Evaluation and improvement of isothermal amplification methods for point-of-need plant disease diagnostics.
A number of isothermal DNA amplification technologies claim to be ideal for point-of-need (PON) applications as they enable reactions to be performed using a single-temperature heat source (e.g. water bath). Thus, we examined several isothermal amplification methods focusing on simplicity, cost, sen...
| Main Authors: | , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2020-01-01
|
| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0235216 |
| _version_ | 1818678430341791744 |
|---|---|
| author | Yiping Zou Michael Glenn Mason Jose Ramon Botella |
| author_facet | Yiping Zou Michael Glenn Mason Jose Ramon Botella |
| author_sort | Yiping Zou |
| collection | DOAJ |
| description | A number of isothermal DNA amplification technologies claim to be ideal for point-of-need (PON) applications as they enable reactions to be performed using a single-temperature heat source (e.g. water bath). Thus, we examined several isothermal amplification methods focusing on simplicity, cost, sensitivity and reproducibility to identify the most suitable method(s) for low resource PON applications. A number of methods were found unsuitable as they either involved multiple temperature incubations, were relatively expensive or required relatively large amounts target DNA for amplification. Among the methods examined, loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) were found to be the most suitable for PON applications as they are both single step methods that provide highly sensitive and reproducible amplifications. The speed of LAMP reactions was greatly enhanced, up to 76%, with the addition of loop primers while the presence of swarm primers and the sequestration of free magnesium ions with nucleotides also enhanced the amplification speed. In contrast, we were unable to enhance RPA's performance from the original published literature. While both RPA and LAMP have some drawbacks, either isothermal technology can reliably be used for on-site diagnostics with minimal equipment. |
| first_indexed | 2024-12-17T09:15:08Z |
| format | Article |
| id | doaj.art-32a902ed1cae4954b65d2b2f4b5fcec9 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-17T09:15:08Z |
| publishDate | 2020-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-32a902ed1cae4954b65d2b2f4b5fcec92022-12-21T21:55:00ZengPublic Library of Science (PLoS)PLoS ONE1932-62032020-01-01156e023521610.1371/journal.pone.0235216Evaluation and improvement of isothermal amplification methods for point-of-need plant disease diagnostics.Yiping ZouMichael Glenn MasonJose Ramon BotellaA number of isothermal DNA amplification technologies claim to be ideal for point-of-need (PON) applications as they enable reactions to be performed using a single-temperature heat source (e.g. water bath). Thus, we examined several isothermal amplification methods focusing on simplicity, cost, sensitivity and reproducibility to identify the most suitable method(s) for low resource PON applications. A number of methods were found unsuitable as they either involved multiple temperature incubations, were relatively expensive or required relatively large amounts target DNA for amplification. Among the methods examined, loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) were found to be the most suitable for PON applications as they are both single step methods that provide highly sensitive and reproducible amplifications. The speed of LAMP reactions was greatly enhanced, up to 76%, with the addition of loop primers while the presence of swarm primers and the sequestration of free magnesium ions with nucleotides also enhanced the amplification speed. In contrast, we were unable to enhance RPA's performance from the original published literature. While both RPA and LAMP have some drawbacks, either isothermal technology can reliably be used for on-site diagnostics with minimal equipment.https://doi.org/10.1371/journal.pone.0235216 |
| spellingShingle | Yiping Zou Michael Glenn Mason Jose Ramon Botella Evaluation and improvement of isothermal amplification methods for point-of-need plant disease diagnostics. PLoS ONE |
| title | Evaluation and improvement of isothermal amplification methods for point-of-need plant disease diagnostics. |
| title_full | Evaluation and improvement of isothermal amplification methods for point-of-need plant disease diagnostics. |
| title_fullStr | Evaluation and improvement of isothermal amplification methods for point-of-need plant disease diagnostics. |
| title_full_unstemmed | Evaluation and improvement of isothermal amplification methods for point-of-need plant disease diagnostics. |
| title_short | Evaluation and improvement of isothermal amplification methods for point-of-need plant disease diagnostics. |
| title_sort | evaluation and improvement of isothermal amplification methods for point of need plant disease diagnostics |
| url | https://doi.org/10.1371/journal.pone.0235216 |
| work_keys_str_mv | AT yipingzou evaluationandimprovementofisothermalamplificationmethodsforpointofneedplantdiseasediagnostics AT michaelglennmason evaluationandimprovementofisothermalamplificationmethodsforpointofneedplantdiseasediagnostics AT joseramonbotella evaluationandimprovementofisothermalamplificationmethodsforpointofneedplantdiseasediagnostics |