Desenvolvimento e otimização de protocolos de PCR em tempo real para o diagnóstico de patógenos emergentes para a aquicultura nacional

Abstract

Outbreaks of infectious diseases are one of the main problems for national aquaculture development. The WSSV and IHHNV viruses, and streptococcal agents have been considered emergent pathogens for Brazilian shrimp, and tilapia farming, respectively. Fast, accurate and sensible diagnostic tools are fundamental for the control of diseases and biosecurity programs. The real-time PCR is a technology which has those characteristics, being an alternative. The aims of this project were: to standard and optimize qPCR assays with hydrolyze probes, containing non fluorescent quencher, for detection of WSSV and IHHNV; to develop a duplex qPCR to detect co-infection cases of WSSV and IHHNV in cultivated whiteleg shrimp (Litopenaeus vannamei); and to develop a duplex qPCR to diagnose Streptococcus agalactiae and Streptococcus dysgalactiae infections in Nile tilapia (Oreochromis niloticus). The use of non fluorescent quenched probe improved significantly the clinical sensitivity of qPCR for WSSV detection. WSSV and IHHNV co-infection cases were successfully diagnosed with the duplex qPCR standardized. The developed duplex qPCR for S. agalactiae and S. dysgalactiae diagnosis presented higher clinical sensitivity than bacteriological assay and PCR. qPCR showed superior performances than other methods used to diagnose those pathogens. The qPCR assays described in the present work showed to be valuable alternatives to diagnose the emergent pathogens for Brazilian aquaculture.