This study is built upon our NCI-funded U54 consortium to study Epigenomic Biomarkers of HIV-Associated Cancers in Nigeria (U54CA221205). In total, 173 cervical tissue samples used in this study were collected between 2018 and 2022 in Nigerian women diagnosed with cervical cancer. The age range was 26 to 80 years old [12]. The use of these samples for this study was covered under IRB approval (STU00207051 at Northwestern University). The samples were obtained at the Jos University Teaching Hospital (JUTH) and Lagos University Teaching Hospital (LUTH) in Nigeria. DNA was extracted from the cervical biopsies using the Qiagen QIAamp DNA Mini Kit and was quantified using Qubit 4.0 fluorometer. DNA samples were stored at − 80℃ until shipment. All DNA samples were shipped in dry ice to Northwestern University and stored at − 80℃.

To further confirm that the Zebra BioDome format will perform well using crude lysed samples, we tested 46 banked vaginal swab samples with both Zebra BioDome format and the standard liquid format by the following procedures: a vaginal swab was added to a tube with lysis buffer. Then, the 5µL sample was added to two test tubes separately by following protocols for Zebra BioDome and standard format respectively. Both tubes were then put into Biorad CFX-96 real-time PCR machine for HPV test (Fig. 1A and 1B).

To overcome the possible contamination hurdles faced with standard lab formats, the Zebra BioDome format is pre-packed in a PCR strip or PCR plate in a liquid format. The primer mix and enzyme mix were pre-aliquoted into the wells, but separated by the middle hydrophobic gel matrix to prevent mixing of the two reagents before samples are added for reaction (Supplemental Fig. 1A). The top gel matrix is used to keep the up-layer reagents from moving during transportation or storage and prevents reagent exposure to the environment, thereby increasing the shelf-life of the reagents. The gel matrix has lower density than water and is semi-solidified at room temperature (up to 40℃) to fix the liquid at the bottom of the wells. However, the gel matrix will liquefy and immediately move to the top of reaction mixture when the reaction starts to prevent any amplicons from leaking into the environment and contaminating the lab (Supplemental Fig. 1B).

The newly designed ScreenFire HPV RS assay Zebra BioDome format can be operated by minimally trained laboratory personnel because the operation involves only one step: adding lysed samples to pre-loaded Zebra BioDome reaction tube strips or plates with pre-aliquoted reagents. This is because the prerequisite PCR lab skills to prevent contamination and manual preparation of the master mix have been eliminated. According to the manufacturer’s unpublished data, the Zebra BioDome matrix can effectively prevent post-amplification contamination as long as the integrity of the matrix barrier is maintained. The hydrogel matrix is hydrophobic and immediately liquifies and moves to the top of the aqueous solution once the reaction starts and seals the reaction liquid from leaking into the environment. The gel matrix is resolidified after the reaction is finished, after which it is ready for disposal. Therefore, the resolidified barrier will prevent the post-amplification materials from leaking out of the tube and contaminating the environment.

A total of 100ng of DNA was prepared in 100 µL of 1X lysis buffer and processed to prepare the samples for hrHPV RS genotyping using both the ScreenFire HPV RS assay standard format and the ScreenFire RS assay Zebra BioDome format by Atila BioSystems, Inc (Atila, Sunnyvale, CA).

For ScreenFire HPV RS assay standard format (Fig. 1A), 20µL of freshly prepared master mix (including reaction mix and primer mix) was aliquoted into each reaction well and then a 5µL prepared DNA sample randomized and blinded in each group was added into each reaction well with master mix in it. Additionally, positive and negative controls were included in the test to monitor the test quality and laboratory contamination respectively. After the plates were sealed, vortexed gently, and centrifuged the plates were loaded into a Biorad CFX-96 real-time PCR machine. The test program was set up at 1 min per cycle at 60℃ for 60 cycles with fluorescence obtained from the following detection channels: CY5 (for HPV16), ROX (for HPV18/45), CY5.5 (for HPV31/33/35/52/58), FAM (for HPV39/51/56/59/68), and HEX (for human beta globin gene as internal control). A sample was considered positive for the corresponding HPV if the signal was detected within 60 min in the viral signal channels (FAM/ROX/Cy5/Cy5.5), regardless of the signal in the HEX channel. If no signal was detected from any of the four HPV channels within 60 min but a signal was present in the HEX fluorescent channel, then the sample was called negative. If no signal was present in any of the five fluorescent channels, the sample is invalid.

For the ScreenFire HPV RS assay Zebra BioDome format (Fig. 1B), 5 µL of the prepared DNA samples randomized and blinded in each group was directly added into the bottom of the pre-packed reaction well as specified by the manufacturer’s instructions. The sealed reaction plates were spun for 10 s to bring all liquid to the bottom then loaded into a Biorad CFX-96 real-time PCR machine. The setup of equipment was the same as described in the ScreenFire HPV RS assay standard format.

The analytic performance of the ScreenFire HPV RS assay Zebra BioDome format was evaluated based on consistency with the ScreenFire HPV RS assay standard format according to positive, negative, and overall agreement as well as unweighted kappa values for four detection groups covering all 13 HPV genotypes (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68). Detection groups were grouped hierarchically based on cervical cancer risk: HPV16 positive, else positive for HPV18/45, else positive for HPV 31/33/35/52/58, else positive for HPV51/59/39/56/68, or else negative.