Loop-Mediated Isothermal Amplification Targeting 18S Ribosomal DNA for Rapid Detection of Acanthamoeba.
10.3347/kjp.2013.51.3.269
- Author:
Hye Won YANG
1
;
Yu Ran LEE
;
Noboru INOUE
;
Bijay Kumar JHA
;
Dinzouna Boutamba Sylvatrie DANNE
;
Hong Kyun KIM
;
Junhun LEE
;
Youn Kyoung GOO
;
Hyun Hee KONG
;
Dong Il CHUNG
;
Yeonchul HONG
Author Information
1. Department of Parasitology and Tropical Medicine, Kyungpook National University School of Medicine, Daegu 700-422, Korea. ychong@knu.ac.kr
- Publication Type:Original Article ; Research Support, Non-U.S. Gov't
- Keywords:
Acanthamoeba;
loop-mediated isothermal amplification (LAMP);
18S rDNA;
rapid detection
- MeSH:
Acanthamoeba/*genetics;
Animals;
Base Sequence;
Humans;
Molecular Sequence Data;
Nucleic Acid Amplification Techniques/*methods;
RNA, Ribosomal, 18S/*genetics;
Sensitivity and Specificity
- From:The Korean Journal of Parasitology
2013;51(3):269-277
- CountryRepublic of Korea
- Language:English
-
Abstract:
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.
