Wax-in-a-Tube: A Simple, Rapid, One-Pot Platform for Molecular Detection

CRISPR technology has emerged as a powerful platform for highly sensitive and specific nucleic acid detection, particularly when coupled with isothermal amplification. However, conventional two-step CRISPR assays still require manual operations, such as shaking, centrifugation, or vortexing, that complicate the workflow and increase the risk of aerosol contamination. Here, we present a wax-in-a-tube (WIAT) platform that enables simple, rapid, one-pot recombinase polymerase amplification (RPA) and CRISPR-based detection by leveraging the phase-change properties of a molded wax separator, thereby eliminating additional manual steps and minimizing contamination risks. Using a molding approach, we directly integrated the wax separator into the reaction tube to physically partition different reaction components. The WIAT platform achieved a detection sensitivity of 10 aM for HSV-2 DNA, comparable to that of standard two-step assays. We further validated its clinical performance using HSV-2 swab samples, demonstrating results comparable to those obtained with PCR. Together, these findings establish the WIAT platform as a simple, rapid, and highly sensitive one-pot RPA-CRISPR assay with strong potential for point-of-care infectious disease detection and early surveillance.