Direct detection of Mycobacterium tuberculosis in clinical samples by a dry methyl green loop-mediated isothermal amplification (LAMP) method

Jeewan Thapa; Bhagwan Maharjan; Meena Malla; Yukari Fukushima; Ajay Poudel; Basu Dev Pandey; Kyoko Hyashida; Stephen V Gordon; Chie Nakajima; Yasuhiko Suzuki

doi:10.1016/j.tube.2019.05.004

Direct detection of Mycobacterium tuberculosis in clinical samples by a dry methyl green loop-mediated isothermal amplification (LAMP) method

Tuberculosis (Edinb). 2019 Jul:117:1-6. doi: 10.1016/j.tube.2019.05.004. Epub 2019 May 21.

Authors

Affiliations

¹ Department of Medical Laboratory Sciences, Faculty of Health Sciences, Hokkaido University, Kita-12 Nishi-5, Kita-ku, Sapporo, Hokkaido, 060-0812, Japan. Electronic address: jeewan@hs.hokudai.ac.jp.
² Division of Bioresources, Hokkaido University Research Center for Zoonosis Control, Kita 20 Nishi 10, Kita-ku, Sapporo, Hokkaido, 001-0020, Japan; German Nepal Tuberculosis Project, Kalimati, Kathmandu, Nepal; Healthy Nepal, Balkhu, Kathmandu, Nepal.
³ Shi-Gan International College of Science and Technology, Kathmandu, Nepal.
⁴ Division of Bioresources, Hokkaido University Research Center for Zoonosis Control, Kita 20 Nishi 10, Kita-ku, Sapporo, Hokkaido, 001-0020, Japan.
⁵ Department of Microbiology, Chitwan Medical College Teaching Hospital, Chitwan, Nepal.
⁶ Ministry of Health and Population, Kathmandu, Nepal.
⁷ Division of Collaboration and Education, Hokkaido University Research Center for Zoonosis Control, Kita 20 Nishi 10, Kita-ku, Sapporo, Hokkaido, 001-0020, Japan.
⁸ UCD School of Veterinary Medicine, University College Dublin, Dublin, D04 W6F6, Ireland; The Global Station for Zoonosis Control, Hokkaido University Global Institution for Collaborative Research and Education, Kita 20 Nishi 10, Kita-ku, Sapporo, Japan.
⁹ Division of Bioresources, Hokkaido University Research Center for Zoonosis Control, Kita 20 Nishi 10, Kita-ku, Sapporo, Hokkaido, 001-0020, Japan; The Global Station for Zoonosis Control, Hokkaido University Global Institution for Collaborative Research and Education, Kita 20 Nishi 10, Kita-ku, Sapporo, Japan.
¹⁰ Division of Bioresources, Hokkaido University Research Center for Zoonosis Control, Kita 20 Nishi 10, Kita-ku, Sapporo, Hokkaido, 001-0020, Japan; The Global Station for Zoonosis Control, Hokkaido University Global Institution for Collaborative Research and Education, Kita 20 Nishi 10, Kita-ku, Sapporo, Japan. Electronic address: suzuki@czc.hokudai.ac.jp.

PMID: 31378262
DOI: 10.1016/j.tube.2019.05.004

Abstract

The purpose of this study was to develop a simple visual methyl green (MeG) based dry loop-mediated isothermal amplification (LAMP) method for early detection of Mycobacterium tuberculosis (MTB) from clinical samples. We identified MeG as an indicator of a positive LAMP reaction, where a positive reaction gave a blue-green color while a negative reaction was colorless. The MeG MTB-LAMP system was further simplified by drying all reagents for ease of use, and was then validated for its ability to diagnose TB directly using Nepalese clinical samples. We evaluated the dry MeG MTB-LAMP with 69 new TB suspected samples from patients that did not have a confirmed history of TB treatment and found the sensitivity in culture positive samples as 92.8% (13/14) and specificity in culture negative samples as 96.3% (53/55). Our LAMP system has the potential to be a point of care test for early diagnosis of active TB in developing countries.

Keywords: Loop mediated isothermal amplification (LAMP); Methyl green; Mycobacterium tuberculosis; Tuberculosis.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Coloring Agents
DNA, Bacterial / analysis
Developing Countries
Humans
Methyl Green
Mycobacterium tuberculosis / genetics
Mycobacterium tuberculosis / isolation & purification*
Nephelometry and Turbidimetry / methods
Nucleic Acid Amplification Techniques / methods*
Point-of-Care Testing
Retrospective Studies
Sensitivity and Specificity
Tuberculosis / diagnosis*

Substances

Coloring Agents
DNA, Bacterial
Methyl Green