Gunderson J H, Goss S H, Coats D W
Department of Biology, Tennessee Technological University, Cookeville 38505, USA.
J Eukaryot Microbiol. 2001 Nov-Dec;48(6):670-5. doi: 10.1111/j.1550-7408.2001.tb00207.x.
A comparison of the small subunit rRNA sequences of a Chesapeake Bay strain of the dinoflagellate Akashiwo sanguinea and the dinoflagellate Amoebophrya sp. parasitizing it revealed several potential target sites that could be used to detect the parasite through in situ hybridization. The fluorescence of probed cells under various conditions of hybridization was measured by using a spot meter on a Nikon UFX-II camera attachment so that the effect of various hybridization parameters on probe binding could be determined. Probes directed against both the junction between helices 8 and 11 and helix 46 could detect the parasite, although the helix 8/11 probe produced a stronger signal under the conditions tested. The fluorescence of the probed cells increased with increasing hybridization time up to approximately twelve hours. The background fluorescence was lower at the wavelengths used to detect Texas Red than at those used to detect fluorescein, so probed cells were more distinct when Texas Red was used as the label. Cells stored in cold paraformaldehyde for a year still bound the probes. Young stages of the parasite could be seen more readily after in situ hybridization than after protargol impregnation.
对切萨皮克湾血红裸甲藻（Akashiwo sanguinea）的一个菌株以及寄生于其上的寄生性裸甲藻（Amoebophrya sp.）的小亚基rRNA序列进行比较，发现了几个潜在的靶位点，可用于通过原位杂交检测该寄生虫。使用尼康UFX-II相机附件上的点测光仪测量在各种杂交条件下探针标记细胞的荧光，以便确定各种杂交参数对探针结合的影响。针对螺旋8和11之间的连接处以及螺旋46的探针都能检测到该寄生虫，尽管在测试条件下螺旋8/11探针产生的信号更强。探针标记细胞的荧光随着杂交时间的增加而增加，直至约12小时。在用于检测德克萨斯红的波长下背景荧光低于用于检测荧光素的波长，因此当使用德克萨斯红作为标记时，探针标记的细胞更清晰。保存在冷多聚甲醛中一年的细胞仍能与探针结合。与原银浸染后相比，原位杂交后更容易看到寄生虫的幼体阶段。
