Development and evaluation of a novel rapid fungal nucleic acid extraction kit

Abstract

Fungal infections can cause different diseases, and nucleic acid extraction is a crucial step in assisting the diagnosis of fungal infections. Traditional methods and current commercial fungal nucleic acid extraction kits are often time-consuming and involve hazardous chemicals. We developed a nucleic acid extraction kit that is safe, rapid, and efficient, designated as FNA-REK (Fungal Nucleic Acid Extraction Kit), which facilitates the simultaneous extraction of DNA and RNA without the necessity for hazardous chemicals, liquid nitrogen, or glass beads. This kit is user-friendly and achieves complete extraction within a mere 10 min. The RNA from the culture of Cryptococcus neoformans, Candida albicans, Candida krusei, Aspergillus flavus, Aspergillus niger and Aspergillus fumigatus. Meanwhile, the RNA from Candida albicans, Candida glabrata and Candida tropicalis of mid-stream urine, vaginal discharge and sputum samples, respectively, Cryptococcus neoformans, genus Aspergillus and Pneumocystis jirovecii of sputum samples, genus Aspergillus of bronchoscopy rinsing fluid were extracted using both the FNA-REK and the BIO-TEK. The concentration and purity of the extracted RNA were measured by nanodrop photometer. The nucleic acid extracted from Cryptococcus neoformans and Candida albicans using FNA-REK showed higher concentration and purity compared to BIO-TEK following a 10^–6 dilution. The maximum coefficients of variation for intra-run and inter-run replicates were 2.26% and 8.44%, respectively, significantly lower than 10%, indicating the high reproducibility of the FNA-REK. The FNA-REK yielded a lower cycle threshold (Ct value) than the BIO-TEK for cultured Candida krusei, Aspergillus niger and Aspergillus fumigatus obtained from clinical laboratory. Also, FNA-REK performed better than BIO-TEK for Candida albicans, Candida glabrata, Candida tropicalis, and genus Aspergillus in sputum samples from patients with severe pneumonia. The FNA-REK is a sensitive and stable nucleic acid extraction kit, offering an economical, efficient, and environmentally sustainable approach for fungal nucleic acid extraction. Its high extraction efficiency provides significant technical support for both laboratory and clinical applications, positioning it as a strong contender to become the most convenient and fastest fungal nucleic acid extraction kit currently available on the market.