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J.Health Sci., 55(5), 820-824, 2009
Specific Detection of Viable Salmonella Cells by an Ethidium Monoazide-Loop Mediated Isothermal Amplification (EMA-LAMP) Method
Yuxia Lu,a Weiqing Yang,b
Lei Shi,a Lin Li,a
Muhammad Jahangir Alam,c Siyuan Guo,a
and Shin-ichi Miyoshi*, d
aCollege of Light Industry and Food Sciences, South China University of Technology, 510640, Guangzhou, China,
bDepartment of Clinical Microbiology, Guangdong Medical College, 524023, Guangzhou, China,
cDepartment of Diagnostic Medicine and Pathobiology, Kansas State University, Manhattan, KS-66506, U.S.A. and
dGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Tsushima-Naka, Okayama 700-8530, Japan
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.
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