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

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...

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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/
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spelling my.um.eprints.284232022-08-04T08:06:47Z http://eprints.um.edu.my/28423/ Real-time loop-mediated isothermal amplification assay for rapid detection of human papillomavirus 16 in oral squamous cell carcinoma Hamzan, Nurul Izzati Ab Rahman, Nurhayu Suraiya, Siti Mohamad, Irfan Kalarakkal, Thomas George Mohamad, Suharni R Medicine (General) RK Dentistry 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. Elsevier 2021-04 Article PeerReviewed Hamzan, Nurul Izzati and Ab Rahman, Nurhayu and Suraiya, Siti and Mohamad, Irfan and Kalarakkal, Thomas George and Mohamad, Suharni (2021) Real-time loop-mediated isothermal amplification assay for rapid detection of human papillomavirus 16 in oral squamous cell carcinoma. Archives of Oral Biology, 124. ISSN 0003-9969, DOI https://doi.org/10.1016/j.archoralbio.2021.105051 <https://doi.org/10.1016/j.archoralbio.2021.105051>. 10.1016/j.archoralbio.2021.105051
institution Universiti Malaya
building UM Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Malaya
content_source UM Research Repository
url_provider http://eprints.um.edu.my/
topic R Medicine (General)
RK Dentistry
spellingShingle R Medicine (General)
RK Dentistry
Hamzan, Nurul Izzati
Ab Rahman, Nurhayu
Suraiya, Siti
Mohamad, Irfan
Kalarakkal, Thomas George
Mohamad, Suharni
Real-time loop-mediated isothermal amplification assay for rapid detection of human papillomavirus 16 in oral squamous cell carcinoma
description 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.
format Article
author Hamzan, Nurul Izzati
Ab Rahman, Nurhayu
Suraiya, Siti
Mohamad, Irfan
Kalarakkal, Thomas George
Mohamad, Suharni
author_facet Hamzan, Nurul Izzati
Ab Rahman, Nurhayu
Suraiya, Siti
Mohamad, Irfan
Kalarakkal, Thomas George
Mohamad, Suharni
author_sort Hamzan, Nurul Izzati
title Real-time loop-mediated isothermal amplification assay for rapid detection of human papillomavirus 16 in oral squamous cell carcinoma
title_short Real-time loop-mediated isothermal amplification assay for rapid detection of human papillomavirus 16 in oral squamous cell carcinoma
title_full Real-time loop-mediated isothermal amplification assay for rapid detection of human papillomavirus 16 in oral squamous cell carcinoma
title_fullStr Real-time loop-mediated isothermal amplification assay for rapid detection of human papillomavirus 16 in oral squamous cell carcinoma
title_full_unstemmed Real-time loop-mediated isothermal amplification assay for rapid detection of human papillomavirus 16 in oral squamous cell carcinoma
title_sort real-time loop-mediated isothermal amplification assay for rapid detection of human papillomavirus 16 in oral squamous cell carcinoma
publisher Elsevier
publishDate 2021
url http://eprints.um.edu.my/28423/
_version_ 1740826013009444864
score 13.211869

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