Room Temperature Detection of Plasma Epstein–Barr Virus DNA with CRISPR–Cas13

To the Editor: Detection of plasma Epstein–Barr virus (EBV)1 DNA is useful for screening and monitoring nasopharyngeal carcinoma (NPC) and other EBV-associated diseases (1–3). However, quantitative PCR requires complex and expensive instrumentation, restricting its use in point-of-care testing and remote regions (4). Thus, a simple, portable, and inexpensive EBV DNA detection assay is needed. Prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated adaptive immune systems contain programmable endonucleases that can be used for CRISPR-based diagnostics (4, 5). Recently, the CRISPR effector Cas13a was combined with recombinase polymerase amplification (RPA) to establish a molecular detection platform, termed specific high-sensitivity enzymatic reporter unlocking (SHERLOCK), providing rapid and analytically sensitive nucleic acid detection (4, 5). To establish a SHERLOCK platform for the detection of EBV DNA, we first purified Leptotrichia wadei Cas13a (LwCas13a) protein. According to a previous study (5), the SHERLOCK platform works at 37 °C. We optimized the RPA primers and occasionally observed that the SHERLOCK platform could work at 25 °C.