Real-time loop-mediated isothermal amplification assay for rapid detection of human papillomavirus 16 in oral squamous cell carcinoma

Hamzan NI 1 , Ab Rahman N 2 , Suraiya S 3 , Mohamad I 4 , George Kalarakkal T 5 , Mohamad S 6

Affiliations 

  • 1 School of Dental Sciences, Universiti Sains Malaysia, Health Campus, 16150, Kubang Kerian, Kelantan, Malaysia
  • 2 Oral Pathology Unit, School of Dental Sciences, Universiti Sains Malaysia, Health Campus, 16150, Kubang Kerian, Kelantan, Malaysia
  • 3 Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Health Campus, 16150, Kubang Kerian, Kelantan, Malaysia
  • 4 Department of Otorhinolaryngology, School of Medical Sciences, Universiti Sains Malaysia, Health Campus, 16150, Kubang Kerian, Kelantan, Malaysia
  • 5 Department of Oral & Maxillofacial Clinical Sciences, Faculty of Dentistry, University of Malaya, 50603, Kuala Lumpur, Malaysia
  • 6 School of Dental Sciences, Universiti Sains Malaysia, Health Campus, 16150, Kubang Kerian, Kelantan, Malaysia. Electronic address: suharni@usm.my
Arch Oral Biol, 2021 Apr;124:105051.
PMID: 33581498 DOI: 10.1016/j.archoralbio.2021.105051

Abstract

OBJECTIVE: The present study established a real-time loop-mediated isothermal amplification (qLAMP) for rapid detection of human papillomavirus subtype 16 (HPV-16) in oral squamous cell carcinoma (OSCC).

METHODS: The qLAMP assay was optimized targeting the HPV-16 E7 gene. The analytical sensitivity and specificity of the assay were determined using HPV-18 (ATCC® 45152D™), HPV-35 (ATCC® 40330™), HPV-43 (ATCC® 40338™) and HPV-56 (ATCC® 40549™) viral strains and oral bacteria. HPV-16 standard curve was constructed for determination of HPV-16 viral load. The diagnostic performance of the assay was evaluated from 63 OSCC patients comprising 63 tissue, 13 saliva and 49 blood samples, in comparison with p16 immunohistochemistry (IHC), in-house PCR and nested PCR assays.

RESULTS: The detection limit of developed LAMP and PCR assays was 4.68 × 101 and 4.68 × 103 copies/μl, respectively. qLAMP assay enabled detection of positive results as early as 23 min at 67 °C. This assay can detect HPV-16 positivity in 23 % (3/13) saliva and 4.8 % (3/63) tissue samples with the viral load ranging from 4.68 × 101 to 4.68 × 104 copies/μl. HPV-16 positivity was not detected in all the blood samples. The sensitivity and specificity of qLAMP were 100 % in comparison with that of p16 IHC and nested PCR.

CONCLUSION: This study reports for the first time on the use of qLAMP assay for detection of HPV-16 in OSCC in both tissue and saliva as the sample matrix which holds promise in improving the diagnostic application owing to its rapidity, simplicity, high sensitivity and specificity.

* Title and MeSH Headings from MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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