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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Faster, Simpler, More-Specific Methods for Improved Molecular Detection of <italic>Phytophthora ramorum</italic> in the Field<xref ref-type="fn" rid="fn1">▿</xref> </title><author><name sortKey="Tomlinson, J A" sort="Tomlinson, J A" uniqKey="Tomlinson J" first="J. A." last="Tomlinson">J. A. Tomlinson</name><affiliation><nlm:aff id="aff0">Central Science Laboratory, Sand Hutton, York YO41 1LZ, United Kingdom</nlm:aff></affiliation></author><author><name sortKey="Barker, I" sort="Barker, I" uniqKey="Barker I" first="I." last="Barker">I. Barker</name><affiliation><nlm:aff id="aff0">Central Science Laboratory, Sand Hutton, York YO41 1LZ, United Kingdom</nlm:aff></affiliation></author><author><name sortKey="Boonham, N" sort="Boonham, N" uniqKey="Boonham N" first="N." last="Boonham">N. Boonham</name><affiliation><nlm:aff id="aff0">Central Science Laboratory, Sand Hutton, York YO41 1LZ, United Kingdom</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">17449689</idno><idno type="pmc">1932743</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1932743</idno><idno type="RBID">PMC:1932743</idno><idno type="doi">10.1128/AEM.00161-07</idno><date when="2007">2007</date><idno type="wicri:Area/Pmc/Corpus">000085</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000085</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Faster, Simpler, More-Specific Methods for Improved Molecular Detection of <italic>Phytophthora ramorum</italic> in the Field<xref ref-type="fn" rid="fn1">▿</xref> </title><author><name sortKey="Tomlinson, J A" sort="Tomlinson, J A" uniqKey="Tomlinson J" first="J. A." last="Tomlinson">J. A. Tomlinson</name><affiliation><nlm:aff id="aff0">Central Science Laboratory, Sand Hutton, York YO41 1LZ, United Kingdom</nlm:aff></affiliation></author><author><name sortKey="Barker, I" sort="Barker, I" uniqKey="Barker I" first="I." last="Barker">I. Barker</name><affiliation><nlm:aff id="aff0">Central Science Laboratory, Sand Hutton, York YO41 1LZ, United Kingdom</nlm:aff></affiliation></author><author><name sortKey="Boonham, N" sort="Boonham, N" uniqKey="Boonham N" first="N." last="Boonham">N. Boonham</name><affiliation><nlm:aff id="aff0">Central Science Laboratory, Sand Hutton, York YO41 1LZ, United Kingdom</nlm:aff></affiliation></author></analytic><series><title level="j">Applied and Environmental Microbiology</title><idno type="ISSN">0099-2240</idno><idno type="eISSN">1098-5336</idno><imprint><date when="2007">2007</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p><italic>Phytophthora ramorum</italic> is the causal agent of sudden oak death. The pathogen also affects a wide range of tree, shrub, and herbaceous species in natural and landscaped environments as well as plants in the nursery industry. A TaqMan real-time PCR method for the detection of this pathogen in the field has been described previously; this paper describes the development of a number of assays based on this method which have various advantages for use in the field. A scorpion real-time PCR assay that is twice as fast as TaqMan was developed, allowing the detection of <italic>P. ramorum</italic> in less than 30 min. Also designed was a loop-mediated isothermal amplification (LAMP) assay, which allowed sensitive and specific detection of <italic>P. ramorum</italic> in 45 min using only a heated block. A positive reaction was identified by the detection of the LAMP product by color change visible to the naked eye.</p></div></front></TEI><pmc article-type="research-article"><pmc-comment>The publisher of this article does not allow downloading of the full text in XML form.