title: Loop-mediated isothermal amplification targeting 18S ribosomal DNA for rapid detection of Acanthamoeba.
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.
authors.affiliation: Department of Parasitology and Tropical Medicine, Kyungpook National University School of Medicine, Daegu 700-422, Korea.
authors.forename: Hye-Won
authors.initials: HW
authors.lastname: Yang
authors.forename: Yu-Ran
authors.initials: YR
authors.lastname: Lee
authors.forename: Noboru
authors.initials: N
authors.lastname: Inoue
authors.forename: Bijay Kumar
authors.initials: BK
authors.lastname: Jha
authors.forename: Dinzouna-Boutamba Sylvatrie
authors.initials: DB
authors.lastname: Danne
authors.forename: Hong-Kyun
authors.initials: HK
authors.lastname: Kim
authors.forename: Junhun
authors.initials: J
authors.lastname: Lee
authors.forename: Youn-Kyoung
authors.initials: YK
authors.lastname: Goo
authors.forename: Hyun-Hee
authors.initials: HH
authors.lastname: Kong
authors.forename: Dong-Il
authors.initials: DI
authors.lastname: Chung
authors.forename: Yeonchul
authors.initials: Y
authors.lastname: Hong
mesh_terms: D000048:Acanthamoeba / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D006801:Humans; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012337:RNA, Ribosomal, 18S / Q000235:genetics*; D012680:Sensitivity and Specificity
chemical_list: D012337:RNA, Ribosomal, 18S
keywords: 18S rDNA; Acanthamoeba; loop-mediated isothermal amplification (LAMP); rapid detection