title: Simultaneous detection and differentiation of human papillomavirus genotypes 6, 11, 16 and 18 by AllGlo quadruplex quantitative PCR.
abstract: 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 10(1) to 10(2) copies/test and a linear detection range from 10(1) to 10(8) 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 (r(2)) of each standard curve was above 0.99 for plasmid templates ranging from 10(3) to 10(7) 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.
authors.affiliation: State Key Laboratory for the Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China.
authors.forename: Daojun
authors.initials: D
authors.lastname: Yu
authors.forename: Yu
authors.initials: Y
authors.lastname: Chen
authors.forename: Shenghai
authors.initials: S
authors.lastname: Wu
authors.forename: Baohong
authors.initials: B
authors.lastname: Wang
authors.forename: Yi-Wei
authors.initials: YW
authors.lastname: Tang
authors.forename: Lanjuan
authors.initials: L
authors.lastname: Li
mesh_terms: D017931:DNA Primers / Q000737:chemistry; D015342:DNA Probes / Q000737:chemistry; D005838:Genotype; D052140:Human papillomavirus 11 / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D052162:Human papillomavirus 16 / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D052161:Human papillomavirus 18 / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D052163:Human papillomavirus 6 / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D027383:Papillomaviridae / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D030361:Papillomavirus Infections / Q000175:diagnosis*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013696:Temperature
chemical_list: D017931:DNA Primers; D015342:DNA Probes