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. 2021 Mar 19;12:1739. doi: 10.1038/s41467-021-21996-6

Fig. 6. Optimizing reaction conditions for enAsCas12a.

Fig. 6

a Evaluation of various experimental conditions, including different concentrations of enAsCas12a and different durations of the cleavage reaction. 1X specifies 65 nM. 2E6 copies of synthetic wild-type SARS-CoV-2 RNA served as input to RT-LAMP. b Detection of wild-type or S254F mutant SARS-CoV-2 sequence using S2 and S6 gRNAs. Different copies of SARS-CoV-2 RNA fragments were used as input to RT-LAMP, which was performed at 65 °C for 15 min. Next, the Cas detection reaction was carried out at 37 °C for 10 min in Buffer 2.1 with DTT before a dipstick was added to each reaction tube. c Systematic testing of different reaction buffers and temperatures for the Cas detection step. Here, enAsCas12a complexed with the S2 gRNA only was utilized in a 50 µl trans-cleavage assay with 2E11 copies of DNA template corresponding to SARS-CoV-2 S-gene. Data represent mean ± s.e.m. (n = 3 [41 °C, 45 °C, 50 °C, 55 °C Tango alone, 60 °C no DTT and NTC], 4 [37 °C, 55 °C all but Tango alone], or 6 [60 °C with DTT] biological replicates). d Preliminary evaluation of our VaNGuard test with leftover patient samples. A Ct value of 30 was estimated to be equivalent to 500 copies of the virus. RT-LAMP was performed at 65 °C for 15 min before the Cas detection reaction was carried out at 37 °C for 10 min in CutSmart with DTT. Each clinical sample was tested twice using dipsticks. e Retesting the pilot set of clinical RNA samples using a fluorescence readout. RT-LAMP was performed at 65 °C for 15 min. Subsequently, 4 μl LAMP products (out of 25 μl) were used for the trans-cleavage assay, which was performed at 37 °C in a real-time instrument where measurements were taken every minute. The fluorescence readings for all six clinically negative samples remained low over the duration of the experiment. Additionally, the fluorescence readings for five out of the six clinically positive samples showed a clear exponential increase with time. The remaining positive sample (RP6), which contained 14 copies of the virus, gave fluorescence signals that were only slightly above those of the negative samples. Source data are available in the Source Data file.