A CRISPR/Cas12a–personal glucose meter biosensor for rapid and quantitative detection of Burkholderia pseudomallei DNA

Abstract

Melioidosis, caused by Burkholderia pseudomallei (B. pseudomallei), is a fatal tropical infectious disease whose diagnosis is hindered by the lack of rapid and accessible detection methods. Here, we present a novel CRISPR/Cas12a-based biosensing platform integrated with a personal glucose meter (PGM) for rapid, quantitative, and amplification-free detection of B. pseudomallei DNA. In this system, target DNA activates Cas12a trans-cleavage, releasing invertase from an invertase-Linker-biotin probe. Subsequent magnetic separation removes the uncleaved complexes, and the invertase remaining in the solution catalyzes the conversion of sucrose to glucose, which is quantitatively measured by the PGM. The assay is completed within 50 min and achieves a detection limit of 0.1 pM with a linear dynamic range from 1 pM to 10 nM. The biosensor exhibits excellent specificity against single- and multi-base mismatched sequences, outstanding precision (CV < 10 %), and strong correlation with qPCR results in clinical samples (recovery: 98 % - 113 %). The reagents remain stable over four weeks of storage (CV = 4.4 %), confirming the robustness of the system. This amplification-free platform, independent of specialized laboratory instrumentation, represents an important step toward accessible molecular diagnostics, demonstrating the potential as a rapid, low-cost, and user-friendly solution for early melioidosis detection and potentially other infectious diseases. From a sensor-engineering standpoint, the CRISPR/Cas12a–PGM architecture constitutes a generalizable chemical/biosensing platform that can be readily reprogrammed for different nucleic-acid targets by simply switching the crRNA.