</pmc-comment>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Appl Environ Microbiol</journal-id><journal-id journal-id-type="publisher-id">aem</journal-id><journal-title>Applied and Environmental Microbiology</journal-title><issn pub-type="ppub">0099-2240</issn><issn pub-type="epub">1098-5336</issn><publisher><publisher-name>American Society for Microbiology</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">17449689</article-id><article-id pub-id-type="pmc">1932743</article-id><article-id pub-id-type="publisher-id">0161-07</article-id><article-id pub-id-type="doi">10.1128/AEM.00161-07</article-id><article-categories><subj-group subj-group-type="heading"><subject>Methods</subject></subj-group></article-categories><title-group><article-title>Faster, Simpler, More-Specific Methods for Improved Molecular Detection of <italic>Phytophthora ramorum</italic> in the Field<xref ref-type="fn" rid="fn1">▿</xref> </article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Tomlinson</surname><given-names>J. A.</given-names></name><xref ref-type="aff" rid="aff0"></xref><xref ref-type="corresp" rid="cor1">*</xref></contrib><contrib contrib-type="author"><name><surname>Barker</surname><given-names>I.</given-names></name><xref ref-type="aff" rid="aff0"></xref></contrib><contrib contrib-type="author"><name><surname>Boonham</surname><given-names>N.</given-names></name><xref ref-type="aff" rid="aff0"></xref></contrib></contrib-group><aff id="aff0">Central Science Laboratory, Sand Hutton, York YO41 1LZ, United Kingdom</aff><author-notes><fn id="cor1"><label>*</label><p>Corresponding author. Mailing address: Central Science Laboratory, Sand Hutton, York YO41 1LZ, United Kingdom. Phone: (44) 1904 462000, ext. 3215. Fax: (44) 1904 462111. E-mail: <email>j.tomlinson@csl.gov.uk</email></p></fn></author-notes><pub-date pub-type="ppub"><month>6</month><year>2007</year></pub-date><pub-date pub-type="epub"><day>20</day><month>4</month><year>2007</year></pub-date><volume>73</volume><issue>12</issue><fpage>4040</fpage><lpage>4047</lpage><history><date date-type="received"><day>22</day><month>1</month><year>2007</year></date><date date-type="accepted"><day>13</day><month>4</month><year>2007</year></date></history><copyright-statement>Copyright © 2007, American Society for Microbiology</copyright-statement><copyright-year>2007</copyright-year><self-uri xlink:title="pdf" xlink:href="zam01207004040.pdf"></self-uri><abstract><p><italic>Phytophthora ramorum</italic> is the causal agent of sudden oak death. The pathogen also affects a wide range of tree, shrub, and herbaceous species in natural and landscaped environments as well as plants in the nursery industry. A TaqMan real-time PCR method for the detection of this pathogen in the field has been described previously; this paper describes the development of a number of assays based on this method which have various advantages for use in the field. A scorpion real-time PCR assay that is twice as fast as TaqMan was developed, allowing the detection of <italic>P. ramorum</italic> in less than 30 min. Also designed was a loop-mediated isothermal amplification (LAMP) assay, which allowed sensitive and specific detection of <italic>P. ramorum</italic> in 45 min using only a heated block. A positive reaction was identified by the detection of the LAMP product by color change visible to the naked eye.</p></abstract></article-meta></front><floats-wrap><fig position="float" id="f1"><label>FIG. 1.</label><caption><p>Primer and probe design for TaqMan, scorpion, and molecular beacon assays. Sequence differences between <italic>P. ramorum</italic> and <italic>P. lateralis</italic> are underlined. The positions of the LNA bases in the primers used for the LNA TaqMan are shown in bold. Sequence accession numbers are AY785958 for <italic>P. ramorum</italic> and AY785952 for <italic>P. lateralis</italic>.</p></caption><graphic xlink:href="zam0120778680001"></graphic></fig><fig position="float" id="f2"><label>FIG. 2.</label><caption><p>Primer design for LAMP assay. Sequence differences between <italic>P. ramorum</italic> and <italic>P. lateralis</italic> are shown in bold. Internal primer FIP is composed of the complementary sequence to F1 plus the sequence F2; internal primer BIP is composed of the sequence B1 plus the complementary sequence to B2. The <italic>P. ramorum</italic> sequence accession number is AY785958.</p></caption><graphic xlink:href="zam0120778680002"></graphic></fig><fig position="float" id="f3"><label>FIG. 3.