This study was conducted in June 2024 at the National HPV Laboratory of the Republic of Türkiye Ministry of Health, General Directorate of Public Health, Microbiology Reference Laboratories and Biological Products Department. The study was planned as two phases, experimental and clinical.

Experimental phase

In the experimental phase of the study, 80 cervical swab samples collected from sexually active women aged 30–65 years were utilized. These samples had already undergone routine nucleic acid isolation and PCR testing in the laboratory, with confirmed positive or negative results.

The samples analyzed in this study were cervical swabs, from which HPV status was determined using PCR. Colposcopy and biopsy were not included as part of the diagnostic protocol for these samples.

After routine testing, the samples were separated, labeled in 2 mL vials of Viral Transport Medium DiaVTM, (SBT, Türkiye), and transported to the laboratory for experimental comparison of methodologies. They were then stored at -80 °C for further use in the study described in this article. The test results obtained using the Molgen PurePrep Pathogen DGX (Molgen, The Netherlands) nucleic acid extraction and multiplex HPV DNA PCR test were compared with the results of the PharmaDirect methodology, as outlined in the study design (Fig. 1).

Fig. 1

Flow chart of different HPV-DNA detection methods compared in the experimental phase of the study. Nucleic Acid Extraction; DNA purification was performed from cervical swab samples. Purified DNA was included in the PCR reaction. PharmaDirect; was added in the PCR reaction along with the cervical swab sample without nucleic acid extraction. DiaVNAT*; A Viral nucleic acid buffer (SBT, Türkiye) used in respiratory tract swabs, was added in the PCR reaction along with the cervical swab sample without nucleic acid extraction. Any; Only the cervical swab sample was included in the PCR reaction, with no additional solutions added

Clinical phase, sample collection and processing

The clinical phase of the study included 1200 sexually active women aged 30–65 years. Cervical swabs were collected from each participant, and the samples were placed in 2 mL of Viral Transport Medium, DiaVTM (SBT, Türkiye). These samples were then transported to the laboratory for use in this study.

Nucleic acid extraction using molgen pureprep pathogen DGX kit

Extraction steps were performed on an automated PurePrep96 (Molgen, The Netherlands) extraction device according to the manufacturer’s recommendations. Extraction of 80 samples as part of routine laboratory work and 1200 samples as part of clinical work was performed in 96-well plates. 50 µl of nucleic acid was extracted from each sample.

Real-time HPV PCR testing with nucleic acid extraction

Extracts were tested with TÜSEB DiaKit HighRisk HPV qPCR diagnostic kit (SBT, Türkiye). TÜSEB DiaKit was developed for the detection of HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 nucleic acids in cervical swab samples. Using this kit, HPV genotypes 16, 18 and 45 are detected in separate fluorescent channels, while other genotypes are tested in a single fluorescent channel.

In accordance with the manufacturer’s recommendations, the main reaction mixture for nucleic acid extracts was prepared as follows; 5 µl Mastermix − 2.5 µl Primer Mix − 2.5 µl nucleic acid extract. The prepared reaction mixture was tested with the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA). The cycling conditions used for PCR assays consisted of 40 cycles of 95 °C for 3 min, followed by 95 °C for 5 s and 60 °C for 10 s, in line with the manufacturer’s recommendations. Negative and positive controls were included in each study.

Real-time HPV PCR testing with Pharmadirect pre-denaturation solution

Samples were tested with TÜSEB DiaKit HighRisk HPV qPCR diagnostic kit. In accordance with the manufacturer’s recommendations, the main reaction mixture used for nucleic samples was prepared as follows; 7.5 µl Mastermix − 2.5 µl Primer Mix − 2.5 µl PharmaDirect − 2.5 µl sample. The prepared reaction mixture was tested using the CFX96 Touch Real-Time PCR Detection System. The PCR cycling conditions included 35 cycles of 95 °C for 3 min, followed by 95 °C for 10 s and 60 °C for 45 s, in accordance with the manufacturer’s recommendations. Both negative and positive controls were included in each assay. For experimental comparison, DiaVNAT was used instead of PharmaDirect in the reaction under the same conditions. As an additional comparison methodology, the study was conducted without adding any solution to the reaction, using the same conditions.

Statistical analysis

In the clinical phase of the study, HPV DNA PCR test results obtained from the two methodologies were combined and presented in a cross-tabulation. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for PharmaDirect solution were calculated by creating a comparative table, considering nucleic acid extraction as the gold standard. The agreement between the test results obtained with both methodologies was analyzed by calculating Cohen’s Kappa values were categorized as follows: below 0.20 indicates insignificant agreement, 0.21–0.40 indicates poor agreement, 0.41–0.60 indicates fair agreement, 0.61–0.80 indicates substantial agreement, and 0.81–1.00 indicates great agreement [18]. Ct mean values obtained in PCR tests performed using both methodologies for HPV genotypes 16, 18, 45 and other high-risk genotypes were compared with the help of Paired Sample T-test.