Detection of bacterial DNA by PCR and reverse hybridization in the 16S rRNA gene with particular reference to neonatal septicemia

Abstract

Aim: The clinical diagnosis of sepsis is difficult, particularly in neonates. It is necessary to develop a rapid and reliable method for detecting bacteria in blood and cerebrospinal fluid (CSF) Polymerase chain reaction (PCR) and reverse hybridization of the 16S rRNA gene would permit fast and sensitive determination of the presence of bacteria and differentiate gram‐positive bacteria from gram‐negative ones in clinical specimens. Methods: We developed a pair of primers according to the gene encoding 16SrRNA found in all bacteria. DNA fragments from different bacterial species and from clinical samples were detected with PCR, and with reverse hybridization using a universal bacterial probe, a gram‐positive probe and a gram‐negative probe. Results: 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 10T‐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 probes hybridized to clinical samples and to known bacterial controls, as predicted by Gram's stain characteristics. Conclusions: 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.

A multiplex real-time PCR assay for rapid detection and differentiation of 25 bacterial and fungal pathogens from whole blood samples

“…The development of rapid diagnostic methods for BSI has been identiWed as an important medical need to supplement conventional blood culture diagnostics and molecular techniques have potential to fulWll this need [1]. Nucleic acid based diagnostic systems, including polymerase chain reaction (PCR) methods as well as the application of DNA and RNA probes are well known sensitive techniques for a more rapid detection and the speciWc identiWcation of pathogens involved in BSI [7][8][9][10][11][12][13][14][15]. From a theoretical point of view, PCR-based diagnostic techniques, therefore, hold promise for sensitive and speciWc detection of target pathogens within much shorter turn around times than usually exercised with conventional blood cultures.…”

Application of broad-range bacterial PCR amplification and direct sequencing on the diagnosis of neonatal sepsis

“…After DNA sequencing of the amplification product, the bacterium can be located in the phylogenetic tree. However, to date, the application of PCR to diagnose bacterial infections in blood samples is limited (15,21,27) and even blood culturing techniques can render unacceptable low sensitivity (30). It is well established that smaller volume of blood directly correlates with lower chances to detect the implicated organism (3,8,17).…”

Early Identification of Sepsis

“…If necessary, Southern blots, dot-blots, or sequencing can be used to positively identify specific PCR products [15]. PCR assays developed for specific detection of pathogens in the blood were described as early as 1993 [14].…”