Ma Biao, Fang Jiehong, Wang Ye, He Haizhen, Dai Mingyan, Lin Wei, Su Wei, Zhang Mingzhou
Clin Lab. 2017 Jan 1;63(1):27-38. doi: 10.7754/Clin.Lab.2016.160325.
Cervical cancer is a common gynecologic malignant tumor and has a great impact on women's health. Human papillomavirus (HPV) is implicated in cervical cancer and precancerous lesions and the two are possibly two stages of disease progression. With the technological development of molecular biology and epidemiology, detection and treatment of HPV has become an important means to prevent cervical cancer.
Here we present a novel, rapid, sensitive and specific isothermal method of recombinase polymerase amplification (RPA), which is established to detect the two most common high-risk human papillomavirus type 16 and type 18 DNA. In this study, we evaluate the efficacy of the RPA assay, incubating clinical specimens of HPV16 and HPV18 using plasmids standard. It operates at constant low temperature without the thermal instrumentation for incubation. The products can be detected via agarose gel electrophoresis assay, reverse dot blot assay, and quantitative real-time assay with SYBR Green I. We assess the diagnostic performance of the RPA assay for detecting of HPV16 and HPV18 in 335 clinical samples from patients suspected of cervical cancer.
The results revealed no cross-reaction with other HPV genotypes and the RPA assay achieve a sensitivity of 100 copies. Compared with TaqMan qPCR, the RPA technique achieves exponential amplification with no need for pretreatment of sample DNA at 37°C for 20 minutes, which reveals more satisfactory performance. The agreement between the RPA and qPCR assays was 97.6% (κ = 0.89) for HPV16 positivity and 98.5% (κ = 0.81) for HPV18 positivity, indicating very good correlation between both tests.
Importantly, the RPA assay was demonstrated to be a useful and powerful method for detection of HPV virus, which therefore may serve as a valuable tool for rapid diagnosis of HPV infection in both commercial and clinical applications.
宫颈癌是常见的妇科恶性肿瘤，对女性健康有重大影响。人乳头瘤病毒（HPV）与宫颈癌及癌前病变有关，二者可能是疾病进展的两个阶段。随着分子生物学和流行病学技术的发展，HPV的检测与治疗已成为预防宫颈癌的重要手段。
在此，我们介绍一种新型、快速、灵敏且特异的重组酶聚合酶扩增（RPA）等温方法，该方法用于检测两种最常见的高危人乳头瘤病毒16型和18型DNA。在本研究中，我们使用质粒标准品评估RPA检测法对HPV16和HPV18临床标本的检测效果。该方法在恒定低温下操作，无需热孵育仪器。产物可通过琼脂糖凝胶电泳检测、反向斑点杂交检测以及SYBR Green I定量实时检测。我们评估了RPA检测法在335例疑似宫颈癌患者临床样本中检测HPV16和HPV18的诊断性能。
结果显示该方法与其他HPV基因型无交叉反应，RPA检测法的灵敏度达到100拷贝。与TaqMan定量聚合酶链反应相比，RPA技术在37°C下无需对样本DNA进行预处理20分钟即可实现指数扩增，表现更为令人满意。RPA检测法与定量聚合酶链反应检测结果在HPV16阳性方面的一致性为97.6%（κ = 0.89），在HPV18阳性方面的一致性为98.5%（κ = 0.81），表明两种检测方法之间具有很好的相关性。
重要的是，RPA检测法被证明是一种检测HPV病毒的有用且强大的方法，因此在商业和临床应用中可能成为快速诊断HPV感染的有价值工具。
