A reverse transcriptase-PCR assay for detecting filarial infective larvae in mosquitoes.
A reverse transcriptase-PCR assay for detecting filarial infective larvae in mosquitoes.
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用于检测蚊子中丝状感染幼虫的逆转录酶-PCR分析。
DOI：
10.1371/journal.pntd.0000251
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发表时间：
2008-06-18
影响因子：
3.8
通讯作者：
Williams SA
中科院分区：
文献类型：
--
作者：
Laney SJ;Buttaro CJ;Visconti S;Pilotte N;Ramzy RM;Weil GJ;Williams SA
现有的蚊子丝状寄生虫DNA分子测定方法无法区分含有任何阶段寄生虫的感染蚊子和含有能够在人类中建立新感染的第三阶段幼虫（L3）的感染蚊子。我们现在报告了基于RT-PCR检测l3激活基因转录物的载体中马来布鲁氏菌分子l3检测方法的发展。以cDNA文库为模板，利用生物信息学方法对马来芽孢杆菌全生命周期EST数据集鉴定出的候选基因进行PCR筛选。利用从感染蚊子中分离的RNA证实了阶段特异性。以猫血为食，每天采集蚊虫，连续14 d。RT-PCR采用对单个候选基因特异的引物进行。由于在较不成熟的幼虫中转录水平较低，许多在L3期强表达的候选基因被排除在外。一种转录物（TC8100，编码一种特殊形式的胶原蛋白）只在含有L3幼虫的蚊子中被检测到。该试验在25只蚊子中检测单个L3。该l3激活基因转录物与一个由所有媒介期丝虫幼虫组成表达的对照转录物（tph-1, accession # U80971）结合，可用于检测蚊虫媒介池中的丝虫传染性。这种一般方法（检测真核病原体的阶段特异性基因转录物）也可用于检测其他媒介传播的寄生虫的感染阶段。全球消除淋巴丝虫病规划于1998年启动，目标是到2020年消除淋巴丝虫病。由于大规模药物给药（MDA）在全球规划中的成功将地方性人群的感染率降至非常低的水平，因此需要开发新的、高灵敏度的方法，通过筛选蚊子是否存在L3感染幼虫来监测传播。目前鉴定L3幼虫的蚊虫解剖方法费力且不敏感，不适合筛选大量蚊子。现有的检测蚊子丝虫DNA的分子分析方法灵敏度高，可以方便地筛选大量媒介。然而，目前基于pcr的方法无法区分含有任何阶段寄生虫的感染蚊子和携带能够在人类中建立新感染的第三阶段幼虫（L3）的感染蚊子。本文首次建立了一种基于RT-PCR检测疟原虫l3激活基因转录物的分子检测方法。这种检测丝虫病期特异性信使RNA的策略也可用于检测其他媒介传播病原体的感染期。
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. 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. 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. The Global Programme for the Elimination of Lymphatic Filariasis (GPELF) was launched in the year 1998 with the goal of eliminating lymphatic filariasis by 2020. As the success of mass drug administration (MDA) in the global program drives the rates of infection in endemic populations to very low levels, the development of new, highly sensitive methods are required for monitoring transmission by screening mosquitoes for the presence of L3 infective larvae. The current method of mosquito dissection to identify L3 larvae is laborious and insensitive and is not amenable to screening large numbers of mosquitoes. Existing molecular assays for the detection of filarial parasite DNA in mosquitoes are sensitive and can easily screen large numbers of vectors. However, current PCR-based methods 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. This paper reports the first development of a molecular L3-detection assay for a filarial parasite in mosquitoes based on RT-PCR detection of an L3-activated gene transcript. This strategy of detecting stage-specific messenger RNA from filarial parasites may also prove useful for detecting infective stages of other vector-borne pathogens.
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PCR和解剖作为监测法氏多尼西斯蚊子的丝状感染的工具。
DOI：
10.1186/1475-2883-5-2
发表时间：
2006-02-24
期刊：
Filaria journal
影响因子：
--
作者：
Plichart, Catherine;Sechan, Yves;Davies, Neil;Legrand, Anne-Marie
通讯作者：
Legrand, Anne-Marie
DOI：
10.1186/1475-2883-3-9
发表时间：
2004-09-03
期刊：
Filaria journal
影响因子：
--
作者：
Lammie, Patrick J;Weil, Gary;Ottesen, Eric
通讯作者：
Ottesen, Eric
DOI：
10.1016/s0169-4758(97)01130-7
发表时间：
1997-10-01
期刊：
PARASITOLOGY TODAY
影响因子：
--
作者：
Weil, GJ;Lammie, PJ;Weiss, N
通讯作者：
Weiss, N
DOI：
10.4269/ajtmh.2007.76.502
发表时间：
2007-03-01
影响因子：
3.3
作者：
Fischer, Peter;Erickson, Sara M.;Weil, Gary J.
通讯作者：
Weil, Gary J.
DOI：
10.4269/ajtmh.1994.51.314
发表时间：
1994-09-01
影响因子：
3.3
作者：
LIZOTTE, MR;SUPALI, T;WILLIAMS, SA
通讯作者：
WILLIAMS, SA