Abstract: Objective To compare the detection sensitivity and infection type range of universal primer SPF1/GP6++ and SPF1/GP6+ polymerase chain reaction(PCR) on human papillomavirus(HPV). Methods Fifteen types of HPV L1 mock target genes were amplified using type-specific primers with full-length HPV16 in plasmid as template and subsequently cloned into pEASY-T1 vector. The obtained recombinant plasmids were serially diluted and mixed with 50ng genomic DNA derived from healthy human blood cells to mimic HPVinfected samples. We compared the sensitivity between SPF1/GP6+ and SPF1/GP6++ general primers and further verified in 68 cases of DNA samples of human cervical cancer tissues. Results Compared with SPF1/GP6+ PCR, SPF1/GP6++ PCR provided advantages of 1 to 2 magnitude orders in the detection sensitivity of HPV 11, 31, 34, 39, 51, 52, 53, 56, 58, 61 and 66; as to the prevalent HPV type 16, 18, 33 and 35, both methods showed similar detection sensitivity. Using SPF1/GP6++ PCR, total HPV positive rate of cervical cancer tissues was 98.5%(67/68), and 11 cases infected with double HPV types and 2 cases with triple HPV types were detected. While the positive rate of HPV infection was 95.6%(65/68) and only 7 cases with double HPV types were detected using SPF1/GP6+ PCR. Conclusion SPF1/GP6++ PCR is established based on SPF1/GP6+ PCR and verified to possess elevated detection sensitivity and expanded range of HPV genotypes.