Development of a Multiplex Assay to Measure the Effects of Shipping and Storage Conditions on the Quality of RNA Used in Molecular Assays for Detection of Viral Haemorrhagic Septicemia Virus

In routine diagnostics, real-time reverse transcriptase quantitative PCR(RT-qPCR) has become a powerful method for fish health screening. Collection, transportation, and storage conditions of specimens could dramatically affect their integrity and could consequently affect RT-qPCR test results. In this study, to assess the expression profile of elongation factor 1 alpha (ELF-1 alpha) gene, head kidney (HK) tissues from Atlantic Salmon Salmo salar were exposed at room temperature, 4 degrees C, -20 degrees C, and -80 degrees C as well as in 70% ethanol for 6, 12, 24, 48, and 72 h. Data showed a significant increase of RT-qPCR cycle threshold (Ct) values for ELF-1 alpha ranging from 14.7 to 26.5 cycles for tissues exposed to room temperature. In order to mimic the sample transportation conditions, different temperatures of storage were used and tissue quality was evaluated using ELF-1 alpha gene expression. Data showed that Ct values for ELF-1 alpha increased significantly when the tissues were transported on ice for 2 h, stored at -20 degrees C, thawed on ice for 6 h, and stored again at -80 degrees C. The HK tissues collected from Atlantic Salmon challenged with viral hemorrhagic septicemia virus (VHSV) through intraperitoneal injection were exposed at room temperature for 0, 6, 12, 24, 48, 72, and 96 h. Data showed a good correlation of values for ELF-1 alpha and VHSV Ct although the ELF-1 alpha mRNA of the host degraded faster than the RNA of VHSV. Based on these data, HK tissues could be transported on ice or ice packs without the quality of the tissue being affected when stored at -80 degrees C upon arrival at the laboratory. In addition, 70% ethanol could be used as a preservative for long-distance transportation. For an efficient diagnostic test, a duplex VHSV-ELF-1 alpha was developed and optimized. Data showed that the sensitivity of the duplex assay for VHSV was similar to the singleplex.