High-Throughput Purification of Viral RNA Based on Novel Aqueous Chemistry for Nucleic Acid Isolation
Extraction protocols using magnetic solid phases offer a high potential for automation. However, commercially available magnetic-bead-based assays either lack the sensitivity required for viral diagnostics or are disproportionately expensive. We developed an aqueous chemistry for extraction of viral...
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| Veröffentlicht in: | Clinical chemistry (Baltimore, Md.) Md.), 2005-07, Vol.51 (7), p.1217-1222 |
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| creator | Hourfar, Michael K Michelsen, Uwe Schmidt, Michael Berger, Annemarie Seifried, Erhard Roth, W. Kurt |
| description | Extraction protocols using magnetic solid phases offer a high potential for automation. However, commercially available magnetic-bead-based assays either lack the sensitivity required for viral diagnostics or are disproportionately expensive.
We developed an aqueous chemistry for extraction of viral nucleic acids from plasma samples by use of common magnetic silica beads. Nucleic acids were bound to the beads at acidic conditions in the presence of a kosmotropic salt and were eluted at a slightly alkaline pH. The method was implemented on a standard pipetting workstation for fully automated extraction of up to 48 samples of 240 muL plasma in 1 batch.
The detection limit of the method was comparable to the spin-column-based QIAamp Viral RNA Mini Kit, which relies on chaotropic salts and binding to a silica membrane, as the comparison method. The 95% detection limit was 23.1 IU per PCR for HIV-1 and 10.7 IU per PCR for hepatitis C virus (HCV). Suitability for clinical routine testing was confirmed in a total of 178 HIV-1- or HCV-positive plasma samples. The method linearity (R(2)) was >0.99 for the viruses evaluated.
Use of reagents without organic solvents allows simple and cost-effective automation of this method on common pipetting robots with low risk of contamination. Performance characteristics of the novel extraction method make it suitable for use in diagnosis of infectious diseases and viral load determinations. |
| doi_str_mv | 10.1373/clinchem.2005.048603 |
| format | Article |
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We developed an aqueous chemistry for extraction of viral nucleic acids from plasma samples by use of common magnetic silica beads. Nucleic acids were bound to the beads at acidic conditions in the presence of a kosmotropic salt and were eluted at a slightly alkaline pH. The method was implemented on a standard pipetting workstation for fully automated extraction of up to 48 samples of 240 muL plasma in 1 batch.
The detection limit of the method was comparable to the spin-column-based QIAamp Viral RNA Mini Kit, which relies on chaotropic salts and binding to a silica membrane, as the comparison method. The 95% detection limit was 23.1 IU per PCR for HIV-1 and 10.7 IU per PCR for hepatitis C virus (HCV). Suitability for clinical routine testing was confirmed in a total of 178 HIV-1- or HCV-positive plasma samples. The method linearity (R(2)) was >0.99 for the viruses evaluated.
Use of reagents without organic solvents allows simple and cost-effective automation of this method on common pipetting robots with low risk of contamination. Performance characteristics of the novel extraction method make it suitable for use in diagnosis of infectious diseases and viral load determinations.</description><identifier>ISSN: 0009-9147</identifier><identifier>EISSN: 1530-8561</identifier><identifier>DOI: 10.1373/clinchem.2005.048603</identifier><identifier>PMID: 15976102</identifier><identifier>CODEN: CLCHAU</identifier><language>eng</language><publisher>Washington, DC: Am Assoc Clin Chem</publisher><subject>Analytical, structural and metabolic biochemistry ; Autoanalysis ; Biological and medical sciences ; Fundamental and applied biological sciences. Psychology ; Hepacivirus - genetics ; HIV-1 - genetics ; Humans ; Hydrogen-Ion Concentration ; Infectious diseases ; Investigative techniques, diagnostic techniques (general aspects) ; Magnetics ; Medical sciences ; Membranes, Artificial ; Nucleic acids ; Organic solvents ; Polymerase Chain Reaction - methods ; Reagent Kits, Diagnostic ; Reagents ; RNA, Viral - isolation & purification ; Robotics ; Salts ; Sensitivity and Specificity ; Silica ; Silicon Dioxide ; Water - chemistry</subject><ispartof>Clinical chemistry (Baltimore, Md.), 2005-07, Vol.51 (7), p.1217-1222</ispartof><rights>2005 INIST-CNRS</rights><rights>Copyright American Association for Clinical Chemistry Jul 2005</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c501t-d7708d9b0f79a19a0351a256f3949bede4c468c85c521a9740410ad9695994c33</citedby><cites>FETCH-LOGICAL-c501t-d7708d9b0f79a19a0351a256f3949bede4c468c85c521a9740410ad9695994c33</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=16905481$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/15976102$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Hourfar, Michael K</creatorcontrib><creatorcontrib>Michelsen, Uwe</creatorcontrib><creatorcontrib>Schmidt, Michael</creatorcontrib><creatorcontrib>Berger, Annemarie</creatorcontrib><creatorcontrib>Seifried, Erhard</creatorcontrib><creatorcontrib>Roth, W. Kurt</creatorcontrib><title>High-Throughput Purification of Viral RNA Based on Novel Aqueous Chemistry for Nucleic Acid Isolation</title><title>Clinical chemistry (Baltimore, Md.)</title><addtitle>Clin Chem</addtitle><description>Extraction protocols using magnetic solid phases offer a high potential for automation. However, commercially available magnetic-bead-based assays either lack the sensitivity required for viral diagnostics or are disproportionately expensive.
