A Systematic Investigation of Parameters Influencing Droplet Rain in the Listeria monocytogenes prfA Assay - Reduction of Ambiguous Results in ddPCR.
The droplet digital polymerase chain reaction (ddPCR) determines DNA amounts based upon the pattern of positive and negative droplets, according to Poisson distribution, without the use of external standards. However, division into positive and negative droplets is often not clear because a part of...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2016-01-01
|
| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC5167268?pdf=render |
| _version_ | 1818608213416738816 |
|---|---|
| author | Anna Kristina Witte Patrick Mester Susanne Fister Matthias Witte Dagmar Schoder Peter Rossmanith |
| author_facet | Anna Kristina Witte Patrick Mester Susanne Fister Matthias Witte Dagmar Schoder Peter Rossmanith |
| author_sort | Anna Kristina Witte |
| collection | DOAJ |
| description | The droplet digital polymerase chain reaction (ddPCR) determines DNA amounts based upon the pattern of positive and negative droplets, according to Poisson distribution, without the use of external standards. However, division into positive and negative droplets is often not clear because a part of the droplets has intermediate fluorescence values, appearing as "rain" in the plot. Despite the droplet rain, absolute quantification with ddPCR is possible, as shown previously for the prfA assay in quantifying Listeria monocytogenes. Nevertheless, reducing the rain, and thus ambiguous results, promotes the accuracy and credibility of ddPCR. In this study, we extensively investigated chemical and physical parameters for optimizing the prfA assay for ddPCR. While differences in the concentration of all chemicals and the dye, quencher and supplier of the probe did not alter the droplet pattern, changes in the PCR cycling program, such as prolonged times and increased cycle numbers, improved the assay. |
| first_indexed | 2024-12-16T14:39:04Z |
| format | Article |
| id | doaj.art-30f535116df4400e99868a27cff3b017 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-16T14:39:04Z |
| publishDate | 2016-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-30f535116df4400e99868a27cff3b0172022-12-21T22:28:00ZengPublic Library of Science (PLoS)PLoS ONE1932-62032016-01-011112e016817910.1371/journal.pone.0168179A Systematic Investigation of Parameters Influencing Droplet Rain in the Listeria monocytogenes prfA Assay - Reduction of Ambiguous Results in ddPCR.Anna Kristina WittePatrick MesterSusanne FisterMatthias WitteDagmar SchoderPeter RossmanithThe droplet digital polymerase chain reaction (ddPCR) determines DNA amounts based upon the pattern of positive and negative droplets, according to Poisson distribution, without the use of external standards. However, division into positive and negative droplets is often not clear because a part of the droplets has intermediate fluorescence values, appearing as "rain" in the plot. Despite the droplet rain, absolute quantification with ddPCR is possible, as shown previously for the prfA assay in quantifying Listeria monocytogenes. Nevertheless, reducing the rain, and thus ambiguous results, promotes the accuracy and credibility of ddPCR. In this study, we extensively investigated chemical and physical parameters for optimizing the prfA assay for ddPCR. While differences in the concentration of all chemicals and the dye, quencher and supplier of the probe did not alter the droplet pattern, changes in the PCR cycling program, such as prolonged times and increased cycle numbers, improved the assay.http://europepmc.org/articles/PMC5167268?pdf=render |
| spellingShingle | Anna Kristina Witte Patrick Mester Susanne Fister Matthias Witte Dagmar Schoder Peter Rossmanith A Systematic Investigation of Parameters Influencing Droplet Rain in the Listeria monocytogenes prfA Assay - Reduction of Ambiguous Results in ddPCR. PLoS ONE |
| title | A Systematic Investigation of Parameters Influencing Droplet Rain in the Listeria monocytogenes prfA Assay - Reduction of Ambiguous Results in ddPCR. |
| title_full | A Systematic Investigation of Parameters Influencing Droplet Rain in the Listeria monocytogenes prfA Assay - Reduction of Ambiguous Results in ddPCR. |
| title_fullStr | A Systematic Investigation of Parameters Influencing Droplet Rain in the Listeria monocytogenes prfA Assay - Reduction of Ambiguous Results in ddPCR. |
| title_full_unstemmed | A Systematic Investigation of Parameters Influencing Droplet Rain in the Listeria monocytogenes prfA Assay - Reduction of Ambiguous Results in ddPCR. |
| title_short | A Systematic Investigation of Parameters Influencing Droplet Rain in the Listeria monocytogenes prfA Assay - Reduction of Ambiguous Results in ddPCR. |
| title_sort | systematic investigation of parameters influencing droplet rain in the listeria monocytogenes prfa assay reduction of ambiguous results in ddpcr |
| url | http://europepmc.org/articles/PMC5167268?pdf=render |
| work_keys_str_mv | AT annakristinawitte asystematicinvestigationofparametersinfluencingdropletraininthelisteriamonocytogenesprfaassayreductionofambiguousresultsinddpcr AT patrickmester asystematicinvestigationofparametersinfluencingdropletraininthelisteriamonocytogenesprfaassayreductionofambiguousresultsinddpcr AT susannefister asystematicinvestigationofparametersinfluencingdropletraininthelisteriamonocytogenesprfaassayreductionofambiguousresultsinddpcr AT matthiaswitte asystematicinvestigationofparametersinfluencingdropletraininthelisteriamonocytogenesprfaassayreductionofambiguousresultsinddpcr AT dagmarschoder asystematicinvestigationofparametersinfluencingdropletraininthelisteriamonocytogenesprfaassayreductionofambiguousresultsinddpcr AT peterrossmanith asystematicinvestigationofparametersinfluencingdropletraininthelisteriamonocytogenesprfaassayreductionofambiguousresultsinddpcr AT annakristinawitte systematicinvestigationofparametersinfluencingdropletraininthelisteriamonocytogenesprfaassayreductionofambiguousresultsinddpcr AT patrickmester systematicinvestigationofparametersinfluencingdropletraininthelisteriamonocytogenesprfaassayreductionofambiguousresultsinddpcr AT susannefister systematicinvestigationofparametersinfluencingdropletraininthelisteriamonocytogenesprfaassayreductionofambiguousresultsinddpcr AT matthiaswitte systematicinvestigationofparametersinfluencingdropletraininthelisteriamonocytogenesprfaassayreductionofambiguousresultsinddpcr AT dagmarschoder systematicinvestigationofparametersinfluencingdropletraininthelisteriamonocytogenesprfaassayreductionofambiguousresultsinddpcr AT peterrossmanith systematicinvestigationofparametersinfluencingdropletraininthelisteriamonocytogenesprfaassayreductionofambiguousresultsinddpcr |