High-risk HPV genotyping assay (qPCR)

Exfoliated cells were collected by swab at cervical foci, placed in sterile glass tube and sealed. Genotyping was performed using a Uterine Cervix Cancer of High-risk HPV Genotype Related Real Time PCR Kit (Liferiver Bio-Tech Corp., San Diego, CA, USA), which is able to detect and distinguish 13 HPV genotypes, including HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. A total of 13 pairs of genotype-specific primers and 13 Taqman probes (Liferiver Bio-Tech Corp.) were used for the detection and genotyping of the genomic DNA of 13 HPV genotypes using qPCR.

In brief, template DNA was prepared from cervical swab samples taken from 15,192 female outpatients using Nucleic Acid Extraction Reagent, which is a component of High-risk HPV Genotype Related Real Time PCR Kit. Primers were provided in the kit and the total reaction volume was 40 µl. Thermal cycling was performed according to the manufacturer's instructions: 94°C for 2 min, followed by 40 cycles of 93°C for 10 sec and 62°C for 31 sec. PCR amplification and hybridization were performed using the Roche LightCycler (Roche Diagnostics, Basel, Switzerland). Positive and negative controls were performed, as provided by in the kit.

Fluorescent signals of cycle 6–15 were used for baseline setting, and threshold line was set just over the highest point of amplification curve of blank, in which H₂O was used as template. Cycle threshold (Ct) value was used as a marker of virus load, Ct value represents the threshold cycle number of qPCR; higher HPV copy number implies more severe HPV infection in patient and higher risk of developing into cervical cancer. If Ct value is <38 and typical amplification curve is shown, a positive result may be reported; one or more HPV genotypes can be detected, which represent simple and multiple infection respectively. HPV virus load can be divided into seven levels, 10²−10⁷ and >10⁷ based on Ct values: When Ct value is <20.0, HPV viral load is >10⁷ copies/10⁴ cells; Ct value is 20.0–23.3, HPV viral load is 10⁷ copies/10⁴ cells; Ct value is 23.4–26.7, HPV viral load is 10⁶ copies/10⁴ cells; Ct value is 26.8–30.1, HPV viral load is 10⁵ copies/10⁴ cells; Ct value is 30.2–33.5, HPV viral load is 10⁴ copies/10⁴ cells; Ct value is 33.6–36.9, HPV viral load is 10³ copies/10⁴ cells; and Ct value is 37.0–40.0, HPV viral load is 10² copies/10⁴ cells. Ct value formula of HPV-positive sample: Ct = Ct_Sample - Ct_IC + 28. If the Ct value of ‘Channel CY5’ in mixture I [which is able to detect HPV16, 56, 31 and internal control (IC)] is <32, which represents IC, and ‘Undetermined’ or ‘No Ct’ is shown in ‘Column Ct’ of all channels except IC, the result was considered to be negative. If the Ct value was between 38.0 and 40.0, the sample was tested repeatedly; if it remained in that interval and a typical S-shaped amplification curve was shown, a result was considered to be positive. If no typical S-shaped curve is observed, a negative result can be reported.