</label><caption><p>Standard curves of <italic>C<sub>T</sub></italic> values for serial dilutions of <italic>P. ramorum</italic> DNA. <italic>C<sub>T</sub></italic> values are mean values for duplicate reactions; error bars show standard deviations. (A) TaqMan real-time PCR. (B) TaqMan real-time PCR with LNA reverse primer. (C) Scorpion real-time PCR. (D) Molecular beacon real-time PCR.</p></caption><graphic xlink:href="zam0120778680003"></graphic></fig><fig position="float" id="f4"><label>FIG. 4.</label><caption><p>LAMP. (A) A dilution series of <italic>P. ramorum</italic> DNA was amplified by LAMP, and the product was visualized by gel electrophoresis. M, marker; lane 1, negative control (water); lanes 2 and 3, 10 ng <italic>P. ramorum</italic> DNA; lanes 4 and 5, 1 ng <italic>P. ramorum</italic> DNA; lanes 6 and 7, 100 pg <italic>P. ramorum</italic> DNA; lanes 8 and 9, 50 pg <italic>P. ramorum</italic> DNA; lanes 10 and 11, 10 pg <italic>P. ramorum</italic> DNA; lanes 12 and 13, 1 pg <italic>P. ramorum</italic> DNA. (B) Cross-reactivity of LAMP assay with <italic>P. lateralis</italic> DNA. M, marker; lane 1, negative control (water); lane 2, positive control (10 ng, <italic>P. ramorum</italic> DNA); lanes 3 and 4, 70 ng <italic>P. lateralis</italic> DNA; lanes 5 and 6, 7 ng <italic>P. lateralis</italic> DNA; lanes 7 and 8, 700 pg <italic>P. lateralis</italic> DNA.</p></caption><graphic xlink:href="zam0120778680004"></graphic></fig><fig position="float" id="f5"><label>FIG. 5.</label><caption><p>LAMP products visualized by adding 2 μl PicoGreen dsDNA reagent at the end of the reaction. The presence of a large amount of LAMP product in positive reaction mixtures causes a color change from orange to yellow. The results shown are for the same reaction mixtures as in Fig. <xref ref-type="fig" rid="f4">4A</xref>. The amount of <italic>Phytophthora ramorum</italic> DNA added to each reaction mixture is indicated for each tube.</p></caption><graphic xlink:href="zam0120778680005"></graphic></fig><fig position="float" id="f6"><label>FIG. 6.</label><caption><p>Real-time monitoring of LAMP reactions. (A) Amplification plots for LAMP amplification of a dilution series of <italic>P. ramorum</italic> DNA. (B) Standard curve of mean Tp values. Tp values are mean values for duplicate reactions; error bars show standard deviations.</p></caption><graphic xlink:href="zam0120778680006"></graphic></fig><fig position="float" id="f7"><label>FIG. 7.</label><caption><p>Speed of real-time PCR assays. Amplification plots showing fluorescence versus time in minutes for the detection of 10 ng <italic>P. ramorum</italic> DNA using TaqMan, molecular beacon, and scorpion real-time PCR.</p></caption><graphic xlink:href="zam0120778680007"></graphic></fig><table-wrap position="float" id="t1"><label>TABLE 1.</label><caption><p>Characteristics of primers and probes used for real-time PCR<xref ref-type="table-fn" rid="t1fn1"><italic>a</italic></xref></p></caption><table frame="hsides" rules="groups"><thead><tr><th colspan="1" rowspan="1" align="center" valign="bottom">Primer/probe</th><th colspan="1" rowspan="1" align="center" valign="bottom">Sequence (5′-3′)</th><th colspan="1" rowspan="1" align="center" valign="bottom">Reporter (5′)</th><th colspan="1" rowspan="1" align="center" valign="bottom">Quencher (3′)</th><th colspan="1" rowspan="1" align="center" valign="bottom">Final concn (μM)</th></tr></thead><tbody><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Pram</italic>-114F</td><td colspan="1" rowspan="1" align="left" valign="top">TCATGGCGAGCGCTTGA</td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">0.3</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Pram</italic>-190R</td><td colspan="1" rowspan="1" align="left" valign="top">AGTATATTCAGTATTTAGGAATGGGTTTAAAAAGT</td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">0.3, 0.7<xref ref-type="table-fn" rid="t1fn5"><italic>e</italic></xref></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Pram</italic>-199R</td><td colspan="1" rowspan="1" align="left" valign="top">CAGAGACTTTCGTCCCCACAGTA</td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">0.3</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Pram</italic>-114F-LNA3</td><td