Simultaneous detection of five shrimp pathogens using a single-tube EvaGreen real-time PCR assay with differential melting temperature

The World Organisation for Animal Health (WOAH) has assessed crustacean diseases, such as infections with white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), decapod iridescent virus 1 (DIV1), and acute hepatopancreatic necrosis disease (AHPND), as listed diseases, and infection with <i>Ecytonucleospora hepatopenaei</i> (EHP) as an emerging disease, all of which significantly threaten the shrimp industry. This study developed a quintuplex EvaGreen-based melting curve real-time PCR method for the simultaneous detection of WSSV, IHHNV, DIV1, AHPND-causing <i>Vibrio</i> (<i>V</i>_AHPND), and EHP. In the specific assay, only the target pathogen demonstrated efficient and detectable amplification, thereby indicating that the method exhibits high specificity. Regarding sensitivity testing, the five pathogens were detected at a concentration of 1.0 × 10¹ copies/μL. Each concentration gradient was evaluated in triplicate, and the coefficient of variation for each gradient remained below 3.38%, thereby affirming that the method demonstrates highly repeatability. We tested the diagnostic sensitivity (DSe) and the diagnostic specificity (DSp) of our method using a total of 800 clinical samples which were gathered from the shrimp farming regions in China. The newly established method in this study demonstrated a DSe above 89.74% for the five pathogens and a DSp of 100%. The quintuplex EvaGreen real-time PCR method developed here offers an accurate and efficient approach for EHP, WSSV, <i>V</i>_AHPND, IHHNV, and DIV1.IMPORTANCECrustacean diseases, such as infections with WSSV, IHHNV, DIV1, <i>V</i>_AHPND, and EHP, pose a significant threat to the global shrimp industry, leading to substantial economic losses. Rapid, accurate, and simultaneous detection of these pathogens is crucial for effective disease management and biosecurity in shrimp farming. In this study, we developed a quintuplex EvaGreen-based melting curve real-time PCR method that enables the simultaneous detection of these five major shrimp pathogens with exceptional specificity, sensitivity, and repeatability. By evaluating 800 clinical samples, our method demonstrated high diagnostic sensitivity and specificity, making it a valuable tool for early pathogen detection and disease control. This novel approach can help mitigate disease outbreaks, improve shrimp farm productivity, and support the sustainable development of the aquaculture industry.