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Article: A six‐plex droplet digital RT‐PCR assay for seasonal influenza virus typing, subtyping, and lineage determination
Title | A six‐plex droplet digital RT‐PCR assay for seasonal influenza virus typing, subtyping, and lineage determination |
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Authors | |
Keywords | digital RT-PCR influenza RT-PCR |
Issue Date | 2020 |
Publisher | Wiley Open Access. The Journal's web site is located at http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1750-2659 |
Citation | Influenza and Other Respiratory Viruses, 2020, v. 14 n. 6, p. 720-729 How to Cite? |
Abstract | Background:
There are two influenza A subtypes (H1 and H3) and two influenza B lineages (Victoria and Yamagata) that currently co-circulate in humans. In this study, we report the development of a six-plex droplet digital RT-PCR (ddRT-PCR) assay that can detect HA and M segments of influenza A (H1, H3, and M) and influenza B (Yamagata HA, Victoria HA, and M) viruses in a single reaction mixture. It can simultaneously detect six different nucleic acid targets in a ddRT-PCR platform.
Methods:
The six-plex ddRT-PCR used in this study is an amplitude-based multiplex assay. The analytical performance of the assay was evaluated. Correlation with standard qRT-PCR methodology was assessed using 55 clinical samples.
Results:
The assay has a wide dynamic range, and it has good reproducibility within and between runs. The limit of quantification of each target in this assay ranged from 15 copies/reaction for influenza B Victoria M gene to 45 copies/reaction for influenza B Yamagata M gene. In addition, this assay can accurately quantify each of these targets in samples containing viral RNAs from two different viruses that were mixed in a highly skewed ratio. Typing, subtyping, and lineage differentiation data of 55 tested clinical respiratory specimens were found to be identical to those deduced from standard monoplex qRT-PCR assays.
Conclusions:
The six-plex ddRT-PCR test was demonstrated to be highly suitable for detecting dual influenza infection cases. This assay is expected to be a useful diagnostic tool for clinical and research use. |
Persistent Identifier | http://hdl.handle.net/10722/304512 |
ISSN | 2023 Impact Factor: 4.3 2023 SCImago Journal Rankings: 1.485 |
PubMed Central ID | |
ISI Accession Number ID |
DC Field | Value | Language |
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dc.contributor.author | Leong, NKC | - |
dc.contributor.author | Chu, DKW | - |
dc.contributor.author | Chu, JTS | - |
dc.contributor.author | Tam, YH | - |
dc.contributor.author | Ip, DKM | - |
dc.contributor.author | Cowling, BJ | - |
dc.contributor.author | Poon, LML | - |
dc.date.accessioned | 2021-09-23T09:01:05Z | - |
dc.date.available | 2021-09-23T09:01:05Z | - |
dc.date.issued | 2020 | - |
dc.identifier.citation | Influenza and Other Respiratory Viruses, 2020, v. 14 n. 6, p. 720-729 | - |
dc.identifier.issn | 1750-2640 | - |
dc.identifier.uri | http://hdl.handle.net/10722/304512 | - |
dc.description.abstract | Background: There are two influenza A subtypes (H1 and H3) and two influenza B lineages (Victoria and Yamagata) that currently co-circulate in humans. In this study, we report the development of a six-plex droplet digital RT-PCR (ddRT-PCR) assay that can detect HA and M segments of influenza A (H1, H3, and M) and influenza B (Yamagata HA, Victoria HA, and M) viruses in a single reaction mixture. It can simultaneously detect six different nucleic acid targets in a ddRT-PCR platform. Methods: The six-plex ddRT-PCR used in this study is an amplitude-based multiplex assay. The analytical performance of the assay was evaluated. Correlation with standard qRT-PCR methodology was assessed using 55 clinical samples. Results: The assay has a wide dynamic range, and it has good reproducibility within and between runs. The limit of quantification of each target in this assay ranged from 15 copies/reaction for influenza B Victoria M gene to 45 copies/reaction for influenza B Yamagata M gene. In addition, this assay can accurately quantify each of these targets in samples containing viral RNAs from two different viruses that were mixed in a highly skewed ratio. Typing, subtyping, and lineage differentiation data of 55 tested clinical respiratory specimens were found to be identical to those deduced from standard monoplex qRT-PCR assays. Conclusions: The six-plex ddRT-PCR test was demonstrated to be highly suitable for detecting dual influenza infection cases. This assay is expected to be a useful diagnostic tool for clinical and research use. | - |
dc.language | eng | - |
dc.publisher | Wiley Open Access. The Journal's web site is located at http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1750-2659 | - |
dc.relation.ispartof | Influenza and Other Respiratory Viruses | - |
dc.rights | This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. | - |
dc.subject | digital RT-PCR | - |
dc.subject | influenza | - |
dc.subject | RT-PCR | - |
dc.title | A six‐plex droplet digital RT‐PCR assay for seasonal influenza virus typing, subtyping, and lineage determination | - |
dc.type | Article | - |
dc.identifier.email | Ip, DKM: dkmip@hku.hk | - |
dc.identifier.email | Cowling, BJ: bcowling@hku.hk | - |
dc.identifier.email | Poon, LML: llmpoon@hkucc.hku.hk | - |
dc.identifier.authority | Tam, YH=rp01881 | - |
dc.identifier.authority | Ip, DKM=rp00256 | - |
dc.identifier.authority | Cowling, BJ=rp01326 | - |
dc.identifier.authority | Poon, LML=rp00484 | - |
dc.description.nature | published_or_final_version | - |
dc.identifier.doi | 10.1111/irv.12769 | - |
dc.identifier.pmid | 32519796 | - |
dc.identifier.pmcid | PMC7578307 | - |
dc.identifier.scopus | eid_2-s2.0-85086155421 | - |
dc.identifier.hkuros | 325221 | - |
dc.identifier.volume | 14 | - |
dc.identifier.issue | 6 | - |
dc.identifier.spage | 720 | - |
dc.identifier.epage | 729 | - |
dc.identifier.isi | WOS:000547924200001 | - |
dc.publisher.place | United Kingdom | - |