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Abstract
American Society for Microbiology The 104th General Meeting(2004.5.23-27)
Development of Method for Loop-Mediated Isothermal Amplification (LAMP) Assay for Specific, Sensitive and Rapid Detection for Listeria monocytogenes
Manabu Yoshino, Tadashi Kojima, Yoshinori Ohta, Masanari Ikedo
Eiken Chemical Co., Ltd., Tochigi, JAPAN.
Background:
The need for a simple, rapid and reliable method for detection L.monocytogenes in food products is paramount to maintain safety. In order to detect L.monocytogenes specifically and simply, we designed and evaluated a novel DNA amplification method using Loop-mediated isothermal amplification (LAMP). The LAMP method synthesizes a large amount of DNA with high specificity, sensitivity and rapidity under isothermal condition at 65 C. 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 increase in turbidity due to an abundance of by-product, pyrophosphate.

Methods:
The primers to recognize iap (invasion associated protein) gene were designed to detect almost serotypes of L.monocytogenes. The amplification reaction was carried out in a reaction mixture containing Bst DNA polymerase, dNTPs and buffer optimized. The specificity of this LAMP method was confirmed with 39 L.monocytogenes strains comprising 10 different serotypes (1/2a, 1/2b, 1/2c, 3a, 3b, 4a, 4b, 4c, 4d, 4e) and unknown. And also 15 other species of L.monocytogenes and 113 non-Listeria strains were tested for specificity. The detection limit of the assay was tested with strain of L.monocytogenes serotype 4b. And for the practical use, we examined about extraction methods from half-Fraser medium with food as pre-enrichment culture.

Results:
All of L.monocytogenes strains showed significant amplification within 40 minutes, while all of other strains were never amplified until 120 minutes incubation. The detection limit of this method showed 6 to 60 colony forming unit per test. And the extraction from pre-enrichment culture using EXTRAGEN II Kit (Tosoh, Japan) was available with simple procedure and high efficiency.

Conclusions:
The LAMP method was able to detect almost serotypes of L.monocytogenes with high specificity, rapidity and simple procedure. This method is very useful to detect L.monocytogenes in food samples.

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