A reverse transcriptase-PCR assay for detecting filarial infective larvae in mosquitoes.

<h4>Background</h4>Existing molecular assays for filarial parasite DNA in mosquitoes cannot distinguish between infected mosquitoes that contain any stage of the parasite and infective mosquitoes that harbor third stage larvae (L3) capable of establishing new infections in humans. We now...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Sandra J Laney, Caitlin J Buttaro, Sabato Visconti, Nils Pilotte, Reda M R Ramzy, Gary J Weil, Steven A Williams
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2008
Materias:	Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270
Acceso en línea:	https://doaj.org/article/9577879a77924192a45507106a3e454d
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:9577879a77924192a45507106a3e454d
record_format	dspace
spelling	oai:doaj.org-article:9577879a77924192a45507106a3e454d2021-11-25T06:32:32ZA reverse transcriptase-PCR assay for detecting filarial infective larvae in mosquitoes.1935-27271935-273510.1371/journal.pntd.0000251https://doaj.org/article/9577879a77924192a45507106a3e454d2008-06-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/18560545/?tool=EBIhttps://doaj.org/toc/1935-2727https://doaj.org/toc/1935-2735<h4>Background</h4>Existing molecular assays for filarial parasite DNA in mosquitoes cannot distinguish between infected mosquitoes that contain any stage of the parasite and infective mosquitoes that harbor third stage larvae (L3) capable of establishing new infections in humans. We now report development of a molecular L3-detection assay for Brugia malayi in vectors based on RT-PCR detection of an L3-activated gene transcript.<h4>Methodology/principal findings</h4>Candidate genes identified by bioinformatics analysis of EST datasets across the B. malayi life cycle were initially screened by PCR using cDNA libraries as templates. Stage-specificity was confirmed using RNA isolated from infected mosquitoes. Mosquitoes were collected daily for 14 days after feeding on microfilaremic cat blood. RT-PCR was performed with primer sets that were specific for individual candidate genes. Many promising candidates with strong expression in the L3 stage were excluded because of low-level transcription in less mature larvae. One transcript (TC8100, which encodes a particular form of collagen) was only detected in mosquitoes that contained L3 larvae. This assay detects a single L3 in a pool of 25 mosquitoes.<h4>Conclusions/significance</h4>This L3-activated gene transcript, combined with a control transcript (tph-1, accession # U80971) that is constitutively expressed by all vector-stage filarial larvae, can be used to detect filarial infectivity in pools of mosquito vectors. This general approach (detection of stage-specific gene transcripts from eukaryotic pathogens) may also be useful for detecting infective stages of other vector-borne parasites.Sandra J LaneyCaitlin J ButtaroSabato ViscontiNils PilotteReda M R RamzyGary J WeilSteven A WilliamsPublic Library of Science (PLoS)articleArctic medicine. Tropical medicineRC955-962Public aspects of medicineRA1-1270ENPLoS Neglected Tropical Diseases, Vol 2, Iss 6, p e251 (2008)
institution	DOAJ
collection	DOAJ
language	EN
topic	Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270
spellingShingle	Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 Sandra J Laney Caitlin J Buttaro Sabato Visconti Nils Pilotte Reda M R Ramzy Gary J Weil Steven A Williams A reverse transcriptase-PCR assay for detecting filarial infective larvae in mosquitoes.
description	<h4>Background</h4>Existing molecular assays for filarial parasite DNA in mosquitoes cannot distinguish between infected mosquitoes that contain any stage of the parasite and infective mosquitoes that harbor third stage larvae (L3) capable of establishing new infections in humans. We now report development of a molecular L3-detection assay for Brugia malayi in vectors based on RT-PCR detection of an L3-activated gene transcript.<h4>Methodology/principal findings</h4>Candidate genes identified by bioinformatics analysis of EST datasets across the B. malayi life cycle were initially screened by PCR using cDNA libraries as templates. Stage-specificity was confirmed using RNA isolated from infected mosquitoes. Mosquitoes were collected daily for 14 days after feeding on microfilaremic cat blood. RT-PCR was performed with primer sets that were specific for individual candidate genes. Many promising candidates with strong expression in the L3 stage were excluded because of low-level transcription in less mature larvae. One transcript (TC8100, which encodes a particular form of collagen) was only detected in mosquitoes that contained L3 larvae. This assay detects a single L3 in a pool of 25 mosquitoes.<h4>Conclusions/significance</h4>This L3-activated gene transcript, combined with a control transcript (tph-1, accession # U80971) that is constitutively expressed by all vector-stage filarial larvae, can be used to detect filarial infectivity in pools of mosquito vectors. This general approach (detection of stage-specific gene transcripts from eukaryotic pathogens) may also be useful for detecting infective stages of other vector-borne parasites.
format	article
author	Sandra J Laney Caitlin J Buttaro Sabato Visconti Nils Pilotte Reda M R Ramzy Gary J Weil Steven A Williams
author_facet	Sandra J Laney Caitlin J Buttaro Sabato Visconti Nils Pilotte Reda M R Ramzy Gary J Weil Steven A Williams
author_sort	Sandra J Laney
title	A reverse transcriptase-PCR assay for detecting filarial infective larvae in mosquitoes.
title_short	A reverse transcriptase-PCR assay for detecting filarial infective larvae in mosquitoes.
title_full	A reverse transcriptase-PCR assay for detecting filarial infective larvae in mosquitoes.
title_fullStr	A reverse transcriptase-PCR assay for detecting filarial infective larvae in mosquitoes.
title_full_unstemmed	A reverse transcriptase-PCR assay for detecting filarial infective larvae in mosquitoes.
title_sort	reverse transcriptase-pcr assay for detecting filarial infective larvae in mosquitoes.
publisher	Public Library of Science (PLoS)
publishDate	2008
url	https://doaj.org/article/9577879a77924192a45507106a3e454d
work_keys_str_mv	AT sandrajlaney areversetranscriptasepcrassayfordetectingfilarialinfectivelarvaeinmosquitoes AT caitlinjbuttaro areversetranscriptasepcrassayfordetectingfilarialinfectivelarvaeinmosquitoes AT sabatovisconti areversetranscriptasepcrassayfordetectingfilarialinfectivelarvaeinmosquitoes AT nilspilotte areversetranscriptasepcrassayfordetectingfilarialinfectivelarvaeinmosquitoes AT redamrramzy areversetranscriptasepcrassayfordetectingfilarialinfectivelarvaeinmosquitoes AT garyjweil areversetranscriptasepcrassayfordetectingfilarialinfectivelarvaeinmosquitoes AT stevenawilliams areversetranscriptasepcrassayfordetectingfilarialinfectivelarvaeinmosquitoes AT sandrajlaney reversetranscriptasepcrassayfordetectingfilarialinfectivelarvaeinmosquitoes AT caitlinjbuttaro reversetranscriptasepcrassayfordetectingfilarialinfectivelarvaeinmosquitoes AT sabatovisconti reversetranscriptasepcrassayfordetectingfilarialinfectivelarvaeinmosquitoes AT nilspilotte reversetranscriptasepcrassayfordetectingfilarialinfectivelarvaeinmosquitoes AT redamrramzy reversetranscriptasepcrassayfordetectingfilarialinfectivelarvaeinmosquitoes AT garyjweil reversetranscriptasepcrassayfordetectingfilarialinfectivelarvaeinmosquitoes AT stevenawilliams reversetranscriptasepcrassayfordetectingfilarialinfectivelarvaeinmosquitoes
_version_	1718413695577489408

A reverse transcriptase-PCR assay for detecting filarial infective larvae in mosquitoes.

Ejemplares similares