SMALL-SUBUNIT RIBOSOMAL-RNA GENE SEQUENCE OF MINCHINIA-TEREDINIS (HAPLOSPORIDIA, HAPLOSPORIDIIDAE) AND A SPECIFIC DNA-PROBE AND PCR PRIMERSFOR 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 wa s amplified by PCR, cloned, and sequenced. The sequence of M. teredini s SSU rDNA was aligned with that of Haplosporidium nelsoni and various protists in GenBank. A 22-base oligonucleotide probe unique to in. te redinis, designated MIN702, was commercially synthesized and tested fo r sensitivity and specificity. In dot-blot hybridizations the probe de tected 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 furt her tested for specificity with in situ hybridizations on AFA-fixed, p araffin-embedded tissue sections. The probe hybridized well with iii. teredinis plasmodia and immature spores, but poorly with mature spores . The probe did not hybridize with shipworm tissue or with the haplosp oridians Haplosporidium louisiana from mud crabs (Panopeus spp.) or Il . nelsoni and Ii. 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 a ble to detect 10 fg of the cloned gene and also detected the presence of M. teredinis DNA in shipworms in which infections were observed mic roscopically. The 536-bp amplification product was not obtained in one Teredo sp. or in one Bankia gouldi, both categorized as uninfected af ter microscopic inspection. The DNA probe and PCR primers appear to be specific for M. teredinis and should be useful as diagnostic tools an d for life cycle investigations. (C) 1995 Academic Press, Inc.