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

Abstract

BackgroundHuman 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.MethodsThe 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.ResultsThe 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.ConclusionsAllGlo 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.

Rapid and combined detection of Mycoplasma pneumoniae, Epstein–Barr virus and human cytomegalovirus using AllGlo quadruplex quantitative PCR

“…AllGlo technology, the latest generation of quantitative fluorescent probes, enables the creation of simple and efficient high-throughput diagnostics with good reproducibility and high sensitivity and specificity (Yu et al, 2012). This technology has been widely used for agricultural and medical studies, e.g.…”

“…With permission from the outpatients and inpatients, 300 clinical specimens, including 130 sputum specimens and 170 nasopharyngeal swab specimens, were collected from Hangzhou First People's Hospital, China. According to Yu et al (2012), DNA was extracted from all samples (details are given in S1.1, available in the online Supplementary Material).…”

“…The indicators of the quadruplex qPCR noted by Yu et al (2012), including sensitivity, specificity, repeatability and standard curves, were analysed (details in S1.4).…”

“…Nevertheless, in current reports of qPCR methods three pathogens (MP, EBV and HCMV) and an internal control in a single tube have not yet been simultaneously detected. In this study, novel AllGlo probes (Alle Logic Biosciences), which yield higher sensitivity and specificity than traditional TaqMan probes (Bai et al, 2014;Yu et al, 2012;Zhang et al, 2014), were designed and used to develop a multiplex qPCR method that rapidly, reliably and simultaneously detected and identified the three pathogens in a single tube in clinical samples. Importantly, we introduced a human housekeeping gene, GAPDH, as an internal control to strengthen the surveillance and control of detection quality.…”

“…This difference was probably due to competitive effects of increasing the amount of nucleic acid templates, whether capable of participating in qPCR or not, which indicated the quadruplex qPCR reaction system with a mixed plasmid template might exhibit mutual interference among the primers and probes. However, the difference of one C t value did not affect the sensitivity of AllGlo quadruplex qPCR and it was considered acceptable in daily clinical practice.AllGlo technology, the latest generation of quantitative fluorescent probes, enables the creation of simple and efficient high-throughput diagnostics with good reproducibility and high sensitivity and specificity (Yu et al, 2012). This technology has been widely used for agricultural and medical studies, e.g.…”