Simple Identification of Human Taenia Species by Multiplex Loop-Mediated Isothermal Amplification in Combination with Dot Enzyme-Linked Immunosorbent Assay.
Simple Identification of Human Taenia Species by Multiplex Loop-Mediated Isothermal Amplification in Combination with Dot Enzyme-Linked Immunosorbent Assay.
复制标题
DOI：
10.4269/ajtmh.15-0829
复制
发表时间：
2016-06
期刊：
影响因子：
--
通讯作者：
A. Nkouawa;Y. Sako;M. Okamoto;A. Ito
中科院分区：
文献类型：
--
作者：
A. Nkouawa;Y. Sako;M. Okamoto;A. Ito
为了区分猪带绦虫、牛带绦虫和亚洲带绦虫，最近开发了针对细胞色素c氧化酶亚基1基因的环介导等温扩增（LAMP）方法，该方法具有敏感性、特异性和有效性。然而，为了实现鉴别，一个标本需要三种反应混合物，分别含有每种带绦虫的引物，这既复杂又耗时，而且增加了交叉污染的风险。本研究利用多重LAMP （mLAMP）结合dot酶联免疫吸附试验（dot- elisa）建立了一种简单的人带绦虫鉴别方法。mLAMP中分别使用异硫氰酸荧光素（FITC）、地高辛（DIG）和四甲基罗丹明（TAMRA）标记的猪胸草、saginata和亚洲胸草的正向内引物和生物素标记的反向内引物。mLAMP试验在单个试管中成功地扩增了每个相应的靶基因。此外，每个物种的mLAMP产物很容易通过带有FITC， DIG或TAMRA特异性抗体的dot-ELISA进行区分。mLAMP法结合dot-ELISA法将使人带绦虫种类的鉴定更简单、更容易、更实用。
For differential detection of Taenia solium, Taenia saginata, and Taenia asiatica, loop-mediated isothermal amplification (LAMP) assay targeting the cytochrome c oxidase subunit 1 gene has been recently developed and shown to be sensitive, specific, and effective. However, to achieve differential identification, one specimen requires three reaction mixtures containing a primer set of each Taenia species separately, which is complex and time consuming and increases the risk of cross-contamination. In this study, we developed a simple differential identification of human Taenia species using multiplex LAMP (mLAMP) in combination with dot enzyme-linked immunosorbent assay (dot-ELISA). Forward inner primers of T. solium, T. saginata, and T. asiatica labeled with fluorescein isothiocyanate (FITC), digoxigenin (DIG), and tetramethylrhodamine (TAMRA), respectively, and biotin-labeled backward inner primers were used in mLAMP. The mLAMP assay succeeded in specific amplification of each respective target gene in a single tube. Furthermore, the mLAMP product from each species was easily distinguished by dot-ELISA with an antibody specific for FITC, DIG, or TAMRA. The mLAMP assay in combination with dot-ELISA will make identification of human Taenia species simpler, easier, and more practical.