Method for the detection and multiple, simultaneous quantification of pathogens by polymerase chain reaction in real time

摘要

A method for the detection and multiple, simultaneous quantification of any combination of Listeria sp, Staphylococcus aureus, Campylobacter jejuni and / or Escherichia coli O157-H7, in one or másmuestras assay using amplification reaction múltiplexempleando the polymerase chain reaction (PCR) RealTime, the method steps are (a) extracting DNA present in the sample or test samples; (B) preparing a reaction mixture specific for the pathogens to detect and quantify such that lamezcla reaction contains the necessary reagents for enzymatic amplification of DNA extracted and identification of pathogens to detect and quantify; (C) amplifying by PCR multiplex amplification reaction used, the reaction mixture; and (d) simultaneously determining the presence or absence and quantification of pathogens in the sample or test samples; elmétodo is unique in that (i) mixing reacciónpara enzymatic amplification of DNA has sets of pairs of oligonucleotide primers identified as SEQ ID NO: 1 ySEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 5, SEQ ID NO: 7 and SEQ IDNO: 8, and SEQ ID NO: 10 and SEQ ID NO: 11, and probes with 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 in any combination is determined by a fluorescent signal or fluorescence emission specific for each pathogen.