Detection of Streptococcus pneumoniae DNA by using polymerase chain reaction and microwell hybridization with Europium-labelled probes.
The present paper describes a novel modification of polymerase chain reaction (PCR) for the detection of Streptococcus pneumoniae DNA in clinical specimens. PCR was based on the detection of a 209-base pair segment of the S. pneumoniae pneumolysin gene. For the demonstration of the amplification product, microwell hybridization with a Europium-labelled oligonucleotide probe complementary to a biotinylated strand of the PCR product was performed, and the presence of the PCR product was monitored by time-resolved fluorescence (TRF) of the Europium chelate. The sensitivity of the assay for purified S. pneumoniae DNA was 50 fg DNA corresponding to 20 genome equivalents of S. pneumoniae DNA. The efficiency of the hybridization step was monitored by using known amounts of synthetic target oligonucleotides as standards. Sensitivity of 3 x 10(8) molecules per individual reaction well was achieved with a 30-min attachment time and a 3-h hybridization time. Detection of PCR-amplified products by the microwell hybridization technique and TRF was compared to agarose gel electrophoresis in 50 middle ear fluid samples obtained from children with acute otitis media. The agarose gel and TRF detection methods identified all culture-positive samples, but both were also positive for 55% of the culture-negative samples. The results suggest that the detection of amplified PCR products by microwell hybridization using Europium-labelled oligonucleotides is a reliable method for the demonstration of the pneumolysin gene fragment. Furthermore, the method is suitable for automation and, thus, for testing high numbers of samples. The clinical significance of the PCR findings remains to be studied.
Rintamäki
S
S
National Public Health Institute (KTL), P.O. Box 310, 90101, Oulu, Finland.
Saukkoriipi
A
A
Salo
P
P
Takala
A
A
Leinonen
M
M
D001431:Bacteriological Techniques / Q000379:methods; D004269:DNA, Bacterial / Q000032:analysis*; D004587:Electrophoresis, Agar Gel; D005063:Europium / Q000737:chemistry*; D005069:Evaluation Studies as Topic; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013296:Streptococcus pneumoniae / Q000235:genetics / Q000302:isolation & purification*
D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D005063:Europium