PCR-based assays for detection of Streptococcus pneumoniae serotypes 3, 14, 19F and 23F in respiratory specimens

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Abstract
Current culture-based assays are insensitive for detection of simultaneous respiratory tract colonization by more than one pneumococcal serotype. Separate single-tube, nested PCR-based assays have been developed to detect Streptococcus pneumoniae serotypes 3, 14, 19F and 23F by amplifying unique DNA sequences in the capsular polysaccharide gene cluster of each serotype. Pairs of 27–32-base outer primers and 20–21-base inner primers and a 20–22-base probe were designed to amplify and detect a 200–221-base sequence by dot blotting using the labelled probe. Sensitivity of the assays was 0.01–10 fg using chromosomal DNA and ⩽ 1 viable cell using DNA extracted from exponential-phase bacteria. Each serotype-specific assay detected chromosomal DNA from all of five to ten clinical isolates of the homologous type and did not detect DNA sequences from any of 190–204 strains from 51–52 different serotypes or 28 non-pneumococcal bacterial strains. Sixteen throat swabs from children that had been cultured for S. pneumoniae were tested in PCR assays following DNA extraction. All of six that grew S. pneumoniae serotype 3, 14, 19F or 23F were positive in the PCR assay for the homologous serotype (and in a PCR assay for sequences in lytA, present in all pneumococci) and were negative in assays for other serotypes. Of eight culture-negative specimens in children not receiving antimicrobials, three were positive for both the lytA assay and an assay for one of the four serotypes, suggesting true positive results; in three others all five PCR assays were negative and, in the remaining two, the lytA assay was positive but each of the four assays for individual serotypes was negative, suggesting either false-positive results or presence of DNA sequences from an S. pneumoniae serotype other than 3, 14, 19F or 23F. These preliminary clinical data suggest that these PCR-based assays are sensitive and specific for detection of individual serotypes of pneumococci and may be used with respiratory tract specimens.