Sun Yidan, Tang Danni, Li Nan, Wang Yudong, Yang Meimei, Shen Chao
College of Life Sciences, Wuhan University, Wuhan, China.
China Center for Type Culture Collection, Wuhan University, Wuhan, China.
Bio Protoc. 2024 Dec 5;14(23):e5122. doi: 10.21769/BioProtoc.5122.
The quality of cellular products used in biological research can impact the accuracy of results. Epstein-Barr virus (EBV) is a latent virus that spreads extensively worldwide, and cell lines used in experiments may carry EBV and pose an infection risk. The presence of EBV in a single cell line can contaminate other cell lines used in the same laboratory, affecting experimental results. Existing tests to detect EBV can be divided into three categories: nucleic acid assays, serological assays, and in situ hybridization assays. However, most methods are time-consuming, expensive, and not conducive to high-volume clinical screening. Therefore, a simple system that allows for the rapid detection of EBV in multiple contexts, including both cell culture and tissue samples, remains necessary. In our research, we developed EBV detection systems: (1) a polymerase chain reaction (PCR)-based detection system, (2) a recombinase polymerase amplification (RPA)-based detection system, and (3) a combined RPA-lateral flow assay (LFA) detection system. The minimum EBV detection limits were 1 × 10 copy numbers for the RPA-based and RPA-LFA systems and 1 × 10 copy numbers for the PCR-based system. Both the PCR and RPA detection systems were applied to 192 cell lines, and the results were consistent with those of the assays specified in industry standards. A total of 10 EBV-positive cell lines were identified. The combined RPA-LFA system is simple to operate, allowing for rapid result visualization. This system can be implemented in laboratories and cell banks as part of a daily quality control strategy to ensure cell quality and experimental safety and may represent a potential new technique for the rapid detection of EBV in clinical samples. Key features • Establishes RPA and RPA-LFA detection systems for EBV. • The RPA-LFA detection system can visualize the results in as short as 15 min. • The specificity of the RPA and RPA-LFA assay systems for the detection of EBV is validated. • The minimum EBV detection limit of the RPA and RPA-LFA systems is 1 × 10 copies.
用于生物学研究的细胞产品质量会影响结果的准确性。爱泼斯坦-巴尔病毒（EBV）是一种在全球广泛传播的潜伏病毒，实验中使用的细胞系可能携带EBV并带来感染风险。单个细胞系中存在EBV会污染同一实验室使用的其他细胞系，从而影响实验结果。现有的检测EBV的方法可分为三类：核酸检测、血清学检测和原位杂交检测。然而，大多数方法耗时、昂贵，且不利于大规模临床筛查。因此，仍需要一种简单的系统，能够在多种情况下快速检测EBV，包括细胞培养和组织样本。在我们的研究中，我们开发了EBV检测系统：（1）基于聚合酶链反应（PCR）的检测系统，（2）基于重组酶聚合酶扩增（RPA）的检测系统，以及（3）组合的RPA-侧向流动分析（LFA）检测系统。基于RPA和RPA-LFA的系统的最低EBV检测限为1×10拷贝数，基于PCR的系统为1×10拷贝数。PCR和RPA检测系统均应用于192个细胞系，结果与行业标准规定的检测结果一致。共鉴定出10个EBV阳性细胞系。组合的RPA-LFA系统操作简单，结果可视化迅速。该系统可作为日常质量控制策略的一部分在实验室和细胞库中实施，以确保细胞质量和实验安全，并且可能代表一种在临床样本中快速检测EBV的潜在新技术。关键特性 • 建立了用于EBV的RPA和RPA-LFA检测系统。 • RPA-LFA检测系统可在短短15分钟内实现结果可视化。 • 验证了RPA和RPA-LFA检测系统检测EBV的特异性。 • RPA和RPA-LFA系统的最低EBV检测限为1×10拷贝。
