Establishment of dual priming oligonucleotide (DPO)-based real-time RT-PCR method for detecting infectious hematopoietic necrosis virus

Abstract

Infectious hematopoietic necrosis virus (IHNV) is widely prevalent in the world, causing a large number of deaths in wild and farmed salmonids. Therefore, the establishment of an effective and accurate detection method is extremely important. In this study, the conservation of the N gene of IHNV was analyzed, followed by the design of dual priming oligonucleotide (DPO) for developing the real-time RT-PCR assay. The specificity and sensitivity of the method were analyzed and the results showed that only IHNV was positive and had high specificity in the detection of 8 aquatic animal viruses. The minimum detection limit was 3.62 × 10¹ copies/μL. Meanwhile, in the repeatability analysis, all CVs (coefficient of variation) were less than 1 %, indicating good repeatability. Notably, the presence of three or more base mismatches in the template significantly reduced amplification efficiency or even terminated the reaction completely. Furthermore, DPO primers allow a wider annealing temperature range, and the target gene can be successfully amplified in the annealing temperature range of 45 °C to 65 °C. Finally, the DPO-based real-time RT-PCR method could be used to detect the clinical samples with higher IHNV prevalence rate compared with the traditional RT-PCR. The IHNV detection approach utilizing DPO primers, as established in this study, showed excellent specificity and sensitivity, thus providing technical support for rapid and accurate identification of IHNV and facilitating the further multiplex real-time PCR establishment.