Development of a polymerase chain reaction assay to detect the presence of Streptococcus pneumoniae DNA.
In this study, we have developed a chemically sensitive and specific polymerase chain reaction (PCR) assay to detect the presence of Streptococcus pneumoniae genomic DNA. The target DNA sequence was a 322-base pair segment of the S. pneumoniae DNA polymerase I gene (pol I). PCR products of pure cultures of a set of pneumococcal serotypes commonly associated with human infection could be amplified in water and in blood cultures of clinical isolates containing S. pneumoniae. We were able to detect 2 fg of purified S. pneumoniae DNA. There were no false-positive reactions when the assay was performed on samples containing the following clinically encountered bacteria: Haemophilus influenzae type B, Neisseria meningitidis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas spp. nontypeable H. influenzae, Staphylococcus aureus, coagulase-negative staphylococci, and Streptococcus pyogenes. The addition of EDTA and citrate-anticoagulated whole blood to the PCR reaction mixture inhibited the PCR assay, whereas the addition of lithium heparin, sodium heparin, and sodium polyanetholesulfonate-anticoagulated whole blood to PCR reaction mixture did not interfere with the ability to detect the presence of S. pneumoniae DNA.
Friedland
L R
LR
Department of Pediatrics, Children's Hospital Medical Center, Cincinnati, OH 45229-3039.
Menon
A G
AG
Reising
S F
SF
Ruddy
R M
RM
Hassett
D J
DJ
D001483:Base Sequence; D004269:DNA, Bacterial / Q000032:analysis*; D004587:Electrophoresis, Agar Gel; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013296:Streptococcus pneumoniae / Q000235:genetics*
D004269:DNA, Bacterial