Development and Assessment of a Single Tube Internally Controlled Multiplex PCR Assay to Detect Different Pathogenic Bacteria Involved in Blood Stream Infections

Bloodstream infections are associated with high morbidity and mortality. Delayed etiological diagnosis and inadequate antimicrobial therapy are associated with treatment failures. This study describes the development and assessment of a new multiplex PCR that includes an Internal Control (IC) for the assurance of the whole workflow from the extraction of the DNA until the revelation of the amplicons. A unique sequence was chosen for each pathogen and used for primer design. Primers for amplification of Enterobacteriaceae, Enterococcus spp, Staphylococcus spp, Acinetobacter baumanii and IC were designed and tested for sensitivity and specificity on the basis of their standard strains. The multiplex PCR showed a sensitivity ranging from 1 to 100 target copies per reaction or 50 to 100 colony forming unit (CFU) per ml to the whole blood depending on the bacterial species. The specificity of this method was elevated and no false positive amplification was identified for 17 different species other than the target microorganisms. Moreover, the detection of the IC was observed in the concentration as low as 1 copy per reaction. The correct co-amplification of IC for each single bacterial species showed a correct whole workflow procedure starting from the extraction step. This new assay permits a rapid and accurate detection of some pathogenic microorganisms, that are among the most commonly detected ones in blood stream infections in Iran, with a simple and cost-effective method which includes the use of an internal control to validate the whole procedure thus avoiding false negative results.