The development of RT-RPA and CRISPR-Cas12a based assay for sensitive detection of infectious hematopoietic necrosis virus (IHNV).

Infectious hematopoietic necrosis virus (IHNV) is an economically important virus causing significant mortalities among wild and cultured salmonid fish worldwide. Rapid and sensitive diagnostic methods of IHNV are crucial for timely controlling infections. For better detection of IHNV, we have established a detection technology based on the reverse transcription and recombinase polymerase amplification (RT-RPA) and CRISPR/Cas12a to detect the N gene of IHNV in two steps. Following the screening of primer pairs, the reaction temperature and time for RPA were optimized to be 41 °C and 35 min, respectively, and the CRISPR/Cas12a reaction was performed at 37 °C for 15 min. The whole detection procedure including can be accomplished within one hour, with a detection sensitivity of about 9.5 copies/µL. The detection method exhibited high specificity with no cross-reaction to the other Novirhabdoviruses HIRRV and VHSV, allowing naked-eye interpretation of the results through lateral flow or fluorescence under ultraviolet light. Overall, our results demonstrated that the developed RT-RPA-Cas12a-mediated assay is a rapid, specific and sensitive detection method for routine and on-site detection of IHNV, which shows a great application promise for the prevention of IHNV infections.

The complete mitochondrial genome of Holothuria fuscocinerea (Jaeger,1833).

In this study, the complete mitochondrial genome of Holothuria fuscocinerea was sequenced on an Illumina platform and assembled using NovoPlasty v. 2.7.1. It was submitted to NCBI GenBank and is available with accession number MN542416. The genome was 15,827 bp in size and contains 22 tRNA genes, 12 protein-coding genes, and 2 rRNA genes. The composition of A + T in Holothura spinifera mtDNA was 60.30%. Except ND6 and 5 tRNAs, the others are not on the H-strand. The phylogenetic relationship of 13 species of sea cucumber were analyzed using the neighbor-joining method by software MEGA5.0. Holothuria fuscocinerea was most closely related to Holothuria polii.