Quantification of the bacterial agent of necrotizing hepatopancreatitis (NHP-B) by real-time PCR and comparison of survival and NHP load of two shrimp populations

A real-time quantitative PCR (qPCR) assay was developed using a TaqMan probe to detect and quantify the necrotizing hepatopancreatitis bacterium (NHP-B) in Penaeus vannamei. A pair of primers which amplify a 67 bp DNA fragment and a TaqMan probe were selected from the 16 S rRNA gene of NHP-B genome. A positive control plasmid DNA was used to demonstrate that the NHP qPCR assay has a detection limit of 100 copies and a log-linear range up to 10(8) copies. An NHP-B challenge test using two different populations, Colombia (COL) and Specific Pathogen Free KONA line (KONA), was conducted. Higher final survival and a better survival curve were found in the COL population. No differences in NHP-B bacterial load were found in hepatopancreas (HP) analyzed from the survivors from each population. Sensitivity of the qPCR test was much higher than the conventional PCR (100 copies vs 1 x 10(5) copies). (C) 2010 Elsevier B.V. All rights reserved.