Developmentof Real Time Rt-Pcr And Molecular Characterization For Detection of Infectious Myonecrosis Virus (IMNV) on Whiteleg Shrimp (Litopenaeus vannamei)

Abstract

Myonecrosis infection in Whiteleg shrimp (Litopenaeus vannamei) causes decline in total shrimp production in Indonesia over the past six years. Myonecrosis infections caused by viruses of the family Totiviradae, size 40 nm, a dsRNA virus, has 7560 nucleotides with a pattern of chronic infection, persistent and progressive infection. Early detection IMNV on Whiteleg shrimp cultivation is necessary for further handling. This study aimed to develope qRT-PCR assay for IMNV, spesifically to detect characterize of local IMNV isolates, determine the limit of detection and diagnostic sensitivity of the assay, and clone the ORF1 gene fragment into plasmid. Whiteleg shrimp samples were collected from farms in Lampung and Situbondo during April to October 2012. Clinical signs of infection myonecrosis were loss of transparency in the muscle tissue and necrosis of the 6th abdominal segment, which indicate early stages of myonecrosis infection. Nucleotide sequence analyses clone of Lampung and Situbondo isolates obtained in this study in comparison to Brazil (GenBank accession no.AY570982) and previous Indonesian isolated showed 99% similarity on average. Detection of IMNV utilizing qRT-PCR techniques resulted in the limit detection of 101 copies RNA per μl total RNA for qRT-PCR (TaqMan probe and IQ Real) and 103 copies RNA per μl total RNA for nested RT-PCR method. Investigation on diagnostic sensitivity of PCR techniques done on number clinical and sub clinical samples showed that qRT-PCR are more sensitive than nested PCR. Both IQ RealTM and TaqMan probe have comparable sensitivity in IMNV detection indicating that this in house developed qRT-PCR test could be used as alternative methods to IQ RealTM kit in Indonesia. Recombinant DNA plasmid containing IMNV ORF1 gene fragment was constructed. This IMNV plasmid could serve as a continuous source fragment ORF1 RNA for preparation of positive control for the the developing IMNV detection techniques by real time RT-PCR.