colspan="1" rowspan="1" align="left" valign="top">AGTATATTCAGTATTTAGGAATGGGTTTAAAAAG<bold>T</bold><xref ref-type="table-fn" rid="t1fn2"><italic>b</italic></xref></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">0.3</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Pram</italic>-190R-LNA1</td><td colspan="1" rowspan="1" align="left" valign="top">TCATGGCGAGCGCTTG<bold>A</bold><xref ref-type="table-fn" rid="t1fn2"><italic>b</italic></xref></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">0.3</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Pram</italic> TaqMan probe</td><td colspan="1" rowspan="1" align="left" valign="top">TTCGGGTCTGAGCTAGTAG</td><td colspan="1" rowspan="1" align="left" valign="top">FAM</td><td colspan="1" rowspan="1" align="left" valign="top">BHQ1</td><td colspan="1" rowspan="1" align="char" char="." valign="top">0.1</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Pram</italic> scorpion</td><td colspan="1" rowspan="1" align="left" valign="top"><bold>CTACTAGCTCAGACCCGAA</bold>[HEG]TCATGGCGAGCGCTTGA<xref ref-type="table-fn" rid="t1fn3"><italic>c</italic></xref></td><td colspan="1" rowspan="1" align="left" valign="top">FAM</td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">0.4</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Pram</italic> quencher</td><td colspan="1" rowspan="1" align="left" valign="top">TTCGGGTCTGAGCTAGTAG</td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top">DDQ</td><td colspan="1" rowspan="1" align="char" char="." valign="top">1.6</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Pram</italic> molecular beacon</td><td colspan="1" rowspan="1" align="left" valign="top"><bold>CGCGGCG</bold>TGAGCTAGTAGCCCACTTTTTAAACCCATTCCT<bold>CGCCGCG</bold><xref ref-type="table-fn" rid="t1fn4"><italic>d</italic></xref></td><td colspan="1" rowspan="1" align="left" valign="top">FAM</td><td colspan="1" rowspan="1" align="left" valign="top">Dabcyl</td><td colspan="1" rowspan="1" align="char" char="." valign="top">0.3</td></tr></tbody></table><table-wrap-foot><fn id="t1fn1"><label>a</label><p>FAM, 6-carboxyfluorescein; BHQ1, black hole quencher 1 (Biosearch Technologies, Novato, CA); DDQ, deep dark quencher 1 (Eurogentec, Seraing, Belgium); Dabcyl, 4-(4′-dimethylaminophenylazo)benzoic acid.</p></fn><fn id="t1fn2"><label>b</label><p>LNA bases are shown in bold.</p></fn><fn id="t1fn3"><label>c</label><p>The Scorpion primer consists of a probe sequence (complementary to the target sequence) (shown in bold) plus a primer sequence separated by a hexaethylene glycol (HEG) PCR blocker.</p></fn><fn id="t1fn4"><label>d</label><p>Molecular Beacon arm sequences are shown in bold.</p></fn><fn id="t1fn5"><label>e</label><p>The final concentration was 0.3 with the TaqMan assay and 0.7 with the scorpion assay.</p></fn></table-wrap-foot></table-wrap><table-wrap position="float" id="t2"><label>TABLE 2.</label><caption><p><italic>Phytophthora</italic> sp. isolates used for specificity testing</p></caption><table frame="hsides" rules="groups"><thead><tr><th colspan="1" rowspan="1" align="center" valign="bottom"><italic>Phytophthora</italic> sp.</th><th colspan="1" rowspan="1" align="center" valign="bottom">Isolate</th></tr></thead><tbody><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>P. boehmeriae</italic></td><td colspan="1" rowspan="1" align="left" valign="top">P 6950<xref ref-type="table-fn" rid="t2fn1"><italic>a</italic></xref></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>P. botryosa</italic></td><td colspan="1" rowspan="1" align="left" valign="top">P 6945<xref ref-type="table-fn" rid="t2fn1"><italic>a</italic></xref></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>P. cactorum</italic></td><td colspan="1" rowspan="1" align="left" valign="top">CSL 2151<xref ref-type="table-fn" rid="t2fn3"><italic>c</italic></xref></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>P. cambivora</italic></td><td colspan="1" rowspan="1" align="left" valign="top">CBS 376.61<xref