评估一种无需 DNA 纯化的环介导等温扩增（LAMP）检测方法，用于检测和亚分型口咽鳞状细胞癌（OPSCC）高危型人乳头瘤病毒（HPV）。
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.
机构信息
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.
出版信息
BMC Cancer. 2018 Feb 8;18(1):166. doi: 10.1186/s12885-018-4087-1.
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 10, 10, 10, and 10 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.
背景
尽管传统危险因素有所下降，但口咽鳞状细胞癌（OPSCC）的发病率仍在上升。人乳头瘤病毒（HPV），特别是亚型 16、18、31 和 35，被认为是高危病因。HPV 阳性癌症的预后明显优于可比分期的 HPV 阴性癌症，并且可能受益于不同的治疗方案。目前，HPV 相关致癌作用是通过免疫组织化学（IHC）染色 p16（一种肿瘤抑制基因）间接建立的，或通过聚合酶链反应（PCR）直接检测活检组织中的 HPV DNA。环介导等温扩增（LAMP）比 IHC 更准确，比 PCR 更快，并且成本显著降低。在之前的工作中，我们表明，针对 HPV 16 和 18 的亚型特异性 HPV LAMP 检测与纯 DNA 上的 PCR 表现相似。在这项研究中，我们在无需 DNA 纯化的情况下检查了这种 LAMP 检测的性能。
方法
我们使用针对 HPV 16 和 18 的已建立的 LAMP 检测引物，以及针对 HPV 31 和 35 的新引物。测试了 LAMP 反应条件，以确认最小病毒拷贝数检测阈值。然后直接在未经 DNA 纯化的不同人细胞系样本上进行 LAMP。
结果
我们的 LAMP 检测可以分别检测到 HPV 16、18、31 和 35 的质粒 DNA 的 10、10、10 和 10 个拷贝。所有引物组均为亚型特异性，无交叉扩增。我们的 LAMP 检测还可以可靠地扩增无需 DNA 分离和纯化的样本中的特定 HPV 亚型 DNA。
结论
高危 OPSCC HPV 亚型特异性 LAMP 引物组表现出极佳的临床相关最小拷贝数检测阈值和易于读取的系统。无需纯化即可直接从样本中扩增说明了该检测的稳健性，以及所使用的引物。这进一步支持 HPV 型特异性 LAMP 检测，并且可以进一步开发这些特定的引物组和检测方法来检测资源和实验室有限环境中的 OPSCC 中的 HPV，甚至是床边检测。
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