Real time hr hpv assay

Subsequent hr-HPV genotyping was performed using the Abbott Real-Time hr-HPV assay according to the manufacturer’s instructions (Abbott GmbH & Co. KG, Wiesbaden, Germany).^{22 (link)} The Abbott Real Time hr-HPV assay qualitatively detects DNA from 14 different hr-HPV genotypes using a modified GP5+/6+ primer mix consisting of three forward primers and two reverse primers that target the conserved L1 region of HPV and an internal control primer pair that targets a human β-globin sequence. The assay provides specific probes that are differentially labeled to qualitatively detect HPV-16 and HPV-18, and an evaluation of the human beta-globin internal control. Probes for hr-HPV genotypes HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-66 and HPV-68 are labeled with the same dye, and the presence of any one or combination of these genotypes is reported as “other hr-HPV” detected.^{22 (link)} Ten known hr-HPV-positive and 10 hr-HPV-negative control samples were genotyped according to a previously published protocol to validate the assay.^{21 (link)}

Tissue sections of 20-μm thicknesses were then incubated in xylene within 24 hr after arrival in the laboratory and left overnight to ensure complete removal of paraffin wax, based on a previously published protocol.^{21 (link)} All 126 specimens had adequate tissue volume for optimal nucleic acid extraction (DNA), and tissues remained intact after sectioning. Tissue adequacy refers to the amount of epithelium with which histological evaluation of invasive cervical cancer can be rendered accurately for diagnosis. After tissue rehydration, samples were centrifuged at 3,500 rpm, enzymatically digested with proteinase K and transferred into an Abbott Real-Time hr-HPV assay (Abbott GmbH & Co. KG, Wiesbaden, Germany) reaction vessel for DNA isolation using the mSample Preparation System DNA kit with the Abbott m2000sp instrument according to the manufacturer’s instructions.^{22 (link)}