrefs="cit22">22</citref> and helicase-dependent amplification (HDA)<citref idrefs="cit14">14</citref> are the base technologies for the next generation of POC-NAT assays being developed by Eiken Chemical and BioHelix, respectively. The integration of these amplification technologies with detection methods that do not require expensive and sophisticated machines for reading of detection signals (UV lamp-illuminated fluorescence or turbidity and lateral-flow detection for LAMP and HDA, respectively) maintains their suitability for POC applications. Although their circumvention of the use of thermal cyclers and expensive and sophisticated instrumentation is important for POC testing, these systems do not have an integrated sample preparation step, instead relying on manual sample preparation by conventional methods for target capture and nucleic acid extraction.</p><p>Microarray-based nucleic acid detection systems and platforms with the potential for integration into multiplex POC-NAT assays include the Verigene<sup>®</sup> Biobarcode™ microarray system developed by Nanosphere,<citref idrefs="cit8 cit9">8,9</citref> the SC1000 electrochemical sensor array developed by GeneFluidics,<citref idrefs="cit23 cit24">23,24</citref> NanoChip<sup>®</sup> developed by Nanogen,<citref idrefs="cit10 cit12">10,12</citref> and microarrays developed by Clondiag.<citref idrefs="cit25">25</citref> Many other nucleic acid microarray and microfluidic technologies and products exist; however, they are designed for laboratory-based use and are therefore not suitable for field POC testing, especially in resource-limited settings.</p><p>The Verigene<sup>®</sup> system from Nanosphere Inc uses a combination of microfluidic processing and optical nanoprobe detection technologies in a microarray chip-based format. The core of the Nanosphere Verigene<sup>®</sup> detection technology is a bio-barcode amplification (BCA) method<citref idrefs="cit8">8</citref> utilizing oligonucleotide probes conjugated to gold nanoparticles (13 nm). Magnetic microparticles conjugated to oligonucleotide probes are used for hybridization-based capture of target nucleic acid and washing of unbound reactants. A scanometric method that measures the scattered light from the nanogold-labelled spots is used to generate detection signals on a DNA-chip.<citref idrefs="cit9">9</citref> The spotted chip is further exposed to silver enhancement solution for signal enhancement. Spots that develop are then read with the Verigene<sup>®</sup> identification system. Amongst the advantages of the Verigene<sup>®</sup> Biobarcode™ detection system is its sensitivity that is comparable with PCR-based methods, its ability to detect non-amplified nucleic acid in a multiplexed format. Assay reagents are not packed into the disposable cartridge but are loaded onto the Verigene<sup>®</sup> auto processing system. The system requires off-line sample preparation. A new generation Verigene<sup>®</sup> Mobile system designed to perform all assay procedures is under development.</p><p>GeneFludics’ electrochemical sensor SC1000 is another microarray detection platform providing non-amplified nucleic acid detection of bacterial 16S rRNA. The core module of the detection platform is an electrochemical sensor array. Picomolar amperometric detection is achieved through sandwich of a target bacterial 16S rRNA with a DNA capture probe immobilized on the surface of the sensor and a DNA detector probe coupled to an oxidoreductase enzymatic transducer, <it>e.g.</it> horseradish peroxidase. Electron transfer to an oxidised mediator substrate tetramethylbenzidine generates the measured amperometric signal.<citref idrefs="cit23">23</citref> When combined with a microfluidics-based sample preparation module, the GeneFluidics electrochemical sensor could serve as a base for a fully integrated POC system for the rapid diagnosis of urinary tract infections.<citref idrefs="cit24">24</citref></p><p>The NanoChip<sup>®</sup> DNA microarray platform developed by Nanogen is a silicon chip containing an array of 100 or 400 sites.<citref idrefs="cit10 cit12">10,12</citref> The chips are housed in a plastic cover with a fluidics chamber for applying solutions and have the unique ability for individual control of each test site connected to an electrode. This enables selective migration, concentration and binding of DNA from the overlying solution. The sample preparation and PCR amplification with biotin-tagged primers are performed external to the NanoChip<sup>®</sup> platform. After denaturation, the PCR product is added to the surface of the chip. The instrument then applies positive charge to one of the test sites, resulting in rapid attraction and local concentration of the negatively-charged sample. The biotinylated DNA binds to a streptavidin-containing gel covering each test site. The bound double-stranded PCR products are denatured with a weak basic solution and the unbound strand is washed away. Detection of DNA is achieved by hybridization with target-specific fluorescent probes. It takes about 14 hours for the NanoChip<sup>®</sup> 400 System to process 400 samples. The integrated NanoChip<sup>®</sup> reader scans the chip using a built-in programmable laser system. The built-in data analysis software analyses the data and provides results in the form of a histogram and results/statistics tables.<citref idrefs="cit10 cit12">10,12</citref> Based on their active microelectronics microarray technology, Nanogen is planning to develop a sample-to-answer system intended for POC use that will also integrate sample preparation in disposable cartridges.</p><p>Clondiag's miniaturized ArrayTube (AT) platform also requires off-line sample preparation. After individual sample preparation and biotin labelling during multiplex DNA amplification, the target solution is added into the ArrayTube for hybridization analysis with the specific probe molecules immobilized on the array chip. The specific interaction pattern on the array is visualised applying a colorimetric substrate staining reaction catalysed by horseradish peroxidase conjugated to streptavidin and bound to target DNA molecules <it>via</it> streptavidin–biotin interaction. The resulting colorimetric precipitation pattern images are detected with the ArrayTube reader.