Digene High-Risk HPV HC2 DNA (Qiagen, Germantown, MD) screens for 13 HR-HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, or 68.
The High-Risk HPV HC2 DNA test uses nucleic acid hybridization, signal amplification, and chemiluminescence detection of the signal. The hybridization and amplification reactions occur on and are read on a microplate.
Step 1
Denaturation of isolated DNA: A base solution is added to a liquid sample of cervical cells, and HPV DNA (if present) is denatured. Denaturation converts the dsDNA to ssDNA strands.
Step 2
Hybridization: Strands of HPV RNA sequences (RNA probes) complementary to HR-HPV types are added. The probes are annealed or hybridized by complementary base-pairing of the ssDNA. RNA: DNA hybrids are created.
The DNA target sequence in the following diagrams (Hybrid Capture 2 and Cervista) is contrived to depict base pairing, hybridization, and detection. Actual target sequences and probes would also be longer.
Step 3
The solution of RNA: DNA hybrids were added to a microplate well coated with capture antibodies specific for RNA: DNA hybrids. The antibodies captured the RNA: DNA hybrids and attached them to the microplate wells.
Step 4
Multiple antibodies for RNA: DNA hybrids conjugated with alkaline phosphatase are added. These labeled antibodies attach to the hybrids and form sandwiches. The three components of each sandwich are capture antibody, hybrid, and labeled antibody.
The microplate is washed to remove any unbound alkaline phosphatase-labeled antibodies. Since multiple labeled antibodies attach to each hybrid, the signal is amplified 3000 fold.
Note: Omitting the wash step will leave unbound alkaline phosphatase-labeled antibodies in the microplate wells of the negative specimens and the negative control. When the substrate is added, these will produce chemiluminescence, the positive specimen, and the positive control.
Step 5
A chemiluminescence substrate is added. Enzymes and substrates produce a light-producing chemical reaction, which a luminometer measures.
