Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2013

Development of field-based loop mediated isothermal amplification (LAMP) methods for the detection of enteric pathogens in a developing setting (#304)

Kevin Soli 1 , Monalisa Kas 1 , Tobias Maure 1 , Masahiro Umezaki 2 , Ayako Morita 2 , Katsura Igai 2 , Andrew Greenhill 3 , Peter Siba 1 , Paul Horwood 1
  1. Papua New Guinea Institute of Medical Research, Goroka, EHP, Papua New Guinea
  2. School of Medicine, The University of Tokyo, Tokyo, Japan
  3. School of Applied Sciences and Engineering, Monash University, Gippsland, Victoria, Australia

Enteric illnesses are a leading cause of morbidity and mortality in developing countries. Diagnosis of enteric infections is important as a broad range of aetiological agents are involved; thus optimal treatment regimens differ. Rapid diagnostic methods that are sensitive, specific and inexpensive are needed for routine diagnostic purposes and for disease outbreak investigations in resource-poor settings. Loop-mediated isothermal amplification (LAMP) assays can facilitate the diagnosis of enteric diseases through single-temperature amplification of nucleic acid, thus reducing the system complexity of PCR-based methods. In order to develop a LAMP assay suitable for use in resource-poor settings, we have a) evaluated colorimetric dyes to increase visibility of positive results; and b) developed a field-based LAMP instrument that does not require electricity. We evaluated SYBR Green, hydroxy napthol blue (HNB) and propidium iodide in laboratory-based LAMP assays for the detection of three important enteric pathogens: Vibrio cholerae, Shigella spp. and Salmonella spp. HBN and SYBR Green were highly effective, enabling detection of DNA (through colour change) visible to the unaided eye in DNA dilutions comparable to real-time PCR (within 1 log). In early trials our field-LAMP instrument has demonstrated similar levels of sensitivity to our dedicated laboratory-based LAMP instrument. These adaptations to the LAMP assay make it well suited to routine diagnostic applications in regional hospitals and healthcare settings in low-income countries, where electricity supply is often unreliable and maintenance of complex scientific equipment is challenging. In addition, such an assay could be well suited to field applications.