Cargando…
Recombinase polymerase amplification assay combined with a dipstick-readout for rapid detection of Mycoplasma ovipneumoniae infections
Mycoplasma ovipneumoniae infects both sheep and goats causing pneumonia resulting in considerable economic losses worldwide. Current diagnosis methods such as bacteriological culture, serology, and PCR are time consuming and require sophisticated laboratory setups. Here we report the development of...
Autores principales: | , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2021
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7861559/ https://www.ncbi.nlm.nih.gov/pubmed/33539437 http://dx.doi.org/10.1371/journal.pone.0246573 |
_version_ | 1783647109048696832 |
---|---|
author | Gupta, Sandeep K. Deng, Qing Gupta, Tanushree B. Maclean, Paul Jores, Joerg Heiser, Axel Wedlock, D. Neil |
author_facet | Gupta, Sandeep K. Deng, Qing Gupta, Tanushree B. Maclean, Paul Jores, Joerg Heiser, Axel Wedlock, D. Neil |
author_sort | Gupta, Sandeep K. |
collection | PubMed |
description | Mycoplasma ovipneumoniae infects both sheep and goats causing pneumonia resulting in considerable economic losses worldwide. Current diagnosis methods such as bacteriological culture, serology, and PCR are time consuming and require sophisticated laboratory setups. Here we report the development of two rapid, specific and sensitive assays; an isothermal DNA amplification using recombinase polymerase amplification (RPA) and a real-time PCR for the detection of M. ovipneumoniae. The target for both assays is a specific region of gene WP_069098309.1, which encodes a hypothetical protein and is conserved in the genome sequences of ten publicly available M. ovipneumoniae strains. The RPA assay performed well at 39°C for 20 min and was combined with a lateral flow dipstick (RPA-LFD) for easy visualization of the amplicons. The detection limit of the RPA-LFD assay was nine genome copies of M. ovipneumoniae per reaction and was comparable to sensitivity of the real-time PCR assay. Both assays showed no cross-reaction with 38 other ovine and caprine pathogenic microorganisms and two parasites of ruminants, demonstrating a high degree of specificity. The assays were validated using bronchoalveolar lavage fluid and nasal swab samples collected from sheep. The positive rate of RPA-LFD (97.4%) was higher than the real-time PCR (95.8%) with DNA as a template purified from the clinical samples. The RPA assay was significantly better at detecting M. ovipneumoniae in clinical samples compared to the real-time PCR when DNA extraction was omitted (50% and 34.4% positive rate for RPA-LFD and real-time PCR respectively). The RPA-LFD developed here allows easy and rapid detection of M. ovipneumoniae infection without DNA extraction, suggesting its potential as a point-of-care test for field settings. |
format | Online Article Text |
id | pubmed-7861559 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-78615592021-02-12 Recombinase polymerase amplification assay combined with a dipstick-readout for rapid detection of Mycoplasma ovipneumoniae infections Gupta, Sandeep K. Deng, Qing Gupta, Tanushree B. Maclean, Paul Jores, Joerg Heiser, Axel Wedlock, D. Neil PLoS One Research Article Mycoplasma ovipneumoniae infects both sheep and goats causing pneumonia resulting in considerable economic losses worldwide. Current diagnosis methods such as bacteriological culture, serology, and PCR are time consuming and require sophisticated laboratory setups. Here we report the development of two rapid, specific and sensitive assays; an isothermal DNA amplification using recombinase polymerase amplification (RPA) and a real-time PCR for the detection of M. ovipneumoniae. The target for both assays is a specific region of gene WP_069098309.1, which encodes a hypothetical protein and is conserved in the genome sequences of ten publicly available M. ovipneumoniae strains. The RPA assay performed well at 39°C for 20 min and was combined with a lateral flow dipstick (RPA-LFD) for easy visualization of the amplicons. The detection limit of the RPA-LFD assay was nine genome copies of M. ovipneumoniae per reaction and was comparable to sensitivity of the real-time PCR assay. Both assays showed no cross-reaction with 38 other ovine and caprine pathogenic microorganisms and two parasites of ruminants, demonstrating a high degree of specificity. The assays were validated using bronchoalveolar lavage fluid and nasal swab samples collected from sheep. The positive rate of RPA-LFD (97.4%) was higher than the real-time PCR (95.8%) with DNA as a template purified from the clinical samples. The RPA assay was significantly better at detecting M. ovipneumoniae in clinical samples compared to the real-time PCR when DNA extraction was omitted (50% and 34.4% positive rate for RPA-LFD and real-time PCR respectively). The RPA-LFD developed here allows easy and rapid detection of M. ovipneumoniae infection without DNA extraction, suggesting its potential as a point-of-care test for field settings. Public Library of Science 2021-02-04 /pmc/articles/PMC7861559/ /pubmed/33539437 http://dx.doi.org/10.1371/journal.pone.0246573 Text en © 2021 Gupta et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Gupta, Sandeep K. Deng, Qing Gupta, Tanushree B. Maclean, Paul Jores, Joerg Heiser, Axel Wedlock, D. Neil Recombinase polymerase amplification assay combined with a dipstick-readout for rapid detection of Mycoplasma ovipneumoniae infections |
title | Recombinase polymerase amplification assay combined with a dipstick-readout for rapid detection of Mycoplasma ovipneumoniae infections |
title_full | Recombinase polymerase amplification assay combined with a dipstick-readout for rapid detection of Mycoplasma ovipneumoniae infections |
title_fullStr | Recombinase polymerase amplification assay combined with a dipstick-readout for rapid detection of Mycoplasma ovipneumoniae infections |
title_full_unstemmed | Recombinase polymerase amplification assay combined with a dipstick-readout for rapid detection of Mycoplasma ovipneumoniae infections |
title_short | Recombinase polymerase amplification assay combined with a dipstick-readout for rapid detection of Mycoplasma ovipneumoniae infections |
title_sort | recombinase polymerase amplification assay combined with a dipstick-readout for rapid detection of mycoplasma ovipneumoniae infections |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7861559/ https://www.ncbi.nlm.nih.gov/pubmed/33539437 http://dx.doi.org/10.1371/journal.pone.0246573 |
work_keys_str_mv | AT guptasandeepk recombinasepolymeraseamplificationassaycombinedwithadipstickreadoutforrapiddetectionofmycoplasmaovipneumoniaeinfections AT dengqing recombinasepolymeraseamplificationassaycombinedwithadipstickreadoutforrapiddetectionofmycoplasmaovipneumoniaeinfections AT guptatanushreeb recombinasepolymeraseamplificationassaycombinedwithadipstickreadoutforrapiddetectionofmycoplasmaovipneumoniaeinfections AT macleanpaul recombinasepolymeraseamplificationassaycombinedwithadipstickreadoutforrapiddetectionofmycoplasmaovipneumoniaeinfections AT joresjoerg recombinasepolymeraseamplificationassaycombinedwithadipstickreadoutforrapiddetectionofmycoplasmaovipneumoniaeinfections AT heiseraxel recombinasepolymeraseamplificationassaycombinedwithadipstickreadoutforrapiddetectionofmycoplasmaovipneumoniaeinfections AT wedlockdneil recombinasepolymeraseamplificationassaycombinedwithadipstickreadoutforrapiddetectionofmycoplasmaovipneumoniaeinfections |