Molecular epidemiological approach to determine S.mutans

描述（由申请人提供）：龋齿是儿童最常见的慢性疾病。每年，由于与牙齿有关的疾病而损失的学时超过5 100万学时，用于治疗这种疾病的费用超过400亿美元。流行病学和临床研究表明，变形链球菌，特别是变形链球菌是与龋齿相关的主要微生物病原体。鉴定变形链球菌最常用的技术是在选择性培养基上培养。该方法的主要局限性包括对唾液中变形链球菌的检测不足，特别是当变形链球菌存在于低水平时；形态的变化取决于所使用的介质；而且成本高，劳动密集。迄今为止，最可靠的快速和特异性鉴定细菌种类的技术是PCR。但对于变形链球菌，缺乏物种特异性探针和引物仍然限制了对变形链球菌流行和定植的高通量研究。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，快速、准确、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)鉴定突变链球菌基因组中潜在的独特序列，为临床标本中突变链球菌的检测提供物种特异性的探针和引物。(2)验证探针的有效性，证明该探针和引物具有较高的灵敏度和特异性。(3)与常规培养方法比较种特异性探针和引物。从这些实验中，我们应该能够获得定义明确的S. mutans特异性探针和引物，并证明新开发的探针与培养方法所得结果的优越性。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，从而提高我们对龋齿多微生物病因的理解，在疾病发展之前确定儿童的潜在风险，并评估龋齿干预措施的有效性。新分子工具的应用将对改善儿童口腔健康产生重大影响。

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.