Microbial Diversity and Genetic Characterization

拟议的研究预计将产生有关构成龋齿相关牙菌斑生物膜的细菌生物群组成的重要新信息。 这些信息的长期利益应该引导研究人员在解决龋齿病因的基础上制定龋齿的诊断和预防策略。 该提案将重点关注患有严重龋齿（称为幼儿期龋齿 (ECC)）的儿童。 使用强大的梯度电泳技术将 16S rDNA 标记物与菌斑生物膜中的细菌阵列分离。 这些凝胶应该显示 ECC 和无龋儿童的微生物群落之间的差异。 反过来，这种分析将使我们能够识别或近似那些与龋齿相关的细菌，其中一些可能无法培养。 另一个需要测试的假设是变形链球菌菌株或整个龋齿生物膜在引起疾病的能力方面是否存在差异。减法 DNA 杂交将用于发现变形链球菌或易患龋齿的儿童牙菌斑中存在的独特遗传位点。 消减DNA杂交的进一步发展将实现我们的总体目标，即从整个牙菌斑中表征龋齿活性生物膜内的一组遗传位点，而不考虑首先培养特定细菌的限制。 这将为后续研究奠定基础，在后续研究中可以编译和测试一组 DNA 探针，这将有助于预测特定儿童是否有患龋齿的风险。 了解这些基因位点的功能以及它们产生的细菌宿主将为了解龋齿的病因提供重要信息。 此外，拥有疾病的遗传标记可以消除从牙菌斑中培养细菌的昂贵且不精确的做法。 拟议的研究可能会对这些更严重的龋齿产生影响，从而最终预防龋齿。

The proposed studies are anticipated to yield important new information as to the composition of the bacterial biota that comprises the caries-associated plaque biofilm. The long-term benefit of such information should lead researchers to devising both diagnostic and preventative strategies for dental caries based on addressing its etiological agents. This proposal will focus on children with a severe forms of dental caries called Early Childhood Caries (ECC). Using a powerful technique of gradient electrophoresis will be used to separate 16S rDNA markers from an array of bacteria in plaque biofilms. These gels should show differences between the microfloras of ECC and caries-free children. This profiling, in turn, will allow us to identify or approximate those bacteria, some likely to be uncultivable, associated with caries. Another hypothesis to be tested is whether strains of mutans streptococci, or the entire caries-biofilm differ in their ability to cause disease. Subtraction DNA hybridization will be used to discover unique genetic loci present in mutans streptococci or dental plaques of caries-prone children. Further development of subtraction DNA hybridization will lead to our overall objective, i.e., to characterize from whole plaque a constellation of genetic loci within the caries-active biofilm, irrespective of the limitation of first cultivating specific bacteria. This will set the groundwork for subsequent studies in which a set of DNA probes can be compiled and tested, which will be useful for predicting whether a particular child is at risk for caries. Knowing the function of these genetic loci and the bacterial host from which they arise will give important information as to the causation of caries. Moreover, having genetic markers for disease may eliminate the costly and imprecise practice of cultivating bacteria from dental plaque. The research proposed will likely impact on these more serious forms of caries, leading to its eventual prevention.