Spiroplasma 16S rDNA in TSE Brain Tissues

描述(改编自申请人的摘要)：本病的发病机制 传染性海绵状脑病(TSE)，包括克氏病 人类的雅各布病(CJD)和绵羊的瘙痒病仍然是一个谜。在这 应用我们提出的证据为螺旋体属，a。 无壁原核生物，带TSE。我们展示了螺旋体的聚合酶链式反应扩增。 TSE感染脑组织中的16S rDNA(19例)，而对照组脑组织中未检测到16S rDNA(0 所以)。对扩增的PCR产物进行直接测序证实了这一点 在所有19个东京证交所的大脑中都发现了螺旋体样DNA。我们的证据不是 必然与PrPres的参与冲突，PrPres是一种抗蛋白酶的宿主 在TSE的发病机制中，派生蛋白被称为Prion，因为 有证据表明还有另一个因素参与其中，我们建议这是一个 细菌。我们的策略将是通过将 TSE中螺旋体核糖体基因的全部序列，然后进行统计学筛选 大量的TSE病例。在目标1中，我们将使用以下组合 通用Molliccut16S rDNA寡核苷酸引物及 螺旋体特异性16S rDNA引物的聚合酶链式反应鉴定接近完全 TSE中螺旋体16S RNA基因的检测聚合酶链式反应产物的直接测序将 确定螺旋体样核糖体基因的未知相邻部分 参与TSE，从中我们将设计新的寡核苷酸引物SET/S 这将检测所有螺旋体菌株，包括那些与TSE有关的菌株。 在目标2中，我们将使用新的探测器/S筛选相当数量的东京证交所 病例，包括25例可获得的CJI人间病例和105例瘙痒病感染和 正常的羊脑。另外100个大脑样本，既有TSE感染的，也有 法线，将由合作者作为随机编码样本发送。聚合酶链式反应 所有情况下的扩增产物都将进行测序，以确定准确的 螺旋体的性质。牵涉其中。尽管螺旋体在TSE中的作用 不能从这些实验中确定，这种微生物在 所有TSE病例和非对照病例都将为开发 TSE测试

DESCRIPTION (Adapted from applicant's abstract): The pathogenesis of the transmissible spongiform encephalopathies (TSE), which include Creutzfeldt Jakob disease (CJD) in humans and scrapie in sheep, remains an enigma. In this application we present evidence for the association of Spiroplasma sp., a wall-less prokaryote, with TSE. We have shown PCR amplification of Spiroplasma 16S rDNA in TSE-infected brain tissues (19) and not in control brains (0 of SO). Direct sequencing of the amplified PCR products has confirmed the presence of Spiroplasma-like DNA in all 19 of the TSE brains. Our evidence is not necessarily in conflict with involvement of a PrPres, a protease resistant host derived protein referred to as the prion, in the pathogenesis of TSE, since there is evidence that another factor is involved, which we propose to be a bacterium. Our strategy will be to optimize our probe by characterizing the entire Spiroplasma ribosomal gene in TSE and, then, screen a statistically significant number of TSE cases. In Aim 1, we will utilize a combination of universal Mollicute 16S rDNA oligonucleotide primers along with Spiroplasma-specific 16S rDNA primers to identify by PCR the near complete Spiroplasma 16S RNA gene in TSE. Direct sequencing of the PCR products will ascertain the unknown adjacent portions of the Spiroplasma-like ribosomal gene involved in TSE, from which we will design new oligonucleotide primer set/s that will detect all Spiroplasma strains, including those associated with TSE. In Aim 2 we will screen, with the new probe/s, a sizable collection of TSE cases, including 25 available CJI) human cases and 105 scrapie-infected and normal sheep brains. An additional 100 brain samples, both TSE-infected and normals, are to be sent by collaborators as randomly coded samples. The PCR amplified products in all instances will be sequenced to determine the precise nature of the Spiroplasma sp. involved. Although the role of Spiroplasma in TSE cannot be determined from these experiments, the presence of this microbe in all cases of TSE and not in controls would provide the basis for developing a test for TSE