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

• Main Authors: Hamzan, Nurul Izzati, Ab Rahman, Nurhayu, Suraiya, Siti, Mohamad, Irfan, Kalarakkal, Thomas George, Mohamad, Suharni
• Format: Article
• Published: Elsevier 2021
• Subjects: R Medicine (General); RK Dentistry
• Online Access: http://eprints.um.edu.my/28423/

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 (R) 45152D T), HPV-35 (ATCC (R) 40330 T), HPV-43 (ATCC (R) 40338 T) and HPV-56 (ATCC (R) 40549 T) 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 x 10(1) and 4.68 x 10(3) copies/mu l, respectively. qLAMP assay enabled detection of positive results as early as 23 min at 67 degrees 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 x 101 to 4.68 x 104 copies/mu l. 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.