Serial Quantitative PCR Assay for Detection, Species Discrimination, and Quantification of Leishmania spp. in Human Samples

Detalhes bibliográficos
Autor(a) principal: Weirather, Jason L.
Data de Publicação: 2011
Outros Autores: Jeronimo, Selma M. B., Gautam, Shalini, Sundar, Shyam, Kang, Mitchell, Kurtz, Melissa A., Haque, Rashidul, Schriefer, Nicolaus Albert Borges, Talhari, Sinésio, Carvalho Filho, Edgar Marcelino de, Donelson, John E., Wilson, Mary E.
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Repositório Institucional da UFBA
Texto Completo: http://repositorio.ufba.br/ri/handle/ri/18027
Resumo: Texto completo: acesso restrito. p. 3892–3904
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spelling Weirather, Jason L.Jeronimo, Selma M. B.Gautam, ShaliniSundar, ShyamKang, MitchellKurtz, Melissa A.Haque, RashidulSchriefer, Nicolaus Albert BorgesTalhari, SinésioCarvalho Filho, Edgar Marcelino deDonelson, John E.Wilson, Mary E.2015-08-19T16:35:59Z20110095-1137http://repositorio.ufba.br/ri/handle/ri/18027v. 49, n. 11Texto completo: acesso restrito. p. 3892–3904The Leishmania species cause a variety of human disease syndromes. Methods for diagnosis and species differentiation are insensitive and many require invasive sampling. Although quantitative PCR (qPCR) methods are reported for leishmania detection, no systematic method to quantify parasites and determine the species in clinical specimens is established. We developed a serial qPCR strategy to identify and rapidly differentiate Leishmania species and quantify parasites in clinical or environmental specimens. SYBR green qPCR is mainly employed, with corresponding TaqMan assays for validation. The screening primers recognize kinetoplast minicircle DNA of all Leishmania species. Species identification employs further qPCR set(s) individualized for geographic regions, combining species-discriminating probes with melt curve analysis. The assay was sufficient to detect Leishmania parasites, make species determinations, and quantify Leishmania spp. in sera, cutaneous biopsy specimens, or cultured isolates from subjects from Bangladesh or Brazil with different forms of leishmaniasis. The multicopy kinetoplast DNA (kDNA) probes were the most sensitive and useful for quantification based on promastigote standard curves. To test their validity for quantification, kDNA copy numbers were compared between Leishmania species, isolates, and life stages using qPCR. Maxicircle and minicircle copy numbers differed up to 6-fold between Leishmania species, but the differences were smaller between strains of the same species. Amastigote and promastigote leishmania life stages retained similar numbers of kDNA maxi- or minicircles. Thus, serial qPCR is useful for leishmania detection and species determination and for absolute quantification when compared to a standard curve from the same Leishmania species.Submitted by Edileide Reis (leyde-landy@hotmail.com) on 2014-04-02T16:24:44Z No. of bitstreams: 1 Edgar M. Carvalho.pdf: 1690161 bytes, checksum: 927df0e53a83a88ee4994f5e3a66203e (MD5)Approved for entry into archive by Flávia Ferreira (flaviaccf@yahoo.com.br) on 2015-08-19T16:35:59Z (GMT) No. of bitstreams: 1 Edgar M. Carvalho.pdf: 1690161 bytes, checksum: 927df0e53a83a88ee4994f5e3a66203e (MD5)Made available in DSpace on 2015-08-19T16:35:59Z (GMT). No. of bitstreams: 1 Edgar M. Carvalho.pdf: 1690161 bytes, checksum: 927df0e53a83a88ee4994f5e3a66203e (MD5) Previous issue date: 2011http://dx.doi.org/ 10.1128/JCM.r00764-11reponame:Repositório Institucional da UFBAinstname:Universidade Federal da Bahia (UFBA)instacron:UFBALeishmaniaPolymerase Chain ReactionSerial Quantitative PCR Assay for Detection, Species Discrimination, and Quantification of Leishmania spp. in Human SamplesJournal of Clinical Microbiologyinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/article10000-01-01info:eu-repo/semantics/openAccessengORIGINALEdgar M. Carvalho.pdfEdgar M. Carvalho.pdfapplication/pdf1690161https://repositorio.ufba.br/bitstream/ri/18027/1/Edgar%20M.%20Carvalho.pdf927df0e53a83a88ee4994f5e3a66203eMD51LICENSElicense.txtlicense.txttext/plain1345https://repositorio.ufba.br/bitstream/ri/18027/2/license.txt0d4b811ef71182510d2015daa7c8a900MD52TEXTEdgar M. Carvalho.pdf.txtEdgar M. 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dc.title.pt_BR.fl_str_mv Serial Quantitative PCR Assay for Detection, Species Discrimination, and Quantification of Leishmania spp. in Human Samples
dc.title.alternative.pt_BR.fl_str_mv Journal of Clinical Microbiology
title Serial Quantitative PCR Assay for Detection, Species Discrimination, and Quantification of Leishmania spp. in Human Samples
spellingShingle Serial Quantitative PCR Assay for Detection, Species Discrimination, and Quantification of Leishmania spp. in Human Samples
Weirather, Jason L.
