Simultaneous Detection and Differentiation of Human Papillomavirus Genotypes 6, 11, 16 and 18 by AllGlo Quadruplex Quantitative PCR

Background

Human papillomaviruses (HPV) are classified into high-risk HPV and low-risk HPV. The most common high-risk HPV types in cervical cancer are HPV 16 and 18, and the most common low-risk types causing genital warts are HPV 6 and HPV 11. In this study, applying novel AllGlo fluorescent probes, we established a quadruplex quantitative PCR method to simultaneously detect and differentiate HPV 6, 11, 16 and 18 in a single tube.

Methods

The specificity, the sensitivity, the detection limit, the reproducibility and the standard curve of this method were examined. Finally, clinical samples that had been tested previously by TaqMan PCR and HPV GenoArray (GA) test were used to verify the accuracy and sensitivity of the method.

Results

The assay has a sensitivity of 10¹ to 10² copies/test and a linear detection range from 10¹ to 10⁸ copies/test. The mean amplification efficiencies for HPV 6, 11, 16, and 18 were 0.97, 1.10, 0.93 and 1.20, respectively, and the mean correlation coefficient (r²) of each standard curve was above 0.99 for plasmid templates ranging from 10³ to 10⁷ copies/test. There was 100% agreement between the AllGlo quadruplex quantitative PCR, HPV GA test and TaqMan uniplex qPCR methods.

Conclusions

AllGlo quadruplex quantitative PCR in a single tube has the advantages of relatively high throughput, good reproducibility, high sensitivity, high specificity, and a wide linear range of detection. The convenient single tube format makes this assay a powerful tool for the studies of mixed infections by multiple pathogens, viral typing and viral load quantification.