Streptococcus pneumoniae subsp. serotyping

Silva, M. E. P., Oliveira, J. R., Carvalho, A. G., Santos, D. G., Lima, N. C. S., Santos, F. A. G., Taborda, R. L. M., Rodrigues, R. S., Dall’Acqua, D. S. V. & Matos, N. B., 2022, Colonization by Streptococcus pneumoniae among children in Porto Velho, Rondônia, Western Brazilian Amazon, Brazilian Journal of Biology (e 260617) 82, pp. 1-11 : 3

2.3. S. pneumoniae serotyping through multiplex PCR

The S. pneumoniae isolates were subjected to genomic DNA extraction using the phenol/chloroform method and then stored at -20 °C for further use in the investigation of serotypes ( Sambrook et al., 1989).

Capsular serotyping of S. pneumoniae was performed using multiplex PCR described previously by Pai et al. (2006). For each reaction, the following components were used: 1 µL of DNA from the bacteria of interest in the research, 15.8 µL of autoclaved MiliQ water, 2.0 µL of DNTP (2.5 mM), 2.0 µL of Buffer (10x PCR 2.5 mM), 2.0 µL of MgCl2 (50 mM), 2.0 µL (10 p/mol) of primers specific for each serotype and 0.2 µL (50 mM) of Taq DNA polymerase (Invitrogen) at a final volume of 25 µL. Amplification conditions for each serotype were performed as described previously by Coskun-Ari et al. (2012).