Development of PCRSeqTyping—a novel molecular assay for typing of Streptococcus pneumoniae

Geetha Nagaraj, Feroze Ganaie, Vandana Govindan, Kadahalli Lingegowda Ravikumar

Precise serotyping of pneumococci is essential for vaccine development, to better understand the pathogenicity and trends of drug resistance. Currently used conventional and molecular methods of serotyping are expensive and time-consuming, with limited coverage of serotypes. An accurate and rapid serotyping method with complete coverage of serotypes is an urgent necessity. This study describes the development and application of a novel technology that addresses this need. Polymerase chain reaction (PCR) was performed, targeting 1061 bp cpsB region, and the amplicon was subjected to sequencing. The sequence data was analyzed using the National Centre for Biotechnology Information database. For homologous strains, a second round of PCR, sequencing, and data analysis was performed targeting 10 group-specific genes located in the capsular polysaccharide region. Ninety-one pneumococcal reference strains were analyzed with PCRSeqTyping and compared with Quellung reaction using Pneumotest Kit (SSI, Denmark). A 100% correlation of PCRSeqTyping results was observed with Pneumotest results. Fifty-nine reference strains were uniquely identified in the first step of PCRSeqTyping. The remaining 32 homologous strains out of 91 were also uniquely identified in the second step. This study describes a PCRSeqTyping assay that is accurate and rapid, with high reproducibility. This assay is amenable for clinical testing and does not require culturing of the samples. It is a significant improvement over other methods because it covers all pneumococcal serotypes, and it has the potential for use in diagnostic laboratories and surveillance studies.