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We evaluated the performance of a new real-time PCR assay \u2013 targeting SP2020 (putative transcriptional regulator gene) \u2013 and compared its performance with the assays previously described. A collection of 150 pneumococci, 433 non-pneumococci and 240 polymicrobial samples (obtained from nasopharynx, oropharynx, and saliva; 80 from each site) was tested. SP2020 and <jats:italic>lytA<\/jats:italic>-CDC assays had the best performance (sensitivity of 100% for each compared to 95.3% for <jats:italic>piaB<\/jats:italic>). The specificity for <jats:italic>lytA<\/jats:italic> and <jats:italic>piaB<\/jats:italic> was 99.5% and for SP2020 was 99.8%. Misidentifications occurred for the three genes: <jats:italic>lytA<\/jats:italic>, <jats:italic>piaB<\/jats:italic> and SP2020 were found in non-pneumococcal strains; <jats:italic>piaB<\/jats:italic> was absent in some pneumococci including a serotype 6B strain. Combining <jats:italic>lytA<\/jats:italic> and SP2020 assays resulted in no misidentifications. Most polymicrobial samples (88.8%) yielded concordant results for the three molecular targets. The remaining samples seemed to contain non-typeable pneumococci (0.8%), and non-pneumococci positive for <jats:italic>lytA<\/jats:italic> (1.7%) or SP2020 (8.7%). We propose that combined detection of both <jats:italic>lytA<\/jats:italic>-CDC and SP2020 is a powerful strategy for the identification of pneumococcus either in pure cultures or in polymicrobial samples.<\/jats:p>","DOI":"10.1038\/s41598-019-39791-1","type":"journal-article","created":{"date-parts":[[2019,3,1]],"date-time":"2019-03-01T11:06:43Z","timestamp":1551438403000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":72,"title":["Identification of Streptococcus pneumoniae by a real-time PCR assay targeting SP2020"],"prefix":"10.1038","volume":"9","author":[{"given":"D\u00e9bora A.","family":"Tavares","sequence":"first","affiliation":[]},{"given":"Sara","family":"Handem","sequence":"additional","affiliation":[]},{"given":"Ricardo J.","family":"Carvalho","sequence":"additional","affiliation":[]},{"given":"A. 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