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

[...]quantitative PCR requires complex and expensive instrumentation, restricting its use in point-of-care testing and remote regions (4). [...]a simple, portable, and inexpensive EBV DNA detection assay is needed. 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. The SHERLOCK detection assay was carried out according to a previous study with modifications (5).A single 100-^L combined reaction assay consisted of 0.48 ^mol/L forward primer, 0.48 ^mol/L reverse primer, 1X RPA rehydration buffer (TwistDx), varying amounts of input DNA, 22.5 nmol/L CRISPR RNA (crRNA), 45 nmol/L LwCas13a recombinant protein, 2.5 ptL of murine RNase inhibitor (New England Biolabs), 125 nmol/L substrate reporter (RNase alert v2), 2 mmol/L ATP, 2 mmol/L GTP, 2 mmol/L UTP, 2 mmol/L CTP (Yeasen), 1 ^LofT7 polymerase (New England Biolabs), 14 mmol/L MgAc (TwistDx), and 5 mmol/L MgCl2 (Invitrogen). The results showed a Pearson correlation coefficient of R2 = 0.9314, P < 0.0001 (Fig. 1) with the quantitative PCR data, supporting that the performance of the room temperature SHERLOCK platform is comparable with that of quantitative PCR. Because the SHERLOCK platform could be carried out at room temperature, the instruments used throughout the assay could be simpler, less expensive, and more convenient.