<citref idrefs="cit25">25</citref></p><p>In summary, although they are intended for POC and field-testing, the new NAT platforms are not appropriate to applications in resource-limited settings. Most of these assays require an additional non-integrated sample preparation step, whereas some also require separate amplification.<citref idrefs="cit11">11</citref> In addition, many of them are currently under development and are not yet validated. A summary of the POC-NAT systems under development and validation is presented in <tableref idrefs="tab1">Table 1</tableref>.</p><table-entry id="tab1"><title>Summary of POC-NAT systems and platforms</title><table><tgroup cols="6"><colspec colname="1" colwidth="6.82pi"></colspec><colspec colname="2" colwidth="9.66pi"></colspec><colspec colname="3" colwidth="7.41pi"></colspec><colspec colname="4" colwidth="6.00pi"></colspec><colspec colname="5" colwidth="9.33pi"></colspec><colspec colname="6" colwidth="6.81pi"></colspec><thead><row><entry colname="1" morerows="1">Company</entry><entry namest="2" nameend="3">Technology</entry><entry colname="4" morerows="1">Highly instrument-dependent</entry><entry colname="5" morerows="1">Sample preparation method</entry><entry colname="6" morerows="1">Integrated sample preparation</entry></row><row><entry colname="2">Amplification</entry><entry colname="3">Detection</entry></row></thead><tbody><row><entry>Cepheid</entry><entry>Real-time PCR</entry><entry>Fluorescence</entry><entry>Yes</entry><entry>Affinity-based target capture and sonication</entry><entry>Yes</entry></row><row><entry namest="1" nameend="6"> </entry></row><row><entry>IQuum</entry><entry>Liat™ Tube, real-time PCR</entry><entry>Fluorescence</entry><entry>Yes</entry><entry>Magnetic silica particle-based extraction</entry><entry>Yes</entry></row><row><entry namest="1" nameend="6"> </entry></row><row><entry>Enigma Diagnostics</entry><entry>Real-time PCR</entry><entry>Fluorescence</entry><entry>Yes</entry><entry>Sonication and magnetic bead separation</entry><entry>Yes</entry></row><row><entry namest="1" nameend="6"> </entry></row><row><entry>PATH, UW, Micronics, and Nanogen</entry><entry>PCR</entry><entry>Microfluidic Lab-on-a-card, lateral-flow</entry><entry>Yes</entry><entry>Affinity-based target capture and solid-phase nucleic acid extraction</entry><entry>Yes</entry></row><row><entry namest="1" nameend="6"> </entry></row><row><entry>HandyLab</entry><entry>PCR</entry><entry>Microfluidic cartridge, fluorescence</entry><entry>Yes</entry><entry>Cationic bead extraction, enzyme treatment</entry><entry>Yes</entry></row><row><entry namest="1" nameend="6"> </entry></row><row><entry>Eiken Chemical</entry><entry>Loop-mediated amplification (LAMP)</entry><entry>Real-time fluorescence or turbidimetry</entry><entry>Yes</entry><entry>Column-based, manual</entry><entry>No</entry></row><row><entry namest="1" nameend="6"> </entry></row><row><entry colname="1" morerows="1">BioHelix</entry><entry colname="2" morerows="1">Helicase-dependent amplification (HDA)</entry><entry colname="3">Dipstick-based</entry><entry colname="4">No</entry><entry colname="5" morerows="1">Column- or filter-based, manual</entry><entry colname="6" morerows="1">No</entry></row><row><entry colname="3">Real-time fluorescence</entry><entry colname="4">Yes</entry></row></tbody></tgroup></table></table-entry></section><section><title>Assessment of technologies and procedures for nucleic acid extraction related to POC-NAT application</title><p>In view of the need for fully integrated NAT systems and assays suitable for POC testing in resource-limited settings, we have undertaken an assessment of commercially available technologies and procedures for the isolation of nucleic acid with human immunodeficiency virus-type 1 (HIV-1) RNA as a model analyte.</p><p>Extraction of viral nucleic acid from human plasma is one of the most challenging feats of nucleic acid extraction because of the low copy number of viral particles and the abundance of inhibitory substances in samples derived from human blood. To evaluate existing technologies for sample preparation, EDTA-anticoagulated plasma from HIV-1-infected individuals as a test system were used because they provide the best stability of HIV-1 RNA levels in whole blood stored at room temperature as reported previously.<citref idrefs="cit26">26</citref> Diagnosis of HIV-1 infection and monitoring of viral load are important applications of POC-NAT, especially in resource-limited settings of developing countries, in which 95% of the estimated 40 million infected individuals worldwide reside.<citref idrefs="cit4 cit5">4,5</citref> Access to equipment required by currently available extraction procedures in such settings is limited, if not totally infeasible. Determination of the ease of use and suitability of procedures for extraction of HIV-1 nucleic acid for POC testing in resource-limited settings is therefore imperative. We have assessed commercially available technologies and procedures for the isolation of nucleic acid with human immunodeficiency virus-type 1 (HIV-1) RNA as a model analyte. These technologies include: (1) solid-phase extraction in the presence of a chaotropic reagent with silica or glass;<citref idrefs="cit27 cit28">27,28</citref> (2) liquid-phase extraction with acid guanidinium thiocyanate–phenol–chloroform;<citref idrefs="cit29">29</citref> (3) single-tube liquid-phase extraction with a chaotropic denaturing salt;<citref idrefs="cit30">30</citref> (4) solid-phase extraction with anti-chaotropic salt and glass fibers (Invisorb<sup>®</sup> Technology);<citref idrefs="cit31">31</citref> (5) FTA<sup>®</sup> Technology,<citref idrefs="cit32">32</citref> which relies on filter matrices impregnated with a patented formula that lyses cell membranes and immobilizes nucleic acid; (6) extraction with an ion-exchange chelating resin;<citref idrefs="cit33">33</citref> and (7) ChargeSwitch<sup>®</sup> Technology, which circumvents the use of organic solvents and chaotropic salts.