High-Risk HPV Hybrid Capture (HC) 2 DNA Test

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High-Risk HPV Hybrid Capture (HC) 2 DNA Test

Digene High-Risk HPV HC2 DNA (Qiagen, Germantown, MD) screens for the presence of 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 with signal amplification and chemiluminescence detection of signal. Hybridization and amplificathion reactions occur in 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 sequesnces (RNA probes) complementary to HR-HPV types are added. The probes anneal or hybridize by complementary base-pairing to the ssDNA. RNA:DNA hybrids are created.
The DNA target sequence in each of 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 greater length.
Step 3
The solution of RNA:DNA hybrids added to a microplate well coated with capture antibodies specific for RNA:DNA hypbrids. RNA:DNA hybrids are captured by the antibodies and become attached 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 componenets of each sandwich are: capture antibody: hybrid: labeled antibodies.
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 mciroplate wells of the negative specimens and the negative control. When the substrate is added, these will produce chemiluminescence along with the positive specimsn and the positive control.
Step 5
A chemiluminescence substrate is added. A light-producing chemical reaction occurs between enzymes and substrates and a luminometer measures the light.