The introduce of a new modified Nested RT-PCR molecular technique for rapid diagnosis of Viral Nervous Necrosis (VNN)

Abstract

Viral Nervous Necrosis (VNN) is considered as one of the most important threats for mariculture and aquaculture in the world and up to now, it has been affecting more than 70 fish species. This study was aimed to detect and identify fish betanodavirus, the causative agent of VNN, using nested RT-PCR from brain tissue of Caspian Sea Mullet. Forty samples of subclinical golden grey mullet fish (range: 50-250 gr) were collected from the coastal areas of Caspian Sea in 2016. The target organ (brain) was excised and the reverse transcription (cDNA synthesis) was carried out followed by the RNA extraction. In the present study, detecting of the virus was performed with Nested RT-PCR using two pairs of primers F2/R3 and NF2/NR3 in two steps. After optimization of PCR, all of the forty specimens were negative by the first-step PCR (RT-PCR) while 20 out of 40 grey mullet were positive using Nested RT-PCR (second-step PCR) and a strong band of the expected size (300 bp) was clearly observed. Therefore, it seems that Nested RT-PCR is a method of choice for rapid detection of the low level of infection and in asymptomatic carrier fish. In addition, the result of sequencing revealed that the virus belonged to the RGNNV genotype.