Evaluation of a novel TaqMan probe-based real-time polymerase chainreaction (PCR) assay for detection and quantitation of red sea breamiridovirus

Fisheries and Aquatic Sciences

卷:24

Min Ji Koo¹  Hee Ju Choi²  Joon Gyu Min²  Min Jae Kim²  Min Jeong Kim²  Kwang Il Kim²  Guk Hyun Kim² 

[1] ;

[2] Department of Aquatic Life Medicine,Pukyong National University, ;

关键词: red sea bream iridovirus;    taqman probe;    real-time polymerase chain reaction (pcr);    diagnostic sensitivity;    diagnostic specificity;   

DOI  :  10.47853/FAS.2021.e34

来源: DOAJ

【 摘 要 】

The red sea bream iridovirus (RSIV) belonging to genusMegalocytivirus is responsible for red sea bream iridoviraldisease (RSIVD) in marine and freshwater fishes. Although several diagnosticassays for RSIV have been developed, diagnostic sensitivity (DSe) andspecificity (DSp) of real-time polymerase chain reaction (PCR) assays are notyet evaluated. In this study, we developed a TaqMan probe-based real-time PCRmethod and evaluated its DSe and DSp. To detect RSIV, the probe and primers weredesigned based on consensus sequences of the major capsid protein (MCP) genesfrom megalocytiviruses including RSIV, infectious spleen and kidney necrosisvirus (ISKNV), and turbot reddish body iridovirus (TRBIV). The probe and primerswere shown to be specific for RSIV, ISKNV, and TRBIV-types megalocytiviruses. A95% limit of detection (LOD95%) was determined to be 5.3 viral genomecopies/μL of plasmid DNA containing the MCP gene from RSIV. The DSe andDSp of the developed real-time PCR assay for field samples (n = 112) werecompared with those of conventional PCR assays and found to be 100% and 95.2%,respectively. The quantitative results for SYBR Green and TaqMan probe-basedreal-time PCR were not significantly different. The TaqMan probe-based real-timePCR assay for RSIV may be used as an appropriate diagnostic tool for qualitativeand quantitative analysis.

【 授权许可】

Unknown