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Improvement and Evaluation of Loop-Mediated Isothermal Amplification for Rapid Detection of Toxoplasma gondii Infection in Human Blood Samples

Loop-mediated isothermal amplification (LAMP), an attractive DNA amplification method, was developed as a valuable tool for the rapid detection of Toxoplasma gondii. In this study, species-specific LAMP primers were designed by targeting the AF146527 sequence, which was a conserved sequence of 200-...

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Autores principales: Sun, Xi-meng, Ji, Yong-sheng, Liu, Xian-yong, Xiang, Mei, He, Guang, Xie, Li, Suo, Jing-xia, Suo, Xun
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5215908/
https://www.ncbi.nlm.nih.gov/pubmed/28056092
http://dx.doi.org/10.1371/journal.pone.0169125
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author Sun, Xi-meng
Ji, Yong-sheng
Liu, Xian-yong
Xiang, Mei
He, Guang
Xie, Li
Suo, Jing-xia
Suo, Xun
author_facet Sun, Xi-meng
Ji, Yong-sheng
Liu, Xian-yong
Xiang, Mei
He, Guang
Xie, Li
Suo, Jing-xia
Suo, Xun
author_sort Sun, Xi-meng
collection PubMed
description Loop-mediated isothermal amplification (LAMP), an attractive DNA amplification method, was developed as a valuable tool for the rapid detection of Toxoplasma gondii. In this study, species-specific LAMP primers were designed by targeting the AF146527 sequence, which was a conserved sequence of 200- to 300-fold repetitive 529 bp fragment of T.gondii. LAMP reaction system was optimized so that it could detect the minimal DNA sample such as a single tachyzoite or 10 copies of recombinant plasmid. No cross-reactivity was found when using DNA from other parasites as templates. Subsequently, a total of 200 human blood samples were directly investigated by two diagnostic methods, LAMP and conventional PCR. Fourteen of 200 (7%) samples were positive for Toxoplasma by LAMP (the primers developed in this study), whereas only 5 of 200 (2.5%) were proved positive by conventional PCR. The procedure of the LAMP assay was very simple, as the reaction would be carried out in a single tube under isothermal conditions at 64°C and the result would be read out with 1 h (as early as 35 min with loop primers). Thus, this method has the advantages of rapid amplification, simple operation, and easy detection and would be useful for rapid and reliable clinical diagnosis of acute toxoplasmosis, especially in developing countries.
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spelling pubmed-52159082017-01-19 Improvement and Evaluation of Loop-Mediated Isothermal Amplification for Rapid Detection of Toxoplasma gondii Infection in Human Blood Samples Sun, Xi-meng Ji, Yong-sheng Liu, Xian-yong Xiang, Mei He, Guang Xie, Li Suo, Jing-xia Suo, Xun PLoS One Research Article Loop-mediated isothermal amplification (LAMP), an attractive DNA amplification method, was developed as a valuable tool for the rapid detection of Toxoplasma gondii. In this study, species-specific LAMP primers were designed by targeting the AF146527 sequence, which was a conserved sequence of 200- to 300-fold repetitive 529 bp fragment of T.gondii. LAMP reaction system was optimized so that it could detect the minimal DNA sample such as a single tachyzoite or 10 copies of recombinant plasmid. No cross-reactivity was found when using DNA from other parasites as templates. Subsequently, a total of 200 human blood samples were directly investigated by two diagnostic methods, LAMP and conventional PCR. Fourteen of 200 (7%) samples were positive for Toxoplasma by LAMP (the primers developed in this study), whereas only 5 of 200 (2.5%) were proved positive by conventional PCR. The procedure of the LAMP assay was very simple, as the reaction would be carried out in a single tube under isothermal conditions at 64°C and the result would be read out with 1 h (as early as 35 min with loop primers). Thus, this method has the advantages of rapid amplification, simple operation, and easy detection and would be useful for rapid and reliable clinical diagnosis of acute toxoplasmosis, especially in developing countries. Public Library of Science 2017-01-05 /pmc/articles/PMC5215908/ /pubmed/28056092 http://dx.doi.org/10.1371/journal.pone.0169125 Text en © 2017 Sun 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
Sun, Xi-meng
Ji, Yong-sheng
Liu, Xian-yong
Xiang, Mei
He, Guang
Xie, Li
Suo, Jing-xia
Suo, Xun
Improvement and Evaluation of Loop-Mediated Isothermal Amplification for Rapid Detection of Toxoplasma gondii Infection in Human Blood Samples
title Improvement and Evaluation of Loop-Mediated Isothermal Amplification for Rapid Detection of Toxoplasma gondii Infection in Human Blood Samples
title_full Improvement and Evaluation of Loop-Mediated Isothermal Amplification for Rapid Detection of Toxoplasma gondii Infection in Human Blood Samples
title_fullStr Improvement and Evaluation of Loop-Mediated Isothermal Amplification for Rapid Detection of Toxoplasma gondii Infection in Human Blood Samples
title_full_unstemmed Improvement and Evaluation of Loop-Mediated Isothermal Amplification for Rapid Detection of Toxoplasma gondii Infection in Human Blood Samples
title_short Improvement and Evaluation of Loop-Mediated Isothermal Amplification for Rapid Detection of Toxoplasma gondii Infection in Human Blood Samples
title_sort improvement and evaluation of loop-mediated isothermal amplification for rapid detection of toxoplasma gondii infection in human blood samples
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5215908/
https://www.ncbi.nlm.nih.gov/pubmed/28056092
http://dx.doi.org/10.1371/journal.pone.0169125
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