Stokes N A, Siddall M E, Burreson E M
School of Marine Science, Virginia Institute of Marine Science, College of William and Mary, Gloucester Point 23062, USA.
J Invertebr Pathol. 1995 May;65(3):300-8. doi: 10.1006/jipa.1995.1046.
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.
米氏蛀木菌是美国中大西洋沿岸钻木软体动物（船蛆）——船蛆属（Teredo spp.）的一种病原体。从米氏蛀木菌孢子中提取基因组DNA，通过聚合酶链式反应（PCR）扩增、克隆并测序小亚基（SSU）核糖体DNA。将米氏蛀木菌SSU rDNA序列与GenBank中尼尔森单孢子虫（Haplosporidium nelsoni）及各种原生生物的序列进行比对。合成了一种米氏蛀木菌特有的22个碱基的寡核苷酸探针，命名为MIN702，并对其敏感性和特异性进行检测。在斑点杂交中，该探针能检测到500 pg克隆的米氏蛀木菌rDNA。该探针不与船蛆属或尼尔森单孢子虫的克隆SSU rDNA杂交。利用原位杂交技术在乙醇-甲醛-冰醋酸（AFA）固定、石蜡包埋的组织切片上进一步检测该探针的特异性。该探针与米氏蛀木菌的原质团和未成熟孢子杂交良好，但与成熟孢子杂交较差。该探针不与船蛆组织杂交，也不与来自泥蟹（Panopeus spp.）的路易斯安那单孢子虫（Haplosporidium louisiana）、来自弗吉尼亚牡蛎（Crassostrea virginica）的尼尔森单孢子虫和科氏单孢子虫（H. costale）杂交。当用作PCR引物时，该探针和另一个18个碱基的寡核苷酸扩增出米氏蛀木菌SSU rRNA基因的一个536 bp片段。PCR检测能够检测到10 fg的克隆基因，还能检测到在显微镜下观察到有感染的船蛆中米氏蛀木菌DNA的存在。在一个船蛆属物种和一个古氏船蛆（Bankia gouldi）中未获得536 bp的扩增产物，经显微镜检查二者均归类为未感染。DNA探针和PCR引物似乎对米氏蛀木菌具有特异性，应可作为诊断工具用于生命周期研究。
