A novel multiplex real-time PCR assay for the detection and quantification of HPV16/18 and HSV1/2 in cervical cancer screening

Mol Cell Probes. 2012 Apr;26(2):66-72. doi: 10.1016/j.mcp.2012.01.003. Epub 2012 Jan 25.


Infection with human papillomavirus (HPV), particularly HPV16 and HPV18, is the main cause of invasive cervical cancer, although other factors such as herpes simplex virus (HSV) may act in conjunction with HPV in this context. To explore the possibility of developing a system for rapid diagnosis and clinical screening of cervical cancer, we developed a multiplex real-time PCR assay that can simultaneously detect and quantify HPV16/18 and HSV1/2. To evaluate its possibilities and practical uses, 177 samples collected from patients with suspected HPV and HSV infection in exfoliated cervical cells, genital herpes or labial herpes were tested by multiplex real-time PCR and compared with results obtained by DNA sequencing. Each virus was detected over a range from 1.0 × 10(1) to 1.0 × 10(7) copies/reaction. The clinical sensitivity was 100% for HPV16/18 and HSV1/2. The clinical specificity was 97.1% for HPV16, 98.1% for HPV18, 97.0% for HSV1 and 96.0% for HSV2. The kappa value was 0.96 for HPV16, 0.92 for HPV18, 0.94 for HSV1 and 0.93 for HSV2, when DNA sequencing was used as the reference standard. In summary, this novel multiplex real-time PCR allows the rapid and specific detection of HPV16/18 and HSV1/2, as well as coinfection with HPV and HSV, in clinical samples. In the future, this multiplex real-time PCR assay will assist in cervical cancer screening, viral treatment evaluation and epidemiological studies in which high throughput analysis is required.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Coinfection / diagnosis*
  • Coinfection / virology
  • Early Detection of Cancer / methods*
  • Female
  • Herpes Genitalis / diagnosis*
  • Herpes Genitalis / virology
  • Herpesvirus 1, Human / genetics
  • Herpesvirus 2, Human / genetics
  • Human papillomavirus 16 / genetics
  • Human papillomavirus 18 / genetics
  • Humans
  • Molecular Diagnostic Techniques / methods
  • Multiplex Polymerase Chain Reaction* / standards
  • Papillomavirus Infections / diagnosis*
  • Papillomavirus Infections / virology
  • Real-Time Polymerase Chain Reaction* / standards
  • Reference Standards
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Sequence Analysis, DNA
  • Uterine Cervical Neoplasms / diagnosis*
  • Uterine Cervical Neoplasms / virology
  • Viral Load