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Assessing the performance of a Loop Mediated Isothermal Amplification (LAMP) assay for the detection and subtyping of high-risk suptypes of Human Papilloma Virus (HPV) for Oropharyngeal Squamous Cell Carcinoma (OPSCC) without DNA purification.
BMC Cancer. 2018 02 08; 18(1):166.BC

Abstract

BACKGROUND

Oropharyngeal Squamous Cell Carcinoma (OPSCC) is increasing in incidence despite a decline in traditional risk factors. Human Papilloma Virus (HPV), specifically subtypes 16, 18, 31 and 35, has been implicated as the high-risk etiologic agent. HPV positive cancers have a significantly better prognosis than HPV negative cancers of comparable stage, and may benefit from different treatment regimens. Currently, HPV related carcinogenesis is established indirectly through Immunohistochemistry (IHC) staining for p16, a tumour suppressor gene, or polymerase chain reaction (PCR) that directly tests for HPV DNA in biopsied tissue. Loop mediated isothermal amplification (LAMP) is more accurate than IHC, more rapid than PCR and is significantly less costly. In previous work we showed that a subtype specific HPV LAMP assay performed similar to PCR on purified DNA. In this study we examined the performance of this LAMP assay without DNA purification.

METHODS

We used LAMP assays using established primers for HPV 16 and 18, and new primers for HPV 31 and 35. LAMP reaction conditions were tested on serial dilutions of plasmid HPV DNA to confirm minimum viral copy number detection thresholds. LAMP was then performed directly on different human cell line samples without DNA purification.

RESULTS

Our LAMP assays could detect 105, 103, 104, and 105 copies of plasmid DNA for HPV 16, 18, 31, and 35, respectively. All primer sets were subtype specific, with no cross-amplification. Our LAMP assays also reliably amplified subtype specific HPV DNA from samples without requiring DNA isolation and purification.

CONCLUSIONS

The high risk OPSCC HPV subtype specific LAMP primer sets demonstrated, excellent clinically relevant, minimum copy number detection thresholds with an easy readout system. Amplification directly from samples without purification illustrated the robust nature of the assay, and the primers used. This lends further support HPV type specific LAMP assays, and these specific primer sets and assays can be further developed to test for HPV in OPSCC in resource and lab limited settings, or even bedside testing.

Authors+Show Affiliations

Undergraduate Medical Education, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB, Canada.Section of Otolaryngology - Head and Neck Surgery, Department of Surgery, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada.Department of Microbiology, Immunology and Infectious Diseases, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada.Department of Microbiology, Immunology and Infectious Diseases, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada.Ohlson Research Initiative, Arnie Charbonneau Cancer Institute, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada.Department of Pathology and Laboratory Medicine, Cumming School of Medicine, University of Calgary and Calgary Laboratory Services, Calgary, AB, Canada.Section of Otolaryngology - Head and Neck Surgery, Department of Surgery, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada. Ohlson Research Initiative, Arnie Charbonneau Cancer Institute, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada.Department of Microbiology, Immunology and Infectious Diseases, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada. vanmarle@ucalgary.ca. Snyder Institute for Chronic Diseases, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada. vanmarle@ucalgary.ca.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

