Duplex real-time quantitative PCR for simultaneous detection and quantification of Pratylenchus neglectus and P. thornei
Pratylenchus neglectus and P. thornei are economically important migratory plant endoparasitic nematodes and are widely found in a wide variety of crops. Based on 28 s rDNA D2D3 expansion region, a duplex real-time quantitative (qPCR) assay was developed to simultaneously detect and quantify these t...
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description | Pratylenchus neglectus
and
P. thornei
are economically important migratory plant endoparasitic nematodes and are widely found in a wide variety of crops. Based on 28 s rDNA D2D3 expansion region, a duplex real-time quantitative (qPCR) assay was developed to simultaneously detect and quantify these two nematodes. The qPCR system included two pairs of primers and two Taqman probes, that is the
P. neglectus
-specific primer pair 28sPnF5/28sPnR7 and probe pntaq3 and the
P. thornei
-specific primer pair 28sPtF1/28sPtR1 and probe pttaq3. The optimal conditions of the qPCR assay were 20 μL reaction volume comprising 10 μL THUNDERBIRD Probe qPCR Mix, 0.2 μM of primer 28sPnF5 and 28sPnR7, 0.25 μM of primer 28sPtF1and 28sPtR1, 0.1 μM of probe pntaq3 and 0.08 μM of probe pttaq3, 1 μL template DNA. And the amplification program were 95 °C for 2 min, 40 cycles of 95 °C for 10s, 65 °C for 25 s. The specificity of the qPCR assay was confirmed by the lack of fluorescence signal from non-target nematode populations including seven other
Pratylenchus
species and five other nematode species. The assay was very sensitive as it can detect a single target nematode and a single target nematode mixed with up to 100 individuals of non-target nematodes. A dilution series from
P. neglectus
and
P. thornei
DNAs resulted in two standard curves respectively showing a highly significant linearity between the Cycle threshold (
Ct
) values and the dilution rates. This study is the first to provide a molecular tool for simultaneous detection and quantification of
P. neglectus
and
P. thornei. |
doi_str_mv | 10.1007/s10658-020-01999-7 |
format | Article |
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and
P. thornei
are economically important migratory plant endoparasitic nematodes and are widely found in a wide variety of crops. Based on 28 s rDNA D2D3 expansion region, a duplex real-time quantitative (qPCR) assay was developed to simultaneously detect and quantify these two nematodes. The qPCR system included two pairs of primers and two Taqman probes, that is the
P. neglectus
-specific primer pair 28sPnF5/28sPnR7 and probe pntaq3 and the
P. thornei
-specific primer pair 28sPtF1/28sPtR1 and probe pttaq3. The optimal conditions of the qPCR assay were 20 μL reaction volume comprising 10 μL THUNDERBIRD Probe qPCR Mix, 0.2 μM of primer 28sPnF5 and 28sPnR7, 0.25 μM of primer 28sPtF1and 28sPtR1, 0.1 μM of probe pntaq3 and 0.08 μM of probe pttaq3, 1 μL template DNA. And the amplification program were 95 °C for 2 min, 40 cycles of 95 °C for 10s, 65 °C for 25 s. The specificity of the qPCR assay was confirmed by the lack of fluorescence signal from non-target nematode populations including seven other
Pratylenchus
species and five other nematode species. The assay was very sensitive as it can detect a single target nematode and a single target nematode mixed with up to 100 individuals of non-target nematodes. A dilution series from
P. neglectus
and
P. thornei
DNAs resulted in two standard curves respectively showing a highly significant linearity between the Cycle threshold (
Ct
) values and the dilution rates. This study is the first to provide a molecular tool for simultaneous detection and quantification of
P. neglectus
and
P. thornei.</description><identifier>ISSN: 0929-1873</identifier><identifier>EISSN: 1573-8469</identifier><identifier>DOI: 10.1007/s10658-020-01999-7</identifier><language>eng</language><publisher>Dordrecht: Springer Netherlands</publisher><subject>Agriculture ; Assaying ; Biomedical and Life Sciences ; Dilution ; DNA probes ; Ecology ; Economic importance ; Fluorescence ; Life Sciences ; Linearity ; Nematodes ; Plant Pathology ; Plant Sciences ; Pratylenchus neglectus ; Real time ; Target detection</subject><ispartof>European journal of plant pathology, 2020-05, Vol.157 (1), p.185-196</ispartof><rights>Koninklijke Nederlandse Planteziektenkundige Vereniging 2020</rights><rights>Koninklijke Nederlandse Planteziektenkundige Vereniging 2020.