PCR assays to detect individual pneumococcal serotypes

描述(申请人提供)：背景肺炎链球菌(肺炎球菌)是人类的主要病原体。咽部携带的肺炎球菌是传播和感染的宿主。根据多糖胶囊，有90种抗原性不同的血清型。抗囊膜抗体提供特定类型的免疫。2000年，肺炎球菌结合疫苗获得许可，这是一种含有来自7个血清型的衣壳抗原的疫苗，显著降低了婴儿侵袭性感染的发生率，目前已在美国所有婴儿中接种。疫苗接受者减少了疫苗血清型的携带，但明显增加了其他血清型的携带。从呼吸道标本中培养肺炎球菌的标准方法灵敏度有限，通常只检测到一种血清型，尽管携带一种以上血清型的情况很常见。需要更灵敏的测试来检测咽部样本中存在的所有血清型。 该项目的广泛和长期目标是研究所有血清型肺炎球菌的携带率以及疫苗对携带率的影响。 特指的目的是开发敏感和特异的基于聚合酶链式反应的检测方法，以检测占临床标本中99%的肺炎球菌的30个血清型/血清组的DNA。比较这些检测方法与标准培养法和小鼠注射法检测临床咽部标本中存在的所有血清型的敏感性和特异性。 研究计划对于总共占临床分离株99%的30个血清型(或血清组)中的每一个，都将确定包膜多糖生物合成簇中的血清型/血清组特异性基因和序列。使用这些序列，将为这30个人中的每一个开发敏感的基于聚合酶链式反应的分析。将使用90个血清型的菌株的DNA来测试每一种检测的特异性，这些菌株是其他定植于咽部的细菌。检测呼吸道样本中所有肺炎球菌血清型的敏感性将与标准培养和小鼠接种进行比较。 意义这些以聚合酶链式反应为基础的分析将允许更灵敏地检测呼吸道样本中存在的所有血清型，从而更全面地了解定殖化的流行病学。它们将被用来检验这样一个假设，即在接种疫苗和未接种疫苗的婴儿中，非疫苗血清型的定植流行率是相似的。此外，它们对于确定当前和未来肺炎球菌疫苗对特定血清型携带者的影响至关重要。

DESCRIPTION (provided by applicant): Background Streptococcus pneumoniae (pneumococcus) is a major human pathogen. Pneumococci carried in the pharynx are the reservoir for both transmission and infection. There are 90 antigenically distinct serotypes based on the polysaccharide capsule. Anti-capsular antibodies provide type-specific immunity. In 2000, a pneumococcal conjugate vaccine, a vaccine containing capsular antigens from seven serotypes that markedly reduces the incidence of invasive infections in infants, was licensed and is now given to all U.S. infants. Vaccine recipients have reduced carriage of vaccine serotypes but an apparent increase in carriage of other serotypes. Standard methods for growing pneumococci from respiratory specimens have a limited sensitivity and generally detect only a single serotype although carriage of more than one serotype is common. There is a need for more sensitive tests that detect all serotypes present in pharyngeal specimens. The broad, long-term objective of this project is to study the carriage of all serotypes of pneumococci and the effect of vaccines on carriage. Specific Aims To develop sensitive and specific PCR-based assays to detect DNA from 30 serotypes/serogroups that account for 99% of pneumococci in clinical specimens. To compare the sensitivity and specificity of these assays with standard culture and mouse injection for detection of all serotypes present in clinical pharyngeal specimens. Research Plan For each of 30 serotypes (or serogroups) that collectively comprise 99% of clinical isolates, serotype/serogroup-specific genes & sequences in the capsular polysaccharide biosynthetic cluster will be identified. Using these sequences sensitive PCR-based assays will be developed for each of the 30. The specificity of each assay will be tested using DNA from strains of the 90 serotypes & other bacteria that colonize the pharynx. The sensitivity for detecting all serotypes of pneumococci in respiratory specimens will be compared with standard culture & mouse inoculation. Significance These PCR-based assays will allow more sensitive detection of all serotypes present in respiratory specimens resulting in a more complete understanding of the epidemiology of colonization. They will be used to test the hypothesis that the prevalence of colonization with non-vaccine serotypes is similar in vaccinated and unvaccinated infants. Furthermore, they will be critical for determining the effects of current and future pneumococcal vaccines on serotype-specific carriage.