[Assessment of two DNA extraction methods to amplify the pneumolysin gene (PLY) from blood culture samples of Streptococcus pneumoniae].
BACKGROUND
Streptococcus pneumoniae is a common etiologic agent of invasive respiratory infections among children under 5 years of age and older adults. Isolation rates of S. pneumoniae by traditional culture techniques are low.


OBJECTIVE
To study the sensitivity and specificity of two different DNA extraction methods to amplify the ply gene, applied to three different types of blood culture broths, experimentally inoculated with S. pneumoniae.


METHODS
DNA was extracted from the cultures using an organic method or a technique that consists in dilution, washing with NaOH and concentration of the sample. This was followed by PCR amplification of a 355 pb fragment of the pneumolysin gene (ply).


RESULTS
The organic DNA extraction method inhibited the PCR reaction at all concentrations studied (0.6 to 10(6) colony forming units/mL). Using the NaOH extraction, ply gene amplification was positive in all three blood culture broths, but only at concentrations of 10(3) colony forming units/mL, or higher. Using the same DNA extraction method, PCR was negative when the broths were inoculated with seven other related bacterial species, which results in a 100% specificity.


CONCLUSIONS
Detection of S. pneumoniae by amplification of ply gene from blood cultures using the protocol of NaOH for DNA extraction is specific and provides results in a short lapse. However, the diagnostic sensitivity is not optimal, which limits its clinical use.
Programa de Microbiología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile.
Carolina
C
Hernández
Claudia
C
Durán
María Teresa
MT
Ulloa
Valeria
V
Prado
D001426:Bacterial Proteins / Q000235:genetics*; D001431:Bacteriological Techniques; D003470:Culture Media; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D011008:Pneumococcal Infections / Q000175:diagnosis; D016133:Polymerase Chain Reaction / Q000592:standards; D012680:Sensitivity and Specificity; D013296:Streptococcus pneumoniae / Q000235:genetics / Q000302:isolation & purification*; D013301:Streptolysins / Q000235:genetics*
D001426:Bacterial Proteins; D003470:Culture Media; D004269:DNA, Bacterial; D013301:Streptolysins; C032290:plY protein, Streptococcus pneumoniae