Real-time PCR detection of Plasmodium directly from whole blood and filter paper samples
Abstract Background Real-time PCR is a sensitive and specific method for the analysis of Plasmodium DNA. However, prior purification of genomic DNA from blood is necessary since PCR inhibitors and quenching of fluorophores from blood prevent efficient amplification and detection of PCR products. Met...
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ftdoajarticles:oai:doaj.org/article:6f451aaa9c044c41a5f0c3cbf9bd83fe 2023-05-15T15:06:37+02:00 Real-time PCR detection of Plasmodium directly from whole blood and filter paper samples Maestre Amanda Agudelo Olga M Arango Eliana Martin Kimberly A Taylor Brian J Yanow Stephanie K 2011-08-01T00:00:00Z https://doi.org/10.1186/1475-2875-10-244 https://doaj.org/article/6f451aaa9c044c41a5f0c3cbf9bd83fe EN eng BMC http://www.malariajournal.com/content/10/1/244 https://doaj.org/toc/1475-2875 doi:10.1186/1475-2875-10-244 1475-2875 https://doaj.org/article/6f451aaa9c044c41a5f0c3cbf9bd83fe Malaria Journal, Vol 10, Iss 1, p 244 (2011) Arctic medicine. Tropical medicine RC955-962 Infectious and parasitic diseases RC109-216 article 2011 ftdoajarticles https://doi.org/10.1186/1475-2875-10-244 2022-12-31T13:44:39Z Abstract Background Real-time PCR is a sensitive and specific method for the analysis of Plasmodium DNA. However, prior purification of genomic DNA from blood is necessary since PCR inhibitors and quenching of fluorophores from blood prevent efficient amplification and detection of PCR products. Methods Reagents designed to specifically overcome PCR inhibition and quenching of fluorescence were evaluated for real-time PCR amplification of Plasmodium DNA directly from blood. Whole blood from clinical samples and dried blood spots collected in the field in Colombia were tested. Results Amplification and fluorescence detection by real-time PCR were optimal with 40× SYBR ® Green dye and 5% blood volume in the PCR reaction. Plasmodium DNA was detected directly from both whole blood and dried blood spots from clinical samples. The sensitivity and specificity ranged from 93-100% compared with PCR performed on purified Plasmodium DNA. Conclusions The methodology described facilitates high-throughput testing of blood samples collected in the field by fluorescence-based real-time PCR. This method can be applied to a broad range of clinical studies with the advantages of immediate sample testing, lower experimental costs and time-savings. Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic Malaria Journal 10 1 244 |
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Directory of Open Access Journals: DOAJ Articles |
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English |
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Arctic medicine. Tropical medicine RC955-962 Infectious and parasitic diseases RC109-216 |
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Arctic medicine. Tropical medicine RC955-962 Infectious and parasitic diseases RC109-216 Maestre Amanda Agudelo Olga M Arango Eliana Martin Kimberly A Taylor Brian J Yanow Stephanie K Real-time PCR detection of Plasmodium directly from whole blood and filter paper samples |
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Arctic medicine. Tropical medicine RC955-962 Infectious and parasitic diseases RC109-216 |
description |
Abstract Background Real-time PCR is a sensitive and specific method for the analysis of Plasmodium DNA. However, prior purification of genomic DNA from blood is necessary since PCR inhibitors and quenching of fluorophores from blood prevent efficient amplification and detection of PCR products. Methods Reagents designed to specifically overcome PCR inhibition and quenching of fluorescence were evaluated for real-time PCR amplification of Plasmodium DNA directly from blood. Whole blood from clinical samples and dried blood spots collected in the field in Colombia were tested. Results Amplification and fluorescence detection by real-time PCR were optimal with 40× SYBR ® Green dye and 5% blood volume in the PCR reaction. Plasmodium DNA was detected directly from both whole blood and dried blood spots from clinical samples. The sensitivity and specificity ranged from 93-100% compared with PCR performed on purified Plasmodium DNA. Conclusions The methodology described facilitates high-throughput testing of blood samples collected in the field by fluorescence-based real-time PCR. This method can be applied to a broad range of clinical studies with the advantages of immediate sample testing, lower experimental costs and time-savings. |
format |
Article in Journal/Newspaper |
author |
Maestre Amanda Agudelo Olga M Arango Eliana Martin Kimberly A Taylor Brian J Yanow Stephanie K |
author_facet |
Maestre Amanda Agudelo Olga M Arango Eliana Martin Kimberly A Taylor Brian J Yanow Stephanie K |
author_sort |
Maestre Amanda |
title |
Real-time PCR detection of Plasmodium directly from whole blood and filter paper samples |
title_short |
Real-time PCR detection of Plasmodium directly from whole blood and filter paper samples |
title_full |
Real-time PCR detection of Plasmodium directly from whole blood and filter paper samples |
title_fullStr |
Real-time PCR detection of Plasmodium directly from whole blood and filter paper samples |
title_full_unstemmed |
Real-time PCR detection of Plasmodium directly from whole blood and filter paper samples |
title_sort |
real-time pcr detection of plasmodium directly from whole blood and filter paper samples |
publisher |
BMC |
publishDate |
2011 |
url |
https://doi.org/10.1186/1475-2875-10-244 https://doaj.org/article/6f451aaa9c044c41a5f0c3cbf9bd83fe |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
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Arctic |
op_source |
Malaria Journal, Vol 10, Iss 1, p 244 (2011) |
op_relation |
http://www.malariajournal.com/content/10/1/244 https://doaj.org/toc/1475-2875 doi:10.1186/1475-2875-10-244 1475-2875 https://doaj.org/article/6f451aaa9c044c41a5f0c3cbf9bd83fe |
op_doi |
https://doi.org/10.1186/1475-2875-10-244 |
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Malaria Journal |
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10 |
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1 |
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244 |
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1766338193431461888 |