Small subunit ribosomal RNA gene sequence of Minchinia teredinis (Haplosporidia: Haplosporidiidae) and a specific DNA probe and PCR primers for its detection.
Minchinia teredinis is a pathogen of wood-boring molluscs (shipworms), Teredo spp., along the middle Atlantic coast of the U.S. Genomic DNA was extracted from M. teredinis spores and small subunit (SSU) rDNA was amplified by PCR, cloned, and sequenced. The sequence of M. teredinis SSU rDNA was aligned with that of Haplosporidium nelsoni and various protists in GenBank. A 22-base oligonucleotide probe unique to M. teredinis, designated MIN702, was commercially synthesized and tested for sensitivity and specificity. In dot-blot hybridizations the probe detected 500 pg of cloned M. teredinis rDNA. The probe did not hybridize with cloned SSU rDNA of Teredo spp. or H. nelsoni. The probe was further tested for specificity with in situ hybridizations on AFA-fixed, paraffin-embedded tissue sections. The probe hybridized well with M. teredinis plasmodia and immature spores, but poorly with mature spores. The probe did not hybridize with shipworm tissue or with the haplosporidians Haplosporidium louisiana from mud crabs (Panopeus spp.) or H. nelsoni and H. costale from Crassostrea virginica. The probe and a second 18-base oligonucleotide, when used as PCR primers, amplified a 536-bp fragment of the M. teredinis SSU rRNA gene. The PCR assay was able to detect 10 fg of the cloned gene and also detected the presence of M. teredinis DNA in shipworms in which infections were observed microscopically. The 536-bp amplification product was not obtained in one Teredo sp. or in one Bankia gouldi, both categorized as uninfected after microscopic inspection. The DNA probe and PCR primers appear to be specific for M. teredinis and should be useful as diagnostic tools and for life cycle investigations.
Stokes
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NA
School of Marine Science, Virginia Institute of Marine Science, College of William and Mary, Gloucester Point 23062, USA.
Siddall
M E
ME
Burreson
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EM
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D015342:DNA Probes; D016054:DNA, Protozoan / Q000032:analysis; D056890:Eukaryota / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D008974:Mollusca / Q000166:cytology / Q000469:parasitology*; D016133:Polymerase Chain Reaction / Q000379:methods; D016053:RNA, Protozoan / Q000032:analysis; D012336:RNA, Ribosomal, 16S / Q000032:analysis
D017931:DNA Primers; D015342:DNA Probes; D016054:DNA, Protozoan; D016053:RNA, Protozoan; D012336:RNA, Ribosomal, 16S