Rao S Nageswara, Nath B Surendra, Bhuvaneswari G, Urs S Raje
Seribiotech Research Laboratory, CSB Campus, Carmelram Post, Kodathi, Bangalore 560 035, India.
J Invertebr Pathol. 2007 Nov;96(3):193-204. doi: 10.1016/j.jip.2007.05.001. Epub 2007 May 13.
Random amplification of polymorphic DNA polymerase chain reaction (RAPD-PCR) and pathological, morphological and ultrastructural characterization were used to differentiate seven new microsporidian isolates infecting the mulberry silkworm, Bombyx mori. The pathogenicity observed was dose-dependent and differed from each of the microsporidian isolates; the NIK-4m was found to be more virulent than other isolates. However, all the isolates, except NIK-4m, showed heavy gonadal infection and vertical transmission in the infected silkworms. Differences in the spore shape ranging from oval to elongate were observed, and the polar filament has 8-16 coils arranged in one or two rows. Of the 80 decamer random primers tested, 50 generated reproducible RAPD profiles and yielded a total of 600 fragments, of which 594 were polymorphic (99%). Forty nine RAPD primers produced 179 unique genetic markers, whose presence or absence differed among the microsporidians, albeit with varied efficiency of polymorphism detection. The degree of band sharing was used to evaluate genetic distances between different microsporidian isolates and to construct a phylogenetic tree using Dice coefficients. Cluster analysis based on Dice coefficients resulted in the formation of one major cluster consisting of NIK-1s, NIAP-7g, NIK-2r and NIK-5d and NIK-4m in the other; while NIAP-6p was intermediate between these two. NIK-8b and NITN-9n were found to be entirely different from others. Reproducible RAPD patterns of all microsporidian isolates enabled us to differentiate the microsporidian isolates. The results demonstrate that besides ultrastructural studies, RAPD-PCR can be a useful and reliable tool to detect polymorphism, genetic relationships, and for the identification of the microsporidians. In addition, DNA fingerprints generated in this process have potential applications as diagnostic tools for identification of different microsporidia with considerable accuracy.
采用随机扩增多态性DNA聚合酶链反应（RAPD-PCR）以及病理学、形态学和超微结构特征分析，对感染家蚕（Bombyx mori）的7种新微孢子虫分离株进行鉴别。观察到的致病性呈剂量依赖性，且每种微孢子虫分离株的致病性有所不同；发现NIK-4m比其他分离株的毒性更强。然而，除NIK-4m外，所有分离株在受感染的家蚕中均表现出严重的性腺感染和垂直传播。观察到孢子形状从椭圆形到细长形存在差异，极丝有8 - 16个呈一排或两排排列的螺旋。在所测试的80种十聚体随机引物中，50种产生了可重复的RAPD图谱，共产生600个片段，其中594个具有多态性（99%）。49种RAPD引物产生了179个独特的遗传标记，这些标记在微孢子虫中的存在与否各不相同，尽管多态性检测效率有所差异。利用条带共享程度评估不同微孢子虫分离株之间的遗传距离，并使用Dice系数构建系统发育树。基于Dice系数的聚类分析形成了一个主要聚类，其中包括NIK-1s、NIAP-7g、NIK-2r和NIK-5d，另一个聚类包括NIK-4m；而NIAP-6p介于这两者之间。发现NIK-8b和NITN-9n与其他分离株完全不同。所有微孢子虫分离株可重复的RAPD模式使我们能够区分这些微孢子虫分离株。结果表明，除了超微结构研究外，RAPD-PCR可以成为检测多态性、遗传关系以及鉴定微孢子虫的有用且可靠的工具。此外，在此过程中产生的DNA指纹图谱作为诊断工具，具有以相当高的准确性鉴定不同微孢子虫的潜在应用价值。
