Specific detection of viable Salmonella cells by an ethidium monoazide-loop mediated isothermal amplification (EMA-LAMP) method

Abstract

The persistence of DNA after the cell death causes a major issue in aspects of medical or biological studies. The signal from viable bacterial cells cannot be distinguished from the dead cells in the conventional DNA-based detection methods. In the present study, the loop-mediated isothermal amplification (LAMP) method combined with the ethidium monoazide (EMA) treatment was applied for specific detection of viable, but not dead, Salmonella cells. For this method (EMA-LAMP), we designed a series of primers, which recognize six distinct sequences of the target invA gene conserved in Salmonella. The invA gene of the viable cells was remarkably amplified within 1 hr when as small amounts as 100 fg of DNA was subjected to EMA-LAMP. Because EMA selectively penetrated into the dead cells and bound covalently to DNA, the gene of the dead cells could not be amplified. This study offers a novel DNA-based method to distinguish the viable bacterial cells from the dead cells. ©2009 The Pharmaceutical Society of Japan.