Comparison of SPF10 real-time PCR and conventional PCR in combination with the INNO-LiPA HPV Genotyping Extra assay for the detection and typing of human papillomavirus in cervical samples.

The novel SPF10 real-time PCR assay allows the simultaneous amplification and detection of the HPV target. That way, LiPA analysis of the HPV-negative samples can be avoided, reducing workload and cost. This study aims to evaluate the performance of the SPF10 real-time PCR in combination with the LiPA assay for HPV detection and typing in cervical samples. Thirty-nine cervical samples were subjected to the SPF10 conventional PCR in combination with the LiPA assay. Subsequently, the SPF10 real-time PCR was performed to enable the comparison between the SPF10 conventional and the real-time PCR results. In case of discrepancy, the samples were subjected to the CLART HPV2 assay. As a result, 27 out of 39 samples were identified as HPV-positive by the SPF10 real-time PCR and were genotyped further by the LiPA assay. Twenty samples (74.1%) showed an absolute agreement between the conventional and real-time SPF10 PCR (concordant), three (11.1%) displayed additional or fewer types (compatible), two (7.4%) did not show any similarity between both assays (discordant) and the remaining two (7.4%) were LiPA-negative. The two assays showed an excellent strength of agreement for individual (κ=0.932) and multiple genotype detection (κ=0.834). In conclusion, the two SPF10 PCR methods are comparable. Therefore, the SPF10 real-time PCR with subsequent LiPA could be used for the detection and genotyping of HPV in cervical samples.

A real-time PCR approach based on SPF10 primers and the INNO-LiPA HPV Genotyping Extra assay for the detection and typing of human papillomavirus.

The SPF10 PCR targets a conserved 65bp region of the HPV L1 gene for broad-spectrum amplification. The LiPA assay allows subsequent genotyping of the HPV amplicons. This study aims to develop a SPF10 real-time PCR to achieve simultaneous amplification and detection of the HPV target. That way, LiPA analysis of the HPV-negative samples can be avoided, reducing workload and cost. The real-time PCR shows an analytical sensitivity of 29.7 copies for HPV 6, 16, 18 and 31 and an HPV-specific melting peak. Thirty-one HPV DNA plasmids were genotyped correctly using the SPF10 real-time PCR in combination with the LiPA. Here, the LiPA assay was performed at an increased hybridisation temperature (49.5°C) in combination with a reduced amplicon volume (1µl) to avoid cross-reactivity. In conclusion, the SPF10 real-time PCR proves to be very sensitive and generates amplicons, which are compatible with the LiPA.

WITHDRAWN: A real-time PCR approach based on SPF10 primers and the INNO-LiPA HPV Genotyping Extra assay for the detection and typing of human papillomavirus.

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Cervical cytology biobanking: quality of DNA from archival cervical Pap-stained smears.

AIMS: Cervical cytology biobanking is a feasible concept in cervical pathology and could be an indispensable tool for fundamental and applied molecular biological research. PCR is a powerful molecular technique that can be performed on a variety of cervical sample types including Pap-stained cervical smears. However, since the quality of DNA from such specimens is inferior to that from fresh tissue, the correct processing methods are required. This study evaluates three commercial isolation methods and one digestion procedure for their ability to obtain DNA suitable for PCR from fixed and stained Pap smears. METHODS: The High Pure PCR Template Preparation kit, the NucliSENS easyMAG system, the QIAamp DNA Mini Kit and crude proteinase K digestion were used to obtain DNA for subsequent PCR applications. Amplification of beta-globin was performed to verify the presence and integrity of target DNA. The influence of PCR inhibitors and extent of DNA fragmentation were analysed. RESULTS: All commercial DNA isolation techniques provided DNA suitable for PCR amplification, and DNA isolated from 10-year-old archival smears yielded amplicons up to 400 base pairs. Conversely, crude proteinase K digestion limited the amplicon size to 300 bp and did not consistently yield amplifiable digests, as these were contaminated with PCR-inhibiting factors and debris. CONCLUSION: The study indicates that commercial DNA isolation techniques are suitable for PCR amplification of DNA isolated from archival smears, yielding amplicons up to 400 base pairs. Proteinase K digestion is not suitable to obtain amplifiable DNA from fixed and stained Pap-stained smears.

Comparison of MY09/11 consensus PCR and type-specific PCRs in the detection of oncogenic HPV types.

The causal relationship between persistent infection with high-risk HPV and cervical cancer has resulted in the development of HPV DNA detection systems. The widely used MY09/11 consensus PCR targets a 450bp conserved sequence in the HPV L1 gene, and can therefore amplify a broad spectrum of HPV types. However, limitations of these consensus primers are evident, particularly in regard to the variability in detection sensitivity among different HPV types. This study compared MY09/11 PCR with type-specific PCRs in the detection of oncogenic HPV types. The study population comprised 15, 774 patients. Consensus PCR failed to detect 522 (10.9%) HPV infections indicated by type-specific PCRs. A significant correlation between failure of consensus PCR and HPV type was found. HPV types 51, 68 and 45 were missed most frequently. The clinical relevance of the HPV infections missed by MY09/11 PCR was reflected in the fraction of cases with cytological abnormalities and in follow-up, showing 104 (25.4%) CIN2+ cases. The MY09/11 false negativity could be the result of poor sensitivity, mismatch of MY09/11 primers or disruption of L1 target by HPV integration or DNA degradation. Furthermore, MY09/11 PCR lacked specificity for oncogenic HPVs. Diagnostic accuracy of the PCR systems, in terms of sensitivity (MY09/11 PCR: 87.9%; type-specific PCRs: 98.3%) and specificity (MY09/11 PCR: 38.7%; type-specific PCRs: 76.14%), and predictive values for histologically confirmed CIN2+, suggest that type-specific PCRs could be used in a clinical setting as a reliable screening tool.

Correlation between laminin-5 immunohistochemistry and human papillomavirus status in squamous cervical carcinoma.

BACKGROUND: Human papillomavirus (HPV) plays a critical role in the carcinogenesis of squamous cervical carcinoma. Integration of viral DNA into the host genome is a major contributing factor to malignant transformation. Viral load may influence integration. AIMS: To compare HPV status (type, viral load, integration status) between normal samples, carcinoma in situ and invasive carcinoma in order to elucidate the role of HPV in progression to invasive lesions. METHODS: The study population comprised 10 biopsy samples from each diagnostic group. Laminin-5 immunohistochemistry was performed to distinguish invasive carcinoma from non-invasive high-grade lesions. Real-time PCR was used to detect specific HPV types, viral load and integrated HPV, with quantification of viral E2 and E6 genes. RESULTS: Invasive carcinomas contained a higher number of laminin-5 immunoreactive cells as compared to non-invasive lesions. Almost all samples contained HPV, with a higher viral load and copy number of HPV16 integrated in E2 in cases of laminin-5 immunoreactivity and cases of invasive carcinoma. High HPV16 viral load was associated with more integrated copies in E2. CONCLUSIONS: HPV is important in progression from carcinoma in situ to invasive carcinoma. Viral load and HPV integration influence the development of cervical cancer towards invasiveness. Overall HPV status may be more predictive of patient outcome and may influence patient management.