Multiplex and quantifiable detection of nucleic acid from pathogenic fungi using padlock probes, generic real time PCR and specific suspension array readout
A new concept for multiplex detection and quantification of microbes is here demonstrated on a range of infectious fungal species. Padlock probe methodology in conjunction with qPCR and Luminex™ technology was used for simultaneous detection of ten fungal species in one single experiment. By combini...
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| Veröffentlicht in: | Journal of microbiological methods 2009-08, Vol.78 (2), p.195-202 |
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| creator | Eriksson, Ronnie Jobs, Magnus Ekstrand, Charlotta Ullberg, Måns Herrmann, Björn Landegren, Ulf Nilsson, Mats Blomberg, Jonas |
| description | A new concept for multiplex detection and quantification of microbes is here demonstrated on a range of infectious fungal species. Padlock probe methodology in conjunction with qPCR and Luminex™ technology was used for simultaneous detection of ten fungal species in one single experiment. By combining the multiplexing properties of padlock probes and Luminex™ detection with the well established quantitative characteristics of qPCR, quantitative microbe detection was done in 10-plex mode.
A padlock probe is an oligonucleotide that via a ligation reaction forms circular DNA when hybridizing to specific target DNA. The region of the padlock probe that does not participate in target DNA hybridization contains generic primer sequences for amplification and a tag sequence for Luminex™ detection. This was the fundament for well performing multiplexing. Circularized padlock probes were initially amplified by rolling circle amplification (RCA), followed by a SybrGreen™ real time PCR which allowed an additive quantitative assessment of target DNA in the sample. Detection and quantification of amplified padlock probes were then done on color coded Luminex™ microspheres carrying anti-tag sequences. A novel technique, using labeled oligonucleotides to prevent reannealing of amplimers by covering the flanks of the address sequence, improved the signal to noise ratio in the detection step considerably. The method correctly detected fungi in a variety of clinical samples and offered quantitative information on fungal nucleic acid. |
| doi_str_mv | 10.1016/j.mimet.2009.05.016 |
| format | Article |
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A padlock probe is an oligonucleotide that via a ligation reaction forms circular DNA when hybridizing to specific target DNA. The region of the padlock probe that does not participate in target DNA hybridization contains generic primer sequences for amplification and a tag sequence for Luminex™ detection. This was the fundament for well performing multiplexing. Circularized padlock probes were initially amplified by rolling circle amplification (RCA), followed by a SybrGreen™ real time PCR which allowed an additive quantitative assessment of target DNA in the sample. Detection and quantification of amplified padlock probes were then done on color coded Luminex™ microspheres carrying anti-tag sequences. A novel technique, using labeled oligonucleotides to prevent reannealing of amplimers by covering the flanks of the address sequence, improved the signal to noise ratio in the detection step considerably. The method correctly detected fungi in a variety of clinical samples and offered quantitative information on fungal nucleic acid.</description><identifier>ISSN: 0167-7012</identifier><identifier>ISSN: 1872-8359</identifier><identifier>EISSN: 1872-8359</identifier><identifier>DOI: 10.1016/j.mimet.2009.05.016</identifier><identifier>PMID: 19490930</identifier><identifier>CODEN: JMIMDQ</identifier><language>eng</language><publisher>Amsterdam: Elsevier B.V</publisher><subject>Biological and medical sciences ; Colony Count, Microbial - methods ; DNA, Fungal - genetics ; DNA, Fungal - isolation & purification ; Fundamental and applied biological sciences. Psychology ; Fungi - classification ; Fungi - genetics ; Fungi - isolation & purification ; Humans ; MEDICIN ; MEDICINE ; Microbiology ; Multiplex detection ; Multiplex detektion av patogena agens ; Mycological methods and techniques used in mycology ; Mycology ; Mycoses - diagnosis ; Nucleic acid hybridization ; Oligonucleotide Probes - genetics ; Padlock probe ; Pathogenic fungi ; Polymerase Chain Reaction - methods ; Real time PCR ; Rolling circle amplification ; Sensitivity and Specificity ; Suspension array</subject><ispartof>Journal of microbiological methods, 2009-08, Vol.78 (2), p.195-202</ispartof><rights>2009 Elsevier B.V.