<citref idrefs="cit34">34</citref> ChargeSwitch<sup>®</sup> Technology provides a switchable positive charge on a solid surface of magnetic particles or microtitre plates that bind negatively-charged nucleic acid selectively under optimized low pH buffered conditions. Impurities are washed away in an aqueous wash buffer. The charge on the binding surface is switched off and the nucleic acid is released for elution in low salt elution buffer with higher pH buffer.</p><subsect1><title>Viral standards and clinical specimens</title><p>WHO International Standard HIV-1 (NIBSC code 97/656) from the National Institute for Biological Standards and Controls (NIBSC, Potters Bar, UK) was used to construct the reference curve for reverse transcription and quantitative PCR (RT-qPCR) analysis. EDTA-anticoagulated plasma samples from blood donors at the Komfo Anokye Teaching Hospital Blood Bank in Kumasi, Ghana, were assayed. These specimens were tested with rapid tests for antibodies to HIV-1 and to HIV-2, and their viral load was determined as previously described.<citref idrefs="cit35">35</citref> The research use of these specimens was approved by the committee on human research publication and ethics of the University of Science and the Technology School of Medical Sciences in Kumasi.</p></subsect1><subsect1><title>Nucleic acid extraction</title><p>Extraction of HIV-1 RNA from the plasma specimens was performed with the following nucleic acid extraction reagents or kits according to the instructions of the manufacturers: NucliSens<sup>®</sup> isolation kit (bioMérieux, Boxtel, The Netherlands), QIAamp Viral RNA Mini kit (Qiagen, Hilden, Germany), High Pure Viral Nucleic Acid kit (Roche, Mannheim, Germany), FTA kit (Whatman, Kent, UK), TRI Reagent<sup>®</sup> BD (Sigma-Aldrich, St Louis, MO), TRI<scp>zol</scp><sup>®</sup> LS (Invitrogen, Carlsbad, CA), ViralXpress™ RNA/DNA extraction reagent (Chemicon, Millipore, Temecula, CA), ChargeSwitch<sup>®</sup> EasyPlex™ Viral RNA/DNA (Invitrogen, Carlsbad, CA), RTP<sup>®</sup> DNA/RNA Virus Mini kit (Invitek, Berlin, Germany), and Chelex 100 InstaGene™ Matrix (BioRad, Hercules, CA). Nucleic acid extracts were stored at −80 °C until analysis. To allow comparison among the different sample preparation methods, equal amounts of input sample (200 μL) were used per assay. Extracts derived from the WHO International Standard and negative samples were also used as positive and negative controls, respectively, for nucleic acid extraction and RT-qPCR.</p></subsect1><subsect1><title>RT-qPCR</title><p>A TaqMan technology-based, real-time qPCR assay was used for evaluation of the efficiency of viral nucleic acid extraction as previously described<citref idrefs="cit35">35</citref> but with primer sets for non-HIV targets omitted from the amplification reaction. In brief, amplification was performed with a Brilliant™ Two-Step Quantitative RT-PCR Core Reagent kit and an Mx4000<sup>®</sup> Multiplex Quantitative PCR System (Stratagene, La Jolla, CA). The sequences of the primers and detection probes were based on the long terminal repeat region of the HIV-1 genome. The fluorogenic probe was labelled at the 5′-end with VIC<sup>®</sup> and at the 3′-end with 6-carboxy-<it>N</it>-tetramethylrhodamine, both of which were obtained from Proligo France SAS (Paris, France). The WHO International Standard extracted with the High Pure Viral Nucleic Acid kit (Roche) was used to construct reference curves for quantification. Each data point represents the mean of quadruplicates from each of four test runs.</p></subsect1><subsect1><title>Summary of results</title><p>An overview of the commercial kits and technologies for nucleic acid extraction tested with HIV-1 RNA as a model target is presented in <tableref idrefs="tab2">Table 2</tableref>. In addition to the efficiency of nucleic acid extraction, other characteristics important for the application of technologies to POC testing in resource-limited settings were addressed, including complexity, cost, requirement for equipment, refrigeration, or additional reagents, and duration of the entire procedure. Most of the technologies require a heating block and a centrifuge or magnetic rack. Furthermore, the kit reagents require cold-chain transport and storage below room temperature. All kit procedures required the supply of additional reagents and disposables, and preparation of working solutions. Cost per extraction is also high, even without the cost of the additional reagents and consumables required for the extraction procedure. Most of the procedures need to be performed by a technically skilled user.</p><table-entry id="tab2"><title>Overview of nucleic acid extraction procedures</title><table><tgroup cols="9"><colspec colname="1" colwidth="6.72pi"></colspec><colspec colname="2" colwidth="7.71pi"></colspec><colspec colname="3" colwidth="5.55pi"></colspec><colspec colname="4" colwidth="6.44pi"></colspec><colspec colname="5" colwidth="5.56pi"></colspec><colspec colname="6" colwidth="7.00pi"></colspec><colspec colname="7" colwidth="6.82pi"></colspec><colspec colname="8" colwidth="6.04pi"></colspec><colspec colname="9" colwidth="5.28pi"></colspec><thead><row><entry>Extraction kit</entry><entry>Technology</entry><entry>Number of steps</entry><entry>Equipment</entry><entry>Duration of procedure/min</entry><entry>Refrigeration required</entry><entry>User skills required<fnoteref idrefs="tab2fna"></fnoteref></entry><entry>Additional reagents required</entry><entry>Cost (£)<fnoteref idrefs="tab2fnb"></fnoteref></entry></row></thead><tfoot><row><entry namest="1" nameend="9"><footnote id="tab2fna">Level of technical skills required: H = high, M = medium, L = low.</footnote><footnote id="tab2fnb">Cost per preparation based on catalogue list price in the United Kingdom; cost of additional reagents and consumables not included.