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Acta Trop. 2016 Jan;153:116-9. doi: 10.1016/j.actatropica.2015.10.013. Epub 2015 Oct 26.

A rapid molecular diagnosis of cutaneous leishmaniasis by colorimetric malachite green-loop-mediated isothermal amplification (LAMP) combined with an FTA card as a direct sampling tool.

Author information

1
Laboratory of Parasitology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
2
Sección de Entomología, Instituto de Medicina Tropical "Daniel A. Carrión" y Departamento Académico de Microbiología Médica, Facultad de Medicina Humana, Universidad Nacional Mayor de San Marcos, Lima, Peru; Laboratorio de Entomología, Instituto Nacional de Salud, Lima, Peru.
3
Laboratorio de Metaxenicas, Laboratorio Regional de Salud Pública, Dirección Regional de Salud Ayacucho, Peru.
4
Laboratorio Regional de Salud Pública, Dirección Regional de Salud Madre de Dios, Peru.
5
Laboratorio de Biología Molecular y Genética, Facultad de Ciencias Biológicas, Universidad Nacional Pedro Ruiz Gallo, Lambayeque, Peru.
6
Department of Microbiology, Faculty of Life Sciences, Graduate School of Health Sciences, Kumamoto University, Kumamoto, Japan.
7
Department of Dermatology, Faculty of Medicine, University of the Ryukyus, Okinawa, Japan.
8
Servicio Nacional de Erradicacion de la Malaria (SNEM), Ministerio de Salud Publica, Ecuador.
9
Centro de Biomedicina, Facultad de Medicina, Universidad Central del Ecuador, Quito, Ecuador.
10
Centro de Biomedicina, Facultad de Medicina, Universidad Central del Ecuador, Quito, Ecuador; Prometeo, Secretaría Nacional de Educacion Superior, Ciencia, Tecnologia e Innovacion (SENESCYT), Ecuador; Department of Parasitology, Kochi Medical School, Kochi University, Kochi, Japan.
11
Laboratory of Parasitology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan. Electronic address: hkato@vetmed.hokudai.ac.jp.

Abstract

Leishmaniasis remains one of the world's most neglected diseases, and early detection of the infectious agent, especially in developing countries, will require a simple and rapid test. In this study, we established a quick, one-step, single-tube, highly sensitive loop-mediated isothermal amplification (LAMP) assay for rapid detection of Leishmania DNA from tissue materials spotted on an FTA card. An FTA-LAMP with pre-added malachite green was performed at 64°C for 60min using a heating block and/or water bath and DNA amplification was detected immediately after incubation. The LAMP assay had high detection sensitivity down to a level of 0.01 parasites per μl. The field- and clinic-applicability of the colorimetric FTA-LAMP assay was demonstrated with 122 clinical samples collected from patients suspected of having cutaneous leishmaniasis in Peru, from which 71 positives were detected. The LAMP assay in combination with an FTA card described here is rapid and sensitive, as well as simple to perform, and has great potential usefulness for diagnosis and surveillance of leishmaniasis in endemic areas.

KEYWORDS:

FTA; LAMP; Leishmania; Malachite green; Peru

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