长牡蛎尼氏单孢子虫的初步研究

Primary study on Haplosporidium nelsoni in Crassostrea gigas

采集大连大窑湾养殖区的长牡蛎(Crassostrea gigas),抽取其血淋巴液在显微镜下观察,观察到尼氏单孢子虫(Hap-losporidium nelsoni)原生质体的类似物。对抽取的血淋巴液进行总基因组DNA的提取,将扩增单孢子虫(Haplosporidiosis)SSU rDNA区域的引物对HAP-F2和HAP-R2进行改进,经过特异性试验证明其特异性很好,得到引物对HAP-F和HAP-R,应用此对引物进行聚合酶链式反应(PCR),结果扩增出239bp条带。将PCR产物进行测序,经序列比对分析确定为尼氏单孢子虫。同时,采用尼氏单孢子虫的特异性DNA探针MSX1347对抽取的长牡蛎血淋巴液进行原位杂交,结果也检测到了尼氏单孢子虫。

Hemolymph extracted from Crassostrea gigas in Dayaowan Bay was examined by the microscope and Haplosporidium-like plasmodia stages were found in Hemolymph. DNA was extracted from the infected oyster Hemolymph for polymerase chain reaction （PCR） amplification of the haplosporidian. The primer HAP-F and HAP-R, which was modification of Hap-F2 and HAP-F2 previously reported, was specific to H. nelsoni by experiments and was applied to amplify short regions of small subunit ribosomal DNA （ SSU rDNA ） from H. nelsoni. PCR amplification of genomic DNA with primer HAP-R and HAP-F yielded the expected-sized product from the primer pair targeting the region for 239 bp. This PCR product was sequenced and it was identical to the corresponding SSU rDNA region of H. nelsoni. The Haplosporidium nelsoni-specific DNA probes MSXl347 hybridized with the C. gigas parasite.