# Rapid and Sensitive Detection of Candida albicans Using Microfluidic-Free Droplet Digital Non-Amplification Dependent CRISPR/Cas12a Assay

## TL;DR

A new CRISPR-based method called NaPddCas detects Candida albicans DNA rapidly and sensitively without amplification or microfluidic devices.

## Contribution

Introduces a microfluidic-free, droplet-based CRISPR/Cas12a assay for amplification-free detection of Candida albicans DNA.

## Key findings

- NaPddCas detects Candida albicans DNA at concentrations several orders of magnitude lower than bulk CRISPR/Cas12a methods.
- The assay shows high specificity against non-target bacterial and fungal species.
- Successfully applied to clinical vaginal secretion samples for detection of Candida albicans.

## Abstract

Candida albicans is a major fungal pathogen associated with vulvovaginal candidiasis, and rapid, sensitive detection remains challenging, particularly in amplification-free formats. Here, we report NaPddCas, a microfluidic-free, droplet-based CRISPR/Cas12a detection strategy for qualitative identification of Candida albicans DNA. Unlike conventional bulk CRISPR assays, NaPddCas partitions the reaction mixture into vortex-generated polydisperse droplets, enabling spatial confinement of Cas12a activation events and effective suppression of background fluorescence. This compartmentalization substantially enhances detection sensitivity without nucleic acid amplification or microfluidic devices. Using plasmid and genomic DNA templates, NaPddCas achieved reliable detection at concentrations several orders of magnitude lower than bulk CRISPR/Cas12a reactions. The assay further demonstrated high specificity against non-target bacterial and fungal species and was successfully applied to clinical vaginal secretion samples. Importantly, NaPddCas is designed as a qualitative or semi-qualitative droplet-dependent digital detection method rather than a quantitative digital assay. Owing to its simplicity, sensitivity, and amplification-free workflow, NaPddCas represents a practical approach for laboratory-based screening of Candida albicans infections.