DAO 108:251-259 (2014)  -  DOI: https://doi.org/10.3354/dao02720

Validation of a quantitative PCR assay for detection and quantification of ‘Candidatus Xenohaliotis californiensis’

Carolyn S. Friedman*, Nate Wight, Lisa M. Crosson, Samuel J. White, Robyn M. Strenge

School of Aquatic and Fishery Sciences, University of Washington, Box 355020, Seattle, Washington 98195, USA
*Corresponding author:

ABSTRACT: 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.


KEY WORDS: Withering syndrome · Rickettsia-like organism · WS-RLO · Validation · Abalone · Haliotis spp.


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Cite this article as: Friedman CS, Wight N, Crosson LM, White SJ, Strenge RM (2014) Validation of a quantitative PCR assay for detection and quantification of ‘Candidatus Xenohaliotis californiensis’. Dis Aquat Org 108:251-259. https://doi.org/10.3354/dao02720

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