Human subject approval from the University of Michigan was obtained to use residual amniocentesis samples that were to be discarded. To be eligible for the study, pregnant women had to be undergoing clinically indicated amniocentesis and have intact membranes. The only information on the women available to the investigators was age, race/ethnicity, and number of fetuses present at time of sampling. All clinical assays related to chromosomal assessment of the fetus had been completed, and the residual amniotic fluid and amniotic cell culture pellets were retrieved from the cytogenetics laboratory.
Amniotic fluid samples (8–12 ml) were spun at 4500 rpm for 30 minutes. After decanting the supernatant fluid, the pellet was suspended in 150 μL of TrisEDTA buffer. The cultured cell pellets were resuspended in 200 μL PBS. A 150 μL volume of each sample was extracted using the Roche MagNA Pure LC instrument and the DNA isolation kit I resulting in a final volume of 150 μL.
We used a direct PCR method followed by a nested PCR method to detect HPV. The Roche line blot assay, based on L1 consensus PCR with biotinylated PGMY09/11 primer sets and β-globin as an internal control for sample amplification [17
] was used as previously described, with 10 μL extract in each 50 μL reaction. All samples were HPV gel-band-negative and Roche Prototype Strip Assay-negative after 40 cycles (reagents provided as a gift from Roche Molecular Systems, Inc., Pleasanton, CA).
For the nested reaction, five microliters of each L1 amplicon was added to a PCR reaction mix containing GP5+/GP6+ [19
] primers (1 μM each) and run for an additional 40 cycles under the following conditions: 40 cycles of 94°C for 45 s, 48°C for 4 s, 38°C for 30 s, 42°C for 5 s, 66°C for 5 s, and 71°C for 1.5 min. This was followed by a final extension of 10 min at 72°C. Fifteen μL from each sample was analyzed on a 2% agarose gel.
The HPV assays were repeated using separate extractions, increased volumes of extracts in the PCR assays and re-amplification of L1 consensus PCR products with PGMY primers.
The data was analyzed using SPSS 11 with frequency distributions of study participants by age, race, and ethnicity by HPV status.