Single-Plex Quantitative Assays for the Detection and Quantification of Most Pneumococcal Serotypes

Streptococcus pneumoniae globally kills more children than any other infectious disease every year. A prerequisite for pneumococcal disease and transmission is colonization of the nasopharynx. While the introduction of pneumococcal conjugate vaccines has reduced the burden of pneumococcal disease, understanding the impact of vaccination on nasopharyngeal colonization has been hampered by the lack of sensitive quantitative methods for the detection of >90 known S. pneumoniae serotypes. In this work, we developed 27 new quantitative (q)PCR reactions and optimized 26 for a total of 53 qPCR reactions targeting pneumococcal serotypes or serogroups, including all vaccine types. Reactions proved to be target-specific with a limit of detection of 2 genome equivalents per reaction. Given the number of probes required for these assays and their unknown shelf-life, the stability of cryopreserved reagents was evaluated. Our studies demonstrate that two-year cryopreserved probes had similar limit of detection as freshly-diluted probes. Moreover, efficiency and limit of detection of 1-month cryopreserved, ready-to-use, qPCR reaction mixtures were similar to those of freshly prepared mixtures. Using these reactions, our proof-of-concept studies utilizing nasopharyngeal samples (N=30) collected from young children detected samples containing ≥2 serotypes/serogroups. Samples colonized by multiple serotypes/serogroups always had a serotype that contributes at least 50% of the pneumococcal load. In addition, a molecular approach called S6-q(PCR)2 was developed and proven to individually detect and quantify epidemiologically-important serogroup 6 strains including 6A, 6B, 6C and 6D. This technology will be useful for epidemiological studies, diagnostic platforms and to study the pneumobiome.

肺炎链球菌（Streptococcus pneumoniae）每年在全球范围内导致的儿童死亡人数超过其他任何传染病。鼻咽部定植是引发肺炎球菌疾病及传播的必要前提。尽管肺炎球菌结合疫苗的推广应用已减轻了肺炎球菌疾病的负担，但由于缺乏可灵敏定量检测90余种已知肺炎链球菌血清型的方法，学界对疫苗接种如何影响鼻咽部定植的认知仍受限于此。本研究中，我们开发了27种全新的定量（q）PCR体系，并优化了26种，最终共计获得53种可靶向肺炎球菌血清型或血清群的qPCR体系，覆盖所有疫苗相关型别。经检验，这些体系具备靶标特异性，单反应体系的检测限可达2个基因组当量。考虑到此类检测所需探针的数量及未知的货架期，我们评估了冻存试剂的稳定性。研究结果显示，冻存两年的探针与新鲜稀释的探针具有相当的检测限。此外，冻存1个月的即用型qPCR反应混合液的扩增效率与检测限，均与新鲜配制的混合液相当。利用这些体系，我们针对幼儿采集的共30份鼻咽样本开展了概念验证研究，成功检出了含至少2种血清型/血清群的样本。存在多种血清型/血清群定植的样本中，总有一种血清型的菌量占肺炎球菌总载量的50%及以上。此外，本研究还开发了一种名为S6-q(PCR)2的分子检测方法，经证实可分别检测并定量6A、6B、6C、6D等具有流行病学重要性的6群菌株。该技术可应用于流行病学研究、诊断平台开发以及肺炎菌群组学研究。