Predicting the sensitivity and specificity of published real-time PCR assays
Identifieur interne : 000486 ( Pmc/Curation ); précédent : 000485; suivant : 000487Predicting the sensitivity and specificity of published real-time PCR assays
Auteurs : Gordon H. Lemmon [États-Unis] ; Shea N. Gardner [États-Unis]Source :
- Annals of Clinical Microbiology and Antimicrobials [ 1476-0711 ] ; 2008.
Abstract
In recent years real-time PCR has become a leading technique for nucleic acid detection and quantification. These assays have the potential to greatly enhance efficiency in the clinical laboratory. Choice of primer and probe sequences is critical for accurate diagnosis in the clinic, yet current primer/probe signature design strategies are limited, and signature evaluation methods are lacking.
We assessed the quality of a signature by predicting the number of true positive, false positive and false negative hits against all available public sequence data. We found real-time PCR signatures described in recent literature and used a BLAST search based approach to collect all hits to the primer-probe combinations that should be amplified by real-time PCR chemistry. We then compared our hits with the sequences in the NCBI taxonomy tree that the signature was designed to detect.
We found that many published signatures have high specificity (almost no false positives) but low sensitivity (high false negative rate). Where high sensitivity is needed, we offer a revised methodology for signature design which may designate that multiple signatures are required to detect all sequenced strains. We use this methodology to produce new signatures that are predicted to have higher sensitivity and specificity.
We show that current methods for real-time PCR assay design have unacceptably low sensitivities for most clinical applications. Additionally, as new sequence data becomes available, old assays must be reassessed and redesigned. A standard protocol for both generating and assessing the quality of these assays is therefore of great value. Real-time PCR has the capacity to greatly improve clinical diagnostics. The improved assay design and evaluation methods presented herein will expedite adoption of this technique in the clinical lab.
Url:
DOI: 10.1186/1476-0711-7-18
PubMed: 18817537
PubMed Central: 2566554
Links toward previous steps (curation, corpus...)
- to stream Pmc, to step Corpus: Pour aller vers cette notice dans l'étape Curation :000486
Links to Exploration step
PMC:2566554Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Predicting the sensitivity and specificity of published real-time PCR assays</title>
<author><name sortKey="Lemmon, Gordon H" sort="Lemmon, Gordon H" uniqKey="Lemmon G" first="Gordon H" last="Lemmon">Gordon H. Lemmon</name>
<affiliation wicri:level="1"><nlm:aff id="I1">Center for Structural Biology, Vanderbilt University, 465 21st Ave. South, BIOSCI/MRB III suite 5140, Nashville, TN 37240, USA</nlm:aff>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>Center for Structural Biology, Vanderbilt University, 465 21st Ave. South, BIOSCI/MRB III suite 5140, Nashville, TN 37240</wicri:regionArea>
</affiliation>
</author>
<author><name sortKey="Gardner, Shea N" sort="Gardner, Shea N" uniqKey="Gardner S" first="Shea N" last="Gardner">Shea N. Gardner</name>
<affiliation wicri:level="1"><nlm:aff id="I2">Pathogen Bioinformatics, Lawrence Livermore National Laboratory, PO Box 808, L-174, Livermore, CA 94551, USA</nlm:aff>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>Pathogen Bioinformatics, Lawrence Livermore National Laboratory, PO Box 808, L-174, Livermore, CA 94551</wicri:regionArea>
</affiliation>
</author>
</titleStmt>
<publicationStmt><idno type="wicri:source">PMC</idno>
<idno type="pmid">18817537</idno>
<idno type="pmc">2566554</idno>
<idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2566554</idno>
<idno type="RBID">PMC:2566554</idno>
<idno type="doi">10.1186/1476-0711-7-18</idno>
<date when="2008">2008</date>
<idno type="wicri:Area/Pmc/Corpus">000486</idno>
<idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000486</idno>
<idno type="wicri:Area/Pmc/Curation">000486</idno>
<idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Curation">000486</idno>
</publicationStmt>
<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Predicting the sensitivity and specificity of published real-time PCR assays</title>
<author><name sortKey="Lemmon, Gordon H" sort="Lemmon, Gordon H" uniqKey="Lemmon G" first="Gordon H" last="Lemmon">Gordon H. Lemmon</name>