</rights><rights>2015 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c528t-15358c61609d7f442d52657a85c6aeb93120762a7cc120390ae7ad8014c7be323</citedby><cites>FETCH-LOGICAL-c528t-15358c61609d7f442d52657a85c6aeb93120762a7cc120390ae7ad8014c7be323</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S0167701209001729$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>230,314,776,780,881,3537,27901,27902,65306</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=21782192$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/19490930$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttps://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-124441$$DView record from Swedish Publication Index$$Hfree_for_read</backlink><backlink>$$Uhttps://urn.kb.se/resolve?urn=urn:nbn:se:du-4148$$DView record from Swedish Publication Index$$Hfree_for_read</backlink><backlink>$$Uhttp://kipublications.ki.se/Default.aspx?queryparsed=id:119198767$$DView record from Swedish Publication Index$$Hfree_for_read</backlink></links><search><creatorcontrib>Eriksson, Ronnie</creatorcontrib><creatorcontrib>Jobs, Magnus</creatorcontrib><creatorcontrib>Ekstrand, Charlotta</creatorcontrib><creatorcontrib>Ullberg, Måns</creatorcontrib><creatorcontrib>Herrmann, Björn</creatorcontrib><creatorcontrib>Landegren, Ulf</creatorcontrib><creatorcontrib>Nilsson, Mats</creatorcontrib><creatorcontrib>Blomberg, Jonas</creatorcontrib><title>Multiplex and quantifiable detection of nucleic acid from pathogenic fungi using padlock probes, generic real time PCR and specific suspension array readout</title><title>Journal of microbiological methods</title><addtitle>J Microbiol Methods</addtitle><description>A new concept for multiplex detection and quantification of microbes is here demonstrated on a range of infectious fungal species. Padlock probe methodology in conjunction with qPCR and Luminex™ technology was used for simultaneous detection of ten fungal species in one single experiment. By combining the multiplexing properties of padlock probes and Luminex™ detection with the well established quantitative characteristics of qPCR, quantitative microbe detection was done in 10-plex mode.
A padlock probe is an oligonucleotide that via a ligation reaction forms circular DNA when hybridizing to specific target DNA. The region of the padlock probe that does not participate in target DNA hybridization contains generic primer sequences for amplification and a tag sequence for Luminex™ detection. This was the fundament for well performing multiplexing. Circularized padlock probes were initially amplified by rolling circle amplification (RCA), followed by a SybrGreen™ real time PCR which allowed an additive quantitative assessment of target DNA in the sample. Detection and quantification of amplified padlock probes were then done on color coded Luminex™ microspheres carrying anti-tag sequences. A novel technique, using labeled oligonucleotides to prevent reannealing of amplimers by covering the flanks of the address sequence, improved the signal to noise ratio in the detection step considerably. 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Psychology</subject><subject>Fungi - classification</subject><subject>Fungi - genetics</subject><subject>Fungi - isolation & purification</subject><subject>Humans</subject><subject>MEDICIN</subject><subject>MEDICINE</subject><subject>Microbiology</subject><subject>Multiplex detection</subject><subject>Multiplex detektion av patogena agens</subject><subject>Mycological methods and techniques used in mycology</subject><subject>Mycology</subject><subject>Mycoses - diagnosis</subject><subject>Nucleic acid hybridization</subject><subject>Oligonucleotide Probes - genetics</subject><subject>Padlock probe</subject><subject>Pathogenic fungi</subject><subject>Polymerase Chain Reaction - methods</subject><subject>Real time PCR</subject><subject>Rolling circle amplification</subject><subject>Sensitivity and Specificity</subject><subject>Suspension