Characterization of the rRNA Locus of Pfiesteria piscicida and Development of Standard and Quantitative PCR-Based Detection Assays Targeted to the Nontranscribed Spacer

AUTOR(ES)

Saito, Keiko

FONTE

American Society for Microbiology

RESUMO

Pfiesteria piscicida is a heterotrophic dinoflagellate widely distributed along the middle Atlantic shore of the United States and associated with fish kills in the Neuse River (North Carolina) and the Chesapeake Bay (Maryland and Virginia). We constructed a genomic DNA library from clonally cultured P. piscicida and characterized the nontranscribed spacer (NTS), small subunit, internal transcribed spacer 1 (ITS1), 5.8S region, ITS2, and large subunit of the rRNA gene cluster. Based on the P. piscicida ribosomal DNA sequence, we developed a PCR-based detection assay that targets the NTS. The assay specificity was assessed by testing clonal P. piscicida and Pfiesteria shumwayae, 35 additional dinoflagellate species, and algal prey (Rhodomonas sp.). Only P. piscicida and nine presumptive P. piscicida isolates tested positive. All PCR-positive products yielded identical sequences for P. piscicida, suggesting that the PCR-based assay is species specific. The assay can detect a single P. piscicida zoospore in 1 ml of water, 10 resting cysts in 1 g of sediment, or 10 fg of P. piscicida DNA in 1 μg of heterologous DNA. An internal standard for the PCR assay was constructed to identify potential false-negative results in testing of environmental sediment and water samples and as a competitor for the development of a quantitative competitive PCR assay format. The specificities of both qualitative and quantitative PCR assay formats were validated with >200 environmental samples, and the assays provide simple, rapid, and accurate methods for the assessment of P. piscicida in water and sediments.

Documentos Relacionados

• Genus- and Species-Specific PCR-Based Detection of Dairy Propionibacteria in Environmental Samples by Using Primers Targeted to the Genes Encoding 16S rRNA
• Rapid Identification of Penicillium marneffei by PCR-Based Detection of Specific Sequences on the rRNA Gene
• Identification of Medically Important Yeasts Using PCR-Based Detection of DNA Sequence Polymorphisms in the Internal Transcribed Spacer 2 Region of the rRNA Genes
• Detection and Quantification of Dehalogenimonas and “Dehalococcoides” Populations via PCR-Based Protocols Targeting 16S rRNA Genes▿ †
• PCR-based preparation of 23S rRNA-targeted group-specific polynucleotide probes.