Cargando…
A versatile isothermal amplification assay for the detection of leptospires from various sample types
BACKGROUND: Leptospirosis is a zoonotic disease caused by bacteria of the genus Leptospira that affects both humans and animals worldwide. Early detection of the pathogen in humans is crucial for early intervention and control of the progression of the disease to a severe state. It is also vitally i...
Autores principales: | , , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
PeerJ Inc.
2022
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8918162/ https://www.ncbi.nlm.nih.gov/pubmed/35291487 http://dx.doi.org/10.7717/peerj.12850 |
_version_ | 1784668674670460928 |
---|---|
author | Othman, Shuhaidah Lee, Pui-Yuei Lam, Jia-Yong Philip, Noraini Azhari, Nurul Natasya Affendy, Norliza Bahtiar Masri, Siti Norbaya Neela, Vasantha Kumari Mohd-Taib, Farah Shafawati Chee, Hui-Yee |
author_facet | Othman, Shuhaidah Lee, Pui-Yuei Lam, Jia-Yong Philip, Noraini Azhari, Nurul Natasya Affendy, Norliza Bahtiar Masri, Siti Norbaya Neela, Vasantha Kumari Mohd-Taib, Farah Shafawati Chee, Hui-Yee |
author_sort | Othman, Shuhaidah |
collection | PubMed |
description | BACKGROUND: Leptospirosis is a zoonotic disease caused by bacteria of the genus Leptospira that affects both humans and animals worldwide. Early detection of the pathogen in humans is crucial for early intervention and control of the progression of the disease to a severe state. It is also vitally important to be able to detect the presence of the pathogen in carrier animals to control the spread of the disease from the environment. Here we developed a simple and rapid loop-mediated isothermal amplification (LAMP) assay targeting the leptospiral secY gene. RESULTS: Several reaction conditions of the LAMP reaction were optimized to ensure efficient amplification of the target DNA. The sensitivity of the developed LAMP assay obtained using a pure Leptospira culture was 2 × 10(4) copies of genomic DNA per reaction (equivalent to 0.1 ng) for a 40-minute reaction time. No cross-reactions were observed in the LAMP reaction against a series of non-leptospiral bacteria, indicating a specific reaction. The applicability of the LAMP assay was demonstrated on human blood and urine specimens collected from suspected leptospirosis patients and rat kidney specimens collected from suspected leptospirosis outbreak areas and high-risk areas. The developed LAMP assay demonstrated a higher detection rate for leptospiral DNA compared with the polymerase chain reaction (PCR) assay, possibly due to the presence of inhibitory substances, especially in rat kidney specimens, to which the PCR method is more susceptible. The present findings also highlight the importance of urine sample collection from patients for routine monitoring of the disease. CONCLUSIONS: In short, the developed LAMP assay can serve as a feasible alternative tool for the diagnosis of leptospirosis and be used for epidemiological and environmental surveillance of the disease, considering its robustness, rapidity, sensitivity, and specificity, as demonstrated in this study. |
format | Online Article Text |
id | pubmed-8918162 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | PeerJ Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-89181622022-03-14 A versatile isothermal amplification assay for the detection of leptospires from various sample types Othman, Shuhaidah Lee, Pui-Yuei Lam, Jia-Yong Philip, Noraini Azhari, Nurul Natasya Affendy, Norliza Bahtiar Masri, Siti Norbaya Neela, Vasantha Kumari Mohd-Taib, Farah Shafawati Chee, Hui-Yee PeerJ Microbiology BACKGROUND: Leptospirosis is a zoonotic disease caused by bacteria of the genus Leptospira that affects both humans and animals worldwide. Early detection of the pathogen in humans is crucial for early intervention and control of the progression of the disease to a severe state. It is also vitally important to be able to detect the presence of the pathogen in carrier animals to control the spread of the disease from the environment. Here we developed a simple and rapid loop-mediated isothermal amplification (LAMP) assay targeting the leptospiral secY gene. RESULTS: Several reaction conditions of the LAMP reaction were optimized to ensure efficient amplification of the target DNA. The sensitivity of the developed LAMP assay obtained using a pure Leptospira culture was 2 × 10(4) copies of genomic DNA per reaction (equivalent to 0.1 ng) for a 40-minute reaction time. No cross-reactions were observed in the LAMP reaction against a