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Human papillomavirus (HPV) DNA screening benefits cervical cancer diagnosis, but a few HPV infections result in cancer. Assays that predict cancer are desirable. A potential biomarker is the ratio of HPV E6–7 over E2 transcripts, which may increase during early cancer progression. Modified hybrid-capture technology detected, in separate wells, HPV E6–7 or E2 mRNA of HPV 16 or HPV 18 in samples. The limit of detection was approximately 1000 copies of in vitro transcribed RNA with linear dynamic range approximately four logs. No cross-reactivity between HPV 16 and HPV 18 mRNAs was detected. RNA of SiHa cells was stable in clinical specimen pools for 67 days, as determined by RT-PCR. The ratio of HPV E6–7:E2 mRNAs was relatively high for cancer cells lines, SiHa, Caski and HeLa cells preserved in clinical specimen pools. A broad distribution of HPV 16 E6–7:E2 mRNA ratio was detected in a small set of clinical specimens with various histological diagnoses. Some specimen ratios were so high for cancer cell lines, but the significance of the results needs to be determined. This method may help determine the pattern of gene expression in HPV-related disease or in other systems.

A novel DNA detection assay comprising Hybrid Capture® sample preparation, GP5+/6+ PCR with modifications and Luminex® 100 detection was developed and applied to genotyping of human papillomavirus (HPV) in cervical samples. Target-specific sample preparation was performed using magnetic beads conjugated with Hybrid Capture® (HC) antibody. DNA–RNA hybrids were formed between DNA target and RNA probes and captured on HC-beads. DNA on magnetic beads was amplified without elution using consensus GP5+/6+ PCR and then genotyped on Luminex® beads using hybridization probes for the 17 high-risk HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73, 82 and an internal control. This new sequence-specific Hybrid Capture® sample preparation is fast, efficient and allows direct HPV genotyping by PCR. Compared to traditional non-sequence-specific sample preparation methods, HC sample preparation demonstrated slightly better detection of multiple HPV infections. The clinical utility of this method was demonstrated on cervical samples positive for HR HPV by the Hybrid Capture® 2 (hc2) screening assay.

The information about the range and distribution of Human papillomavirus load in clinical specimens is important for the design of accurate clinical tests. The amount of Human papillomavirus in cervical specimens was estimated using the digene HC2 HPV DNA Test® (QIAGEN). This semi-quantitative assay is based on linear signal amplification with an analytical limit-of-detection of approximately 2500 virus copies per assay and 3–4 log dynamic range. The dynamic range of the assay was extended by a serial dilution strategy. Two large sets of positive specimens (n = 501 and 569) were analyzed and 9–11% of specimens was estimated to contain more than 7 × 107 copies of virus. The viral load was also assessed for an assortment of specimens with known cytology diagnoses (n = 9435) and histological diagnoses (n = 2056). The percentage of specimens with more than 7 × 107 copies of virus was estimated to be 0.89 for normal cells, 4.2 for atypical cells (unknown significance), 14.31 for cells of low-grade lesions and 22.24 for cells of high-grade lesions. The viral load increased with disease severity, but its broad distribution may not support its use as a disease biomarker. This information is important for assay design and automation, where cross-reactivity and sample-to-sample contamination must be addressed rigorously.