Leishmania
Polymerase Chain Reaction
title_short Serial Quantitative PCR Assay for Detection, Species Discrimination, and Quantification of Leishmania spp. in Human Samples
title_full Serial Quantitative PCR Assay for Detection, Species Discrimination, and Quantification of Leishmania spp. in Human Samples
title_fullStr Serial Quantitative PCR Assay for Detection, Species Discrimination, and Quantification of Leishmania spp. in Human Samples
title_full_unstemmed Serial Quantitative PCR Assay for Detection, Species Discrimination, and Quantification of Leishmania spp. in Human Samples
title_sort Serial Quantitative PCR Assay for Detection, Species Discrimination, and Quantification of Leishmania spp. in Human Samples
author Weirather, Jason L.
author_facet Weirather, Jason L.
Jeronimo, Selma M. B.
Gautam, Shalini
Sundar, Shyam
Kang, Mitchell
Kurtz, Melissa A.
Haque, Rashidul
Schriefer, Nicolaus Albert Borges
Talhari, Sinésio
Carvalho Filho, Edgar Marcelino de
Donelson, John E.
Wilson, Mary E.
author_role author
author2 Jeronimo, Selma M. B.
Gautam, Shalini
Sundar, Shyam
Kang, Mitchell
Kurtz, Melissa A.
Haque, Rashidul
Schriefer, Nicolaus Albert Borges
Talhari, Sinésio
Carvalho Filho, Edgar Marcelino de
Donelson, John E.
Wilson, Mary E.
author2_role author
author
author
author
author
author
author
author
author
author
author
dc.contributor.author.fl_str_mv Weirather, Jason L.
Jeronimo, Selma M. B.
Gautam, Shalini
Sundar, Shyam
Kang, Mitchell
Kurtz, Melissa A.
Haque, Rashidul
Schriefer, Nicolaus Albert Borges
Talhari, Sinésio
Carvalho Filho, Edgar Marcelino de
Donelson, John E.
Wilson, Mary E.
dc.subject.por.fl_str_mv Leishmania
Polymerase Chain Reaction
topic Leishmania
Polymerase Chain Reaction
description Texto completo: acesso restrito. p. 3892–3904
publishDate 2011
dc.date.issued.fl_str_mv 2011
dc.date.accessioned.fl_str_mv 2015-08-19T16:35:59Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://repositorio.ufba.br/ri/handle/ri/18027
dc.identifier.issn.none.fl_str_mv 0095-1137
dc.identifier.number.pt_BR.fl_str_mv v. 49, n. 11
identifier_str_mv 0095-1137
v. 49, n. 11
url http://repositorio.ufba.br/ri/handle/ri/18027
dc.language.iso.fl_str_mv eng
language eng
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.source.pt_BR.fl_str_mv http://dx.doi.org/ 10.1128/JCM.r00764-11
dc.source.none.fl_str_mv reponame:Repositório Institucional da UFBA
instname:Universidade Federal da Bahia (UFBA)
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instname_str Universidade Federal da Bahia (UFBA)
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reponame_str Repositório Institucional da UFBA
collection Repositório Institucional da UFBA
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