Development of a rapid assay for the diagnosis of Myxobolus cerebralis in fish and oligochaetes using loop-mediated isothermal amplification.
A loop-mediated isothermal amplification assay was developed for the rapid detection of Myxobolus cerebralis in both fish and oligochaete hosts. The assay was optimized to amplify parasitic DNA by incubation with Bst DNA polymerase and a set of six specially constructed primers at 65 degrees C for 60 min. The amplification products were detected visually using SYBR Green I dye which gave identical results to gel electrophoresis analysis. Parasite DNA was detected from infected oligochaetes, and from the anal fin, caudal fin, dorsal fin and operculum of clinically infected fish. This 'Myxo-LAMP' assay has a detection limit similar to that of a polymerase chain reaction assay (10(-6)), but is more rapid and only requires a water bath for amplification and is therefore practical for simple and rapid diagnosis of infected tissue.
El-Matbouli
M
M
Institute of Zoology, Fish Biology and Fish Diseases, Faculty of Veterinary Medicine, University of Munich, Munich, Germany. elmatbouli@zoofisch.vetmed.uni-muenchen.de
Soliman
H
H
D000818:Animals; D001483:Base Sequence; D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D004587:Electrophoresis, Agar Gel / Q000662:veterinary; D056890:Eukaryota / Q000235:genetics*; D005393:Fish Diseases / Q000175:diagnosis* / Q000469:parasitology*; D005399:Fishes; D025202:Molecular Diagnostic Techniques / Q000379:methods / Q000662:veterinary; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods* / Q000662:veterinary*; D009835:Oligochaeta / Q000469:parasitology*; D009930:Organic Chemicals; D011529:Protozoan Infections, Animal / Q000175:diagnosis*; D011804:Quinolines; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000662:veterinary
D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D012337:RNA, Ribosomal, 18S; C098022:SYBR Green I