Quantitative PCR for detection and quantification of Veronaea botryosa in fish and environmental samples

被引:5
|
作者
Yazdi, Zeinab [1 ]
Griffin, Matt J. [2 ]
Pierezan, Felipe [3 ]
Eetemadi, Ameen [4 ]
Shahin, Khalid [1 ]
Soto, Esteban [1 ]
机构
[1] Univ Calif Davis, Sch Vet Med, Dept Med & Epidemiol, Davis, CA 95616 USA
[2] Mississippi State Univ, Coll Vet Med, Dept Pathobiol & Populat Med, Stoneville, MS 38776 USA
[3] Univ Fed Minas Gerais, Sch Vet Med, BR-31279901 Belo Horizonte, MG, Brazil
[4] Univ Calif Davis, Dept Comp Sci, Davis, CA 95616 USA
基金
美国食品与农业研究所;
关键词
Fluid belly; White sturgeon; Molecular; Fungus; Phaeohyphomycosis; CHAIN-REACTION ASSAY; REAL-TIME PCR; GENETIC DIVERSITY; EDWARDSIELLA-ICTALURI; DNA EXTRACTION; FORMALIN; QPCR; AMPLIFICATION; HERPESVIRUS; INFECTIONS;
D O I
10.3354/dao03582
中图分类号
S9 [水产、渔业];
学科分类号
0908 ;
摘要
Systemic phaeohyphomycosis, aka 'fluid belly', is one of the most important emergent diseases in sturgeon Acipenser spp. aquaculture. The etiologic agent is the saprobic, dematiaceous fungus Veronaea botryosa. Effective vaccines and chemotherapeutic treatments are currently unavailable. Additionally, the fungus is a slow-growing organism, taking from 10-15 d for colonies to be observed in agar media. To this end, a specific quantitative PCR (qPCR) targeting the V. botryosa beta-tubulin gene was developed and validated. The specificity of the assay to V. botryosa was initially confirmed in silico and in vivo against common fungal fish pathogens, including closely related members of the order Chaetothyriales (Exophiala spp.) and other black pigmented fungi (Alternaria spp. and Cladosporium spp.), as well as tissues from uninfected sturgeon. The assay possessed high clinical specificity (100%) and clinical sensitivity (74%) in detecting V. botryosa DNA in splenic tissues from laboratory-infected sturgeon. Using V. botryosa genomic DNA as a template, the limit of detection was equivalent to 10 conidia, and the method was found suitable for the detection of fungal DNA in fresh and formalin-fixed tissues. In addition, the presence of non-target DNA from white sturgeon did not influence assay sensitivity. The developed qPCR assay is a sensitive, specific, and rapid diagnostic method for the detection and quantification of V. botryosa DNA from white sturgeon tissues.
引用
收藏
页码:175 / 185
页数:11
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