One-Pot CRISPR/Cas12a Assay Based on Ultrashort HDA for Ultrasensitive and Universal Nucleic Acid Detection

Isothermal amplification techniques, such as helicase-dependent amplification (HDA) combined with CRISPR, are cutting-edge approaches for nucleic acid detection. In this work, we developed a novel ultrashort mesophilic HDA (termed usHDA) for rapid, highly sensitive nucleic acid amplification at 37 °C and constructed a one-pot usHDA-CRISPR/Cas12 assay. The usHDA is specifically designed for rapid amplification of ultrashort sequences (about 40 nt) at 37 °C within 30 min. This usHDA-CRISPR/Cas12a detection can be completed within 1 h, achieving a limit of detection (LOD) of 5 aM. When tested on 58 clinical specimens from patients infected with respiratory pathogens, this assay identified 41 positive and 17 negative samples for influenza A virus. This assay achieved 100% sensitivity, 100% specificity, and a perfect receiver operating characteristic curve (area under the curve value = 1.00; <i>n</i> = 58) compared with PCR analysis. Furthermore, 24 samples of Staphylococcus infection were detected using usHDA-CRISPR/Cas12a, and the same 100% sensitivity and specificity were achieved. These findings highlighted the strong applicability of our proposed assay for universal nucleic acid detection.