Stokes N A, Burreson E M
School of Marine Science, Virginia Institute of Marine Science, College of William and Mary, Gloucester Point 23062, USA.
J Eukaryot Microbiol. 1995 Jul-Aug;42(4):350-7. doi: 10.1111/j.1550-7408.1995.tb01593.x.
Haplosporidium nelsoni is a pathogen of the eastern oyster, Crassostrea virginica, along the middle Atlantic coast of the U.S. Genomic DNA was extracted from H. nelsoni plasmodia and small subunit (SSU) rDNA was amplified by PCR, cloned and sequenced. The sequence of H. nelsoni SSU rDNA was aligned with that of another haplosporidian, Minchinia teredinis, and with SSU rDNA data of C. virginica and various protists in GenBank. A 21-base oligonucleotide unique to H. nelsoni, designated MSX1347, was commercially synthesized and tested for sensitivity and specificity. In dot blot hybridizations the probe detected 100 pg of cloned H. nelsoni rDNA and the presence of H. nelsoni in 1 microgram of genomic DNA from an infected oyster. It did not hybridize with 1 microgram of genomic DNA from uninfected C. virginica or with cloned SSU rDNA of M. teredinis. The probe was further tested for specificity with in situ hybridizations on AFA-fixed, paraffin-embedded tissue sections. The probe hybridized well with H. nelsoni plasmodia and immature spores, but poorly with mature spores. The probe did not hybridize with oyster tissue, with other common oyster parasites such as P. marinus or Nematopsis sp., or with the haplosporidians Haplosporidium louisiana from mud crabs (Panopeus spp.), Haplosporidium costale from C. virginica or M. teredinis from shipworms (Teredo spp.).
尼尔森单孢子虫是美国中大西洋沿岸地区东部牡蛎（弗吉尼亚牡蛎）的一种病原体。从尼尔森单孢子虫的多核体中提取基因组DNA，通过聚合酶链反应（PCR）扩增、克隆并测序小亚基（SSU）核糖体DNA。将尼尔森单孢子虫SSU rDNA的序列与另一种单孢子虫——船蛆单孢子虫的序列进行比对，并与GenBank中弗吉尼亚牡蛎和各种原生生物的SSU rDNA数据进行比对。一种尼尔森单孢子虫特有的21碱基寡核苷酸，命名为MSX1347，进行了商业合成，并测试了其敏感性和特异性。在斑点杂交中，该探针能检测到100 pg克隆的尼尔森单孢子虫rDNA，以及感染牡蛎1微克基因组DNA中尼尔森单孢子虫的存在。它不与未感染的弗吉尼亚牡蛎的1微克基因组DNA杂交，也不与船蛆单孢子虫的克隆SSU rDNA杂交。该探针在经乙醇-甲醛-冰醋酸（AFA）固定、石蜡包埋的组织切片上进行原位杂交，进一步测试其特异性。该探针与尼尔森单孢子虫的多核体和未成熟孢子杂交良好，但与成熟孢子杂交效果较差。该探针不与牡蛎组织、其他常见的牡蛎寄生虫（如美人鱼虫或类线虫属）杂交，也不与来自泥蟹（豆蟹属）的路易斯安那单孢子虫、来自弗吉尼亚牡蛎的肋状单孢子虫或来自船蛆（船蛆属）的船蛆单孢子虫杂交。