</footnote></entry></row></tfoot><tbody><row><entry>High Pure Viral Nucleic Acid (Roche)</entry><entry>Chaotropic reagent, silica-glass extraction</entry><entry align="char" char=".">21</entry><entry>Heater, microfuge</entry><entry align="char" char=".">20</entry><entry>Yes</entry><entry>M</entry><entry>Yes</entry><entry align="char" char=".">2.18</entry></row><row><entry namest="1" nameend="9"> </entry></row><row><entry>QIAamp Viral RNA (Qiagen)</entry><entry>Chaotropic reagent, silica-glass extraction</entry><entry align="char" char=".">24</entry><entry>Microfuge</entry><entry align="char" char=".">20</entry><entry>Yes</entry><entry>M</entry><entry>Yes</entry><entry align="char" char=".">2.82</entry></row><row><entry namest="1" nameend="9"> </entry></row><row><entry>NucliSens<sup>®</sup> (bioMérieux)</entry><entry>Chaotropic reagent, silica-glass extraction</entry><entry align="char" char=".">30</entry><entry>Heater, microfuge</entry><entry align="char" char=".">40</entry><entry>Yes</entry><entry>H</entry><entry>Yes</entry><entry align="char" char=".">4.58</entry></row><row><entry namest="1" nameend="9"> </entry></row><row><entry>FTA (Whatman)</entry><entry>FTA paper</entry><entry align="char" char=".">10</entry><entry>Puncher, microfuge</entry><entry align="char" char=".">>120</entry><entry>No</entry><entry>M</entry><entry>Yes</entry><entry align="char" char=".">1.76</entry></row><row><entry namest="1" nameend="9"> </entry></row><row><entry>TRI Reagent<sup>®</sup> BD (Sigma)</entry><entry>Organic solvent, chaotropic salt extraction</entry><entry align="char" char=".">18</entry><entry>Microfuge</entry><entry align="char" char=".">60</entry><entry>Yes</entry><entry>H</entry><entry>Yes</entry><entry align="char" char=".">1.27</entry></row><row><entry namest="1" nameend="9"> </entry></row><row><entry>TRI<scp>zol</scp><sup>®</sup> LS (Invitrogen)</entry><entry>Organic solvent, chaotropic salt extraction</entry><entry align="char" char=".">18</entry><entry>Microfuge</entry><entry align="char" char=".">60</entry><entry>Yes</entry><entry>H</entry><entry>Yes</entry><entry align="char" char=".">1.41</entry></row><row><entry namest="1" nameend="9"> </entry></row><row><entry>ViralXpress™ (Millipore)</entry><entry>Chaotropic salt, alcohol precipitation</entry><entry align="char" char=".">14</entry><entry>Microfuge</entry><entry align="char" char=".">30</entry><entry>Yes</entry><entry>M</entry><entry>Yes</entry><entry align="char" char=".">1.54</entry></row><row><entry namest="1" nameend="9"> </entry></row><row><entry>ChargeSwitch<sup>®</sup> (Invitrogen)</entry><entry>ChargeSwitch<sup>®</sup></entry><entry align="char" char=".">24</entry><entry>Heater, microfuge or magnet</entry><entry align="char" char=".">30</entry><entry>Yes</entry><entry>H</entry><entry>Yes</entry><entry align="char" char=".">1.15</entry></row><row><entry namest="1" nameend="9"> </entry></row><row><entry>RTP<sup>®</sup> DNA/RNA (Invitek)</entry><entry>Anti-chaotropic salt, glass fiber</entry><entry align="char" char=".">19</entry><entry>Heater, microfuge</entry><entry align="char" char=".">30</entry><entry>No</entry><entry>M</entry><entry>Yes</entry><entry align="char" char=".">2.60</entry></row><row><entry namest="1" nameend="9"> </entry></row><row><entry>Chelex (BioRad)</entry><entry>Chelating resin</entry><entry align="char" char=".">7</entry><entry>Heater, microfuge</entry><entry align="char" char=".">20</entry><entry>Yes</entry><entry>L</entry><entry>Yes</entry><entry align="char" char="."><1.00</entry></row><row><entry namest="1" nameend="9"> </entry></row></tbody></tgroup></table></table-entry><p>The efficiency of nucleic acid extraction was evaluated by RT-qPCR analysis of HIV-1-positive specimens with various viral loads from Africa (<figref idrefs="fig2">Fig. 2</figref>). Results from replicate extractions of low viral load samples (<it>ca.</it> 10<sup>3</sup> copies mL<sup>−1</sup>), performed by two independent and technically skilled users in quadruplicates have also given the indication about reliability and reproducibility of the extraction procedures. Overall, solid-phase extraction with silica or glass in the presence of a chaotropic reagent proved to be the most consistent and efficient procedure (<figref idrefs="fig2">Fig. 2</figref>). Despite its advantages, this technology is the most expensive (up to £4.58 per test), entails multiple (>20), complex, and time-consuming steps, and requires instrumentation. These drawbacks limit the application of this technology to POC testing in resource-limited settings.</p><figure xsrc="b705672a-f2.tif" id="fig2" pos="float"><title>RT-qPCR analysis of the nucleic acid template extracted from two HIV seropositive plasma samples with a low (1.8 × 10<sup>3</sup> copies mL<sup>−1</sup>, yellow) or high (4 × 10<sup>5</sup> copies mL<sup>−1</sup>, blue) viral load with the use of commercially available technologies for sample preparation.</title></figure><p>Liquid-phase extraction with phenol–chloroform and a chaotropic salt was among the most time-consuming procedures (<it>ca.</it> 1 h), requiring more than 15 steps. It showed variable extraction efficiency, failing to reproducibly extract viral RNA from samples with an HIV-1 titer of <10<sup>3</sup> copies mL<sup>−1</sup> (data not shown). This extraction method also requires a high level of technical skill to separate the organic phase from the aqueous phase and is therefore prone to inconsistency with regard to downstream amplification as a result of carryover of inhibitory agents (guanidinium salts and organic solvents).</p><p>ViralXpress™, a single-tube extraction procedure based on a chaotropic denaturing salt was among the cheapest (£1.54 per test) of the technologies assessed and was relatively simple to perform and rapid (<it>ca.</it> 30 min). However, it showed a relatively low efficiency and reliability of extraction. The FTA method required more than 2 h to complete and the yield of HIV-1 RNA was below the detection limit of RT-qPCR (data not shown). Although relatively simple to perform, Chelex-based extraction also yielded an amount of HIV-1 RNA that was below the detection limit of RT-qPCR, probably because of insufficient separation of nucleic acid from inhibitory substances, as indicated by a low 260/280 nm absorbance ratio.