Design and construction of a single tube, quantitative endpoint, LATE-PCR multiplex assay for ventilator-associated pneumonia

Abstract

The goal of this study was to develop a molecular diagnostic multiplex assay for the quantitative detection of microbial pathogens commonly responsible for ventilator-associated pneumonia (VAP) and their antibiotic resistance using linear-after-the-exponential polymerase chain reaction (LATE-PCR). This multiplex assay was designed for the quantitative detection and identification of pathogen genomic DNA of methicillin-resistant Staphylococcus aureus (MRSA), Acinetobacter baumannii, Pseudomonas aeruginosa, plus a control target from Lactococcus lactis. After amplification, the single-stranded amplicons were detected simultaneously in the same closed tube by hybridization to low-temperature molecular beacon probes labelled with four differently coloured fluorophores. The resulting hybrids were then analysed by determining the fluorescence intensity of each of the four fluorophores as a function of temperature. This LATE-PCR single tube multiplex assay generated endpoint fluorescent contours that allowed identification of all microbial pathogens commonly responsible for VAP, including MRSA. The assay was quantitative, identifying the pathogens present in the sample, no matter whether there were as few as 10 or as many 100000 target genomes. This assay is rapid, reliable and sensitive and is ready for preclinical testing using samples recovered from patients suffering from ventilator-associated pneumonia.