Identification of Bacteria by Polymerase Chain Reaction Followed by Liquid Chromatography−Mass Spectrometry

Abstract

Bloodstream infections are an important cause of serious morbidity and mortality. Rapid detection and identification of specific pathogens from blood or other clinical specimens could improve the rational use of antimicrobial therapy in clinical medicine and have a great impact on the outcome of patients with systemic infections. Polymerase chain reaction using generic primers was used to amplify genomic DNA of different bacterial strains. The identification was accomplished by measuring the molecular masses of the PCR products using ion-pair reversed-phase high-performance liquid chromatography hyphenated to electrospray ionization mass spectrometry. DNA from 10 bacterial species was amplified by PCR, and the resulting amplification products were analyzed. In all cases, the measured molecular masses of the PCR products matched the theoretical value for the species-specific DNA sequence. However, three pairs of bacteria could not be distinguished since the theoretical difference in amplicon molecular mass was < 1.0 Da (different sequence, same base composition of amplicon). Determination of intra- and interday mass reproducibility resulted in relative standard deviations of 0.0030 and 0.018%, respectively. The limit of detection of the presented method was shown to be 0.5 genome equivalents/PCR. The specificity of the method in a human background was successfully tested by amplifying and analyzing 1000-10000 genome equivalents of Staphylococcus aureus spiked into human plasma.