Development and application of an in situ hybridizationassay for detection of mud crab dicistrovirus-1

Mud crab dicistrovirus-1 was isolated from massive deaths of mud crab and had a strong pathogenicity.Based on the MCDV-1 genome sequences，we designed a pair of primers and obtained a 1 314 bp fragment of MCDV-1gene by RT-PCR. Consequently，probe was prepared using DIG-labeling. The sensitivity and specificity of probe wereanalyzed by dot blot hybridization method. Results showed that this probe could detect a minimum of 8.58×107 copies/μLof MCDV-1 and was specific for MCDV-1 detection，without cross-reaction with MCRV（mud crab reovirus），AbSV（abalone shriveling syndrome-associated virus），AbHV（abalpone herpesvirus），WSSV（white spot syndrome virus），TFV（tigerfrogvirus）and KHV（Koi herpesvirus）. This probe was used to detect MCDV-1 in different tissues of diseasedmud crab by in situ hybridization and positive signals of MCDV-1 were mainly observed in hepatopancreas，ganglion，intestines and esophagus.