Simple Identification of Human Taenia Species by Multiplex Loop-Mediated Isothermal Amplification in Combination with Dot Enzyme-Linked Immunosorbent Assay

Agathe Nkouawa Department of Parasitology, Asahikawa Medical University, Asahikawa, Japan; Medical Research Centre, Institute of Medical Research and Medicinal Plants Studies (IMPM), Ministry of Scientific Research and Innovation, Yaoundé, Cameroon; Center for Human Evolution Modeling Research, Primate Research Institute, Kyoto University, Inuyama, Japan

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Yasuhito Sako Department of Parasitology, Asahikawa Medical University, Asahikawa, Japan; Medical Research Centre, Institute of Medical Research and Medicinal Plants Studies (IMPM), Ministry of Scientific Research and Innovation, Yaoundé, Cameroon; Center for Human Evolution Modeling Research, Primate Research Institute, Kyoto University, Inuyama, Japan

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Munehiro Okamoto Department of Parasitology, Asahikawa Medical University, Asahikawa, Japan; Medical Research Centre, Institute of Medical Research and Medicinal Plants Studies (IMPM), Ministry of Scientific Research and Innovation, Yaoundé, Cameroon; Center for Human Evolution Modeling Research, Primate Research Institute, Kyoto University, Inuyama, Japan

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Akira Ito Department of Parasitology, Asahikawa Medical University, Asahikawa, Japan; Medical Research Centre, Institute of Medical Research and Medicinal Plants Studies (IMPM), Ministry of Scientific Research and Innovation, Yaoundé, Cameroon; Center for Human Evolution Modeling Research, Primate Research Institute, Kyoto University, Inuyama, Japan

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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.

Author Notes

* Address correspondence to Agathe Nkouawa, Department of Parasitology, Asahikawa Medical University, Midorigaoka Higashi 2-1-1-1, Asahikawa, Hokkaido 078-8510, Japan. E-mail: ankouawa@gmail.com
† These authors contributed equally to this work.

Financial support: This work was supported by the Japan Society for the Promotion of Science (JSPS) Postdoctoral Fellowship for Foreign Researchers (no. 26-04103) to Agathe Nkouawa, by the Grant-in-Aid for Scientific Research (B) (15H05273) to Yasuhito Sako, (B) (21406009, 24406011) to Munehiro Okamoto, and (A) (21256003, 24256002) to Akira Ito from JSPS; by the JSPS-Asia/Africa Scientific Platform Fund (2006–2011); and by the Special Coordination Fund for Promoting Science and Technology from the Ministry of Education, Japan (2003–2005 and 2010–2012) to Akira Ito.

Authors' addresses: Agathe Nkouawa, Yasuhito Sako, and Akira Ito, Department of Parasitology, Asahikawa Medical University, Asahikawa, Japan, E-mails: ankouawa@gmail.com, yasusako@asahikawa-med.ac.jp, and akiraito@asahikawa-med.ac.jp. Munehiro Okamoto, Center for Human Evolution Modeling Research, Primate Research Institute, Kyoto University, Inuyama, Japan, E-mail: okamoto.munehiro.6w@kyoto-u.ac.jp.

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