Tsou Jen-Hui, Leng Qixin, Jiang Feng
Department of Pathology, University of Maryland School of Medicine, 10 South Pine Street, Baltimore, MD, USA.
Department of Pathology, University of Maryland School of Medicine, 10 South Pine Street, Baltimore, MD, USA.
Transl Oncol. 2019 Dec;12(12):1566-1573. doi: 10.1016/j.tranon.2019.08.011. Epub 2019 Oct 18.
Emerging CRISPR-based nucleic acid detection shows great promise in molecular diagnosis of diseases. CRISPR-Cas12a can sensitively and specifically detect human papillomavirus (HPV) DNA in anal swabs. However, the current CRISPR-Cas12a system needs auxiliary and expensive equipment, which limit its application as a point-of-care (POC) diagnostic tool. This study aimed to develop CRISPR-Cas12a as a POC test to directly target plasma for circulating HPV DNA detection by immediately reading results with naked eyes. Cell-cultured supernatants of either HPV16- or 18-positive cancer cells were treated with lysis buffer followed by isothermal amplification without DNA isolation. Cas12a, crRNA, and fluorescent-biotin reporters were incubated with the lysates. Our data showed that integrating CRISPR-Cas12a with lateral-flow strips could directly and specifically detect HPV16 and 18 in the liquid samples with the same limit of detection (0.24 fM) as did polymerase chain reaction but requiring less time. Furthermore, the CRISPR-Cas12a system could rapidly detect presence of HPV16 and HPV18 in plasma samples of 13 of 14 and 3 of 10 the patients with histopathological diagnosis of cervical cancer, respectively. Therefore, a CRISPR-Cas12a-based POC system was developed for conveniently detecting circulating nuclei acid targets in body fluids without requiring technical expertise and ancillary machineries.
新兴的基于CRISPR的核酸检测在疾病分子诊断中显示出巨大潜力。CRISPR-Cas12a能够灵敏且特异地检测肛门拭子中的人乳头瘤病毒（HPV）DNA。然而，当前的CRISPR-Cas12a系统需要辅助且昂贵的设备，这限制了其作为即时检测（POC）诊断工具的应用。本研究旨在开发一种CRISPR-Cas12a即时检测方法，通过肉眼直接读取结果，直接针对血浆检测循环中的HPV DNA。将HPV16或18阳性癌细胞的细胞培养上清液用裂解缓冲液处理，随后进行等温扩增，无需进行DNA分离。将Cas12a、crRNA和荧光生物素报告分子与裂解物一起孵育。我们的数据表明，将CRISPR-Cas12a与侧流试纸条整合，能够直接且特异地检测液体样本中的HPV16和18，检测限与聚合酶链反应相同（0.24 fM），但所需时间更短。此外，CRISPR-Cas12a系统能够分别快速检测出14例组织病理学诊断为宫颈癌患者中的13例以及10例中的3例血浆样本中HPV16和HPV18的存在。因此，开发了一种基于CRISPR-Cas12a的即时检测系统，用于方便地检测体液中的循环核酸靶标，无需专业技术知识和辅助设备。