</p><p>The new-generation technologies for solid-phase extraction, Invisorb (RTP<sup>®</sup> DNA/RNA) and ChargeSwitch<sup>®</sup>, circumvent the use of chaotropic salts and organic solvents. The aqueous buffer systems of the ChargeSwitch<sup>®</sup> method are inherently less inhibitory to downstream applications, but this method was less efficient than the classical solid-phase extraction technology. The RTP<sup>®</sup> DNA/RNA method showed extraction efficiency and reproducibility similar to that of the classical methods. This technology is also fully compatible with storage at room temperature, a definite advantage for POC applications in resource-limited settings, where refrigeration and a stable electricity supply are not readily available. In spite of their advantages, however, these new methods are limited by the numerous handling steps and the requirement for heating and centrifugation.</p></subsect1></section><section><title>Conclusion</title><p>Existing procedures for the extraction of nucleic acid from clinical specimens are complex, laborious, time-consuming, and expensive, with most of them also requiring specialized equipment. In their current formats, none of the existing methods and technologies is readily adaptable to testing in resource-limited settings. Extraction procedures should ideally also be effective with samples (such as whole blood instead of plasma) that do not require initial processing steps such as centrifugation or blotting and drying on filter paper (dry blood spots) for storage and transportation to reference laboratories.<citref idrefs="cit36">36</citref> The use of whole blood obtained from a finger or heel prick is desirable for POC or field-testing, but the small volume of such samples and their inherent high level of inhibitors present additional challenges. The existing sample preparation technologies are either not designed for whole blood or do not work with this specimen without compromising extraction efficiency and analyte purity. The development of a simple generic procedure for sample preparation that is suitable for integration into POC-NAT diagnostics without such compromises is thus sorely needed.</p></section></art-body><art-back><ack><p>This study has been supported by the Wellcome Trust, England.</p><p>We thank Dr Annabel Whibley and Fe Magbanua for technical assistance.</p></ack><biblist><citgroup id="cit1"><journalcit><citauth><fname>K.</fname><surname>Soldan</surname></citauth><citauth><fname>J. A.</fname><surname>Barbara</surname></citauth><citauth><fname>M. E.</fname><surname>Ramsay</surname></citauth><citauth><fname>A. J.</fname><surname>Hall</surname></citauth><title>Vox Sang.</title><year>2003</year><volumeno>84</volumeno><pages><fpage>274</fpage><lpage>286</lpage></pages></journalcit></citgroup><citgroup id="cit2"><journalcit><citauth><fname>I.</fname><surname>Loussert-Ajaka</surname></citauth><citauth><fname>T. D.</fname><surname>Ly</surname></citauth><citauth><fname>M. L.</fname><surname>Chaix</surname></citauth><citauth><fname>D.</fname><surname>Ingrand</surname></citauth><citauth><fname>S.</fname><surname>Saragosti</surname></citauth><citauth><fname>A. M.</fname><surname>Courouce</surname></citauth><citauth><fname>F.</fname><surname>Brun-Vezinet</surname></citauth><citauth><fname>F.</fname><surname>Simon</surname></citauth><title>Lancet</title><year>1994</year><volumeno>343</volumeno><pages><fpage>1393</fpage><lpage>1394</lpage></pages></journalcit></citgroup><citgroup id="cit3"><journalcit><citauth><fname>J.-P.</fname><surname>Allain</surname></citauth><title>Vox Sang.</title><year>2004</year><volumeno>86</volumeno><pages><fpage>83</fpage><lpage>91</lpage></pages></journalcit></citgroup><citgroup id="cit4"><citation><title>Reports on the global AIDS epidemic</title>, <citpub>UNAIDS/WHO</citpub>, <year>2006</year>; <url url="http://www.unaids.org/en/HIV_data/2006globalreport/default.asp">http://www.unaids.org/en/HIV_data/2006globalreport/default.asp</url> (accessed 1 October 2007)</citation></citgroup><citgroup id="cit5"><journalcit><citauth><fname>S. A.</fname><surname>Fiscus</surname></citauth><citauth><fname>B.</fname><surname>Cheng</surname></citauth><citauth><fname>S. M.</fname><surname>Crowe</surname></citauth><citauth><fname>L.</fname><surname>Demeter</surname></citauth><citauth><fname>Ch. J.</fname><surname>Jennings</surname></citauth><citauth><fname>V.</fname><surname>Miller</surname></citauth><citauth><fname>R.</fname><surname>Respess</surname></citauth><citauth><fname>W.</fname><surname>Stevens</surname></citauth><citauth><surname>the forum for Collaborative HIV Research Alternative Viral Load Assay Working Group</surname></citauth><title>PLOS Med.</title><year>2006</year><volumeno>3</volumeno><pages><fpage>1743</fpage><lpage>1750</lpage></pages></journalcit></citgroup><citgroup id="cit6"><journalcit><citauth><fname>P.</fname><surname>Yager</surname></citauth><citauth><fname>Th.</fname><surname>Edwards</surname></citauth><citauth><fname>E.</fname><surname>Fu</surname></citauth><citauth><fname>K.</fname><surname>Helton</surname></citauth><citauth><fname>K.</fname><surname>Nelson</surname></citauth><citauth><fname>M. R.</fname><surname>Tam</surname></citauth><citauth><fname>B. H.</fname><surname>Weigl</surname></citauth><title>Nature</title><year>2006</year><volumeno>442</volumeno><pages><fpage>412</fpage><lpage>418</lpage></pages></journalcit></citgroup><citgroup id="cit7"><journalcit><citauth><fname>D.</fname><surname>Walt</surname></citauth><title>Science</title><year>2005</year><volumeno>308</volumeno><pages><fpage>217</fpage><lpage>219</lpage></pages></journalcit></citgroup><citgroup id="cit8"><journalcit><citauth><fname>J.-M.</fname><surname>Nam</surname></citauth><citauth><fname>C. S.</fname><surname>Thaxton</surname></citauth><citauth><fname>C. A.</fname><surname>Mirkin</surname></citauth><title>Science</title><year>2003</year><volumeno>301</volumeno><pages><fpage>1884</fpage><lpage>1886</lpage></pages></journalcit></citgroup><citgroup id="cit9"><journalcit><citauth><fname>T. A.</fname><surname>Taton</surname></citauth><citauth><fname>C. A.</fname><surname>Mirkin</surname></citauth><citauth><fname>R. L.</fname><surname>Letsinger</surname></citauth><title>Science</title><year>2000</year><volumeno>289</volumeno><pages><fpage>1757</fpage><lpage>1760</lpage></pages></journalcit></citgroup><citgroup id="cit10"><journalcit><citauth><fname>D.