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Development and evaluation of a Loop Mediated Isothermal Amplification (LAMP) technique for the detection of hookworm (Necator americanus) infection in fecal samples
BACKGROUND: Hookworm infection is a major concern in sub-Saharan Africa, particularly in children and pregnant women. Necator americanus and Ancylostoma duodenale are responsible for this condition. Hookworm disease is one of the Neglected tropical diseases (NTDs) that are targeted for elimination t...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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BioMed Central
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4636844/ https://www.ncbi.nlm.nih.gov/pubmed/26546069 http://dx.doi.org/10.1186/s13071-015-1183-9 |
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author | Mugambi, Robert Muriuki Agola, Eric L. Mwangi, Ibrahim N. Kinyua, Johnson Shiraho, Esther Andia Mkoji, Gerald M. |
author_facet | Mugambi, Robert Muriuki Agola, Eric L. Mwangi, Ibrahim N. Kinyua, Johnson Shiraho, Esther Andia Mkoji, Gerald M. |
author_sort | Mugambi, Robert Muriuki |
collection | PubMed |
description | BACKGROUND: Hookworm infection is a major concern in sub-Saharan Africa, particularly in children and pregnant women. Necator americanus and Ancylostoma duodenale are responsible for this condition. Hookworm disease is one of the Neglected tropical diseases (NTDs) that are targeted for elimination through global mass chemotherapy. To support this there is a need for reliable diagnostic tools. The conventional diagnostic test, Kato-Katz that is based on microscopic detection of parasite ova in faecal samples, is not effective due to its low sensitivity that is brought about mainly by non-random distribution of eggs in stool and day to day variation in egg output. It is tedious, cumbersome to perform and requires experience for correct diagnosis. LAMP-based tests are simple, relatively cheap, offer greater sensitivity, specificity than existing tests, have high throughput capability, and are ideal for use at the point of care. METHODS: We have developed a LAMP diagnostic test for detection of hookworm infection in faecal samples. LAMP relies on auto cycling strand displacement DNA synthesis performed at isothermal temperature by Bst polymerase and a set of 4 specific primers. The primers used in the LAMP assay were based on the second Internal Transcribed Spacer (ITS-2) region and designed using Primer Explorer version 4 Software. The ITS-2 region of the ribosomal gene (rDNA) was identified as a suitable target due to its low mutation rates and substantial differences between species. DNA was extracted directly from human faecal samples, followed by LAMP amplification at isothermal temperature of 63 °C for 1 h. Amplicons were visualized using gel electrophoresis and SYBR green dye. Both specificity and sensitivity of the assay were determined. RESULTS: The LAMP based technique developed was able to detect N. americanus DNA in faecal samples. The assay showed 100 % specificity and no cross-reaction was observed with other helminth parasites (S. mansoni, A. lumbricoides or T. trichiura). The developed LAMP assay was 97 % sensitive and DNA at concentrations as low as 0.4 fg were amplified. CONCLUSION: The LAMP assay developed is an appropriate diagnostic method for the detection of N. americanus DNA in human stool samples because of its simplicity, low cost, sensitivity, and specificity. It holds great promise as a useful diagnostic tool for use in disease control where infection intensities have been significantly reduced. |
format | Online Article Text |
id | pubmed-4636844 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-46368442015-11-08 Development and evaluation of a Loop Mediated Isothermal Amplification (LAMP) technique for the detection of hookworm (Necator americanus) infection in fecal samples Mugambi, Robert Muriuki Agola, Eric L. Mwangi, Ibrahim N. Kinyua, Johnson Shiraho, Esther Andia Mkoji, Gerald M. Parasit Vectors Research BACKGROUND: Hookworm infection is a major concern in sub-Saharan Africa, particularly in children and pregnant women. Necator americanus and Ancylostoma duodenale are responsible for this condition. Hookworm disease is one of the Neglected tropical diseases (NTDs) that are targeted for elimination through global mass chemotherapy. To support this there is a need for reliable diagnostic tools. The conventional diagnostic test, Kato-Katz that is based on microscopic detection of parasite ova in faecal samples, is not effective due to its low sensitivity that is brought about mainly by non-random distribution of eggs in stool and day to day variation in egg output. It is tedious, cumbersome to perform and requires experience for correct diagnosis. LAMP-based tests are simple, relatively cheap, offer greater sensitivity, specificity than existing tests, have high throughput capability, and are ideal for use at the point of care. METHODS: We have developed a LAMP diagnostic test for detection of hookworm infection in faecal samples. LAMP relies on auto cycling strand displacement DNA synthesis performed at isothermal temperature by Bst polymerase and a set of 4 specific primers. The primers used in the LAMP assay were based on the second Internal Transcribed Spacer (ITS-2) region and designed using Primer Explorer version 4 Software. The ITS-2 region of the ribosomal gene (rDNA) was identified as a suitable target due to its low mutation rates and substantial differences between species. DNA was extracted directly from human faecal samples, followed by LAMP amplification at isothermal temperature of 63 °C for 1 h. Amplicons were visualized using gel electrophoresis and SYBR green dye. Both specificity and sensitivity of the assay were determined. RESULTS: The LAMP based technique developed was able to detect N. americanus DNA in faecal samples. The assay showed 100 % specificity and no cross-reaction was observed with other helminth parasites (S. mansoni, A. lumbricoides or T. trichiura). The developed LAMP assay was 97 % sensitive and DNA at concentrations as low as 0.4 fg were amplified. CONCLUSION: The LAMP assay developed is an appropriate diagnostic method for the detection of N. americanus DNA in human stool samples because of its simplicity, low cost, sensitivity, and specificity. It holds great promise as a useful diagnostic tool for use in disease control where infection intensities have been significantly reduced. BioMed Central 2015-11-06 /pmc/articles/PMC4636844/ /pubmed/26546069 http://dx.doi.org/10.1186/s13071-015-1183-9 Text en © Mugambi et al. 2015 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Research Mugambi, Robert Muriuki Agola, Eric L. Mwangi, Ibrahim N. Kinyua, Johnson Shiraho, Esther Andia Mkoji, Gerald M. Development and evaluation of a Loop Mediated Isothermal Amplification (LAMP) technique for the detection of hookworm (Necator americanus) infection in fecal samples |
title | Development and evaluation of a Loop Mediated Isothermal Amplification (LAMP) technique for the detection of hookworm (Necator americanus) infection in fecal samples |
title_full | Development and evaluation of a Loop Mediated Isothermal Amplification (LAMP) technique for the detection of hookworm (Necator americanus) infection in fecal samples |
title_fullStr | Development and evaluation of a Loop Mediated Isothermal Amplification (LAMP) technique for the detection of hookworm (Necator americanus) infection in fecal samples |
title_full_unstemmed | Development and evaluation of a Loop Mediated Isothermal Amplification (LAMP) technique for the detection of hookworm (Necator americanus) infection in fecal samples |
title_short | Development and evaluation of a Loop Mediated Isothermal Amplification (LAMP) technique for the detection of hookworm (Necator americanus) infection in fecal samples |
title_sort | development and evaluation of a loop mediated isothermal amplification (lamp) technique for the detection of hookworm (necator americanus) infection in fecal samples |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4636844/ https://www.ncbi.nlm.nih.gov/pubmed/26546069 http://dx.doi.org/10.1186/s13071-015-1183-9 |
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