Alhassan Andy, Makepeace Benjamin L, LaCourse Elwyn James, Osei-Atweneboana Mike Y, Carlow Clotilde K S
Division of Genome Biology, New England Biolabs, Ipswich, Massachusetts, United States of America.
Institute of Infection & Global Health, University of Liverpool, Liverpool, United Kingdom.
PLoS One. 2014 Oct 9;9(10):e108927. doi: 10.1371/journal.pone.0108927. eCollection 2014.
Onchocerciasis is a debilitating neglected tropical disease caused by infection with the filarial parasite Onchocerca volvulus. Adult worms live in subcutaneous tissues and produce large numbers of microfilariae that migrate to the skin and eyes. The disease is spread by black flies of the genus Simulium following ingestion of microfilariae that develop into infective stage larvae in the insect. Currently, transmission is monitored by capture and dissection of black flies and microscopic examination of parasites, or using the polymerase chain reaction to determine the presence of parasite DNA in pools of black flies. In this study we identified a new DNA biomarker, encoding O. volvulus glutathione S-transferase 1a (OvGST1a), to detect O. volvulus infection in vector black flies. We developed an OvGST1a-based loop-mediated isothermal amplification (LAMP) assay where amplification of specific target DNA is detectable using turbidity or by a hydroxy naphthol blue color change. The results indicated that the assay is sensitive and rapid, capable of detecting DNA equivalent to less than one microfilaria within 60 minutes. The test is highly specific for the human parasite, as no cross-reaction was detected using DNA from the closely related and sympatric cattle parasite Onchocerca ochengi. The test has the potential to be developed further as a field tool for use in the surveillance of transmission before and after implementation of mass drug administration programs for onchocerciasis.
盘尾丝虫病是一种由丝虫寄生虫盘尾丝虫感染引起的使人衰弱的被忽视热带病。成虫生活在皮下组织，产生大量微丝蚴，微丝蚴会迁移至皮肤和眼睛。该疾病通过蚋属黑蝇传播，黑蝇摄取微丝蚴后，微丝蚴在昆虫体内发育成感染期幼虫。目前，通过捕获和解剖黑蝇以及对寄生虫进行显微镜检查，或使用聚合酶链反应来确定黑蝇群体中是否存在寄生虫DNA来监测传播情况。在本研究中，我们鉴定出一种新的DNA生物标志物，其编码盘尾丝虫谷胱甘肽S-转移酶1a（OvGST1a），用于检测媒介黑蝇中的盘尾丝虫感染。我们开发了一种基于OvGST1a的环介导等温扩增（LAMP）检测方法，通过浊度或羟基萘酚蓝颜色变化可检测到特定靶DNA的扩增。结果表明该检测方法灵敏且快速，能够在60分钟内检测到相当于少于一条微丝蚴的DNA。该检测方法对人体寄生虫具有高度特异性，因为使用来自密切相关且同域分布的牛寄生虫奥氏盘尾丝虫的DNA未检测到交叉反应。该检测方法有潜力进一步开发成为一种现场工具，用于在实施盘尾丝虫病大规模药物给药计划前后监测传播情况。