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c363t-cf4d209550ae7946097121feaa222b0c506be21a9516db376ecb1384f84f4efc3</citedby><cites>FETCH-LOGICAL-c363t-cf4d209550ae7946097121feaa222b0c506be21a9516db376ecb1384f84f4efc3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s10658-020-01999-7$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s10658-020-01999-7$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,780,784,27924,27925,41488,42557,51319</link.rule.ids></links><search><creatorcontrib>Lin, Borong</creatorcontrib><creatorcontrib>Tao, Ye</creatorcontrib><creatorcontrib>Wang, Honghong</creatorcontrib><creatorcontrib>Liao, Jinling</creatorcontrib><creatorcontrib>Zhuo, Kan</creatorcontrib><title>Duplex real-time quantitative PCR for simultaneous detection and quantification of Pratylenchus neglectus and P. thornei</title><title>European journal of plant pathology</title><addtitle>Eur J Plant Pathol</addtitle><description>Pratylenchus neglectus
and
P. thornei
are economically important migratory plant endoparasitic nematodes and are widely found in a wide variety of crops. Based on 28 s rDNA D2D3 expansion region, a duplex real-time quantitative (qPCR) assay was developed to simultaneously detect and quantify these two nematodes. The qPCR system included two pairs of primers and two Taqman probes, that is the
P. neglectus
-specific primer pair 28sPnF5/28sPnR7 and probe pntaq3 and the
P. thornei
-specific primer pair 28sPtF1/28sPtR1 and probe pttaq3. The optimal conditions of the qPCR assay were 20 μL reaction volume comprising 10 μL THUNDERBIRD Probe qPCR Mix, 0.2 μM of primer 28sPnF5 and 28sPnR7, 0.25 μM of primer 28sPtF1and 28sPtR1, 0.1 μM of probe pntaq3 and 0.08 μM of probe pttaq3, 1 μL template DNA. And the amplification program were 95 °C for 2 min, 40 cycles of 95 °C for 10s, 65 °C for 25 s. The specificity of the qPCR assay was confirmed by the lack of fluorescence signal from non-target nematode populations including seven other
Pratylenchus
species and five other nematode species. The assay was very sensitive as it can detect a single target nematode and a single target nematode mixed with up to 100 individuals of non-target nematodes. A dilution series from
P. neglectus
and
P. thornei
DNAs resulted in two standard curves respectively showing a highly significant linearity between the Cycle threshold (
Ct
) values and the dilution rates. This study is the first to provide a molecular tool for simultaneous detection and quantification of
P. neglectus
and
P. thornei.</description><subject>Agriculture</subject><subject>Assaying</subject><subject>Biomedical and Life Sciences</subject><subject>Dilution</subject><subject>DNA probes</subject><subject>Ecology</subject><subject>Economic importance</subject><subject>Fluorescence</subject><subject>Life Sciences</subject><subject>Linearity</subject><subject>Nematodes</subject><subject>Plant Pathology</subject><subject>Plant Sciences</subject><subject>Pratylenchus neglectus</subject><subject>Real time</subject><subject>Target detection</subject><issn>0929-1873</issn><issn>1573-8469</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2020</creationdate><recordtype>article</recordtype><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNp9kNtKAzEQhoMoWKsv4FXA69Qc9tBcSj1CwSJ6HbLZSbtlm22TrNi3N-0WvBMGZhi-bwZ-hG4ZnTBKy_vAaJFPCeWUUCalJOUZGrG8FGSaFfIcjajkkrBpKS7RVQhrmiQp-Qj9PPbbFn6wB92S2GwA73rtYhN1bL4BL2Yf2HYeh2bTt1E76PqAa4hgYtM5rF194m1j9HHVWbzwOu5bcGaVYAfLNtFpOsCLCY6rzjtortGF1W2Am1Mfo6_np8_ZK5m_v7zNHubEiEJEYmxWcyrznGooZVZQWTLOLGjNOa-oyWlRAWda5qyoK1EWYCompplNlYE1Yozuhrtb3-16CFGtu9679FLxjGY8k7ksE8UHyvguBA9WbX2z0X6vGFWHhNWQsEoJq2PC6iCJQQoJdkvwf6f_sX4BM4-AjQ</recordid><startdate>20200501</startdate><enddate>20200501</enddate><creator>Lin, Borong</creator><creator>Tao, Ye</creator><creator>Wang, Honghong</creator><creator>Liao, Jinling</creator><creator>Zhuo, Kan</creator><general>Springer Netherlands</general><general>Springer Nature B.V</general><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7QL</scope><scope>7T7</scope><scope>7U9</scope><scope>7X2</scope><scope>88A</scope><scope>8AO</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>LK8</scope><scope>M0K</scope><scope>M7N</scope><scope>M7P</scope><scope>P64</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope></search><sort><creationdate>20200501</creationdate><title>Duplex real-time quantitative PCR for simultaneous