</fname><surname>Keen-Kim</surname></citauth><citauth><fname>W. W.</fname><surname>Grody</surname></citauth><citauth><fname>C. S.</fname><surname>Richards</surname></citauth><title>Expert Rev. Mol. Diagn.</title><year>2006</year><volumeno>6</volumeno><pages><fpage>287</fpage><lpage>294</lpage></pages></journalcit></citgroup><citgroup id="cit11"><journalcit><citauth><fname>C. A.</fname><surname>Holland</surname></citauth><citauth><fname>F. L.</fname><surname>Kiechle</surname></citauth><title>Curr. Opin. Microbiol.</title><year>2005</year><volumeno>8</volumeno><pages><fpage>504</fpage><lpage>509</lpage></pages></journalcit></citgroup><citgroup id="cit12"><journalcit><citauth><fname>M.</fname><surname>Sanguinetti</surname></citauth><citauth><fname>L.</fname><surname>Novarese</surname></citauth><citauth><fname>B.</fname><surname>Posteraro</surname></citauth><citauth><fname>S.</fname><surname>Ranno</surname></citauth><citauth><fname>E. De</fname><surname>Carolis</surname></citauth><citauth><fname>G.</fname><surname>Pecorini</surname></citauth><citauth><fname>B.</fname><surname>Lucignano</surname></citauth><citauth><fname>F.</fname><surname>Ardito</surname></citauth><citauth><fname>G.</fname><surname>Fadda</surname></citauth><title>J. Clin. Microbiol.</title><year>2005</year><volumeno>43</volumeno><pages><fpage>6189</fpage><lpage>6193</lpage></pages></journalcit></citgroup><citgroup id="cit13"><citation><citauth><fname>Sh.</fname><surname>Chen</surname></citauth>, <citauth><fname>G.</fname><surname>Selecman</surname></citauth> and <citauth><fname>B.</fname><surname>Lemieux</surname></citauth>, <arttitle>‘Expanding rapid nucleic acid testing’</arttitle>, <title>IVD Technology</title>, Medical device link, <citpub>Canon Communications LLC</citpub>, <pubplace>Los Angeles, CA, USA</pubplace>, July, <year>2004</year>, <biblscope>p. 51</biblscope></citation></citgroup><citgroup id="cit14"><journalcit><citauth><fname>M.</fname><surname>Vincent</surname></citauth><citauth><fname>Y.</fname><surname>Xu</surname></citauth><citauth><fname>H.</fname><surname>Kong</surname></citauth><arttitle>‘Helicase-dependent isothermal DNA amplification’</arttitle><title>EMBO Rep.</title><year>2004</year><volumeno>5</volumeno><pages><fpage>795</fpage><lpage>800</lpage></pages></journalcit></citgroup><citgroup id="cit15"><journalcit><citauth><fname>B. H.</fname><surname>Weigl</surname></citauth><citauth><fname>P.</fname><surname>Tarr</surname></citauth><citauth><fname>P.</fname><surname>Yager</surname></citauth><citauth><fname>L.</fname><surname>Dillman</surname></citauth><citauth><fname>R.</fname><surname>Peck</surname></citauth><citauth><fname>S.</fname><surname>Ramachandran</surname></citauth><citauth><fname>M.</fname><surname>Lemba</surname></citauth><citauth><fname>M.</fname><surname>Kokoris</surname></citauth><citauth><fname>M.</fname><surname>Nabavi</surname></citauth><citauth><fname>F.</fname><surname>Battrell</surname></citauth><citauth><fname>D.</fname><surname>Hoekstra</surname></citauth><citauth><fname>E. J.</fname><surname>Klein</surname></citauth><citauth><fname>D. M.</fname><surname>Denno</surname></citauth><title>Proc. SPIE–Int. Soc. Opt. Eng.</title><year>2006</year><volumeno>6112</volumeno><pages><fpage>1</fpage><lpage>11</lpage></pages></journalcit></citgroup><citgroup id="cit16"><citation type="patent"><citauth><fname>K. E.</fname><surname>Petersen</surname></citauth>, <citauth><fname>W. A.</fname><surname>Kovacs</surname></citauth> and <citauth><fname>S. J.</fname><surname>Young</surname></citauth>, <it>US Pat.</it>, 5 958 349, <year>1999</year></citation></citgroup><citgroup id="cit17"><journalcit><citauth><fname>M. T.</fname><surname>Taylor</surname></citauth><citauth><fname>P.</fname><surname>Nguyen</surname></citauth><citauth><fname>J.</fname><surname>Ching</surname></citauth><citauth><fname>K. E.</fname><surname>Petersen</surname></citauth><title>J. Micromech. Microeng.</title><year>2003</year><volumeno>13</volumeno><pages><fpage>201</fpage><lpage>208</lpage></pages></journalcit></citgroup><citgroup id="cit18"><citation type="patent"><citauth><fname>Sh.</fname><surname>Chen</surname></citauth>, <it>US Pat.</it>, 6 780 617, <year>2004</year></citation></citgroup><citgroup id="cit19"><citation type="patent"><citauth><fname>D. J.</fname><surname>Squirrel</surname></citauth> and <citauth><fname>M. A.</fname><surname>Lee</surname></citauth>, <it>Int. Pat. Appl.</it>, WO 2006079814, <year>2006</year></citation></citgroup><citgroup id="cit20"><citation><citauth><fname>B.</fname><surname>Wu</surname></citauth>, <citauth><fname>J. S.</fname><surname>Altaus</surname></citauth>, <citauth><fname>N.</fname><surname>Phadke</surname></citauth>, <citauth><fname>S. N.</fname><surname>Brahmasandra</surname></citauth>, <citauth><fname>K.</fname><surname>Handique</surname></citauth>, <citauth><fname>A.</fname><surname>Kehrer</surname></citauth>, <citauth><fname>G.</fname><surname>Parunak</surname></citauth>, <citauth><fname>C.</fname><surname>Haley</surname></citauth> and <citauth><fname>T.</fname><surname>Springer</surname></citauth>, <it>US Pat. Appl.</it> 20060166233, <year>2006</year></citation></citgroup><citgroup id="cit21"><journalcit><citauth><fname>K.</fname><surname>Handique</surname></citauth><citauth><fname>D. T.</fname><surname>Burke</surname></citauth><citauth><fname>C. H.</fname><surname>Mastrangelo</surname></citauth><citauth><fname>M. A.</fname><surname>Burns</surname></citauth><title>Anal. Chem.</title><year>2001</year><volumeno>73</volumeno><pages><fpage>1831</fpage><lpage>1838</lpage></pages></journalcit></citgroup><citgroup id="cit22"><journalcit><citauth><fname>T.</fname><surname>Notomi</surname></citauth><citauth><fname>H.</fname><surname>Okayama</surname></citauth><citauth><fname>H.</fname><surname>Masubuchi</surname></citauth><citauth><fname>T.</fname><surname>Yonekawa</surname></citauth><citauth><fname>K.</fname><surname>Watanabe</surname></citauth><citauth><fname>N.</fname><surname>Amino</surname></citauth><citauth><fname>T.