Figure 5. Development of the CasPART assay.

(A) Illustrations of the one-pot CasPART assay. (B) Designs of the HIV RT substrate in CasPART to improve assay sensitivity. Delta RFU during 2hr incubation (endpoint RFU minus starting RFU) was used as assay signals. Left, CasPART signals with DNA, DNA-RNA chimeric, and RNA substrates. Middle, assay signals of incubating crRNA-Cas12a with double-stranded DNA with or without poly-T tails next to the activation region. Right, CasPART signals with varied primer lengths in the substrate. Bottom, illustrations of the final substrate design. (C) Performance of CasPART with HIV RT enzyme directly added in the assay. Real-time curves of 5hr incubation and delta RFU during 0.5hr and 5hr incubation can be found in Supplementary Figure S11. (D) Performance of CasPART integrated with direct immunocapture for sample preparation on 25μL HIV-negative plasma spiked with HIV RT enzyme. Statistical significance in panels C and D was determined by unpaired t tests with Welch’s correction and conducted in GraphPad Prism 10. * indicates p < 0.05 and ** indicates p < 0.01.