Performing RT-PCR for HPV Detection
For HPV detection, use FDA-approved nucleic acid amplification tests that detect viral DNA or RNA from cervical specimens, as RT-PCR is not specifically FDA-approved for HPV testing. 1
Approved HPV Testing Methods
FDA-Cleared HPV Tests
- Several FDA-cleared tests are available for HPV detection:
- Cobas 4800 HPV test (Roche Molecular Diagnostics) - detects 14 oncogenic HPV types (16,18,31,33,35,39,45,51,52,56,58,59,66,68) and specifically identifies types 16 and 18 1
- Onclarity HPV test (Becton Dickinson) - also detects 14 oncogenic HPV types with specific identification of types 16 and 18 1
- Hybrid Capture 2 High-Risk HPV DNA test (Qiagen) - detects 13 high-risk HPV types 1
- Cervista HPV High-Risk and HPV 16/18 DNA tests (Hologics) 1
- APTIMA HR HPV test (Gen Probe) - detects viral RNA 1
Sample Collection and Processing
- Collect cervical specimens using appropriate collection devices
- Process samples according to manufacturer's instructions
- For most commercial tests, samples should be processed in a certified laboratory
Important Considerations for HPV Testing
Appropriate Clinical Indications
HPV testing should only be performed for:
- Cervical cancer screening as a primary test (age ≥30)
- Cervical cancer screening with cytology (co-testing)
- Triage of abnormal cervical cytology results
- Follow-up after abnormal screening results
- Follow-up after colposcopy with no CIN 2/3 found
- Follow-up after treatment of cervical precancers 1
Inappropriate Uses of HPV Testing
HPV testing should NOT be performed for:
- Deciding whether to vaccinate against HPV
- Testing for low-risk (non-oncogenic) HPV types (e.g., 6,11)
- Providing care to persons with genital warts or their partners
- Testing persons aged <25 years as part of routine screening
- Testing oral or anal specimens (only FDA-cleared for cervical specimens) 1
Research-Based RT-PCR Methods for HPV
If performing RT-PCR for research purposes (not clinical diagnosis), several approaches have been validated:
Type-Specific Real-Time qPCR
- Enables detection of up to 17 HPV genotypes in multiplex reactions
- Can provide viral load quantification
- Analytical sensitivity of ≤100 copies for all HPV types
- Suitable for high-throughput screening 2
Multiplex Nested PCR-Immunochromatographic Test
- Can rapidly identify HPV 16 and 18 simultaneously
- Amplifies HPV 16 and 18 genotype-specific L1 gene fragments
- Detection limit of approximately 100 copies of viral DNA 3
General Primer-Mediated PCR with Oligonucleotide Probes
- Uses GP5+/GP6+ primers to detect a broad spectrum of HPV genotypes
- Can differentiate between high-risk and low-risk HPV types
- Sensitivity ranges from femtogram to picogram levels depending on HPV type 4
Clinical Algorithm for HPV Testing
Determine if HPV testing is clinically indicated
- Appropriate for women ≥21 years with abnormal Pap results
- Appropriate for women ≥30 years for primary or co-testing screening
- Not appropriate for women <21 years or for STD screening 1
Select appropriate FDA-cleared test based on clinical scenario
- Primary screening: Cobas 4800 or Onclarity HPV test
- Co-testing or triage: Any FDA-cleared HPV test 1
Ensure proper specimen collection and handling
- Cervical specimens only (not oral or anal)
- Follow manufacturer's instructions for collection and transport
Interpret results in clinical context
- HPV 16/18 positive: Consider colposcopy
- Other high-risk HPV positive: Follow management guidelines based on cytology results 1
Common Pitfalls to Avoid
- Do not use RT-PCR tests that are not FDA-cleared for clinical HPV testing
- Do not test specimens other than cervical samples (oral or anal)
- Do not use HPV testing for STD screening
- Do not confuse pelvic examination with HPV testing
- Remember that self-collection for HPV testing is not FDA-cleared or recommended by U.S. medical organizations 1
By following these guidelines, clinicians can ensure appropriate use of HPV testing for cervical cancer screening and management.