Development of a quantitative polymerase chain reaction (qPCR) assay for the detection of dwarf gourami iridovirus (DGIV) and other megalocytiviruses and comparison with the Office International des Epizooties (OIE) reference PCR protocol

Viruses in the genus Megalocytivirus (Family Iridoviridae) have emerged as a source of significant disease and mortality in fish worldwide. Although regarded as exotic in Australia, the importation of ornamental fish provides a transmission pathway for the introduction of a megalocytivirus, dwarf gourami iridovirus (DGIV), which is a strain of infectious spleen and kidney necrosis virus (ISKNV). There are no effective cell lines for isolation of ISKNV-like viruses, particularly those from ornamental fish. Consequently a quantitative polymerase chain reaction (qPCR) assay was developed for the detection and quantification of known megalocytiviruses from fish tissues. A plasmid containing a target DGIV sequence was developed for use as a positive control and also for quantification of the virus in qPCR, and is suitable for inter-laboratory standardisation of the assay. The limit of detection for the qPCR assay was 100 copies and the assay did not cross-react with ranaviruses or other aquatic viral pathogens tested. The method of DNA extraction (magnetic bead or spin column based) had no effect on the sensitivity of the qPCR assay. Analytical sensitivity was comparable to a nested PCR assay which employed the same internal primers; however, compared to the Office International des Epizooties (OIE) reference PCR protocol, sensitivity was approximately 3-4 logs greater. The OIE reference PCR protocol detected only 8% of the samples identified to be positive by qPCR. This rapid, sensitive and specific test with the accompanying plasmid as a reference standard will be of benefit to the national quarantine system as it can be used to screen fish prior to importation as well as while fish are in a quarantine approved premises, as a way of limiting the inadvertent release of DGIV and related megalocytiviruses from Australian quarantine. The assay also detects red sea bream iridovirus (RSIV). (C) 2012 Elsevier B. V. All rights reserved.