Molecular epidemiological approach to determine S.mutans

描述(申请人提供)：龋齿是最常见的儿童慢性疾病。每年有5100多万学时因牙科疾病而减少，超过400亿美元用于治疗这种疾病。流行病学和临床研究表明，变形链球菌，尤其是变形链球菌是引起龋病的主要病原菌。鉴定变形链球菌最常用的技术是选择性培养。该方法的主要局限性包括不能在唾液中检测到变形链球菌，特别是当变形链球菌存在于低水平时；形态因使用的介质而异；而且它成本高昂且劳动密集型。到目前为止，最可靠的快速和特异鉴定细菌种类的技术是聚合酶链式反应。但对于变形链球菌来说，缺乏特定物种的探针和引物继续限制了对变形链球菌流行率和定殖性的高通量研究。因此，本项目的目标是开发高灵敏度和物种特异性的探针和引物，用于基于PCR的检测，以快速、准确和有效地鉴定临床口腔标本中的变形链球菌。我们的目标将通过追求以下具体目标来实现。(1)鉴定变形链球菌基因组中潜在的独特序列，使我们能够开发出检测临床标本中变形链球菌的物种特异性探针和引物。(2)对探针进行验证，证明该种属特异性探针和引物具有较高的敏感性和特异性。(3)种属特异性探针和引物与常规培养方法比较。从这些实验中，我们应该能够获得明确的变形链球菌特异性探针和引物，并证明新开发的探针比培养方法获得的结果更优越。新的分子标记将使我们能够进行变形链球菌感染的分子流行病学研究和高通量研究，以便我们能够更好地了解龋病的多菌病原学，在疾病发展之前确定儿童的风险潜力，并评估龋病干预的有效性。新的分子工具的应用将对改善儿童口腔健康产生实质性的影响。

DESCRIPTION (provided by applicant): Dental caries is the single most common chronic childhood disease. Each year, more than 51 million school hours are lost due to dental-related illness and over $40 billion is spent on the treatment of this disease. Epidemiological and clinical studies have suggested that mutans streptococci, particularly Streptococcus mutans, are the major microbial pathogens associated with dental caries. The most commonly used technique to identify S. mutans is cultivation on selective media. The major limitations of the method include inadequate detection of S. mutans in saliva particularly when S. mutans is present at low levels; morphology varies depending upon the medium used; and it is costly and labor-intensive. To date, the most reliable technique to rapidly and specifically identify bacterial species is PCR. But for S. mutans, the lack of species-specific probes and primers continues to limit high-throughput research on prevalence and colonization of S. mutans. Therefore, the objective of this project is to develop highly sensitive and species-specific probes and primers that can be used in PCR-based assays to rapidly, accurately, and effectively identify S. mutans in clinical oral specimens. Our goal will be accomplished by pursuing the following specific aims. (1) To identify potentially unique sequences in S. mutans genome that will enable us to develop species-specific probes and primers for the detection of S. mutans in the clinical specimens. (2) To validate the probes and to demonstrate the high sensitivity and specificity of the species-specific probes and primers. (3) To compare the species-specific probes and primers with the conventional culture method. From these experiments, we should be able to obtain well-defined S. mutans-specific probes and primers and to prove the superiority of the newly developed probes to the findings obtained from the culture method. The new molecular markers will enable us to conduct molecular epidemiological studies of S. mutans infection and high-throughput research so that we can improve our understanding of the polymicrobial etiology of dental caries, ascertain a child's risk potential prior to disease development, and evaluate the effectiveness of caries interventions. Application of the new molecular tools will have a substantial impact on improving the oral health of children.