Novel peptide nucleic acid-based real-time PCR assay for detection and genotyping of Megalocytivirus

Megalocytivirus, the causative agent for Red Sea Bream Iridoviral Disease (RSIVD), is classified into three genotypes based on its phylogeny: Red Sea Bream Iridovirus (RSIV), Infectious Spleen and Kidney Necrosis Virus (ISKNV), and Turbot Reddish Body Iridovirus (TRBIV). To overcome drawbacks such as limitation of genotypic variant type discrimination and unexpected PCR reaction from an SYBR green-based genotyping method by high-resolution melting analysis, peptide nucleic acid (PNA)-based real-time PCR assay for detecting and genotyping Megalocytivirus was developed. Compared with DNA hybridization, PNA has more suitable hybridization traits that allowed the difference in melting temperature (Tm) even by a single nucleotide mismatch. Based on the consensus sequences of the viral major capsid protein gene, four PNA probes labeled with respective fluorescence at their 3′ ends were designed as reporter molecules. PNA-based real-time PCR assay allowed quantification of Megalocytivirus copies using a synthetic DNA as standard (detection limit of 103 copies) and discrimination of the genotypes based on Tm in a single-tube reaction. Furthermore, the results of this new method were consistent with those of sequencing analysis of Megalocytivirus-infected field and artificial samples, proving its accuracy and efficiency. The novel PNA-based real-time PCR assay could contribute to the detection as well as discrimination of the genotypes of Megalocytivirus in a single simple assay, and could be used as a highly robust diagnostic tool in the field of aquatic animal diseases.