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

Diseases of Aquatic Organisms

Lisa M. Crosson¹  Samuel J. White¹  Nate Wight¹  Carolyn S. Friedman¹  Robyn M. Strenge¹ 

关键词: Withering syndrome;    Rickettsia-like organism;    WS-RLO;    Validation;    Abalone;    Haliotis spp.;   

DOI  :  10.3354/dao02720

学科分类：生物科学（综合）

来源: Inter-Research

【 摘 要 】

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.

【 授权许可】

Unknown