Division of Pediatric Dentistry, Department of Human Development and Fostering, Meikai University School of Dentistry, Saitama, Japan.
Department of Pharmacy, College of Pharmacy, Hanyang University, Ansan, Republic of Korea.
PLoS One. 2021 Feb 16;16(2):e0246699. doi: 10.1371/journal.pone.0246699. eCollection 2021.
Reports of invasive disease due to Streptococcus pneumoniae have declined since the introduction of pneumococcal conjugate vaccines (PCV7 and PCV13). The incidence of invasive diseases due to S. pneumoniae that are not addressed by the vaccines, however, has increased in children and adults, creating a global public health problem. Previously, we established the loop-mediated isothermal amplification (LAMP) method for a PCV13 serotype-specific assay. In the current study, we developed a rapid, simple, and cost-effective assay to detect serotypes in the 23-valent pneumococcal polysaccharide vaccine (PPSV23) using the LAMP method. In this study, LAMP primer sets for serotypes 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F, and 33F of S. pneumoniae were developed. The reactivity, specificity, and sensitivity of LAMP assays were determined and compared to those of conventional PCR. The feasibility of LAMP assays in clinical application in patients with invasive pneumococcal diseases was validated by defining the detection limit of the LAMP assay with bacterial genomic DNA-spiked blood specimens. The specificity of each LAMP assay was determined using 44 serotypes of pneumococcal strains. Their sensitivity was 100 copies per reaction versus 103 to 106 copies per reaction for PCR assays. Using DNA-spiked blood specimens, excluding the LAMP assay that targeted serotype 22F (103 copies per reaction), the limit of detection of the LAMP assay was similar to that with purified DNA as the template (102 copies per reaction), compared with 103 to >106 copies per reaction for PCR assays. In conclusion, a rapid and simple LAMP-based PPSV23-targeted serotype detection assay was developed for use in many countries. This study is the first report of a LAMP-based assay for identification of PPSV23 serotypes. Further evaluation of this assay is needed through surveillance and vaccine efficacy studies.
自从肺炎球菌结合疫苗（PCV7 和 PCV13）问世以来，由肺炎球菌引起的侵袭性疾病报告有所减少。然而，疫苗未涵盖的非侵袭性疾病的发生率在儿童和成人中有所增加，这构成了一个全球性的公共卫生问题。此前，我们建立了用于 PCV13 血清型特异性检测的环介导等温扩增（LAMP）方法。在本研究中，我们开发了一种快速、简单且具有成本效益的 LAMP 方法，用于检测 23 价肺炎球菌多糖疫苗（PPSV23）中的血清型。在这项研究中，我们为肺炎球菌的血清型 2、8、9N、10A、11A、12F、15B、17F、20、22F 和 33F 开发了 LAMP 引物组。我们确定了 LAMP 检测的反应性、特异性和敏感性，并与常规 PCR 进行了比较。通过定义 LAMP 检测的检测限，用含有细菌基因组 DNA 的血样标本验证了 LAMP 检测在侵袭性肺炎球菌病患者临床应用中的可行性。用 44 种肺炎球菌菌株确定了每种 LAMP 检测的特异性。其敏感性为 100 个拷贝/反应，而 PCR 检测的敏感性为 103 至 106 个拷贝/反应。用含有 DNA 的血样标本检测，除了针对血清型 22F 的 LAMP 检测（103 个拷贝/反应）外，LAMP 检测的检测限与用纯化 DNA 作为模板时相似（102 个拷贝/反应），而 PCR 检测的检测限为 103 至>106 个拷贝/反应。总之，我们开发了一种快速简便的基于 LAMP 的 PPSV23 靶向血清型检测方法，适用于许多国家。这是首次报道基于 LAMP 的 PPSV23 血清型鉴定方法。需要通过监测和疫苗效力研究进一步评估该检测方法。
