Development of an 18S rRNA gene-targeted PCR-based diagnostic for the blue crab parasite Hematodinium sp.
Development of an 18S rRNA gene-targeted PCR-based diagnostic for the blue crab parasite Hematodinium sp.
复制标题
DOI：
10.3354/dao049061
复制
发表时间：
2002-04-24
影响因子：
1.4
通讯作者：
Lee, RF
中科院分区：
文献类型：
--
作者：
Gruebl, T;Frischer, ME;Lee, RF
对侵染青蟹的甲藻血藻的18S rRNA基因进行了扩增、克隆和序列测定。该序列与从其他甲藻物种获得的序列具有很高的相似性(在核苷酸水平上为95%)，包括自由生活和共生物种。与生活史相似的其他海洋无脊椎动物的寄生虫和蓝蟹的18S rRNA基因相比，序列相似性要低得多。在比较中华绒毛虫、其他甲鞭毛虫、牡蛎原生动物和青蟹185rRNA基因序列比对的基础上，设计了两套特异扩增18S rRNA基因片段的聚合酶链式反应(PCR)引物。其中一组(HEMAT-F-1483和HEMAT-R-1654)扩增出183BY片段，可用于诊断青蟹血淋巴中是否存在血球线虫。从感染血吸虫的青蟹血淋巴基因组DNA中连续扩增出该片段。将该方法与标准组织学检查方法进行比较，结果表明，该方法是可靠的，其灵敏度是组织学方法的1000倍。PCR诊断的敏感性估计为30万个螃蟹血细胞中有一个寄生虫细胞。初步研究表明，利用聚合酶链式反应诊断技术，血藻。在低盐度(5至10ppt)水域采集的螃蟹中不存在，但在美国佐治亚州东南部河口水域较高盐度环境中常见。
The 18S rRNA gene from Hematodinum sp., a parasitic dinoflagellate that infects blue crabs, was amplified, cloned, and sequenced. The sequence showed a high similarity (95% at the nucleotide level) to sequences obtained from other dinoflagellate species, including both free-living and symbiotic species. Sequence similarity was much lower when compared with parasites of other marine invertebrates with similar life histories and with the 18S rRNA gene from the blue crab. Based on comparison of sequence alignments between Hematodinium, other dinoflagellate species, protozoan pathogens of oysters, and blue crab 185 rRNA gene sequences, 2 sets of PCR primers that specifically amplified fragments of the Hematodinium 18S rRNA gene were developed and tested. One of these primer sets (Hemat-F-1483 and Hemat-R-1654) amplified a 183 by fragment that could be used routinely as a diagnostic test for the presence of Hematodinium in hemolymph from blue crabs. This fragment was consistently amplified from genomic DNA extracted from hemolymph of Hematodinium infected blue crabs. Comparison between the PCR technique and standard histological examination indicated that the PCR technique was reliable and provided 1000 times more sensitivity than the histological methods. The sensitivity of the PCR diagnostic was estimated to be one parasite cell among 300 000 crab hemocytes. Preliminary studies using the PCR diagnostic technique suggest that Hematodinium sp. is absent in crabs collected from waters with low salinity (5 to 10 ppt), but common in crabs from higher salinity environments in estuarine waters from southeastern Georgia (USA).