title: Development of a rapid assay to detect the dinoflagellate Amyloodinium ocellatum using loop-mediated isothermal amplification (LAMP).
abstract: Amyloodinium ocellatum is a highly pathogenic dinoflagellate parasite with global distribution that causes high mortalities in the culture of tropical and sub-tropical marine and estuarine fishes. Diagnosis typically occurs through gross examination following the onset of morbidity, at which point treatment is of limited benefit. In the present study, a new molecular diagnostic tool for the rapid detection of A. ocellatum (AO) was developed using the loop-mediated isothermal amplification method (LAMP). The AO-LAMP assay designed is highly specific using a set of four primers - two outer and two inner primers targeting six different regions on the 5' end of the Small Subunit rDNA region (SSU rDNA) of A. ocellatum. The AO-LAMP assay, optimized for 25-30 min at 62°C, amplified the DNA from A. ocellatum extracted from both water and gill tissue samples and did not amplify DNA from four closely related dinoflagellate sp ecies. The detection limit of the AO-LAMP assay was 10 fg, exceptionally higher than the conventional PCR (1 pg). In addition, the standardized AO-LAMP assay was capable of detecting single tomonts and trophonts; the assay was not affected by the presence of possible inhibitory substances present in environmental water samples or gill samples. The AO-LAMP assay developed in the present study provides a novel useful tool for the simple, rapid and sensitive detection of A. ocellatum in water and gill tissue samples, which could assist in the early detection and improved control of A. ocellatum infections in aquaculture systems.
authors.affiliation: Gulf Coast Research Laboratory, University of Southern Mississippi, Ocean Springs, MS 39564, USA. sarapicon@yahoo.es
authors.forename: Sara M
authors.initials: SM
authors.lastname: Picón-Camacho
authors.forename: William P
authors.initials: WP
authors.lastname: Thompson
authors.forename: Reginald B
authors.initials: RB
authors.lastname: Blaylock
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Lotz
mesh_terms: D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D004141:Dinoflagellida / Q000302:isolation & purification*; D005393:Fish Diseases / Q000175:diagnosis / Q000469:parasitology; D005399:Fishes; D021141:Nucleic Acid Amplification Techniques / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity; D013997:Time Factors; D014867:Water / Q000469:parasitology
chemical_list: D014867:Water; D004247:DNA
keywords: Amyloodiniosis; Diagnostic test; Fish parasite; Velvet disease