Development of a Broad-Range 23S rDNA Real-Time PCR Assay for the Detection and Quantification of Pathogenic Bacteria in Human Whole Blood and Plasma Specimens

Paolo Gaibani,Stefano Novati,Mara Mariconti,Davide Sassera,Vittorio Sambri,Claudio Bandi,Maria Paola Landini,Sonia Bonora,Gloria Bua,Patrizia Mulatto

doi:10.1155/2013/264651

Abstract

Molecular methods are important tools in the diagnosis of bloodstream bacterial infections, in particular in patients treated with antimicrobial therapy, due to their quick turn-around time. Here we describe a new broad-range real-time PCR targeting the 23S rDNA gene and capable to detect as low as 10 plasmid copies per reaction of targeted bacterial 23S rDNA gene. Two commercially available DNA extraction kits were evaluated to assess their efficiency for the extraction of plasma and whole blood samples spiked with different amount of either Staphylococcus aureus or Escherichia coli, in order to find the optimal extraction method to be used. Manual QIAmp extraction method with enzyme pre-treatment resulted the most sensitive for detection of bacterial load. Sensitivity of this novel assay ranged between 10 and 103 CFU per PCR reaction for E. coli and S. aureus in human whole blood samples depending on the extraction methods used. Analysis of plasma samples showed a 10- to 100-fold reduction of bacterial 23S rDNA in comparison to the corresponding whole blood specimens, thus indicating that whole blood is the preferential sample type to be used in this real-time PCR protocol. Our results thus show that the 23S rDNA gene represents an optimal target for bacteria quantification in human whole blood.

Highlights

Blood culture (BC) is the most widely used method for the diagnosis of bloodstream bacterial infections (BSIs) [1]
Alignment of the sequences was performed using the MUSCLE software [21] and manually checked. 28S rDNA sequences from Caenorhabditis elegans, Candida albicans, Drosophila melanogaster, and Homo sapiens were included in the alignment, in order to evaluate the specificity for the bacterial rDNA of the designed primers
In order to validate our in silico findings, the specificity of the novel 23S rDNA-targeted primers was evaluated on a total of 20 Gram-positive and 27 Gramnegative bacterial strains and on five eukaryotic species, from the genus Candida

Summary

Introduction

Blood culture (BC) is the most widely used method for the diagnosis of bloodstream bacterial infections (BSIs) [1]. Several studies reported the development and clinical assessment of broad-range real-time PCR protocols, capable of rapid detection and identification of a vast proportion of cultivable and uncultivable bacteria, from different types of biological samples [4,5,6,7,8,9,10,11,12]. The majority of the broad-range real-time PCRs use a single pair of universal primers to identify bacteria through the amplification of the 16S ribosomal DNA (16S 0072DNA), given the high level of homology of this gene throughout the bacterial species diversity [12].

Objectives

Methods

Results

Conclusion