METHOD FOR THE DETECTION AND THE MULTIPLE AND SIMULTANEOUS QUANTIFICATION OF BACTERIA CAUSING VENTILATOR-ASSOCIATED PNEUMONIA.

摘要

A method for the detection and the multiple and simultaneous quantification of any combination of pathogens causing ventilator-associated pneumonia, such as staphylococus aureus, pseudomonas aeruginosa, acinetobacter baumannii and stenotrophomonas maltophilia, which is performed in one or a plurality of assay samples by means of a multiplex amplification reaction using an in real-time polymerase chain reaction (PCR), the method including the following stages: (a) extracting DNA present in the sample or assay samples; (b) preparing a reaction mixture specific for the pathogens to be detected and quantified, which contains the reactants required for carrying out the enzymatic amplification of the extracted DNA, as well as the identification of the pathogens to be detected and quantified; (c) amplifying the reaction mixture by means of the multiplex amplification reaction using PCR; and (d)simultaneously detecting the presence or absence, and quantification of the pathogens located in th e sample or assay samples; the method is characterized in that (i) the reaction mixture useful for performing the enzymatic amplification of the DNA includes pair sets of oligonucleotide initiators identified as SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 8, and SEQ ID NO: 10 and SEQ ID NO: 11, and probes having oligonucleotide sequences identified as SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9 and SEQ ID NO: 12; (ii) the presence or absence and quantification of said pathogens located in any combination is determined by a specific fluorescent signal or emission emitted by each pathogen.