detection and quantification of Pratylenchus neglectus and P. thornei</title><author>Lin, Borong ; Tao, Ye ; Wang, Honghong ; Liao, Jinling ; Zhuo, Kan</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c363t-cf4d209550ae7946097121feaa222b0c506be21a9516db376ecb1384f84f4efc3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2020</creationdate><topic>Agriculture</topic><topic>Assaying</topic><topic>Biomedical and Life Sciences</topic><topic>Dilution</topic><topic>DNA probes</topic><topic>Ecology</topic><topic>Economic importance</topic><topic>Fluorescence</topic><topic>Life Sciences</topic><topic>Linearity</topic><topic>Nematodes</topic><topic>Plant Pathology</topic><topic>Plant Sciences</topic><topic>Pratylenchus neglectus</topic><topic>Real time</topic><topic>Target detection</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Lin, Borong</creatorcontrib><creatorcontrib>Tao, Ye</creatorcontrib><creatorcontrib>Wang, Honghong</creatorcontrib><creatorcontrib>Liao, Jinling</creatorcontrib><creatorcontrib>Zhuo, Kan</creatorcontrib><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Virology and AIDS Abstracts</collection><collection>Agricultural Science Collection</collection><collection>Biology Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>Agricultural & Environmental Science Collection</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Biological Science Collection</collection><collection>Agricultural Science Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biological Science Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><jtitle>European journal of plant pathology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Lin, Borong</au><au>Tao, Ye</au><au>Wang, Honghong</au><au>Liao, Jinling</au><au>Zhuo, Kan</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Duplex real-time quantitative PCR for simultaneous detection and quantification of Pratylenchus neglectus and P. thornei</atitle><jtitle>European journal of plant pathology</jtitle><stitle>Eur J Plant Pathol</stitle><date>2020-05-01</date><risdate>2020</risdate><volume>157</volume><issue>1</issue><spage>185</spage><epage>196</epage><pages>185-196</pages><issn>0929-1873</issn><eissn>1573-8469</eissn><abstract>Pratylenchus neglectus
and
P. thornei
are economically important migratory plant endoparasitic nematodes and are widely found in a wide variety of crops. Based on 28 s rDNA D2D3 expansion region, a duplex real-time quantitative (qPCR) assay was developed to simultaneously detect and quantify these two nematodes. The qPCR system included two pairs of primers and two Taqman probes, that is the
P. neglectus
-specific primer pair 28sPnF5/28sPnR7 and probe pntaq3 and the
P. thornei
-specific primer pair 28sPtF1/28sPtR1 and probe pttaq3. The optimal conditions of the qPCR assay were 20 μL reaction volume comprising 10 μL THUNDERBIRD Probe qPCR Mix, 0.2 μM of primer 28sPnF5 and 28sPnR7, 0.25 μM of primer 28sPtF1and 28sPtR1, 0.1 μM of probe pntaq3 and 0.08 μM of probe pttaq3, 1 μL template DNA. And the amplification program were 95 °C for 2 min, 40 cycles of 95 °C for 10s, 65 °C for 25 s. The specificity of the qPCR assay was confirmed by the lack of fluorescence signal from non-target nematode populations including seven other
Pratylenchus
species and five other nematode species. The assay was very sensitive as it can detect a single target nematode and a single target nematode mixed with up to 100 individuals of non-target nematodes. A dilution series from
P. neglectus
and
P. thornei
DNAs resulted in two standard curves respectively showing a highly significant linearity between the Cycle threshold (
Ct
) values and the dilution rates. This study is the first to provide a molecular tool for simultaneous detection and quantification of
P. neglectus
and
P. thornei.</abstract><cop>Dordrecht</cop><pub>Springer Netherlands</pub><doi>10.1007/s10658-020-01999-7</doi><tpages>12</tpages><oa>free_for_read</oa></addata></record> |
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subjects | Agriculture Assaying Biomedical and Life Sciences Dilution DNA probes Ecology Economic importance Fluorescence Life Sciences Linearity Nematodes Plant Pathology Plant Sciences Pratylenchus neglectus Real time Target detection |
title | Duplex real-time quantitative PCR for simultaneous detection and quantification of Pratylenchus neglectus and P. thornei |
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