ref-type="table-fn" rid="t2fn2"><italic>b</italic></xref></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>P. cinnamomi</italic></td><td colspan="1" rowspan="1" align="left" valign="top">SCRI CIN5<xref ref-type="table-fn" rid="t2fn4"><italic>d</italic></xref></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>P. citricola</italic></td><td colspan="1" rowspan="1" align="left" valign="top">SCRI CIT1<xref ref-type="table-fn" rid="t2fn4"><italic>d</italic></xref></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>P. citrophthora</italic></td><td colspan="1" rowspan="1" align="left" valign="top">IMI 132217<xref ref-type="table-fn" rid="t2fn5"><italic>e</italic></xref></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>P. cryptogea</italic></td><td colspan="1" rowspan="1" align="left" valign="top">SCRI P521<xref ref-type="table-fn" rid="t2fn4"><italic>d</italic></xref></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>P. erythroseptica</italic></td><td colspan="1" rowspan="1" align="left" valign="top">P 7889<xref ref-type="table-fn" rid="t2fn1"><italic>a</italic></xref></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>P. europaea</italic></td><td colspan="1" rowspan="1" align="left" valign="top">CBS 109053<xref ref-type="table-fn" rid="t2fn2"><italic>b</italic></xref></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>P. fragariae</italic> var. <italic>rubi</italic></td><td colspan="1" rowspan="1" align="left" valign="top">FR-163<xref ref-type="table-fn" rid="t2fn6"><italic>f</italic></xref></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>P. gonopodyides</italic></td><td colspan="1" rowspan="1" align="left" valign="top">P 10337<xref ref-type="table-fn" rid="t2fn1"><italic>a</italic></xref></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>P. heveae</italic></td><td colspan="1" rowspan="1" align="left" valign="top">CBS 958.87<xref ref-type="table-fn" rid="t2fn2"><italic>b</italic></xref></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>P. hibernalis</italic></td><td colspan="1" rowspan="1" align="left" valign="top">P 3822<xref ref-type="table-fn" rid="t2fn1"><italic>a</italic></xref></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>P. ilicis</italic></td><td colspan="1" rowspan="1" align="left" valign="top">P 3939<xref ref-type="table-fn" rid="t2fn1"><italic>a</italic></xref></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>P. insolita</italic></td><td colspan="1" rowspan="1" align="left" valign="top">P 6195<xref ref-type="table-fn" rid="t2fn1"><italic>a</italic></xref></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>P. kernoviae</italic></td><td colspan="1" rowspan="1" align="left" valign="top">CSL 2169<xref ref-type="table-fn" rid="t2fn3"><italic>c</italic></xref></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>P. lateralis</italic></td><td colspan="1" rowspan="1" align="left" valign="top">P 1728<xref ref-type="table-fn" rid="t2fn1"><italic>a</italic></xref></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>P. macrochlamydospora</italic></td><td colspan="1" rowspan="1" align="left" valign="top">P 10263<xref ref-type="table-fn" rid="t2fn1"><italic>a</italic></xref></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>P. megasperma</italic></td><td colspan="1" rowspan="1" align="left" valign="top">CBS 320.49<xref ref-type="table-fn" rid="t2fn2"><italic>b</italic></xref></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>P. nemorosa</italic></td><td colspan="1" rowspan="1" align="left" valign="top">P 10288<xref ref-type="table-fn" rid="t2fn1"><italic>a</italic></xref></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>P. nicotianae</italic></td><td colspan="1" rowspan="1" align="left" valign="top">CBS 411.87<xref ref-type="table-fn" rid="t2fn2"><italic>b</italic></xref></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>P. palmivora</italic></td><td colspan="1" rowspan="1" align="left" valign="top">SCRI P488<xref ref-type="table-fn" rid="t2fn4"><italic>d</italic></xref></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>P. pseudosyringae</italic></td><td colspan="1" rowspan="1" align="left" valign="top">P 10444<xref