*2.3. BD Onclarity™ HPV Assay*

The BD Onclarity™ HPV assay (BD, USA), which detects 14 high-risk (HR) genotypes, provides the capability of extended genotyping through individual detection of HPV 31, 51, and 52 (in addition to 16, 18, and 45) and pooled detection of 33/58, 35/39/68, and 56/59/66. An endogenous human beta-globin sequence is detected as a sample validity control, sample extraction, and amplification efficiency [20–22].

All samples were tested following manufacturer instructions: BD Onclarity™ uses 0.5 mL of sample (cervical, vaginal, or urine sample) which is added to a suitable solution produced by BD (the LBC tube) to reach a final volume of 2.2 mL, of which 0.8 mL of sample is automatically taken by the instrument to perform nucleic acid extraction using the extraction chemistry developed by BD (BD FOX ™). The extracted DNA is then eluted to a final volume of 400 microliters, and 50 microliters is automatically pipetted into each of the three wells containing the dried master mix in order to perform real-time PCR.

The clinical cut-off is set to be related to CIN2+ disease, and an algorithm verifies the adequacy of the sample using the amplification of the human beta-globin gene. In particular, the software interprets the amplification curves on the basis of the following threshold cycles (Ct): 38.3 Ct for HPV16, 34.2 Ct for other HR-HPVs, and 34.2 Ct for beta-globin. Samples which were judged invalid were re-tested a second time (9 cervical samples, 2 vaginal swabs, and 4 urines).
