Development and validation of a real‐time PCR assay for the detection of Aeromonas salmonicida

Abstract

A real-time PCR assay using a molecular beacon was developed and validated to detect the vapA (surface array protein) gene in the fish pathogen, Aeromonas salmonicida. The assay had 100% analytical specificity and analytical sensitivities of 5 ± 0 fg (DNA), 2.2 × 10(4) ± 1 × 10(4) CFU g(-1) (without enrichment) and 40 ± 10 CFU g(-1) (with enrichment) in kidney tissue. The assay was highly repeatable and proved to be robust following equivalency testing using a different real-time PCR platform. Following analytical validation, diagnostic specificity was determined using New Zealand farmed Chinook salmon, Oncorhynchus tshawytscha (Walbaum), (n = 750) and pink shubunkin, Carassius auratus (L.) (n = 157). The real-time PCR was run in parallel with culture and all fish tested were found to be negative by both methods for A. salmonicida, resulting in 100% diagnostic specificity (95% confidence interval). The molecular beacon real-time PCR system is specific, sensitive and a reproducible method for the detection of A. salmonicida. It can be used for diagnostic testing, health certification and active surveillance programmes.

Aeromonas salmonicida infection levels in pre‐ and post‐stocked cleaner fish assessed by culture and an amended qPCR assay

“…Introduction of the original A. salmonicida qPCR protocol described by Keeling et al . () to our laboratory required several modifications to reagent concentrations and thermal cycling parameters (see Materials and methods for details). Further, alignment of the original oligonucleotide sequences against all publicly available vapA sequence types (Gulla et al .…”

“…We were unable to consistently detect equally low concentrations of A. salmonicida pure culture DNA as reported by Keeling et al . () (increase in LOD from ~1 to 7–8 genomes per template; Fig. ).…”

“…Prior to A. salmonicida qPCR screening, several modifications to the original assay (Keeling et al . ) were required (see Materials and methods and Results for details). This included the need for a new primer (Table ) due to sequence mismatch against the target gene ( vapA ) of A‐layer type XIV and V strains (the latter thus far exclusively cultured from wrasse; Gulla et al .…”

Development of an Absolute Quantitative Real-Time PCR (qPCR) for the Diagnosis of Aeromonas hydrophila Infections in Fish

“…Notably, the detection of ahaI in tilapia´s kidney experimentally infected with A. hydrophila was only possible using real-time qPCR. to the literature published [16,17].…”

Development and Application of a Quantitative Real‐time Polymerase Chain Reaction Assay for the Detection of Aeromonas salmonicida

“…Recently, there have been some reports on the detection of A . salmonicida by quantitative real‐time PCR (qPCR) based on molecular beacons (Liu et al ; Keeling et al ); however, the detection of A . salmonicida by qPCR based on the fluorescent dye SYBR Green I has not been reported until now.…”