29422018

Citation

Rohatensky, Mitchell G., et al. "Assessing the Performance of a Loop Mediated Isothermal Amplification (LAMP) Assay for the Detection and Subtyping of High-risk Suptypes of Human Papilloma Virus (HPV) for Oropharyngeal Squamous Cell Carcinoma (OPSCC) Without DNA Purification." BMC Cancer, vol. 18, no. 1, 2018, p. 166.
Rohatensky MG, Livingstone DM, Mintchev P, et al. Assessing the performance of a Loop Mediated Isothermal Amplification (LAMP) assay for the detection and subtyping of high-risk suptypes of Human Papilloma Virus (HPV) for Oropharyngeal Squamous Cell Carcinoma (OPSCC) without DNA purification. BMC Cancer. 2018;18(1):166.
Rohatensky, M. G., Livingstone, D. M., Mintchev, P., Barnes, H. K., Nakoneshny, S. C., Demetrick, D. J., Dort, J. C., & van Marle, G. (2018). Assessing the performance of a Loop Mediated Isothermal Amplification (LAMP) assay for the detection and subtyping of high-risk suptypes of Human Papilloma Virus (HPV) for Oropharyngeal Squamous Cell Carcinoma (OPSCC) without DNA purification. BMC Cancer, 18(1), 166. https://doi.org/10.1186/s12885-018-4087-1
Rohatensky MG, et al. Assessing the Performance of a Loop Mediated Isothermal Amplification (LAMP) Assay for the Detection and Subtyping of High-risk Suptypes of Human Papilloma Virus (HPV) for Oropharyngeal Squamous Cell Carcinoma (OPSCC) Without DNA Purification. BMC Cancer. 2018 02 8;18(1):166. PubMed PMID: 29422018.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Assessing the performance of a Loop Mediated Isothermal Amplification (LAMP) assay for the detection and subtyping of high-risk suptypes of Human Papilloma Virus (HPV) for Oropharyngeal Squamous Cell Carcinoma (OPSCC) without DNA purification. AU - Rohatensky,Mitchell G, AU - Livingstone,Devon M, AU - Mintchev,Paul, AU - Barnes,Heather K, AU - Nakoneshny,Steven C, AU - Demetrick,Douglas J, AU - Dort,Joseph C, AU - van Marle,Guido, Y1 - 2018/02/08/ PY - 2016/12/31/received PY - 2018/01/31/accepted PY - 2018/2/10/entrez PY - 2018/2/10/pubmed PY - 2018/8/22/medline KW - HPV KW - LAMP KW - Loop-mediated isothermal amplification KW - OPSCC KW - Oropharynx SP - 166 EP - 166 JF - BMC cancer JO - BMC Cancer VL - 18 IS - 1 N2 - BACKGROUND: Oropharyngeal Squamous Cell Carcinoma (OPSCC) is increasing in incidence despite a decline in traditional risk factors. Human Papilloma Virus (HPV), specifically subtypes 16, 18, 31 and 35, has been implicated as the high-risk etiologic agent. HPV positive cancers have a significantly better prognosis than HPV negative cancers of comparable stage, and may benefit from different treatment regimens. Currently, HPV related carcinogenesis is established indirectly through Immunohistochemistry (IHC) staining for p16, a tumour suppressor gene, or polymerase chain reaction (PCR) that directly tests for HPV DNA in biopsied tissue. Loop mediated isothermal amplification (LAMP) is more accurate than IHC, more rapid than PCR and is significantly less costly. In previous work we showed that a subtype specific HPV LAMP assay performed similar to PCR on purified DNA. In this study we examined the performance of this LAMP assay without DNA purification. METHODS: We used LAMP assays using established primers for HPV 16 and 18, and new primers for HPV 31 and 35. LAMP reaction conditions were tested on serial dilutions of plasmid HPV DNA to confirm minimum viral copy number detection thresholds. LAMP was then performed directly on different human cell line samples without DNA purification. RESULTS: Our LAMP assays could detect 105, 103, 104, and 105 copies of plasmid DNA for HPV 16, 18, 31, and 35, respectively. All primer sets were subtype specific, with no cross-amplification. Our LAMP assays also reliably amplified subtype specific HPV DNA from samples without requiring DNA isolation and purification. CONCLUSIONS: The high risk OPSCC HPV subtype specific LAMP primer sets demonstrated, excellent clinically relevant, minimum copy number detection thresholds with an easy readout system. Amplification directly from samples without purification illustrated the robust nature of the assay, and the primers used. This lends further support HPV type specific LAMP assays, and these specific primer sets and assays can be further developed to test for HPV in OPSCC in resource and lab limited settings, or even bedside testing. SN - 1471-2407 UR - https://www.unboundmedicine.com/medline/citation/29422018/Assessing_the_performance_of_a_Loop_Mediated_Isothermal_Amplification__LAMP__assay_for_the_detection_and_subtyping_of_high_risk_suptypes_of_Human_Papilloma_Virus__HPV__for_Oropharyngeal_Squamous_Cell_Carcinoma__OPSCC__without_DNA_purification_ DB - PRIME DP - Unbound Medicine ER -