Detection of bacterial DNA by PCR and reverse hybridization in the 16s rRNA gene

The clinical diagnosis of sepsis is difficult, particularly in neonates. To devise a rapid and reliable method for identifing bacteria in blood and cerebrospinal fluid (CSF), we developed a pair of primers according to the gene encoding 16 s rRNA, found in all bacteria. DNA fragments from different bacterial species and from clinical samples were detected with polymerase chain reaction (PCR), and with reverse hybridization using a universal bacterial probe, a gram-positive probe and a gram-negative probe. Our results showed that a 371 bp DNA fragment was amplified from 20 different bacterial species. No signal was observed when human DNA and viruses were used as templates. The sensitivity could be improved to 10−12 g. All 26 culture-positive clinical samples (22 blood samples and 4 CSF samples), were positive with PCR. The gram-negative and gram-positive probe hybridized to clinical samples and to known bacterial controls, as predicted by Gram’s stain characteristics. Our results suggest that the method of PCR and reverse hybridization is rapid, sensitive and specific in detecting bacterial infections. This finding may be significant in the clinical diagnosis of sepsis in neonates.