ref-type="table-fn" rid="t2fn1"><italic>a</italic></xref></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>P. quercina</italic></td><td colspan="1" rowspan="1" align="left" valign="top">P 10334<xref ref-type="table-fn" rid="t2fn1"><italic>a</italic></xref></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>P. richardiae</italic></td><td colspan="1" rowspan="1" align="left" valign="top">P 7788<xref ref-type="table-fn" rid="t2fn1"><italic>a</italic></xref></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>P. syringae</italic></td><td colspan="1" rowspan="1" align="left" valign="top">4N0247-6<xref ref-type="table-fn" rid="t2fn6"><italic>f</italic></xref></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>P. uliginosa</italic></td><td colspan="1" rowspan="1" align="left" valign="top">CBS 109055<xref ref-type="table-fn" rid="t2fn2"><italic>b</italic></xref></td></tr></tbody></table><table-wrap-foot><fn id="t2fn1"><label>a</label><p>Provided by Michael Coffey, University of California.</p></fn><fn id="t2fn2"><label>b</label><p>Provided by Centraalbureau voor Schimmelcultures, Utrech, The Netherlands.</p></fn><fn id="t2fn3"><label>c</label><p>Provided by Central Science Laboratory, York, United Kingdom.</p></fn><fn id="t2fn4"><label>d</label><p>Provided by David Cooke, Scottish Crop Research Institute Invergowrie, Dundee, United Kingdom.</p></fn><fn id="t2fn5"><label>e</label><p>Provided by CABI Bioscience, Egham, United Kingdom.</p></fn><fn id="t2fn6"><label>f</label><p>Provided by Laboratoire National de la Protection des Vegataux, Nancy, France.</p></fn></table-wrap-foot></table-wrap><table-wrap position="float" id="t3"><label>TABLE 3.</label><caption><p>Effect of changing TaqMan real-time PCR cycling conditions on <italic>C<sub>T</sub></italic> values for the detection of <italic>P. ramorum</italic> DNA<xref ref-type="table-fn" rid="t3fn1"><italic>a</italic></xref></p></caption><table frame="hsides" rules="groups"><thead><tr><th colspan="1" rowspan="1" align="center" valign="bottom">Cycling conditions</th><th colspan="1" rowspan="1" align="center" valign="bottom">Approximate run time for 40 cycles (min)</th><th colspan="1" rowspan="1" align="center" valign="bottom">Mean <italic>C<sub>T</sub></italic> value</th><th colspan="1" rowspan="1" align="center" valign="bottom">Approximate time to positive result (min)</th></tr></thead><tbody><tr><td colspan="1" rowspan="1" align="left" valign="top">15 s denature, 60 s anneal</td><td colspan="1" rowspan="1" align="char" char="." valign="top">70</td><td colspan="1" rowspan="1" align="char" char="." valign="top">19.37</td><td colspan="1" rowspan="1" align="char" char="." valign="top">39</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">5 s denature, 60 s anneal</td><td colspan="1" rowspan="1" align="char" char="." valign="top">63</td><td colspan="1" rowspan="1" align="char" char="." valign="top">19.93</td><td colspan="1" rowspan="1" align="char" char="." valign="top">37</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">5 s denature, 30 s anneal</td><td colspan="1" rowspan="1" align="char" char="." valign="top">43</td><td colspan="1" rowspan="1" align="char" char="." valign="top">24.03</td><td colspan="1" rowspan="1" align="char" char="." valign="top">30</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">5 s denature, 25 s anneal</td><td colspan="1" rowspan="1" align="char" char="." valign="top">40</td><td colspan="1" rowspan="1" align="char" char="." valign="top">26.98</td><td colspan="1" rowspan="1" align="char" char="." valign="top">30</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">5 s denature, 20 s anneal</td><td colspan="1" rowspan="1" align="char" char="." valign="top">37</td><td colspan="1" rowspan="1" align="char" char="." valign="top">29.38</td><td colspan="1" rowspan="1" align="char" char="." valign="top">30</td></tr></tbody></table><table-wrap-foot><fn id="t3fn1"><label>a</label><p>Approximately 10 ng DNA per reaction mixture.</p></fn></table-wrap-foot></table-wrap></floats-wrap></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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