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 t he coat protein gene (RNA2) of striped jack nervous necrosis virus (SJ NNV), the causative agent of viral nervous necrosis (VNN) of larval st riped jack Pseudocaranx dentex. Based on the sequence data of SJNNV RN A2, 2 forward (F1 and F2) and 3 reverse (R1, R2 and R3) PCR primers we re 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 pr imer sets, F1-R2 and F2-R3, produced the specified 180 bp and 430 bp p roducts. The PCR system, using the F2-R3 primer set, was able to detec t 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 ex tracted from larval striped jack affected with VNN.