crRNA array-mediated CRISPR/Cas12a coupling with dual RPA for highly sensitive detection of Streptomyces aureofaciens Tü117 from hypertension with multi-signal output

Abstract

Accurate and sensitive detection of Streptomyces aureofaciens Tü117 is crucial for hypertension classification and early warning. To achieve this, a dual recombinase polymerase amplification coupled with a crRNA array-mediated CRISPR/Cas12a assay (DR-CAMCas) was developed, enabling multi-signal output for precise identification and detection of S. aureofaciens Tü117. The 16S rDNA and LipReg4 genes of S. aureofaciens Tü117 are amplified simultaneously via one-step dual RPA, activating the crRNA array-mediated CRISPR/Cas12a system to cleave exogenous FQ-reporters, releasing fluorescent signals. DR-CAMCas offers high amplification efficiency, multi-site recognition through crRNA array signal superposition, and the programmability of CRISPR/Cas12a, achieving ultrasensitive detection with a linear range of 10 to 10⁸ cfu/mL and a limit of detection of approximately 3 cfu/mL. DR-CAMCas successfully detected S. aureofaciens Tü117 in fecal samples from high-salt diet-induced hypertensive mice and hypertensive patients, matching qPCR results and demonstrating high reliability and practicality. Additionally, target-induced cleavage of a DNA linker by DR-CAMCas dispersed AuNPs-DNA probes, enabling colorimetric detection. Integrated onto lateral flow sensors, DR-CAMCas allows point-of-care testing via simple visual strip analysis. Its triple signal output meets diverse detection needs, offering a promising tool for diagnosing salt-sensitive hypertension.