We developed an aqueous chemistry for extraction of viral nucleic acids from plasma samples by use of common magnetic silica beads. Nucleic acids were bound to the beads at acidic conditions in the presence of a kosmotropic salt and were eluted at a slightly alkaline pH. The method was implemented on a standard pipetting workstation for fully automated extraction of up to 48 samples of 240 muL plasma in 1 batch.
The detection limit of the method was comparable to the spin-column-based QIAamp Viral RNA Mini Kit, which relies on chaotropic salts and binding to a silica membrane, as the comparison method. The 95% detection limit was 23.1 IU per PCR for HIV-1 and 10.7 IU per PCR for hepatitis C virus (HCV). Suitability for clinical routine testing was confirmed in a total of 178 HIV-1- or HCV-positive plasma samples. The method linearity (R(2)) was >0.99 for the viruses evaluated.
Use of reagents without organic solvents allows simple and cost-effective automation of this method on common pipetting robots with low risk of contamination. Performance characteristics of the novel extraction method make it suitable for use in diagnosis of infectious diseases and viral load determinations.</description><subject>Analytical, structural and metabolic biochemistry</subject><subject>Autoanalysis</subject><subject>Biological and medical sciences</subject><subject>Fundamental and applied biological sciences. 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Kurt</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>High-Throughput Purification of Viral RNA Based on Novel Aqueous Chemistry for Nucleic Acid Isolation</atitle><jtitle>Clinical chemistry (Baltimore, Md.)</jtitle><addtitle>Clin Chem</addtitle><date>2005-07-01</date><risdate>2005</risdate><volume>51</volume><issue>7</issue><spage>1217</spage><epage>1222</epage><pages>1217-1222</pages><issn>0009-9147</issn><eissn>1530-8561</eissn><coden>CLCHAU</coden><abstract>Extraction protocols using magnetic solid phases offer a high potential for automation. However, commercially available magnetic-bead-based assays either lack the sensitivity required for viral diagnostics or are disproportionately expensive.
We developed an aqueous chemistry for extraction of viral nucleic acids from plasma samples by use of common magnetic silica beads. Nucleic acids were bound to the beads at acidic conditions in the presence of a kosmotropic salt and were eluted at a slightly alkaline pH. The method was implemented on a standard pipetting workstation for fully automated extraction of up to 48 samples of 240 muL plasma in 1 batch.
The detection limit of the method was comparable to the spin-column-based QIAamp Viral RNA Mini Kit, which relies on chaotropic salts and binding to a silica membrane, as the comparison method. The 95% detection limit was 23.1 IU per PCR for HIV-1 and 10.7 IU per PCR for hepatitis C virus (HCV). Suitability for clinical routine testing was confirmed in a total of 178 HIV-1- or HCV-positive plasma samples. The method linearity (R(2)) was >0.99 for the viruses evaluated.
Use of reagents without organic solvents allows simple and cost-effective automation of this method on common pipetting robots with low risk of contamination. Performance characteristics of the novel extraction method make it suitable for use in diagnosis of infectious diseases and viral load determinations.</abstract><cop>Washington, DC</cop><pub>Am Assoc Clin Chem</pub><pmid>15976102</pmid><doi>10.1373/clinchem.2005.048603</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Analytical, structural and metabolic biochemistry Autoanalysis Biological and medical sciences Fundamental and applied biological sciences. Psychology Hepacivirus - genetics HIV-1 - genetics Humans Hydrogen-Ion Concentration Infectious diseases Investigative techniques, diagnostic techniques (general aspects) Magnetics Medical sciences Membranes, Artificial Nucleic acids Organic solvents Polymerase Chain Reaction - methods Reagent Kits, Diagnostic Reagents RNA, Viral - isolation & purification Robotics Salts Sensitivity and Specificity Silica Silicon Dioxide Water - chemistry |
| title | High-Throughput Purification of Viral RNA Based on Novel Aqueous Chemistry for Nucleic Acid Isolation |
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