Department of Otolaryngology Head and Neck Surgery, Shengjing Hospital of China Medical University, Shenyang 110004, P.R. China.
J Microbiol Biotechnol. 2021 Apr 28;31(4):610-620. doi: 10.4014/jmb.2012.12047.
PCRThere has been increasing interest in the head and neck squamous cell carcinoma (HNSCC) that is caused by high-risk human papillomavirus (HR-HPV) and has posed a significant challenge to Otolaryngologists. A rapid, sensitive, and reliable method is required for the detection of HR-HPV in clinical specimens to prevent and treat HPV-induced diseases. In this study, a multiple cross-linking spiral amplification (MCLSA) assay was developed for the visual detection of HPV-16. In the MCLSA assay, samples were incubated under optimized conditions at 62°C for 45 min, and after mixing with the SYBR Green I (SGI) dye, the positive amplicons showed bright green fluorescence while the negative amplicons exhibited no obvious change. The specificity test revealed that the developed MCLSA technique had high specificity and could effectively distinguish all five HPV-16 strains from other pathogenic microorganisms. In terms of analytical sensitivity, the limit of detection (LoD) of MCLSA assay was approximately 5.4 × 10 copies/tube, which was 10-fold more sensitive than loop-mediated isothermal amplification (LAMP) and RT-PCR. The detection results of laryngeal cancer specimens collected from 46 patients with suspected HPV infection in the Liaoning region demonstrated that the positive detection rates of MCLSA and hybridized capture 2 kit were 32.61% (15/46). The true positive rate of the MCLSA assay was higher than that of RT-PCR (100% vs. 93.33%) and LAMP (100% vs. 86.67%). Therefore, the MCLSA assay developed in the present study could be a potentially useful tool for the point-of-care (PoC) diagnosis of HR-HPV, especially in resource-limited countries.
聚合酶链反应（PCR）
越来越多的研究关注由高危型人乳头瘤病毒（HR-HPV）引起的头颈部鳞状细胞癌（HNSCC），这对头颈科医生来说是一个重大挑战。为了预防和治疗 HPV 引起的疾病，需要一种快速、敏感和可靠的方法来检测临床标本中的 HR-HPV。本研究开发了一种用于 HPV-16 可视化检测的多重交联螺旋扩增（MCLSA）检测方法。在 MCLSA 检测中，样品在 62°C 下优化条件下孵育 45 分钟，与 SYBR Green I（SGI）染料混合后，阳性扩增产物呈现明亮的绿色荧光，而阴性扩增产物则没有明显变化。特异性试验表明，所开发的 MCLSA 技术具有高度特异性，能够有效区分五种 HPV-16 株与其他致病微生物。在分析灵敏度方面，MCLSA 检测的检测限（LoD）约为 5.4×10 拷贝/管，比环介导等温扩增（LAMP）和 RT-PCR 灵敏 10 倍。对来自中国辽宁地区 46 例疑似 HPV 感染的喉癌标本的检测结果表明，MCLSA 和杂交捕获 2 试剂盒的阳性检出率分别为 32.61%（15/46）。MCLSA 检测的真阳性率高于 RT-PCR（100% vs. 93.33%）和 LAMP（100% vs. 86.67%）。因此，本研究开发的 MCLSA 检测方法可能是 HR-HPV 即时检测（POC）的一种潜在有用工具，特别是在资源有限的国家。
