Detection and surveillance of viral hemorrhagic septicemia virus using real-time RT-PCR. I. Initial comparison of four protocols

Diseases of Aquatic Organisms

Emily R. Cornwell¹  Robert Kim¹  Kathy Toohey-Kurth¹  Mohamed Faisal¹  Andrew E. Goodwin¹  Geoffrey H. Groocock¹  Monica M. Reising¹  Cem Giray¹  Gwenn E. Merry¹  0¹  Isaac Standish¹  Nicholas B. D. Phelps¹  Janet V. Warg¹  Rodman G. Getchell¹  Yan Zhang¹  Travis Clement¹  Angela Cruz¹ 

关键词: VHSV;    Surveillance;    Real-time RT-PCR;    Analytical sensitivity;    Analytical specificity;    Validation;   

DOI  :  10.3354/dao02753

学科分类：生物科学（综合）

来源: Inter-Research

【 摘 要 】

ABSTRACT: 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.

【 授权许可】

Unknown