Janet V. Warg

Eight laboratories worked collectively to evaluate 4 real-time RT-PCR (rRT-PCR) protocols targeting viral hemorrhagic septicemia virus (VHSV) being considered for deployment to a USA laboratory testing network. The protocols utilized previously published primers and probe sets developed for detection and surveillance of VHSV. All participating laboratories received and followed a standard operating protocol for extraction and for each of the rRT-PCR assays. Performance measures specifically evaluated included limit of detection (defined as the smallest amount of analyte in which 95% of the samples are classified as positive), analytical specificity, assay efficiency across genotype representatives, within- and between-plate variation within a laboratory, and variation between laboratories using the same platform, between platforms, and between software versions. This evaluation clearly demonstrated that the TaqMan®-based assay developed by Jonstrup et al. (2013; J Fish Dis 36:9-23) produced the most consistent analytical performance characteristics for detecting all genotypes of VHSV across the 8 participating laboratories.

Two real-time reverse transcription polymerase chain reaction (rRT-PCR) assays under consideration for deployment to multiple testing laboratories across the USA were evaluated for diagnostic sensitivity and specificity on tissue homogenates obtained from natural and experimental viral hemorrhagic septicemia (VHS)-infected fish. Estimates for diagnostic specificity using virus isolation as the reference method were similar between laboratories regardless of the assay. Diagnostic sensitivity estimates of 0.96 (95% CI: 0.95, 0.97) for Jonstrup et al. (2013)'s assay (J Fish Dis 36:9-23) exceeded the diagnostic sensitivity of 0.85 (95% CI: 0.83, 0.87) for Phelps et al. (2012)'s assay (J Aquat Anim Health 24:238-243). The Jonstrup rRT-PCR assay is robust as demonstrated by high sensitivity and specificity estimates across laboratories and can be used as a valuable tool for targeted surveillance and for testing of suspect VHSV samples.

Five spring viremia of carp viruses (SVCV), Rhabdovirus carpio, were isolated in the United States (US) between 2002 and 2004. Single tube reverse transcription-polymerase chain reaction (RT-PCR) was used to generate overlapping cDNA fragments from the US isolates of SVCV. Multiple pairs of specific primers were designed to amplify a portion of the phosphoprotein gene, the matrix gene, and the glycoprotein gene of SVCV genogroup Id (corresponding to nucleotides 2174–4942 of GenBank accession NC_002803). Sequences were proofread and aligned to generate a consensus sequence for each isolate. Phylogenetic analysis of the 2705 nucleotide consensus sequence revealed that all five US isolates belong to SVCV genogroup Ia, Asian origin isolates, and a PCR primer binding site unique to SVCV genogroup Ia was identified.