Internally controlled recombinase-aided amplification (IC-RAA) assays for the detection of human papillomavirus genotypes 16 and 18 using extracted DNA and samples treated with nucleic acid releasing agent

Arch Virol. 2020 Oct;165(10):2241-2247. doi: 10.1007/s00705-020-04722-3. Epub 2020 Jul 17.

Abstract

Cervical cancer is primarily caused by persistent infection with high-risk human papillomavirus (HPV), and 70% of cases are associated with HPV16 and 18 infections. The objective of this study was to establish rapid, simple, and sensitive internally controlled recombinase-aided amplification (IC-RAA) assays for the detection of HPV16 and 18. The assays were performed at 39 ℃ and were completed within 30 min. A total of 277 clinical samples of exfoliated cervical cells were tested by IC-RAA assays and commercial HPV real-time fluorescent PCR kits using extracted DNA and samples treated with nucleic acid releasing agent. The analytical sensitivity of the IC-RAA assay was found to be 10 copies/μL for the detection of HPV16 and 18 when using recombinant plasmids as targets. The optimal concentration of the internal control (IC) plasmid and 18 was 1000 copies/μL for HPV16 and 100 copies/μL for HPV18. The clinical sensitivity of the IC-RAA assays for HPV16 using extracted DNA and samples treated with nucleic acid releasing agent was 98.73% and 97.47%, respectively, with kappa values of 0.977 (P < 0.01) and 0.955 (P < 0.01), respectively, and 100% The specificity in both cases. For HPV18, the sensitivity and specificity were 100%, and the kappa value was 1 for both samples (P < 0.01). The IC-RAA assay is a promising tool for the detection of HPV16 and HPV18, especially in resource-constrained settings.

MeSH terms

  • Adolescent
  • Adult
  • Aged
  • Cervix Uteri / pathology
  • Cervix Uteri / virology
  • DNA Primers / chemical synthesis
  • DNA Primers / genetics
  • DNA, Viral / genetics*
  • Epithelial Cells / pathology
  • Epithelial Cells / virology
  • Female
  • Human papillomavirus 16 / classification
  • Human papillomavirus 16 / genetics*
  • Human papillomavirus 16 / isolation & purification
  • Human papillomavirus 18 / classification
  • Human papillomavirus 18 / genetics*
  • Human papillomavirus 18 / isolation & purification
  • Humans
  • Middle Aged
  • Nucleic Acid Amplification Techniques*
  • Papillomavirus Infections / diagnosis*
  • Papillomavirus Infections / pathology
  • Papillomavirus Infections / virology
  • Reagent Kits, Diagnostic
  • Sensitivity and Specificity
  • Uterine Cervical Neoplasms / diagnosis*
  • Uterine Cervical Neoplasms / pathology
  • Uterine Cervical Neoplasms / virology

Substances

  • DNA Primers
  • DNA, Viral
  • Reagent Kits, Diagnostic