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Abstract
American Society for Microbiology The 104th General Meeting(2004.5.23-27)
Sensitive and Rapid Detection of Coxiella burnetii by Loop-mediated Isothermal Amplification (LAMP),a Novel DNA Amplification Method
Takayoshi Momoda, Motohiko Ogawa, Tadashi Kojima, Yoshinori Ohta, Masanari Ikedo
Eiken Chemical Co., Ltd., Tochigi, JAPAN.
Kozue Sato, Agus Setiyono, Toshiro Kishimoto
National Institute of Infectious Diseases, Tokyo, JAPAN.
Background:
Coxiella burnetii is the etiologic agent of Q fever that known one of zoonotic diseases. The organism has been isolated from domestic animals, wild animals, and human. In Japan, the diagnosis of Q fever is based on isolation of the pathogen and serological test, no simplicity diagnosis is established. In order to detect C. burnetii, we designed and evaluated a novel DNA amplification method, the 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 C (1). This method employs a DNA polymerase with strand displacement activity and a set of four specially designed primers that recognized a total of six distinct sequences on the target DNA. The LAMP reaction can be monitored in real-time by increasing in turbidity due to an abundance of the by-product, pyrophosphate.

Methods:
The primers targeting the com1 gene encoding a 27-kDa outer membrane protein of C. burnetii were designed in order to detect C. burnetii. 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 35 non-Coxiella strains related community-acquired pneumonia. The detection limit of the assay was obtained using a series of tenfold dilution of genomic DNA of C. burnetii, the PCR method targeting the com1 gene (2) was performed as reference simultaneously.

Results:
The 35 non-Coxiella strains were not amplified. The detection limit of this method showed DNA quantity corresponding to 6 cells per test within 60 minutes and same sensitivity was obtained by the PCR method.

Conclusions:
The LAMP method was able to detect C. burnetii with high specificity, rapidity and simple procedure. This method would be one superior simplicity diagnosis of C. burnetii.

(1) Notomi, T. et al. 2000. Nucleic Acids Research. 28:e63.
(2) Zhang, CQ et al. 1998. J Clin Microbiol. 36:77-80.

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