POLYMERASE CHAIN-REACTION (PCR) AMPLIFICATION OF RNA OF STRIPED JACK NERVOUS NECROSIS VIRUS (SJNNV)

The polymerase chain reaction (PCR) was used to amplify a portion of the coat protein gene (RNA2) of striped jack nervous necrosis virus (SJNNV), the causative agent of viral nervous necrosis (VNN) of larval striped jack Pseudocaranx dentex. Based on the sequence data of SJNNV RNA2, 2 forward (F1 and F2) and 3 reverse (R1, R2 and R3) PCR primers were synthesized and the 5 potential target regions were amplified with a combination of these primers. After reverse transcription of genomic RNA extracted from SJNNV and 25 cycles of PCR amplification, products of the expected size were detected separately on agarose gels stained with ethidium bromide. Southern blot hybridization confirmed that all of the amplified products were specific to cDNA of SJNNV RNA2. Two primer sets, F1-R2 and F2-R3, produced the specified 180 bp and 430 bp products. The PCR system, using the F2-R3 primer set, was able to detect 100 fg of SJNNV RNA after 25 cycles and was also able to efficiently amplify the target region of SJNNV gene in the total nucleic acids extracted from larval striped jack affected with VNN.