Padronização de uma PCR em tempo real para identificação de bactérias gram positivas e gram negativas.

Abstract

Introduction: Infections related to health care affect patients hospitalized, mainly in Intensive Care Units (ICU), exposed to a range of pathogenic microorganisms, which promotes the natural selection and, consequently, colonization and/or infection by multiresistant agents. The use of real time PCR for the diagnosis of bacterial infections significantly increases the sensitivity of detection and allows results in a shorter time, facilitating the start of antimicrobial therapy, giving the technique a greater advantage over other methods already existing in the laboratory routine. Objective: Standardize real time PCR for identification of Gram positive and Gram negative bacteria. Methods: Standardization of a real time PCR technique was determined with standard strains of the American Type Culture Collection (ATCC) clinically important in cases of HAIs, including Staphylococcus aureus (ATCC 29213), Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853). The bacterial DNA extraction was performed according to the adaptation of the alkaline wash/lysis technique. For the Universal TaqMan 16S rDNA bacterial PCR reaction, the primers NT-341Fw and 16S-522Rv and probes for Gram positive and Gram negative were used which had their sequences drawn in GenBank. Results: The uniplex qPCR reaction presented an average of 25,28 for the Ct of duplicate samples of the Gram positive bacteria and 25,21 for the duplicate samples of the Gram negative bacteria, while in the Multiplex qPCR, an average of Ct 23,9 for S. aureus (Gram positive) and Ct of 27,15 for the bacteria E. coli and P. aeruginosa (Gram negative).