A total of 289 women with a median age of 41 years (range 16 to 81 years) participated in this study between February 2006 and January 2007. The population was consecutively recruited from the Gynaecology Outpatient Clinic at the Department of Obstetrics and Gynaecology of the Hospital de Santo Espírito de Angra do Heroísmo (HSEAH). The Ethics Committee of Hospital de Santo Espírito de Angra do Heroísmo, approved the study protocol.
Sample material was rinsed into a liquid medium (PreservCyt, Cytyc Corporation), transported to Serviço Especializado de Epidemiologia e Biologia Molecular (SEEBMO) of HSEAH and stored at 4°C before DNA extraction.
DNA extraction and purification
The DNA from cervical samples was extracted and purified, using Papillomavirus Clinical Arrays® (Genomica) kit, according to the manufacturer's instructions. Briefly, after 1 ml cell suspension centrifugation (10 minutes, 12 000 rpm, room temperature), the lysis buffer and the proteinase K (20 mg/ml) were added on the pelleted cells and incubated 2–3 hours ate 56°C in a Thermomixer Comfort Eppendorf. After 10 minutes proteinase K inactivation period at 70°C, DNA purification was performed, using DNA purification columns and the pelleted DNA was resuspended in 100 μl Elution Buffer.
Polymerase chain reaction (PCR)
The amplification reaction was carried out in a total volume of 50 μl, containing 5 μl of extracted DNA and 45 μl of a PCR mix with PGMYO9/PGMY11 generic consensus primers. The PCR mix contains, beside the generic consensus primers, two internal controls that will validate each sample genotyping: genomic DNA control and amplification control. The first control consists in a primer set that amplifies a 892 bp CFTR gene fragment and the second control amplifies 1202 bp of a modified plasmid, using the same primers as the CFTR gene.
The mixture was submitted to 45 cycles of amplification, using a DNA thermalcycler T-Gradient (Biometra). Each cycle included a denaturation step at 94°C for 30 seconds, followed by an annealing step at 55°C for 1 minute and a chain elongation step at 72°C for 90 seconds. Each PCR was initiated with a 9 minutes denaturation step at 95°C and finished by an 8 minutes extension-step at 72°C.
Different laboratory areas were used for sample handling (pre-PCR area), product amplification (PCR area) and HPV genotyping (post-PCR area).
The commercially available Papillomavirus Clinical Arrays® (Genomica) kit was used for HPV DNA genotyping. All samples were analysed for the presence of he following HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66, 68, 70, 71, 72, 73, 81, 82, 83, 84, 85 and 89.
HPV detection was conducted using PGMYO9/PGMY11 general consensus primers designed to amplify a 450 bp HPV L1 gene fragment. This region is used because it is highly conserved between different HPV types but has sufficient variation for the identification of each one .
The detection of the amplified PCR product was performed with a new technological platform using a low density Microarray, anchored in 2 ml tube-AT tube. It allows simultaneous detection of multiple molecular markers in the L1 fragment of 35 different HPV types and in the necessary controls to insure a feasible assay. All process was followed according to the manufacture's standard protocol.
PCR products were marked with biotin and, after amplification, they hybridised with the respective probes in specific known AT tube areas. Biotin binds to streptavidin-peroxidase after incubation. The addition of the TMB substrate (3,3',5,5'-tetrametilbinzidine) generates an insoluble product after hybridisation.
The results were processed by software, which allows detection, interpretation and reporting for each sample.