Validation of a quantitative PCR assay for detection and quantification of 'Candidatus Xenohaliotis californiensis'.
Withering syndrome (WS), a serious disease affecting abalone Haliotis spp., is caused by infection from an intracellular Rickettsia-like organism (WS-RLO). Diagnosis of the disease currently relies on a combination of histological examination and molecular methods (in situ hybridization, standard PCR, and sequence analysis). However, these techniques only provide a semi-quantitative assessment of bacterial load. We created a real-time quantitative PCR (qPCR) assay to specifically identify and enumerate bacterial loads of WS-RLO in abalone tissue, fecal, and seawater samples based on 16S rDNA gene copy numbers. The qPCR assay designed to detect DNA of the WS-RLO was validated according to standards set by the World Organisation for Animal Health. Standard curves derived from purified plasmid dilutions were linear across 7 logs of concentration, and efficiencies ranged from 90.2 to 97.4%. The limit of detection was 3 gene copies per reaction. Diagnostic sensitivity was 100% and specificity was 99.8%. The qPCR assay was robust, as evidenced by its high level of repeatability and reproducibility. This study has shown for the first time that WS-RLO DNA can be detected and quantified in abalone tissue, fecal, and seawater samples. The ability to detect and quantify RLO gene copies in a variety of materials will enable us to better understand transmission dynamics in both farmed and natural environments.
School of Aquatic and Fishery Sciences, University of Washington, Box 355020, Seattle, Washington 98195, USA.
Carolyn S
CS
Friedman
Nate
N
Wight
Lisa M
LM
Crosson
Samuel J
SJ
White
Robyn M
RM
Strenge
D000818:Animals; D049851:Gastropoda / Q000382:microbiology*; D054884:Host-Pathogen Interactions; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012281:Rickettsia / Q000302:isolation & purification*; D012680:Sensitivity and Specificity