Introduction: Staphylococcus aureus, coagulase-negative staphylococci, Enterococcus spp., Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumanii have been found to be the most prevalent bacteremia-causing bacteria in patients with septicemia. Early detection of bloodstream infection (BSI) is crucial in the clinical setting. A multiplex PCR method for identification of these agents in clinical samples has been developed in parallel by conventional microbiological methods. Methodology: The target genes selected for each of the organisms were very specific for designing primers. Design of primers was done using Mega4, Allel ID6, Oligo6, and Oligo analyzer software. The test comprises a universal PCR from the 16S rDNA gene and multiplex PCR from the rpoB, gyrA, sss, and chromosome X (as an internal control). Results: The sensitivity and specificity for universal PCR and multiplex PCR in comparison with BC were 83.87% and 91.58%, and 74.19% and 91.58%, respectively. The positive predictive value (PPV) and the negative predictive value (NPV) for these two PCRs were 76.47% and 94.57%, and 74.19% and 91.58%, respectively. PCR failed to identify bacteria which were found conventionally in only 3.96% and 6.34% of the cases by universal and multiplex PCR (mostly bacteria not included in the PCR cassette). In 6.34% of the cases, multiplex PCR afforded identification of bacteria, but BC showed no bacteria in the sample. Conclusions: The multiplex PCR approach facilitates the detection of bacteremia in blood samples within a few hours. Rapid detection of bacteria by multiplex PCR appears to be a valuable tool, allowing earlier pathogen-adopted antimicrobial therapy in critically ill patients.
Background:Throughout the world, bloodstream infections (BSIs) are associated with high rates of morbidity and mortality. Rapid pathogens identification is central significance for the outcome of the patient than culture techniques for microbial identification. To develop an end point multiplex PCR to identify a group of bacteria including Enterococcus spp., Pseudomons aeruginosa, Staphylococcus spp., Acinetobacter baumannii, 16S rDNA, and Drosophila Melanogaster were used as internal control (IC).Materials and Methods:Design of primers was done using Mega4, Allel ID6, Oligo6 and Oligo analyzer softwares. Genetic targets for primer designing and identification of genus Enterococcus spp., Staphylococcus spp., and species of Acinetobacter baumannii, Pseudomons aeruginosa, included the rpoB, rpoB and gyrA, sss respectively. Then PCR and multiplex PCR were performedResults:The intended specificity was obtained for the bacteria, which used in this study and there wasn't seen any unspecific amplification by the multiplex PCR. The test showed a sensitivity ranging from 1 to 100 target copies per reaction depending on the bacterial species.Conclusions:The presented multiplex PCR offers a rapid and accurate molecular diagnostic tool for simultaneous detection of some pathogenic microorganisms. The IC exists in the multiplex PCR accompanied by other primers in the system, can serve as a simple, cost- effective internal control for the multiplex PCR assay.
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