Development and application of SYBR Green-based qPCR method to detect Vibrio parahaemolyticus causing translucent post-larvae disease in shrimp Litopenaeus vannamei

Abstract

As a dominant species in global aquaculture, Litopenaeus vannamei not only represents one of the three most extensively farmed shrimp varieties, but had also emerged as a core economic species in many countries. However, shrimp farming operations have been persistently plagued by diseases and infections, with multiple highly contagious and lethal pathogens causing devastating economic losses. Among them, translucent post-larvae disease (TPD) was characterized as a novel pathology attributable to a distinct subtype of Vibrio parahaemolyticus (VpTPD), which harbors three virulence genes designated as vhvp-1, vhvp-2 and vhvp-3. In this research, a series of SYBR Green-based qPCR methods were efficiently applied to detect and quantify the vhvp-1, vhvp-2 and vhvp-3 genes of VpTPD. By designing 18 pairs of primers and screening the optimal combination, a highly specific amplification system with vhvp-2-F3/vhvp-2-R3 primer pair was successfully established. Quantitative analysis demonstrated that the limit of detection in this assay for detecting VpTPD (vhvp-2) was 4.46 × 10⁰ copies/μL. Furthermore, the standard curve exhibited excellent linearity (R² = 0.992), and a single distinct peak was observed in the melting curve, indicating the absence of non-target products. Specificity tests confirmed that the method had no amplification reaction against any other common pathogens. In addition, the detection system remained stable at background shrimp DNA concentrations as high as 1500 ng/μL, and achieved 100 % detection rates in practical testing. Compared with traditional methods, these SYBR Green-based qPCR methods developed in this research possessed superior sensitivity, strong specificity and anti-interference ability, which could efficiently detect VpTPD pathogens and provided reliable technical support for large-scale screening and early prevention in shrimp farming.