Molecular diagnostic methods for detection of Thelohania contejeani (Microsporidia), the causative agent of porcelain disease in crayfish

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.