Background

As biological research continues to advance, cells are increasingly at risk of being misidentified or contaminated with other cell types or exogenous factors [1,2] including viruses; such contamination can be difficult to detect and remediate.

Epstein–Barr virus (EBV) is a member of the human herpesvirus family. EBV displays prolonged latency in lymphocytes, interfering with immune functions and potentially inducing cell proliferation and transformation. EBV infection involves many organ systems and is often misdiagnosed or underdiagnosed. Multiple reports in the literature have described the detection of EBV infection in various cell types maintained in cell culture banks [3,4]. These infections can be attributed to the presence of an existing EBV infection during the initial process of cell line establishment, EBV contamination of culturing materials, or improper manipulation by the experimental staff.

The most widely used of the existing EBV assays is PCR, which is a well-established and reliable molecular method. Quantitative PCR (qPCR) can be used to monitor disease progression or therapeutic efficacy by detecting changes in EBV load in the blood [5]. Iso-thermal amplification techniques, such as recombinant enzyme-mediated isothermal amplification and loop-mediated isothermal amplification (LAMP), are easy to perform, have limited equipment requirements, return rapid responses, and display high sensitivity. However, designing primers for use in the LAMP technique is complicated and can be difficult to replicate. In situ hybridization assay to detect Epstein–Barr early RNA (EBER) is the current gold standard for detecting EBV infection in clinic. However, this technique can only be applied to tissue samples and is generally limited to the clinical diagnosis of EBV-related diseases, such as cancer and lymphoma.

Recombinase polymerase amplification (RPA) is a highly sensitive and selective isothermal amplification technique that can be performed at 39 °C and can be used to amplify a large number of samples in a short period of time. RPA, first introduced in 2006 by Niall Armes of ASM Scientific Ltd., UK [6], relies on modified homologous recombination mechanisms. RPA reagents are commercially available, and the basic RPA reaction kit can be augmented by additional commercially available kits that use different probes that can be cleaved by different enzymes: exo (exonuclease III), fpg (formamidopyrimidine DNA Glycosylase), and nfo (endonuclease IV) [7]. The exo and fpg probes are typically used for real-time detection, whereas the nfo probe is typically used for detection systems that use lateral flow dipsticks.

In this study, we combined RPA technology with a lateral flow assay (LFA) to develop an RPA-LFA detection system for application to the rapid and bulk screening of EBV contamination in cell lines stored by cell banks and to conduct regular and daily inspection of cell lines used in biological experiments, ensuring the quality of cell lines and the safety of experimental personnel. This system also demonstrates high potential for clinical adaptation to improve EBV detection in blood and tissue samples.