Real-time PCR is the traditional face of nucleic acid detection in the diagnostic microbiology laboratory and is now generally regarded as robust enough to be widely adopted. Methods based on nucleic acid detection of this type are bringing increased accuracy to diagnosis in areas where culture is difficult and/or expensive, and these methods are often effective partners to other rapid molecular diagnostic tools such as matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS). This change in practice has particularly affected the recognition of viruses and fastidious or antibiotic-exposed bacteria, but has been also shown to be effective in the recognition of troublesome or specialised phenotypes such as antiviral resistance and transmissible antibiotic resistance in the Enterobacteriaceae. Quantitation and high-intensity sequencing (of multiple whole genomes) has brought new opportunities as well as new challenges to the microbiology community. Diagnostic microbiologists currently training might be expected to deal less with the culture-based techniques of the last half-century than with the high-volume data and complex analyses of the next.