</fname><surname>Hase</surname></citauth><title>Nucleic Acids Res.</title><year>2000</year><volumeno>28</volumeno><pages><fpage>e63</fpage></pages></journalcit></citgroup><citgroup id="cit23"><journalcit><citauth><fname>V.</fname><surname>Gau</surname></citauth><citauth><fname>S.</fname><surname>Ma</surname></citauth><citauth><fname>H.</fname><surname>Wang</surname></citauth><citauth><fname>J.</fname><surname>Tsukuda</surname></citauth><citauth><fname>J.</fname><surname>Kibler</surname></citauth><citauth><fname>D. A.</fname><surname>Haake</surname></citauth><title>Methods</title><year>2005</year><volumeno>37</volumeno><pages><fpage>73</fpage><lpage>83</lpage></pages></journalcit></citgroup><citgroup id="cit24"><journalcit><citauth><fname>J. C.</fname><surname>Liao</surname></citauth><citauth><fname>M.</fname><surname>Mastali</surname></citauth><citauth><fname>V.</fname><surname>Gau</surname></citauth><citauth><fname>M. A.</fname><surname>Suchard</surname></citauth><citauth><fname>A. K.</fname><surname>Moller</surname></citauth><citauth><fname>D. A.</fname><surname>Brickner</surname></citauth><citauth><fname>J. T.</fname><surname>Babbitt</surname></citauth><citauth><fname>Y.</fname><surname>Li</surname></citauth><citauth><fname>J.</fname><surname>Gornbein</surname></citauth><citauth><fname>E. M.</fname><surname>Landaw</surname></citauth><citauth><fname>E. R. B.</fname><surname>McCabe</surname></citauth><citauth><fname>B. M.</fname><surname>Churchill</surname></citauth><citauth><fname>D. A.</fname><surname>Haake</surname></citauth><title>J. Clin. Microbiol.</title><year>2006</year><volumeno>44</volumeno><pages><fpage>561</fpage><lpage>570</lpage></pages></journalcit></citgroup><citgroup id="cit25"><journalcit><citauth><fname>K.</fname><surname>Ballmer</surname></citauth><citauth><fname>B. M.</fname><surname>Korczak</surname></citauth><citauth><fname>P.</fname><surname>Kuhnert</surname></citauth><citauth><fname>P.</fname><surname>Slickers</surname></citauth><citauth><fname>R.</fname><surname>Ehricht</surname></citauth><citauth><fname>H.</fname><surname>Hachler</surname></citauth><title>J. Clin. Microbiol.</title><year>2007</year><volumeno>45</volumeno><pages><fpage>370</fpage><lpage>379</lpage></pages></journalcit></citgroup><citgroup id="cit26"><journalcit><citauth><fname>R. E.</fname><surname>Dickover</surname></citauth><citauth><fname>S. A.</fname><surname>Herman</surname></citauth><citauth><fname>K.</fname><surname>Saddiq</surname></citauth><citauth><fname>D.</fname><surname>Wafer</surname></citauth><citauth><fname>M.</fname><surname>Dillon</surname></citauth><citauth><fname>Y. J.</fname><surname>Bryson</surname></citauth><title>J. Clin. Microbiol.</title><year>1998</year><volumeno>36</volumeno><pages><fpage>1070</fpage><lpage>1073</lpage></pages></journalcit></citgroup><citgroup id="cit27"><journalcit><citauth><fname>R.</fname><surname>Boom</surname></citauth><citauth><fname>C. J.</fname><surname>Sol</surname></citauth><citauth><fname>M. M.</fname><surname>Salimans</surname></citauth><citauth><fname>C. L.</fname><surname>Jansen</surname></citauth><citauth><fname>P. M.</fname><surname>Wertheim-van Dillen</surname></citauth><citauth><fname>J.</fname><surname>Noordaa</surname></citauth><title>J. Clin. Microbiol.</title><year>1990</year><volumeno>28</volumeno><pages><fpage>495</fpage><lpage>503</lpage></pages></journalcit></citgroup><citgroup id="cit28"><journalcit><citauth><fname>B.</fname><surname>Vogelstein</surname></citauth><citauth><fname>D.</fname><surname>Gillespie</surname></citauth><title>Proc. Natl. Acad. Sci. U. S. A.</title><year>1979</year><volumeno>76</volumeno><pages><fpage>615</fpage><lpage>619</lpage></pages></journalcit></citgroup><citgroup id="cit29"><journalcit><citauth><fname>P.</fname><surname>Chomczynski</surname></citauth><citauth><fname>N.</fname><surname>Sacchi</surname></citauth><title>Anal. Biochem.</title><year>1987</year><volumeno>162</volumeno><pages><fpage>156</fpage><lpage>159</lpage></pages></journalcit></citgroup><citgroup id="cit30"><citation><citauth><fname>R. S.</fname><surname>Davis</surname></citauth>, <citauth><fname>J. M.</fname><surname>Warren</surname></citauth> and <citauth><fname>P. A.</fname><surname>Schneider</surname></citauth>, <arttitle>‘Evaluation of Chemicon's single-tube nucleic acid extraction reagent ViralXpress™’</arttitle>, presented at The Clinical Virology Symposium, 2000, held at Clearwater, Florida, USA; <url url="http://www.chemicon.com/Resource/abstracts/molbio/viral_abstract.asp">http://www.chemicon.com/Resource/abstracts/molbio/viral_abstract.asp</url> (accessed 1 October 2007)</citation></citgroup><citgroup id="cit31"><citation type="patent"><citauth><fname>T.</fname><surname>Hillebrand</surname></citauth> and <citauth><fname>P.</fname><surname>Bendzko</surname></citauth>, <it>US Pat.</it>, 6 699 987, <year>2004</year></citation></citgroup><citgroup id="cit32"><citation type="patent"><citauth><fname>L. A.</fname><surname>Burgoyne</surname></citauth>, <it>US Pat.</it>, 5 496 562, <year>1996</year>; <it>US Pat.</it>, 6 294 203, <year>2001</year>; <it>US Pat.</it>, 6 322 983, <year>2001</year></citation></citgroup><citgroup id="cit33"><journalcit><citauth><fname>T.</fname><surname>Mygind</surname></citauth><citauth><fname>S.</fname><surname>Birkelund</surname></citauth><citauth><fname>N. H.</fname><surname>Birkebæk</surname></citauth><citauth><fname>L.</fname><surname>Østergaard</surname></citauth><citauth><fname>J. S.</fname><surname>Jensen</surname></citauth><citauth><fname>G.</fname><surname>Christiansen</surname></citauth><title>BMC Microbiol.</title><year>2002</year><volumeno>2</volumeno><link type="doi">10.1186/1471-2180-2-17</link></journalcit></citgroup><citgroup id="cit34"><citation type="patent"><citauth><fname>M. J.</fname><surname>Baker</surname></citauth>, <it>Eur. Pat.</it>, 1 036 082, <year>2000</year></citation></citgroup><citgroup id="cit35"><journalcit><citauth><fname>D.</fname><surname>Candotti</surname></citauth><citauth><fname>J.</fname><surname>Temple</surname></citauth><citauth><fname>S.</fname><surname>Owusu-Ofori</surname></citauth><citauth><fname>J.-P.