Fast and visual detection of nucleic acids using a one-step RPA-CRISPR detection (ORCD) system unrestricted by the PAM.

CRISPR-Cas12a (Cpf1) is widely used for pathogen detection. However, most Cas12a nucleic acid detection methods are limited by a PAM sequence requirement. Moreover, preamplification and Cas12a cleavage are separate. Here, we developed a one-step RPA-CRISPR detection (ORCD) system unrestricted by the PAM sequence with high sensitivity and specificity that offers one-tube, rapid, and visually observable detection of nucleic acids. In this system, Cas12a detection and RPA amplification are performed simultaneously, without separate preamplification and product transfer steps, and 0.2 copies/µL of DNA and 0.4 copies/µL of RNA can be detected. In the ORCD system, the activity of Cas12a is the key to the nucleic acid detection; specifically, reducing Cas12a activity increases the sensitivity of ORCD assay detection of the PAM target. Furthermore, by combining this detection technique with a nucleic acid extraction-free method, our ORCD system can be used to extract, amplify and detect samples within 30 min, as verified with tests of 82 Bordetella pertussis clinical samples with a sensitivity and specificity of 97.30% and 100% compared with PCR. We also tested 13 SARS-CoV-2 samples with RT-ORCD, and the results were consistent with RT-PCR.

Quantification of MicroRNAs or Viral RNAs with Microelectrode Sensors Enabled by Electrochemical Signal Amplification.

Quantification of circulating microRNAs (miRNAs) or viral RNAs is of great significance because of their broad relevance to human health. Currently, quantitative reverse transcription polymerase chain reaction (qRT-PCR), as well as microarray and gene sequencing, are considered mainstream techniques for miRNA identification and quantitation and the gold standard for SARS-CoV2 detection in the COVID-19 pandemic. However, these laboratory techniques are challenged by the low levels and wide dynamic range (from aM to nM) of miRNAs in a physiological sample, as well as the difficulty in the implementation in point-of-care settings. Here, we describe a one-step label-free electrochemical sensing technique by assembling self-folded multi-stem DNA-redox probe structure on gold microelectrodes and introducing a reductant, tris(2-carboxyethyl) phosphine hydrochloride (TCEP), in the detection buffer solution to achieve ultrasensitive detection with a detection limit of 0.1 fM that can be further improved if needed.