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Evaluation of the stability of lyophilized loop-mediated isothermal amplification reagents for the detection of Coxiella burnetii

Coxiella burnetii, the causative pathogen for Q fever, is an obligate intracellular bacterium and designated as a biosafety level 3 agent. Detection and quantification of the bacteria with conventional culturing methods is time-consuming and poses significant health risks. Polymerase chain reaction...

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Autores principales: Chen, Hua-Wei, Ching, Wei-Mei
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5639046/
https://www.ncbi.nlm.nih.gov/pubmed/29057336
http://dx.doi.org/10.1016/j.heliyon.2017.e00415
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author Chen, Hua-Wei
Ching, Wei-Mei
author_facet Chen, Hua-Wei
Ching, Wei-Mei
author_sort Chen, Hua-Wei
collection PubMed
description Coxiella burnetii, the causative pathogen for Q fever, is an obligate intracellular bacterium and designated as a biosafety level 3 agent. Detection and quantification of the bacteria with conventional culturing methods is time-consuming and poses significant health risks. Polymerase chain reaction (PCR)-based assays have been developed for detecting C. burnetii and could provide rapid diagnosis. However, they require specialized equipment, including a cold chain for PCR reagents that maintains their stability during storage and transport. These requirements limit the advantage of PCR-based methods, especially in resource-limited areas. Previously, we had developed a lyophilized loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii. To simplify and improve this assay, the reagents for the LAMP assay and the detecting reagent, SYBR green, were lyophilized together. The stability of the lyophilized reagents was evaluated by measuring changes in detection limit for plasmid DNA encoding a C. burnetii gene upon storage at 4 °C, 25 °C, or 37 °C. Our data indicate that the lyophilized reagents remain stable for 24 months when stored at 4 °C, 28 days at 25 °C, and 2 days at 37 °C. This improved LAMP assay can be easily performed in a simple water bath or heating block. The stability at ambient temperature, the simplicity of assay procedure, and the availability of low cost equipment make this method ideal for use in resource-limited settings where Q fever is endemic.
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spelling pubmed-56390462017-10-20 Evaluation of the stability of lyophilized loop-mediated isothermal amplification reagents for the detection of Coxiella burnetii Chen, Hua-Wei Ching, Wei-Mei Heliyon Article Coxiella burnetii, the causative pathogen for Q fever, is an obligate intracellular bacterium and designated as a biosafety level 3 agent. Detection and quantification of the bacteria with conventional culturing methods is time-consuming and poses significant health risks. Polymerase chain reaction (PCR)-based assays have been developed for detecting C. burnetii and could provide rapid diagnosis. However, they require specialized equipment, including a cold chain for PCR reagents that maintains their stability during storage and transport. These requirements limit the advantage of PCR-based methods, especially in resource-limited areas. Previously, we had developed a lyophilized loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii. To simplify and improve this assay, the reagents for the LAMP assay and the detecting reagent, SYBR green, were lyophilized together. The stability of the lyophilized reagents was evaluated by measuring changes in detection limit for plasmid DNA encoding a C. burnetii gene upon storage at 4 °C, 25 °C, or 37 °C. Our data indicate that the lyophilized reagents remain stable for 24 months when stored at 4 °C, 28 days at 25 °C, and 2 days at 37 °C. This improved LAMP assay can be easily performed in a simple water bath or heating block. The stability at ambient temperature, the simplicity of assay procedure, and the availability of low cost equipment make this method ideal for use in resource-limited settings where Q fever is endemic. Elsevier 2017-10-06 /pmc/articles/PMC5639046/ /pubmed/29057336 http://dx.doi.org/10.1016/j.heliyon.2017.e00415 Text en © 2017 Published by Elsevier Ltd. http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Article
Chen, Hua-Wei
Ching, Wei-Mei
Evaluation of the stability of lyophilized loop-mediated isothermal amplification reagents for the detection of Coxiella burnetii
title Evaluation of the stability of lyophilized loop-mediated isothermal amplification reagents for the detection of Coxiella burnetii
title_full Evaluation of the stability of lyophilized loop-mediated isothermal amplification reagents for the detection of Coxiella burnetii
title_fullStr Evaluation of the stability of lyophilized loop-mediated isothermal amplification reagents for the detection of Coxiella burnetii
title_full_unstemmed Evaluation of the stability of lyophilized loop-mediated isothermal amplification reagents for the detection of Coxiella burnetii
title_short Evaluation of the stability of lyophilized loop-mediated isothermal amplification reagents for the detection of Coxiella burnetii
title_sort evaluation of the stability of lyophilized loop-mediated isothermal amplification reagents for the detection of coxiella burnetii
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5639046/
https://www.ncbi.nlm.nih.gov/pubmed/29057336
http://dx.doi.org/10.1016/j.heliyon.2017.e00415
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