Early and rapid identification of disease-causing pathogens, particularly those responsible for serious epidemic diseases, is a key element in the prevention of outbreaks and protection of susceptible populations. The detection of pathogen-specific nucleic acids has proven to be an invaluable tool in the diagnostic field. The advancement of technology involving the integration of amplification and signal detection systems has increased diagnostic capability, enabled development of robust, standard quantification techniques, as well as sequence analysis, by exposing products of the polymerase chain reaction (PCR) to a thermal gradient, i.e. melting and hybridisation curves. The most recent molecular technologies provide precision or broad detection facility, which is crucial to finding low level viraemia, distinct subtypes of interest, or mutants. In order for the development of new technologies and assays to proceed, a detailed knowledge of the diagnostic requirements is needed to create fit-for-purpose tools for the detection and discrimination of present and emerging diseases. The development of fit-for-purpose tools only makes sense if they can be transferred to, and applied in, appropriate laboratories and the field to provide reliable results using the most efficient methods for recognition of pathogens thereby allowing effective control measures to be employed as soon as possible. The prime objective in using these diagnostic tools should be to detect the pathogen at the earliest stage of the disease to prevent its spread.