PlexProbes enhance qPCR multiplexing by discriminating multiple targets in each fluorescent channel.
The probe technology described in this paper facilitates detection and discrimination of multiple targets in a single fluorescent channel during PCR. This provides a strategy for doubling the number of targets that can be analysed simultaneously on existing PCR instruments. These probes are referred...
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2022-01-01
|
| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0263329 |
| _version_ | 1811332330974871552 |
|---|---|
| author | Nicole Hasick Ryung Rae Kim Yin Xu Simon Bone Andrea Lawrence Claire Gibbs Nathan Danckert Alison Todd |
| author_facet | Nicole Hasick Ryung Rae Kim Yin Xu Simon Bone Andrea Lawrence Claire Gibbs Nathan Danckert Alison Todd |
| author_sort | Nicole Hasick |
| collection | DOAJ |
| description | The probe technology described in this paper facilitates detection and discrimination of multiple targets in a single fluorescent channel during PCR. This provides a strategy for doubling the number of targets that can be analysed simultaneously on existing PCR instruments. These probes are referred to as PlexProbes and produce fluorescence that can be switched 'on' or 'off' in the presence of target by manipulating the temperature. During PCR, fluorescence can be measured at multiple temperatures allowing discrimination of specific targets at defined temperatures. In a single fluorescent channel, a model duplex assay allowed either real-time or endpoint detection of Chlamydia trachomatis (CT) at 52°C and end-point detection of Neisseria gonorrhoeae (GC) at 74°C. Using this model system, as few as 40 copies of each specific target could be detected as single infection or co-infection, regardless of the presence or absence of the other target. A PlexProbe prototype assay for sexually transmitted infections (PP-STI) which simultaneously enables detection and differentiation of six targets using only three fluorescent channels was then constructed and evaluated. The PP-STI assay detects GC (2 gene targets), CT, Mycoplasma genitalium (MG), Trichomonas vaginalis (TV) and an internal control (IC). To evaluate assay performance, a panel of archived clinical samples (n = 337) were analysed using PP-STI and results compared to those obtained with a commercially available diagnostic assay. The overall agreement between results obtained with the PP-STI assay and the reference test was greater than 99.5%. PlexProbes offer a method of detecting more targets from a single diagnostic test, empowering physicians to make evidence-based treatment decisions while conserving time, labour, sample volume and reagent costs. |
| first_indexed | 2024-04-13T16:34:26Z |
| format | Article |
| id | doaj.art-b3540f2cb88f4c6db166fb100761138d |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-13T16:34:26Z |
| publishDate | 2022-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-b3540f2cb88f4c6db166fb100761138d2022-12-22T02:39:28ZengPublic Library of Science (PLoS)PLoS ONE1932-62032022-01-01173e026332910.1371/journal.pone.0263329PlexProbes enhance qPCR multiplexing by discriminating multiple targets in each fluorescent channel.Nicole HasickRyung Rae KimYin XuSimon BoneAndrea LawrenceClaire GibbsNathan DanckertAlison ToddThe probe technology described in this paper facilitates detection and discrimination of multiple targets in a single fluorescent channel during PCR. This provides a strategy for doubling the number of targets that can be analysed simultaneously on existing PCR instruments. These probes are referred to as PlexProbes and produce fluorescence that can be switched 'on' or 'off' in the presence of target by manipulating the temperature. During PCR, fluorescence can be measured at multiple temperatures allowing discrimination of specific targets at defined temperatures. In a single fluorescent channel, a model duplex assay allowed either real-time or endpoint detection of Chlamydia trachomatis (CT) at 52°C and end-point detection of Neisseria gonorrhoeae (GC) at 74°C. Using this model system, as few as 40 copies of each specific target could be detected as single infection or co-infection, regardless of the presence or absence of the other target. A PlexProbe prototype assay for sexually transmitted infections (PP-STI) which simultaneously enables detection and differentiation of six targets using only three fluorescent channels was then constructed and evaluated. The PP-STI assay detects GC (2 gene targets), CT, Mycoplasma genitalium (MG), Trichomonas vaginalis (TV) and an internal control (IC). To evaluate assay performance, a panel of archived clinical samples (n = 337) were analysed using PP-STI and results compared to those obtained with a commercially available diagnostic assay. The overall agreement between results obtained with the PP-STI assay and the reference test was greater than 99.5%. PlexProbes offer a method of detecting more targets from a single diagnostic test, empowering physicians to make evidence-based treatment decisions while conserving time, labour, sample volume and reagent costs.https://doi.org/10.1371/journal.pone.0263329 |
| spellingShingle | Nicole Hasick Ryung Rae Kim Yin Xu Simon Bone Andrea Lawrence Claire Gibbs Nathan Danckert Alison Todd PlexProbes enhance qPCR multiplexing by discriminating multiple targets in each fluorescent channel. PLoS ONE |
| title | PlexProbes enhance qPCR multiplexing by discriminating multiple targets in each fluorescent channel. |
| title_full | PlexProbes enhance qPCR multiplexing by discriminating multiple targets in each fluorescent channel. |
| title_fullStr | PlexProbes enhance qPCR multiplexing by discriminating multiple targets in each fluorescent channel. |
| title_full_unstemmed | PlexProbes enhance qPCR multiplexing by discriminating multiple targets in each fluorescent channel. |
| title_short | PlexProbes enhance qPCR multiplexing by discriminating multiple targets in each fluorescent channel. |
| title_sort | plexprobes enhance qpcr multiplexing by discriminating multiple targets in each fluorescent channel |
| url | https://doi.org/10.1371/journal.pone.0263329 |
| work_keys_str_mv | AT nicolehasick plexprobesenhanceqpcrmultiplexingbydiscriminatingmultipletargetsineachfluorescentchannel AT ryungraekim plexprobesenhanceqpcrmultiplexingbydiscriminatingmultipletargetsineachfluorescentchannel AT yinxu plexprobesenhanceqpcrmultiplexingbydiscriminatingmultipletargetsineachfluorescentchannel AT simonbone plexprobesenhanceqpcrmultiplexingbydiscriminatingmultipletargetsineachfluorescentchannel AT andrealawrence plexprobesenhanceqpcrmultiplexingbydiscriminatingmultipletargetsineachfluorescentchannel AT clairegibbs plexprobesenhanceqpcrmultiplexingbydiscriminatingmultipletargetsineachfluorescentchannel AT nathandanckert plexprobesenhanceqpcrmultiplexingbydiscriminatingmultipletargetsineachfluorescentchannel AT alisontodd plexprobesenhanceqpcrmultiplexingbydiscriminatingmultipletargetsineachfluorescentchannel |