<affiliation wicri:level="1"><nlm:aff id="I1">Center for Structural Biology, Vanderbilt University, 465 21st Ave. South, BIOSCI/MRB III suite 5140, Nashville, TN 37240, USA</nlm:aff>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>Center for Structural Biology, Vanderbilt University, 465 21st Ave. South, BIOSCI/MRB III suite 5140, Nashville, TN 37240</wicri:regionArea>
</affiliation>
</author>
<author><name sortKey="Gardner, Shea N" sort="Gardner, Shea N" uniqKey="Gardner S" first="Shea N" last="Gardner">Shea N. Gardner</name>
<affiliation wicri:level="1"><nlm:aff id="I2">Pathogen Bioinformatics, Lawrence Livermore National Laboratory, PO Box 808, L-174, Livermore, CA 94551, USA</nlm:aff>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>Pathogen Bioinformatics, Lawrence Livermore National Laboratory, PO Box 808, L-174, Livermore, CA 94551</wicri:regionArea>
</affiliation>
</author>
</analytic>
<series><title level="j">Annals of Clinical Microbiology and Antimicrobials</title>
<idno type="eISSN">1476-0711</idno>
<imprint><date when="2008">2008</date>
</imprint>
</series>
</biblStruct>
</sourceDesc>
</fileDesc>
<profileDesc><textClass></textClass>
</profileDesc>
</teiHeader>
<front><div type="abstract" xml:lang="en"><sec><title>Background</title>
<p>In recent years real-time PCR has become a leading technique for nucleic acid detection and quantification. These assays have the potential to greatly enhance efficiency in the clinical laboratory. Choice of primer and probe sequences is critical for accurate diagnosis in the clinic, yet current primer/probe signature design strategies are limited, and signature evaluation methods are lacking.</p>
</sec>
<sec sec-type="methods"><title>Methods</title>
<p>We assessed the quality of a signature by predicting the number of true positive, false positive and false negative hits against all available public sequence data. We found real-time PCR signatures described in recent literature and used a BLAST search based approach to collect all hits to the primer-probe combinations that should be amplified by real-time PCR chemistry. We then compared our hits with the sequences in the NCBI taxonomy tree that the signature was designed to detect.</p>
</sec>
<sec><title>Results</title>
<p>We found that many published signatures have high specificity (almost no false positives) but low sensitivity (high false negative rate). Where high sensitivity is needed, we offer a revised methodology for signature design which may designate that multiple signatures are required to detect all sequenced strains. We use this methodology to produce new signatures that are predicted to have higher sensitivity and specificity.</p>
</sec>
<sec><title>Conclusion</title>
<p>We show that current methods for real-time PCR assay design have unacceptably low sensitivities for most clinical applications. Additionally, as new sequence data becomes available, old assays must be reassessed and redesigned. A standard protocol for both generating and assessing the quality of these assays is therefore of great value. Real-time PCR has the capacity to greatly improve clinical diagnostics. The improved assay design and evaluation methods presented herein will expedite adoption of this technique in the clinical lab.</p>
</sec>
</div>
</front>
<back><div1 type="bibliography"><listBibl><biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct></biblStruct>
</listBibl>
</div1>
</back>
</TEI>
<pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Ann Clin Microbiol Antimicrob</journal-id>
<journal-title>Annals of Clinical Microbiology and Antimicrobials</journal-title>
<issn pub-type="epub">1476-0711</issn>
<publisher><publisher-name>BioMed Central</publisher-name>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">18817537</article-id>
<article-id pub-id-type="pmc">2566554</article-id>
<article-id pub-id-type="publisher-id">1476-0711-7-18</article-id>
<article-id pub-id-type="doi">10.1186/1476-0711-7-18</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Research</subject>
</subj-group>
</article-categories>
<title-group><article-title>Predicting the sensitivity and specificity of published real-time PCR assays</article-title>
</title-group>
<contrib-group><contrib id="A1" contrib-type="author"><name><surname>Lemmon</surname>
<given-names>Gordon H</given-names>
</name>
<xref ref-type="aff" rid="I1">1</xref>
<email>gordon.lemmon@vanderbilt.edu</email>
</contrib>
<contrib id="A2" corresp="yes" contrib-type="author"><name><surname>Gardner</surname>
