Molecular assays target nucleic acid (DNA or RNA) sequences to demonstrate the presence of a particular target(s) within a sample, in contrast to immunoassays, which target proteins. Most commercial molecular assays are based on PCR (the Polymerase Chain Reaction), an enzymatic amplification method that amplifies the target sequence to improve the limit of detection and time to detect. The PCR process involves forward and reverse primers, which define the target region.
Real-Time PCR assays include a third nucleic acid sequence, termed a probe, which binds a region between the amplification primers and is tagged with a fluorescent molecule and a fluorescence quencher. Thermal cycling is key to the PCR process and involves repeated rounds of heating and cooling. The reaction tube is heated to dissociate the double-stranded DNA template. As the reaction tube is cooled, the primers and probe bind to the single stranded DNA. The PCR enzyme synthesizes a new second DNA strand based on the primer “start button” and removes any DNA in it’s path as it proceeds down the strand, thereby dissociating the probe and releasing the fluorescent molecule into solution where it is “seen” by the PCR instrument’s detection element. Changes in the fluorescence properties of the reaction tube indicate that the particular target sequence is present in the sample and is being amplified.
Direct hybridization assays employee coded bead sets, in which each bead set is functionalized with a capture oligo specific for a nucleic acid sequence (referred to as the capture oligonucleotide) in the target that binds the amplicon product of the PCR reaction, thereby demonstrating the presence of the target nucleic acid sequence.
An advantage of real-time PCR is a relatively short time to detect, typically in 15-120 minutes (instrument dependent). An advantage of bead-based assays is that a sample can be interrogated for more targets in a single tube; up to 50 vs. 4 to 6 targets for real-time PCR, which is constrained by overlap in the fluorescence spectrum of available probes.