array</subject><issn>0167-7012</issn><issn>1872-8359</issn><issn>1872-8359</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2009</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFks9u1DAQxiMEokvhCZCQL3BAzWI7cZwcOFRL-SMVgRBwtRx7snibxKkdA32XPiyT7qqcCiePPv3G83n8ZdlTRteMsurVbj24AeY1p7RZU7FG7V62YrXkeV2I5n62QkXmkjJ-lD2KcUcpE0VZP8yOWFM2tCnoKrv-mPrZTT38Jnq05DLpcXad020PxMIMZnZ-JL4jYzI9OEO0cZZ0wQ9k0vMPv4URxS6NW0dSdOMWZdt7c0Gm4FuIJwQJCMgE0D2Z0TH5vPlyMyxOYHCWITFhOcZlkg5BXy2s9Wl-nD3odB_hyeE8zr69Pfu6eZ-ff3r3YXN6nhvB6znHV4naVKyijZVdWXIreCWkroWpNLRNwTiVFdfSGKyKhmqQ2taUlUa2UPDiOMv398ZfMKVWTcENOlwpr506SBdYgRJCSkGRf3knb3UPWtm04CUra4RP7oTfuO-nyoetSkkxXpYlQ_zFHsf9XSaIsxpcNND3egSfoqrQAOe0-i_IKWO4ngLBYg-a4GMM0N1aYFQtSVI7dZMktSRJUaFQw65nh-tTO4D923OIDgLPD4CORvdd0KNx8ZbjTNacNctyX-85wB_86SCoaByMBqwLGC9lvfunkT_il-r4</recordid><startdate>20090801</startdate><enddate>20090801</enddate><creator>Eriksson, Ronnie</creator><creator>Jobs, Magnus</creator><creator>Ekstrand, Charlotta</creator><creator>Ullberg, Måns</creator><creator>Herrmann, Björn</creator><creator>Landegren, Ulf</creator><creator>Nilsson, Mats</creator><creator>Blomberg, Jonas</creator><general>Elsevier B.V</general><general>Elsevier</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7T7</scope><scope>7TM</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>M7N</scope><scope>P64</scope><scope>7X8</scope><scope>ADTPV</scope><scope>AOWAS</scope><scope>DF2</scope></search><sort><creationdate>20090801</creationdate><title>Multiplex and quantifiable detection of nucleic acid from pathogenic fungi using padlock probes, generic real time PCR and specific suspension array readout</title><author>Eriksson, Ronnie ; Jobs, Magnus ; Ekstrand, Charlotta ; Ullberg, Måns ; Herrmann, Björn ; Landegren, Ulf ; Nilsson, Mats ; Blomberg, Jonas</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c528t-15358c61609d7f442d52657a85c6aeb93120762a7cc120390ae7ad8014c7be323</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2009</creationdate><topic>Biological and medical sciences</topic><topic>Colony Count, Microbial - methods</topic><topic>DNA, Fungal - genetics</topic><topic>DNA, Fungal - isolation & purification</topic><topic>Fundamental and applied biological sciences. 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A padlock probe is an oligonucleotide that via a ligation reaction forms circular DNA when hybridizing to specific target DNA. The region of the padlock probe that does not participate in target DNA hybridization contains generic primer sequences for amplification and a tag sequence for Luminex™ detection. This was the fundament for well performing multiplexing. Circularized padlock probes were initially amplified by rolling circle amplification (RCA), followed by a SybrGreen™ real time PCR which allowed an additive quantitative assessment of target DNA in the sample. Detection and quantification of amplified padlock probes were then done on color coded Luminex™ microspheres carrying anti-tag sequences. A novel technique, using labeled oligonucleotides to prevent reannealing of amplimers by covering the flanks of the address sequence, improved the signal to noise ratio in the detection step considerably. The method correctly detected fungi in a variety of clinical samples and offered quantitative information on fungal nucleic acid.</abstract><cop>Amsterdam</cop><pub>Elsevier B.V</pub><pmid>19490930</pmid><doi>10.1016/j.mimet.2009.05.016</doi><tpages>8</tpages></addata></record> |
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| identifier | ISSN: 0167-7012 |
| ispartof | Journal of microbiological methods, 2009-08, Vol.78 (2), p.195-202 |
| issn | 0167-7012 1872-8359 1872-8359 |
| language | eng |
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| source | MEDLINE; Elsevier ScienceDirect Journals |
| subjects | Biological and medical sciences Colony Count, Microbial - methods DNA, Fungal - genetics DNA, Fungal - isolation & purification Fundamental and applied biological sciences. Psychology Fungi - classification Fungi - genetics Fungi - isolation & purification Humans MEDICIN MEDICINE Microbiology Multiplex detection Multiplex detektion av patogena agens Mycological methods and techniques used in mycology Mycology Mycoses - diagnosis Nucleic acid hybridization Oligonucleotide Probes - genetics Padlock probe Pathogenic fungi Polymerase Chain Reaction - methods Real time PCR Rolling circle amplification Sensitivity and Specificity Suspension array |
| title | Multiplex and quantifiable detection of nucleic acid from pathogenic fungi using padlock probes, generic real time PCR and specific suspension array readout |
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