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Abstract
American Society for Microbiology The 103rd General Meeting(2003.5.18-22)
Sensitive and Rapid Detection of Salmonella Species by a Novel DNA Amplification Method, LAMP (Loop-mediated Isothermal Amplification)
M. Yoshino, H. Miyajima, A. Sunada, T. Annaka, J. Nemoto, T. Momoda, Y. Ohta, S. Kohjiya, S. Sadamoto, T. Kojima, M. Ikedo;
Eiken Chemical Co., Ltd., Tochigi, JAPAN.
Background:
Effective prevention of food-borne diseases requires sensitive and rapid surveillance of food supplies for known pathogens. Salmonella species has emerged as a major cause of food-borne illness worldwide. To detect Salmonella species, we designed and evaluated a novel DNA amplification method, Loop-mediated isothermal amplification (LAMP) method. The LAMP method synthesizes a large amount of DNA with high specificity, sensitivity and rapidity under isothermal conditions at 65 ーC (1). This method employs a DNA polymerase with strand displacement activity and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. The LAMP reaction can be monitored in real-time by increasing the fluorescence intensity with intercalating dye using a real-time PCR system, and also recognized as an increase in turbidity due to an abundance of the by-product, pyrophosphate.

Methods:
The primers targeting invA gene were designed to detect a wide range of Salmonella serovar. LAMP was carried out in a reaction mixture containing Bst DNA polymerase large fragment and its appended buffer, dNTPs and betaines. The specificity of this LAMP method was confirmed with 225 Salmonella strains comprising 41 different serovars and untyped isolates, and 80 non-Salmonella strains. The detection limit of the assay was obtained using a series of tenfold dilutions of S. enterica serovar Enteritidis cell suspension.

Results:
All of the 225 strains Salmonella strains showed significant amplification within 30 minutes, while none of the 80 non-Salmonella strains were amplified after 120 minutes incubation. The detection limit of this method showed 6 to 60 colony forming unit per test.

Conclusions:
The LAMP method was able to detect a wide range of Salmonella species with high specificity, rapidity and simple procedure. This method is very useful to detect Salmonella in food samples.

(1) Notomi, T. et al. Nucleic Acids Research. 28:e63

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