Development of a duplex real-time fluorescent quantitative PCR assay for simultaneous detection of white spot syndrome virus and shrimp reference gene

Introduction The white spot syndrome virus (WSSV) is a highly infectious and lethal pathogen that poses a significant threat to the global shrimp farming industry. WSSV can spread rapidly through both horizontal and vertical transmission, leading to mortality rates of up to 100% within 3−10 days post-infection. Due to the absence of effective prevention and treatment measures, developing rapid, accurate, and sensitive detection technologies for WSSV is crucial for timely diagnosis and implementation of control measures to prevent disease spread.

Objective This study aimes to establish a sensitive and specific duplex real-time fluorescent quantitative PCR (qPCR) method for the simultaneous detection of WSSV and an endogenous control gene in shrimp.

Methods First, specific primers and TaqMan probes were designed based on the conserved sequences of the WSSV genome, establishing a single WSSV qPCR detection system. Specificity and repeatability tests confirmed that the method had excellent specificity for WSSV without cross-reactivity, with the coefficient of variation for different gradients of plasmid standards being less than 1.5%, indicating high repeatability. Subsequently, primers and probes for the endogenous control gene of Litopenaeus vannamei were designed and synthesized, and the primer sequences were optimized to establish a duplex qPCR detection system.

Results Sensitivity tests revealed that the detection limit of the duplex system for WSSV was 15 copies·µL⁻¹, with a strong linear relationship between the logarithmic value of the initial template amount and the cycle threshold (Ct) value of WSSV plasmid standards within the range of 6.6 to 6.6×10⁵ copies·µL⁻¹(R²=0.998). Furthermore, the incorporation of the endogenous control gene effectively distinguished false-negative results caused by nucleic acid extraction or operational issues in the samples.

Conclusion In conclusion, the established duplex qPCR detection method for WSSV and the shrimp endogenous control gene exhibits high sensitivity, strong specificity, and good repeatability. This method provides a reliable technical approach for the rapid diagnosis and epidemiological investigation of WSSV, which is crucial for controlling WSSV outbreaks and ensuring the sustainable development of the shrimp farming industry.