PCR assays to detect individual pneumococcal serotypes

描述（由申请人提供）：背景肺炎链球菌（肺炎球菌）是一种主要的人类病原体。咽部携带的肺炎球菌是传播和感染的宿主。基于多糖胶囊，有90种抗原不同的血清型。抗荚膜抗体提供类型特异性免疫。2000年，一种肺炎球菌结合疫苗（一种含有7种血清型荚膜抗原的疫苗，可显著降低婴儿侵袭性感染的发生率）获得许可，目前已接种给所有美国婴儿。疫苗接种者携带疫苗血清型减少，但携带其他血清型的人数明显增加。从呼吸道标本中培养肺炎球菌的标准方法灵敏度有限，通常只能检测到一种血清型，尽管携带一种以上血清型是常见的。需要更灵敏的检测方法来检测咽标本中存在的所有血清型。

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.