Evaluation of a novel TaqMan probe-based real-time polymerase chain reaction (PCR) assay for detection and quantitation of red sea bream iridovirus

Abstract

The red sea bream iridovirus (RSIV) belonging to genus Megalocytivirus is responsible for red sea bream iridoviral disease (RSIVD) in marine and freshwater fishes. Although several diagnostic assays for RSIV have been developed, diagnostic sensitivity (DSe) and specificity (DSp) of real-time polymerase chain reaction (PCR) assays are not yet evaluated. In this study, we developed a TaqMan probe-based real-time PCR method and evaluated its DSe and DSp. To detect RSIV, the probe and primers were designed based on consensus sequences of the major capsid protein (MCP) genes from megalocytiviruses including RSIV, infectious spleen and kidney necrosis virus (ISKNV), and turbot reddish body iridovirus (TRBIV). The probe and primers were shown to be specific for RSIV, ISKNV, and TRBIV-types megalocytiviruses. A 95% limit of detection (LOD 95% ) was determined to be 5.3 viral genome copies/μL of plasmid DNA containing the MCP gene from RSIV. The DSe and DSp of the developed real-time PCR assay for field samples (n = 112) were compared with those of conventional PCR assays and found to be 100% and 95.2%, respectively. The quantitative results for SYBR Green and TaqMan probe-based real-time PCR were not significantly different. The TaqMan probe-based real-time PCR assay for RSIV may be used as an appropriate diagnostic tool for qualitative and quantitative analysis.

Partial validation of a TaqMan quantitative polymerase chain reaction for the detection of the three genotypes of Infectious spleen and kidney necrosis virus

“…WOAH chapter Principles and Methods of Validation of Diagnostic Assays for Infectious Diseases [89]. At present, only two real-time PCR assays have been partially validated with consideration of all three ISKNV genotypes which presents a major gap in detecting this pathogen and preventing the pathogen from spreading further globally [90,91]. The lack of a fully validated qPCR assay highlights the need for an assay that is applicable to surveillance and diagnosis of diseases caused by each of the ISKNV genotypes [49,68,70,92].…”

“…The analytic performance of the ISKNV104R qPCR assay exhibited high coefficient of correlation, efficiency, sensitivity (LOD, was less than 10 copies reaction -1 ), specificity (100%), repeatability, and reproducibility. Recently, a TaqMan qPCR assay was developed targeting the RSIV major capsid protein and was partially validated with an even lower LOD of 2.5 viral genome copies [91]. Validation of diagnostic assays with extremely low limits of detections are important in subclinical cases where the animal may have early or recovering infection.…”

Complete Genome Sequences and Pathogenicity Analysis of Two Red Sea Bream Iridoviruses Isolated from Cultured Fish in Korea

“…Tissue samples (spleen and kidney, 50 mg) were collected from diseased Japanese seabass in Tongyeong and rock bream in Goseong in 2017. To identify RSIV infection, realtime polymerase chain reaction (PCR) [15] was carried out. Briefly, each 20 µL real-time PCR mixture contained 1 µL of DNA, which was extracted using the yesG TM Cell Tissue Mini Kit (GensGen, Busan, Korea), 200 nM each primer and probe (Table A1), 10 µL of the 2× HS Prime qPCR Premix (Genet Bio, Daejeon, Korea), 0.4 µL of the 50× ROX dye, and 5.6 µL of nuclease-free water.…”

“…Healthy rock bream (body length: 8.75 ± 1.95 [mean ± SD]; body weight: 6.79 ± 4.16 g) were obtained from an aquaculture farm in Geoje, Korea, after confirming that they were RSIV-free by PCR, as described in the Manual of Diagnostic Tests for Aquatic Animals for RSIVD [2,28], and by real-time PCR [15] (Table A1). The fish were acclimated in a 500 L aqua tank at 25.0 ± 0.5 • C for 2 weeks and were fed a commercial diet once daily.…”

“…Each day, 50% of rearing water was replaced with temperature-adjusted (25 • C) fresh seawater. To prepare a viral inoculum, viral genome copy numbers of cultured 17SbTy and 17RbGs were determined by real-time PCR [15] with a standard curve constructed using the serial dilutions of a plasmid containing the MCP gene of 17RbGs. In a challenge test, each fish group was intraperitoneally injected with 0.1 mL of 17SbTy (n = 18; 10 4 viral genome copies per fish), 17RbGs (n = 18; 10 4 viral genome copies per fish), or PBS (n = 18; a negative control).…”