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KMID : 0425120130510030269
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Parasites, Hosts and Diseases
2013 Volume.51 No. 3 p.269 ~ p.277
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Loop-Mediated Isothermal Amplification Targeting 18S Ribosomal DNA for Rapid Detection of Acanthamoeba
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Yang Hye-Won
Lee Yu-Ran
Inoue Noboru
Jha Bijay Kumar
Danne Dinzouna-Boutamba Sylvatrie
Kim Hong-Kyun
Lee Jun-Hun
Goo Youn-Kyoung
Kong Hyun-Hee
Chung Dong-Il
Hong Yeon-Chul
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Abstract
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Amoebic keratitis (AK) caused by Acanthamoeba is one of the most serious corneal infections. AK is frequently misdiagnosed initially as viral, bacterial, or fungal keratitis, thus ensuring treatment delays. Accordingly, the early detection of Acanthamoeba would contribute significantly to disease management and selection of an appropriate anti-amoebic therapy. Recently, the loop-mediated isothermal amplification (LAMP) method has been applied to the clinical diagnosis of a range of infectious diseases. Here, we describe a rapid and efficient LAMP-based method targeting Acanthamoeba 18S rDNA gene for the detection of Acanthamoeba using clinical ocular specimens in the diagnosis of AK. Acanthamoeba LAMP assays detected 11 different strains including all AK-associated species. The copy number detection limit for a positive signal was 10 DNA copies of 18S rDNA per reaction. No cross-reactivity with the DNA of fungi or other protozoa was observed. The sensitivity of LAMP assay was higher than those of Nelson primer PCR and JDP primer PCR. In the present study, LAMP assay based on directly heat-treated samples was found to be as efficient at detecting Acanthamoeba as DNA extracted using a commercial kit, whereas PCR was only effective when commercial kit-extracted DNA was used. This study showed that the devised Acanthamoeba LAMP assay could be used to diagnose AK in a simple, sensitive, and specific manner.
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KEYWORD
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Acanthamoeba, loop-mediated isothermal amplification (LAMP), 18S rDNA, rapid detection
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