Yuan Ting, Mukama Omar, Li Zhiyuan, Chen Wei, Zhang Yuxia, de Dieu Habimana Jean, Zhang Yinghui, Zeng Rong, Nie Chengrong, He Zhixu, Zeng Lingwen
Guizhou Provincial Key Laboratory for Regenerative Medicine, Department of Immunology, Guizhou Medical University, Guiyang, China.
Analyst. 2020 Sep 28;145(19):6388-6394. doi: 10.1039/d0an00663g.
Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in the world, and several studies have associated Epstein-Barr virus (EBV) with NPC occurrence and development. EBV-PCR (polymerase chain reaction), in situ hybridization and immunoassays are the most common methods for NPC identification. However, these approaches have drawbacks, which include tedious procedures and false results. Therefore, a rapid, accurate, and sensitive clinical diagnostic method for the prognosis of EBV-related diseases is needed. In this study, we developed a simple and sensitive approach for EBV detection based on the combination of CRISPR-Cas12a and a lateral flow biosensor (LFB). Cas12a exhibits collateral cleavage propensity of both target DNA and any single-stranded(ss) DNA in the vicinity (herein referred to as a reporter). The LFB test line contained an ssDNA probe complementary to the reporter. In the presence of the target, Cas12a trans-cleaved the ssDNA reporter, which resulted in the inability of cleaved sequences to bind the LFB test line. With a PCR pre-amplification of the target (45 min), the assay achieved a sensitivity of 7.1 × 10-14 M (∼42 000 copies per μl) both in plasmid and plasmid-spiked samples. The assay attained a high specificity in the presence of various bacteria and applicability in EBV Burkitt's lymphoma serum samples. This method could be applied for the detection of EBV and other infectious diseases.
鼻咽癌（NPC）是世界上最常见的恶性肿瘤之一，多项研究表明爱泼斯坦-巴尔病毒（EBV）与鼻咽癌的发生和发展有关。EBV-PCR（聚合酶链反应）、原位杂交和免疫测定是鼻咽癌检测最常用的方法。然而，这些方法存在缺点，包括操作繁琐和结果有误。因此，需要一种快速、准确且灵敏的临床诊断方法来预测EBV相关疾病。在本研究中，我们基于CRISPR-Cas12a和侧向流动生物传感器（LFB）的组合开发了一种简单且灵敏的EBV检测方法。Cas12a对靶DNA及附近的任何单链（ss）DNA（在此称为报告分子）均表现出附带切割倾向。LFB检测线包含与报告分子互补的ssDNA探针。在存在靶标的情况下，Cas12a反式切割ssDNA报告分子，导致切割后的序列无法与LFB检测线结合。通过对靶标进行PCR预扩增（45分钟），该检测方法在质粒和加标质粒样本中均达到了7.1×10-14 M（约每微升42000个拷贝）的灵敏度。该检测方法在存在各种细菌的情况下具有高特异性，并且在EBV伯基特淋巴瘤血清样本中具有适用性。这种方法可用于检测EBV和其他传染病。
