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

Nurul Izzati Hamzan; Nurhayu Ab Rahman; Siti Suraiya; Irfan Mohamad; Thomas George Kalarakkal; Suharni Mohamad

doi:10.1016/j.archoralbio.2021.105051

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

Arch Oral Biol. 2021 Apr:124:105051. doi: 10.1016/j.archoralbio.2021.105051. Epub 2021 Jan 28.

Authors

Nurul Izzati Hamzan¹, Nurhayu Ab Rahman², Siti Suraiya³, Irfan Mohamad⁴, Thomas George Kalarakkal⁵, Suharni Mohamad⁶

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.

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.

Keywords: Human papillomavirus; Oral squamous cell carcinoma; Real-time loop-mediated isothermal amplification.

MeSH terms

Carcinoma, Squamous Cell* / diagnosis
Head and Neck Neoplasms*
Human papillomavirus 16 / genetics
Humans
Molecular Diagnostic Techniques
Mouth Neoplasms* / diagnosis
Nucleic Acid Amplification Techniques
Papillomavirus Infections* / diagnosis
Sensitivity and Specificity
Squamous Cell Carcinoma of Head and Neck

Supplementary concepts

LAMP assay