Evaluation of the detection limits of PCR for identification of Mycoplasma pneumoniae in clinical samples.
The detection limits of the polymerase chain reaction (PCR) for Mycoplasma pneumoniae were determined using specimens from persons known to have had M. pneumoniae pneumonia. Four primers were selected from the known sequence of the P1 gene. The primer pair (P1-178 and P1-809) which generates a 631 fragment gave the lowest detection limit. Nineteen of 21 throat swabs, which contained between 0.06 and 2 colony-forming units (CFU) per microlitre, from culture positive patients, were positive by PCR. The fact that M. pneumoniae grows in broth culture in spherules causes problems for determining the number of CFU detected in PCR. Filtering broth cultures through a 0.6 micron polycarbonate filter increased the number of CFUs two-to-ten-fold compared to unfiltered cultures. The lysis method needed to assay throat swabs differed from that necessary for broth cultures in that proteinase K treatment for 18 h increased the detection limit 10- to 100-fold when compared to NaOH digestion.
Leng
Z
Z
Department of Pathobiology, School of Public Health, University of Washington, Seattle 91895.
Kenny
G E
GE
Roberts
M C
MC
D001483:Base Sequence; D015342:DNA Probes; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009177:Mycoplasma pneumoniae / Q000235:genetics* / Q000302:isolation & purification; D010614:Pharynx / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods
D015342:DNA Probes; D004269:DNA, Bacterial