</fname><surname>Allain</surname></citauth><title>J. Virol. Methods</title><year>2004</year><volumeno>118</volumeno><pages><fpage>39</fpage><lpage>47</lpage></pages></journalcit></citgroup><citgroup id="cit36"><journalcit><citauth><fname>S. A.</fname><surname>Fiscus</surname></citauth><citauth><fname>D.</fname><surname>Brambilla</surname></citauth><citauth><fname>L.</fname><surname>Grosso</surname></citauth><citauth><fname>J.</fname><surname>Schock</surname></citauth><citauth><fname>M.</fname><surname>Cronin</surname></citauth><title>J. Clin. Microbiol.</title><year>1998</year><volumeno>36</volumeno><pages><fpage>258</fpage><lpage>260</lpage></pages></journalcit></citgroup></biblist><compoundgrp></compoundgrp><annotationgrp></annotationgrp><datagrp></datagrp><resourcegrp></resourcegrp></art-back></article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo><title>Sample preparation: a challenge in the development of point-of-care nucleic acid-based assays for resource-limited settings</title></titleInfo><titleInfo type="alternative" contentType="CDATA"><title>Sample preparation: a challenge in the development of point-of-care nucleic acid-based assays for resource-limited settings</title></titleInfo><name type="personal"><namePart type="given">Magda Anastassova</namePart><namePart type="family">Dineva</namePart><affiliation>Department of Haematology, University of Cambridge, CB2 2PT, Cambridge, UK</affiliation></name><name type="personal"><namePart type="given">Lourdes</namePart><namePart type="family">Mahilum-TapayPresent address: Diagnostics for the Real World Ltd., Sunnyvale, CA 94085, USA.</namePart><affiliation>Department of Haematology, University of Cambridge, CB2 2PT, Cambridge, UK</affiliation></name><name type="personal"><namePart type="given">Helen</namePart><namePart type="family">Lee</namePart><affiliation>Department of Haematology, University of Cambridge, CB2 2PT, Cambridge, UK</affiliation></name><typeOfResource>text</typeOfResource><genre type="review-article" displayLabel="PER"></genre><originInfo><publisher>The Royal Society of Chemistry.</publisher><dateIssued encoding="w3cdtf">2007</dateIssued><copyrightDate encoding="w3cdtf">2007</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><physicalDescription><internetMediaType>text/html</internetMediaType></physicalDescription><abstract>Currently available nucleic acid testing (NAT)-based assays are complex and time-consuming, and they require expensive instrumentation and dedicated laboratory spaces for sample preparation as well as for amplification and detection of the nucleic acid target. Reagents required for these tests are also expensive and must be transported and stored refrigerated or frozen. These characteristics have limited the use of such assays for point-of-care (POC) testing, especially in resource-poor settings. Efforts to develop simple and rapid NAT-based assays have focused predominantly on the amplification and detection steps, with sample preparation and nucleic acid extraction remaining the bottleneck in the development of NAT systems suitable for POC applications or resource-limited settings. A review of NAT platforms and technologies currently under development and validation for rapid field testing revealed that, in addition to requiring expensive and complex instrumentation, many of these systems also require off-line sample preparation and reagent handling. In their current format, they are therefore not appropriate for POC testing in resource-limited settings. We evaluated several commercially available technologies and procedures for the isolation of nucleic acid with the extraction of HIV-1 RNA from human plasma as a model system. Our results indicate that solid-phase extraction with silica or glass in the presence of a chaotropic salt provides the highest extraction efficiency. However, none of the existing methods and technologies is readily adaptable to a POC system. The integration of sample preparation procedures well suited to NAT-based assays in resource-limited settings therefore remains a challenge.</abstract><note type="footnote" displayLabel="fn1">This paper was published as part of the special issue on the Rapid Diagnostic Testing of Infection and Disease States.</note><note>Point-of-care nucleic acid testing (POC-NAT), a rapidly developing field, can improve healthcare in resource-limited settings. This paper reviews the current status and challenges presented by current POC-NAT systems. [b705672a-ga.tif]</note><note displayLabel="cit4">Reports on the global AIDS epidemic, UNAIDS/WHO, 2006; http://www.unaids.org/en/HIV_data/2006globalreport/default.asp (accessed 1 October 2007)</note><relatedItem type="host"><titleInfo><title>Analyst</title></titleInfo><titleInfo type="abbreviated"><title>Analyst</title></titleInfo><genre type="journal">journal</genre><originInfo><publisher>The Royal Society of Chemistry.</publisher><dateIssued encoding="w3cdtf">2007</dateIssued></originInfo><identifier type="ISSN">0003-2654</identifier><identifier type="eISSN">1364-5528</identifier><identifier type="coden">ANALAO</identifier><identifier type="RSC sercode">AN</identifier><part><date>2007</date><detail type="volume"><caption>vol.</caption><number>132</number></detail><detail type="issue"><caption>no.</caption><number>12</number></detail><extent unit="pages"><start>1193</start><end>1199</end><total>7</total></extent></part></relatedItem><identifier type="istex">EE568A42B9A541FB73C5D09C26A9EFFA741CC966</identifier><identifier type="DOI">10.1039/b705672a</identifier><identifier type="ms-id">b705672a</identifier><accessCondition type="use and reproduction" contentType="copyright">This journal is © The Royal Society of Chemistry</accessCondition><recordInfo><recordContentSource>RSC Journals</recordContentSource><recordOrigin>The Royal Society of Chemistry</recordOrigin></recordInfo></mods></metadata><annexes><json:item><extension>gif</extension><original>true</original><mimetype>image/gif</mimetype><uri>https://api.istex.fr/document/EE568A42B9A541FB73C5D09C26A9EFFA741CC966/annexes/gif</uri></json:item></annexes><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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