Detection assay for HPV16 and HPV18 by loop‑mediated isothermal amplification with lateral flow dipstick tests

Mol Med Rep. 2017 May;15(5):3203-3209. doi: 10.3892/mmr.2017.6370. Epub 2017 Mar 23.

Abstract

Cervical cancer is the third highest cause of death in developing countries and most commonly results from high‑risk human papillomavirus (HR‑HPV) infection. Among HR‑HPV genotypes, HPV16 and HPV18 are the most prevalent in cervical cancers. Therefore, the present study aimed to develop a detection assay for HPV16 and HPV18 infection using loop‑mediated isothermal amplification (LAMP) with lateral flow dipstick (LFD) tests. This assay is a simplified, user‑friendly method for the visual detection of HPV genotypes. DNA was extracted from clinical tissue samples, and HPV genotyping was performed using nested polymerase chain reaction (PCR). The clinical samples were demonstrated to include 44 HPV16‑positive, 18 HPV18‑positive and 80 HPV‑negative samples. All DNA samples were also used as templates for a LAMP reaction (30 min at 65˚C), and subsequently, a fluorescein isothiocyanate‑labelled probe was hybridized with the reaction product. Finally, the LFD test was performed. The sensitivity of the LAMP‑LFD test was higher than LAMP‑turbidity, exhibiting up to 100‑fold higher sensitivity for HPV16 and 10‑fold higher sensitivity for HPV18. All HPV16 and HPV18‑positive samples generated positive results in both assays; however, 22 samples detected as HPV‑negative by LAMP‑turbidity exhibited positive results by LAMP‑LFD test (22 of 80 samples). Therefore, these samples were further examined using quantitative (q)PCR. The results demonstrated that 20 out of the 22 samples designated positive by LAMP‑LFD, but negative by LAMP turbidity, gave a positive result with qPCR, while the remaining 2 samples were negative by qPCR. The present results suggested that LAMP‑LFD provided higher sensitivity than LAMP‑turbidity and nested PCR. Thus, the LAMP‑LFD test developed in the present study might be useful for the detection of HPV16 and HPV18 in local hospitals.

MeSH terms

  • Antibodies / chemistry
  • Antibodies / immunology
  • DNA Probes / chemistry
  • DNA Probes / metabolism
  • DNA, Viral / isolation & purification
  • DNA, Viral / metabolism*
  • Female
  • Genotype
  • Gold / chemistry
  • Human papillomavirus 16 / genetics*
  • Human papillomavirus 16 / isolation & purification
  • Human papillomavirus 18 / genetics*
  • Human papillomavirus 18 / isolation & purification
  • Humans
  • Immunoassay
  • Nucleic Acid Amplification Techniques / methods*
  • Reagent Kits, Diagnostic
  • Real-Time Polymerase Chain Reaction
  • Uterine Cervical Neoplasms / diagnosis
  • Uterine Cervical Neoplasms / virology

Substances

  • Antibodies
  • DNA Probes
  • DNA, Viral
  • Reagent Kits, Diagnostic
  • Gold