Proposta de análise diagnóstica de Vibrio em camarão Penaeus vannamei por PCR em tempo real

Abstract

The intensification of shrimp farming has led to a rise in bacterial diseases, particularly those caused by the Vibrio genus. Among them, Vibrio parahaemolyticus stands out as the etiological agent of Acute Hepatopancreatic Necrosis Disease (AHPND), responsible for high mortality rates in Penaeus vannamei. Given the need for rapid and sensitive diagnostic methods, this study aimed to develop and validate a real-time PCR (qPCR) protocol for the detection and quantification of Vibrio sp. and V. parahaemolyticus. Primers were selected through in silico analysis using the Primer-BLAST tool and validated through sequence alignment with MEGA software. The analysis showed that the primers for V. parahaemolyticus exhibited high specificity, amplifying 229 sequences exclusively from the target species. In vitro assays were conducted with different bacterial concentrations mixed with P. vannamei tissue for DNA extraction and subsequent qPCR analysis. The results demonstrated consistent amplification and melting curves, with high reproducibility and sensitivity. Statistical analysis revealed significant differences between experimental groups, confirming the protocol’s ability to quantify bacterial load variation. Thus, the developed protocol proved to be efficient for the molecular diagnosis of Vibrio sp. and V. parahaemolyticus, representing a promising tool for health monitoring in aquaculture