title: Molecular diagnostic methods for detection of Thelohania contejeani (Microsporidia), the causative agent of porcelain disease in crayfish.
abstract: Diagnosis of Thelohania contejeani in the crayfish Astacus astacus is currently based on observation of gross clinical signs--opaque appearance of the abdomen and whitish colouration of the musculature--and confirmed by microscopic examination of histological sections of muscle. We have developed 2 molecular diagnostic methods for sensitive and rapid detection of porcelain disease in its early stages: PCR and loop-mediated isothermal amplification (LAMP). The PCR test utilises a primer based on the T. contejeani small subunit ssu ribosomal RNA (ssu rRNA) gene and amplified parasite DNA with high specificity and a detection limit of 10(-5) dilution. The LAMP assay involves incubation of the target DNA with a set of 6 primers and Bst DNA polymerase for 60 min at 65 degrees C in a water bath or heating block, followed by visualisation of the reaction products with the SYBR Green I stain; sensitivity of visual detection with SYBR Green I is equivalent to that with agarose gel electrophoresis. The LAMP assay can detect T. contejeani DNA to a dilution of 10(-7). The LAMP assay is 100 times more sensitive than the PCR test and is the method we recommend as an alternative to traditional means of diagnosing T. contejeani.
authors.affiliation: Institute of Zoology, Fish Biology and Fish Diseases, Faculty of Veterinary Medicine, University of Munich, Germany. el-matbouli@lmu.de
authors.forename: M
authors.initials: M
authors.lastname: El-Matbouli
authors.forename: H
authors.initials: H
authors.lastname: Soliman
mesh_terms: D000818:Animals; D003400:Astacoidea / Q000382:microbiology*; D052160:Benzothiazoles; D017931:DNA Primers / Q000737:chemistry; D016054:DNA, Protozoan / Q000032:analysis*; D003959:Diamines; D004587:Electrophoresis, Agar Gel / Q000662:veterinary; D005456:Fluorescent Dyes; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009930:Organic Chemicals; D050175:Pansporablastina / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D011804:Quinolines; D012335:RNA, Ribosomal / Q000235:genetics; D012680:Sensitivity and Specificity
chemical_list: D052160:Benzothiazoles; D017931:DNA Primers; D016054:DNA, Protozoan; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D012335:RNA, Ribosomal; C098022:SYBR Green I