Isolation of pneumococcal DNA from nasopharyngeal samples for real-time, quantitative PCR: comparison of three methods.
BACKGROUND
Real-time PCR is a useful method for detecting and quantifying bacterial DNA in clinical samples. DNA extraction is a crucial step when performing quantitative PCR.


METHODS
We compared three methods, QIAamp. The use of tradenames is for product identification purposes only and does not imply endorsement. DNA Mini kit, MagNAPure trade mark LC DNA Isolation Kit II together with PickPen trade mark magnetic particle transfer device, and KingFisher genomic DNA purification Kit with KingFisher mL instrument, for purification of Streptococcus pneumoniae DNA from 50 nasopharyngeal swab samples, collected into skim milk-tryptone-glucose-glycerin medium. Pneumococcal DNA was detected and quantified by real-time PCR and results were compared to culture findings.


RESULTS
The 22 (44%) pneumococcal culture-positive specimens were all positive by PCR regardless of DNA extraction method used, except that one KingFisher-extracted sample was positive only when repeatedly tested. Additionally, 71%, 57%, and 82% of the culture-negative samples were positive by real-time PCR when DNA was extracted by QIAamp, MagNAPure-PickPen, and KingFisher methods, respectively. The number of genome equivalents detected by real-time PCR varied, but was mainly low in culture-negative samples. The sensitivities of culture and real-time PCR were hence compared by analyzing different dilutions of a pneumococcal suspension. Real-time PCR detected significantly higher numbers of genome equivalents than the numbers of bacteria detected by culture.


CONCLUSIONS
The results indicate that the DNA extraction method used for quantitative PCR should be evaluated and that real-time PCR is more sensitive than bacterial culture for detecting pneumococcus in nasopharyngeal swab samples.
Saukkoriipi
Annika
A
National Public Health Institute, Oulu, Finland. annika.saukkoriipi@ktl.fi
Kaijalainen
Tarja
T
Kuisma
Leena
L
Ojala
Anu
A
Leinonen
Maija
M
D001431:Bacteriological Techniques; D002675:Child, Preschool; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification*; D006801:Humans; D007223:Infant; D009305:Nasopharynx / Q000382:microbiology*; D011008:Pneumococcal Infections / Q000382:microbiology*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013296:Streptococcus pneumoniae / Q000235:genetics*
D004269:DNA, Bacterial