Nucleic acid based tests (NATs) are based on the polymerase chain reaction (PCR) and detect pathogen specific DNA or RNA sequences rather than antigens and antibodies. A large variety of NATs are currently available for diagnostic purposes. PCR is the most well-developed molecular technique to date for detecting a broad spectrum of viral and bacterial pathogens and for profiling antimicrobial resistance, and its use has increased significantly over the past several years (Figure 3A).10, 15
PCR is an enzyme driven process that amplifies short regions of DNA in vitro. When at least a partial sequence of the target DNA is known beforehand, oligonucleotide primers can be synthesized that bind specifically to the target sequences. In PCR, the target DNA is copied by a DNA polymerase enzyme and can then be exponentially amplified through a process involving multiple cycles of heating and cooling in a thermocycler (Figure 4). In reverse transcription PCR (RT-PCR), RNA is reverse transcribed to a single-stranded cDNA, prior to amplification. In quantitative real-time PCR, amplification and detection of amplified products are carried out together in a single reaction vessel. Using different types of fluorescent probes, a fluorescent signal is emitted during each amplification cycle only when target sequences are present. The intensity of the signal increases in proportion to the amount of amplified product.18 In addition to its quantitating ability, real-time PCR is faster and more reproducible than conventional PCR.
Traditional diagnostic techniques used for detecting bacterial and viral pathogens, such as cultures, antigen detection, viral isolation, serologic assays, and direct fluorescent antibody tests are time-consuming and can yield variable results16 that may take days to finalize and report, delaying accurate diagnoses and the implementation of targeted antimicrobial therapies. PCR has a shorter turn-around time and a higher sensitivity for viral pathogens16 and can detect the presence of microbial pathogens far below the level required by other techniques.18 Multiplex PCR, which enables the simultaneous detection of several target sequences by incorporating multiple sets of primers, is being increasingly relied on for the accurate detection of respiratory pathogens (Figure 3B), and several mPCRs have been approved by the US Food and Drug Association (FDA) for that purpose.5