A Modified PCR‐Based Method for Rapid Non‐Radioactive Detection of Clinically Important Pathogens

Abstract

We have devised a sensitive and rapid method for the detection of several bacterial pathogens in clinical specimens using PCR. This method has been named Direct Labeling and Detection Procedure (DLDP) and is based on the direct incorporation of a nonradioactive digoxigenin label (DIG-11-dUTP) into a microbial species-specific gene fragment during amplification. Following amplification, the resulting PCR products are cleansed of nonincorporated DIG-11-dUTP, spotted onto a nylon membrane, fixed by UV-crosslinking and the labeled DNA is visualized by digoxigenin detection reagents. Using cultivated reference bacteria (Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa) we were able to demonstrate a rapid and sensitive detection of < 20 CFU of bacteria in human secretions (sputum, urine, mucous). The present study suggests that DLDP can be used as a reliable method for indication of bacteria in clinical or environmental specimens with the proviso that the selected corresponding oligonucleotide primers provide amplification of strong species-specific genes.

Accuracy of a Polymerase Chain Reaction-based Assay for Detection of Pneumococcal Bacteremia in Children

“…[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] Although we have developed an assay that showed excellent in vitro activity for the detection of S pneumoniae in whole blood, 19 we were unable to achieve similar sensitivity and specificity when using this assay on clinical specimens.…”

“…1-10 A number of recent investigations have applied this technology toward the identification of common bacterial pathogens. [11][12][13][14][15] Although the use of the PCR has offered promise for the rapid identification of serious bacterial infections in children, little has been done to assess its utility in the detection of bacteremia. 16 -18 Streptococcus pneumoniae is the most common cause of invasive bacterial infection in children older than 2 months of age.…”