series of non-leptospiral bacteria, indicating a specific reaction. The applicability of the LAMP assay was demonstrated on human blood and urine specimens collected from suspected leptospirosis patients and rat kidney specimens collected from suspected leptospirosis outbreak areas and high-risk areas. The developed LAMP assay demonstrated a higher detection rate for leptospiral DNA compared with the polymerase chain reaction (PCR) assay, possibly due to the presence of inhibitory substances, especially in rat kidney specimens, to which the PCR method is more susceptible. The present findings also highlight the importance of urine sample collection from patients for routine monitoring of the disease. CONCLUSIONS: In short, the developed LAMP assay can serve as a feasible alternative tool for the diagnosis of leptospirosis and be used for epidemiological and environmental surveillance of the disease, considering its robustness, rapidity, sensitivity, and specificity, as demonstrated in this study. PeerJ Inc. 2022-03-10 /pmc/articles/PMC8918162/ /pubmed/35291487 http://dx.doi.org/10.7717/peerj.12850 Text en ©2022 Othman et al. https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited. |
spellingShingle | Microbiology Othman, Shuhaidah Lee, Pui-Yuei Lam, Jia-Yong Philip, Noraini Azhari, Nurul Natasya Affendy, Norliza Bahtiar Masri, Siti Norbaya Neela, Vasantha Kumari Mohd-Taib, Farah Shafawati Chee, Hui-Yee A versatile isothermal amplification assay for the detection of leptospires from various sample types |
title | A versatile isothermal amplification assay for the detection of leptospires from various sample types |
title_full | A versatile isothermal amplification assay for the detection of leptospires from various sample types |
title_fullStr | A versatile isothermal amplification assay for the detection of leptospires from various sample types |
title_full_unstemmed | A versatile isothermal amplification assay for the detection of leptospires from various sample types |
title_short | A versatile isothermal amplification assay for the detection of leptospires from various sample types |
title_sort | versatile isothermal amplification assay for the detection of leptospires from various sample types |
topic | Microbiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8918162/ https://www.ncbi.nlm.nih.gov/pubmed/35291487 http://dx.doi.org/10.7717/peerj.12850 |
work_keys_str_mv | AT othmanshuhaidah aversatileisothermalamplificationassayforthedetectionofleptospiresfromvarioussampletypes AT leepuiyuei aversatileisothermalamplificationassayforthedetectionofleptospiresfromvarioussampletypes AT lamjiayong aversatileisothermalamplificationassayforthedetectionofleptospiresfromvarioussampletypes AT philipnoraini aversatileisothermalamplificationassayforthedetectionofleptospiresfromvarioussampletypes AT azharinurulnatasya aversatileisothermalamplificationassayforthedetectionofleptospiresfromvarioussampletypes AT affendynorlizabahtiar aversatileisothermalamplificationassayforthedetectionofleptospiresfromvarioussampletypes AT masrisitinorbaya aversatileisothermalamplificationassayforthedetectionofleptospiresfromvarioussampletypes AT neelavasanthakumari aversatileisothermalamplificationassayforthedetectionofleptospiresfromvarioussampletypes AT mohdtaibfarahshafawati aversatileisothermalamplificationassayforthedetectionofleptospiresfromvarioussampletypes AT cheehuiyee aversatileisothermalamplificationassayforthedetectionofleptospiresfromvarioussampletypes AT othmanshuhaidah versatileisothermalamplificationassayforthedetectionofleptospiresfromvarioussampletypes AT leepuiyuei versatileisothermalamplificationassayforthedetectionofleptospiresfromvarioussampletypes AT lamjiayong versatileisothermalamplificationassayforthedetectionofleptospiresfromvarioussampletypes AT philipnoraini versatileisothermalamplificationassayforthedetectionofleptospiresfromvarioussampletypes AT azharinurulnatasya versatileisothermalamplificationassayforthedetectionofleptospiresfromvarioussampletypes AT affendynorlizabahtiar versatileisothermalamplificationassayforthedetectionofleptospiresfromvarioussampletypes AT masrisitinorbaya versatileisothermalamplificationassayforthedetectionofleptospiresfromvarioussampletypes AT neelavasanthakumari versatileisothermalamplificationassayforthedetectionofleptospiresfromvarioussampletypes AT mohdtaibfarahshafawati versatileisothermalamplificationassayforthedetectionofleptospiresfromvarioussampletypes AT cheehuiyee versatileisothermalamplificationassayforthedetectionofleptospiresfromvarioussampletypes |