Low cost extraction and isothermal amplification of DNA for infectious diarrhea diagnosis.

In order to counter the common perception that molecular diagnostics are too complicated to work in low resource settings, we have performed a difficult sample preparation and DNA amplification protocol using instrumentation designed to be operated without wall or battery power. In this work we have...

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Autores principales:	Shichu Huang, Jaephil Do, Madhumita Mahalanabis, Andy Fan, Lei Zhao, Lisa Jepeal, Satish K Singh, Catherine M Klapperich
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/cd30a07b3191436991519cb09787dcf8
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spelling	oai:doaj.org-article:cd30a07b3191436991519cb09787dcf82021-11-18T07:51:32ZLow cost extraction and isothermal amplification of DNA for infectious diarrhea diagnosis.1932-620310.1371/journal.pone.0060059https://doaj.org/article/cd30a07b3191436991519cb09787dcf82013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23555883/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203In order to counter the common perception that molecular diagnostics are too complicated to work in low resource settings, we have performed a difficult sample preparation and DNA amplification protocol using instrumentation designed to be operated without wall or battery power. In this work we have combined a nearly electricity-free nucleic acid extraction process with an electricity-free isothermal amplification assay to detect the presence of Clostridium difficile (C. difficile) DNA in the stool of infected patients. We used helicase-dependent isothermal amplification (HDA) to amplify the DNA in a low-cost, thermoplastic reaction chip heated with a pair of commercially available toe warmers, while using a simple Styrofoam insulator. DNA was extracted from known positive and negative stool samples. The DNA extraction protocol utilized an air pressure driven solid phase extraction device run using a standard bicycle pump. The simple heater setup required no electricity or battery and was capable of maintaining the temperature at 65°C±2°C for 55 min, suitable for repeatable HDA amplification. Experiments were performed to explore the adaptability of the system for use in a range of ambient conditions. When compared to a traditional centrifuge extraction protocol and a laboratory thermocycler, this disposable, no power platform achieved approximately the same lower limit of detection (1.25×10(-2) pg of C. difficile DNA) while requiring much less raw material and a fraction of the lab infrastructure and cost. This proof of concept study could greatly impact the accessibility of molecular assays for applications in global health.Shichu HuangJaephil DoMadhumita MahalanabisAndy FanLei ZhaoLisa JepealSatish K SinghCatherine M KlapperichPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 3, p e60059 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Shichu Huang Jaephil Do Madhumita Mahalanabis Andy Fan Lei Zhao Lisa Jepeal Satish K Singh Catherine M Klapperich Low cost extraction and isothermal amplification of DNA for infectious diarrhea diagnosis.
description	In order to counter the common perception that molecular diagnostics are too complicated to work in low resource settings, we have performed a difficult sample preparation and DNA amplification protocol using instrumentation designed to be operated without wall or battery power. In this work we have combined a nearly electricity-free nucleic acid extraction process with an electricity-free isothermal amplification assay to detect the presence of Clostridium difficile (C. difficile) DNA in the stool of infected patients. We used helicase-dependent isothermal amplification (HDA) to amplify the DNA in a low-cost, thermoplastic reaction chip heated with a pair of commercially available toe warmers, while using a simple Styrofoam insulator. DNA was extracted from known positive and negative stool samples. The DNA extraction protocol utilized an air pressure driven solid phase extraction device run using a standard bicycle pump. The simple heater setup required no electricity or battery and was capable of maintaining the temperature at 65°C±2°C for 55 min, suitable for repeatable HDA amplification. Experiments were performed to explore the adaptability of the system for use in a range of ambient conditions. When compared to a traditional centrifuge extraction protocol and a laboratory thermocycler, this disposable, no power platform achieved approximately the same lower limit of detection (1.25×10(-2) pg of C. difficile DNA) while requiring much less raw material and a fraction of the lab infrastructure and cost. This proof of concept study could greatly impact the accessibility of molecular assays for applications in global health.
format	article
author	Shichu Huang Jaephil Do Madhumita Mahalanabis Andy Fan Lei Zhao Lisa Jepeal Satish K Singh Catherine M Klapperich
author_facet	Shichu Huang Jaephil Do Madhumita Mahalanabis Andy Fan Lei Zhao Lisa Jepeal Satish K Singh Catherine M Klapperich
author_sort	Shichu Huang
title	Low cost extraction and isothermal amplification of DNA for infectious diarrhea diagnosis.
title_short	Low cost extraction and isothermal amplification of DNA for infectious diarrhea diagnosis.
title_full	Low cost extraction and isothermal amplification of DNA for infectious diarrhea diagnosis.
title_fullStr	Low cost extraction and isothermal amplification of DNA for infectious diarrhea diagnosis.
title_full_unstemmed	Low cost extraction and isothermal amplification of DNA for infectious diarrhea diagnosis.
title_sort	low cost extraction and isothermal amplification of dna for infectious diarrhea diagnosis.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/cd30a07b3191436991519cb09787dcf8
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Low cost extraction and isothermal amplification of DNA for infectious diarrhea diagnosis.

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