The development and initial evaluation of a multiplex RT-PCR and high-throughput liquid chip assay for the simultaneous detection of five salmonid virus diseases

Abstract

Viral diseases pose a major threat to salmonids aquaculture, and mixed infections against salmonids are common. Early detection and identification of viral pathogens are crucial for effective prevention and control measures. This study used samples of infectious hematopoietic necrosis virus (IHNV), infectious pancreatic necrosis virus (IPNV), infectious salmon anemia virus (ISAV), salmonid alphavirus (SAV), and viral hemorrhagic septicemia virus (VHSV) to evaluate a multiplex RT-PCR assay developed for the simultaneous detection of these viruses. A liquid chip technique combined with multiplex RT-PCR was developed. The RT-PCR amplification products were tested using a liquid chip probe. The sensitivity of the assay system was evaluated using 10-fold serial dilutions of RNA extracted from IHNV, IPNV, ISAV, SAV, and VHSV with concentrations of 0.1, 0.4, 2, 0.9, and 1.6 pg µL−1, respectively. The assay was highly specific for these different viruses owing to the use of two nested degenerate primers and one probe. No cross-reactions occurred among the viruses nor did any non-specific reactions occur with other pathogens. The proposed method can be used for rapid, accurate, sensitive, and simultaneous detection of five salmon and trout viruses, thereby contributing to the rapid and accurate quarantine of aquatic animal samples.

Keywords

• Salmonids
• viruses
• quarantine
• general multiplex RT-PCR
• Luminex assay