Development and a TaqMan probe-based qPCR assay for Vibrio parahaemolyticus causing translucent post-larvae disease...

Pacific white shrimp, Litopenaeus vannamei, aquaculture industry faces severe threats from translucent post-larvae disease (TPD), caused by the recently identified highly virulent Vibrio parahaemolyticus (VpTPD) harboring the critical vhvp-2 virulence gene. Therefore, establishing a prompt and precise detection method is fundamental for initial diagnosis and the effective management of this disease. This study developed a vhvp-2-targeted TaqMan probe-based qPCR assay for rapid VpTPD detection. Through rigorous optimization, the method achieved a detection limit of 14.64 copies/reaction with high amplification efficiency (E = 109.25%) and strong linear correlation (R² = 0.9916), while demonstrating no cross-reactivity with six major shrimp pathogens. It exhibited robust matrix interference resistance at high background DNA concentrations (≤2000 ng/μL), and clinical validation on 104 samples (100 infected shrimps and 4 healthy shrimps) demonstrated 100% diagnostic sensitivity and specificity, as reflected by the perfect concordance between test results and actual infection status. Comparative analysis with our previously established SYBR Green-based qPCR assay revealed distinct advantages. While the SYBR Green-based assay showed superior sensitivity with limit of detection (LOD) at 10⁰ copies/reaction, the TaqMan-based assay demonstrated enhanced interference resistance and shorter analytical turnaround time. Notably, TaqMan probe synthesis incurred 10-fold higher costs than SYBR Green reagents. These experimental data revealed that the TaqMan probe-based qPCR assay, with its high precision, was ideal for confirmatory laboratory diagnostics, whereas the SYBR Green-based qPCR method offered a cost-effective alternative for large-scale field screening. This dual-method framework provides a comprehensive solution for TPD prevention and detection across diverse aquaculture environments.