title: High-throughput two-step LNA real time PCR assay for the quantitative detection and genotyping of HPV prognostic-risk groups.
abstract: BACKGROUND
  Human papillomavirus (HPV) infection is a necessary event in the development of cervical carcinoma. High risk (HR) HPV genotypes, however, may progress differentially from low grade lesions to malignancy.
  
  
  OBJECTIVE
  The necessity to genotype and quantify HPV-DNA in cervical screening programs, in the follow up post-surgical treatments and in monitoring the effectiveness of HPV vaccination programs, requires access to economical, high-throughput and flexible molecular technologies.
  
  
  METHODS
  A high-throughput two-step LNA real time PCR assay was developed consisting of real time PCR reactions with fluorescent Locked Nucleic Acid (LNA) probes. The first step permits classification into three prognostic-risk groups of nine HR HPV genotypes (16, 18, 31, 33, 35, 45, 52, 56 and 58) most frequently found associated with cervical lesions in Europe. The second step allows us to genotype/quantify the HPV-DNA only when clinical, epidemiological or prophylactic aims exist.
  
  
  RESULTS
  The specificity, repeatability, detection and quantitation limit, and linearity of the assay were evaluated and appear to be in agreement with guidelines for the validation of analytical procedures. The overall genotype concordance on cervical samples between our assay and INNOLiPA test was 94% (k 0.83) indicating good agreement.
  
  
  CONCLUSIONS
  The two-step PCR assay can give much information relative to the predictive value of different HR HPV types and can quantify the genotype-specific viral load. In particular, its ability to detect and quantify nine HR HPV genotypes can help provide more efficient and successful patient care and may be useful for the monitoring of the efficacy of HPV vaccines.
authors.affiliation: Division of Microbiology, University of Bologna, Policlinico S. Orsola-Malpighi, via Massarenti 9, 40138 Bologna, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Leo
authors.forename: Simona
authors.initials: S
authors.lastname: Venturoli
authors.forename: Monica
authors.initials: M
authors.lastname: Cricca
authors.forename: Monica
authors.initials: M
authors.lastname: Musiani
authors.forename: Marialuisa
authors.initials: M
authors.lastname: Zerbini
mesh_terms: D005060:Europe; D005260:Female; D006801:Humans; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics*; D027383:Papillomaviridae / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D030361:Papillomavirus Infections / Q000175:diagnosis* / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D011237:Predictive Value of Tests; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D015345:Oligonucleotide Probes