(1) Oligonucleotides - Two oligonucleotides are used as probes in the Cervista HPV DNA primary reaction.
Probe- strand of nucleotides complementary to the target DNA.
Invader Oligo - strand of nucleotides complementary to one half of target DNA.
(2) Both oligonucleotides are added to isolated and denatured HPV DNA. When the Probe hybridizes with the Invader Oligo already hybridized on the target region, an overlapping structure is created. This overlapping structure from the Probe is referred to as a 5' Flap.
(3) A specific cleavage enzyme cuts away the Probe overlapping structure, releasing the 5' Flap from target region.
During the incubation in the primary reaction, both oligonucleotides hybridize and unhybridize to the target region rapidly. Each time a Probe attaches in the presence of an Invader Oligo, a 5' Flap is created and cleaved. The number of 5' Flaps cleaved is relative to the quantity of target HPV DNA in the sample.
4) In the secondary reaction, the cleaved 5' Flaps attach to a FRET oligonucleotide probe. The FRET probe is curved such that the 5' Flap end nucleotide base pair attaches to the FRET probe in two places. Another cleavage reaction cleaves the 5' Flaps off the FRET probes and excites the fluorophores. The 5' Flaps reanneal and a cycling of the 5' Flaps off and on the FRET probes occurs. The emitted light is detected in a fluorescence plate reader.
Because of the increasing number of 5' Flaps created in the primary reaction and the cycling of the 5' Flaps off and on the FRET probes, the target-specific signal is consequently amplified. During a 4-hour testing period, on average, the two reactions result in a 1-10 million-fold signal amplification.