<given-names>Shea N</given-names>
</name>
<xref ref-type="aff" rid="I2">2</xref>
<email>gardner26@LLNL.gov</email>
</contrib>
</contrib-group>
<aff id="I1"><label>1</label>
Center for Structural Biology, Vanderbilt University, 465 21st Ave. South, BIOSCI/MRB III suite 5140, Nashville, TN 37240, USA</aff>
<aff id="I2"><label>2</label>
Pathogen Bioinformatics, Lawrence Livermore National Laboratory, PO Box 808, L-174, Livermore, CA 94551, USA</aff>
<pub-date pub-type="collection"><year>2008</year>
</pub-date>
<pub-date pub-type="epub"><day>25</day>
<month>9</month>
<year>2008</year>
</pub-date>
<volume>7</volume>
<fpage>18</fpage>
<lpage>18</lpage>
<ext-link ext-link-type="uri" xlink:href="http://www.ann-clinmicrob.com/content/7/1/18"></ext-link>
<history><date date-type="received"><day>17</day>
<month>6</month>
<year>2008</year>
</date>
<date date-type="accepted"><day>25</day>
<month>9</month>
<year>2008</year>
</date>
</history>
<permissions><copyright-statement>Copyright © 2008 Lemmon and Gardner; licensee BioMed Central Ltd.</copyright-statement>
<copyright-year>2008</copyright-year>
<copyright-holder>Lemmon and Gardner; licensee BioMed Central Ltd.</copyright-holder>
<license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/2.0"><p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/2.0"></ext-link>
), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>
<pmc-comment>
Lemmon
H
Gordon
gordon.lemmon@vanderbilt.edu
Predicting the sensitivity and specificity of published real-time PCR assays
2008 Annals of Clinical Microbiology and Antimicrobials 7(1): 18-. (2008) 1476-0711(2008)7:1<18> urn:ISSN:1476-0711 </pmc-comment>
</license>
</permissions>
<abstract><sec><title>Background</title>
<p>In recent years real-time PCR has become a leading technique for nucleic acid detection and quantification. These assays have the potential to greatly enhance efficiency in the clinical laboratory. Choice of primer and probe sequences is critical for accurate diagnosis in the clinic, yet current primer/probe signature design strategies are limited, and signature evaluation methods are lacking.</p>
</sec>
<sec sec-type="methods"><title>Methods</title>
<p>We assessed the quality of a signature by predicting the number of true positive, false positive and false negative hits against all available public sequence data. We found real-time PCR signatures described in recent literature and used a BLAST search based approach to collect all hits to the primer-probe combinations that should be amplified by real-time PCR chemistry. We then compared our hits with the sequences in the NCBI taxonomy tree that the signature was designed to detect.</p>
</sec>
<sec><title>Results</title>
<p>We found that many published signatures have high specificity (almost no false positives) but low sensitivity (high false negative rate). Where high sensitivity is needed, we offer a revised methodology for signature design which may designate that multiple signatures are required to detect all sequenced strains. We use this methodology to produce new signatures that are predicted to have higher sensitivity and specificity.</p>
</sec>
<sec><title>Conclusion</title>
<p>We show that current methods for real-time PCR assay design have unacceptably low sensitivities for most clinical applications. Additionally, as new sequence data becomes available, old assays must be reassessed and redesigned. A standard protocol for both generating and assessing the quality of these assays is therefore of great value. Real-time PCR has the capacity to greatly improve clinical diagnostics. The improved assay design and evaluation methods presented herein will expedite adoption of this technique in the clinical lab.</p>
</sec>
</abstract>
</article-meta>
</front>
</pmc>
</record>
Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Wicri/Sante/explor/CovidV2/Data/Pmc/Curation
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 000486 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Pmc/Curation/biblio.hfd -nk 000486 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien |wiki= Wicri/Sante |area= CovidV2 |flux= Pmc |étape= Curation |type= RBID |clé= PMC:2566554 |texte= Predicting the sensitivity and specificity of published real-time PCR assays }}
Pour générer des pages wiki
HfdIndexSelect -h $EXPLOR_AREA/Data/Pmc/Curation/RBID.i -Sk "pubmed:18817537" \ | HfdSelect -Kh $EXPLOR_AREA/Data/Pmc/Curation/biblio.hfd \ | NlmPubMed2Wicri -a CovidV2
![]() | This area was generated with Dilib version V0.6.33. | ![]() |