Development of a real-time enzymatic recombinase amplification assay for rapid detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in shrimp Penaeus vannamei.

ABSTRACT

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is classified as a reportable crustacean disease by the World Organisation for Animal Health (WOAH), which causes poor growth in Penaeus vannamei. According to genome sequence alignment analysis, enzymatic recombinase amplification (ERA) primers and probe were designed based on the ORF1 region of IHHNV, and a real-time ERA assay for IHHNV detection (IHHNV-ERA) was established. The experimental results show that IHHNV-F2/IHHNV-R2 and IHHNV-Probe can effectively amplify the target gene, and the sensitivity is 1.4 × 101 copies/µL within 14.97 ± 0.19 min, while the qPCR using primers 309F/309R could reach the detection limit of 1.4 × 101 copies/µL within 21.76 ± 0.63 min, and the sensitivity results of one-step PCR could be as low as 1.4 copies/µL with expense of time and false positives. The IHHNV-ERA system can effectively amplify the target gene at 42 ℃ within 20 min, and has no cross-reaction with white spot syndrome virus (WSSV), Ecytonucleospora hepatopenaei (EHP), Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (VpAHPND), and healthy shrimp genomic DNA. Test results of practical samples showed that the detection rate of IHHNV-ERA (18/20) was better than the industrial standard qPCR assay (17/20). Compared with the existing technology, the useful results of this detection assay are (1) get rid of the dependence on the thermal cycle instrument in the PCR process; (2) the experimental procedure is simple, time-consuming and fast; (3) the detection sensitivity is high. This study provides an ERA based detection assay for IHHNV, which can be used not only for the rapid detection of IHHNV infection, but also for the field screening of pathogens. This assay can also be applied to clinical inspection, customs detection, enterprise quality inspection and other fields, and has obvious practical application value.