A TaqMan probe-based RT-qPCR for the detection of brine shrimp reovirus 1

Abstract

Brine shrimp (Artemia) are widely utilized as a vital live feed in the aquaculture industry. Recently, we found a novel reovirus - brine shrimp reovirus 1 (BSR1) in brine shrimp. Considering the potential impact of reovirus on the industry, it is necessary to develop a highly sensitive and specific detection method in advance for identifying BSR1. In this study, we established a real-time quantitative reverse transcription PCR (RT-qPCR) method based on TaqMan probe. The detection limit of this method is as low as 4.4 × 10¹ copies/reaction. The standard curve between 4.4 × 10¹ to 4.4 × 10⁹ copies/reaction showed a high correlation coefficient (R² = 0.997). This newly developed method was used to screen for BSR1 in brine shrimp cyst samples from Africa, Asia, Europe, Oceania, South America and North America, and the results showed that BSR1 was detected in samples from Asia and North America. The brine shrimp cyst samples tested covered seven different species, with BSR1-positive samples found in A. franciscana, A. urmiana, A. sinica, and parthenogenetic Artemia lineage. The diagnostic sensitivity (DSe) and diagnostic specificity (DSp) values of the newly developed RT-qPCR method, when compared to the high-throughput sequencing, were determined to be 100 % and 88 %. This newly developed RT-qPCR method can serve as a crucial tool for detecting BSR1, facilitating early warning and swift response to viruses carried by brine shrimp, thereby effectively mitigating the risk of pathogen transmission to cultured animals.