Application of the Signaling Probe-Based DNA Microarray to Pathogenic Fungi Detection
Authors: Maemura Chika , Hirakawa Yuko , Murakami Eiichi , Yoshino Tomoko , Matsunaga Tadashi , Tanaka Tsuyoshi , Taguchi Tomoyuki 
Published: December 2024

Background and Purpose

Microbiological testing is crucial for quality control in the production of foods and pharmaceuticals. Biochemical identification has been conventionally employed to detect and identify microorganisms. However, it requires time-consuming and labor-intensive processes as well as the skilled operators. To overcome the above problems, we have developed a novel DNA detection system; i.e. the signaling probe-based DNA microarray system towards pathogen detection1). The proposed system, which can eliminate labeling and washing steps, allows a simple and rapid detection of pathogenic bacteria genes. However, the proposed system has been only applied for bacterial gene detection based on 16S rDNA, but not for other pathogens including fungi. In this study, the signaling probe-based DNA microarray system was applied to the detection of three kinds of fungi, Aspergillus niger, Candida albicans, and Chaetomium globosum, common contaminants in food production.

Materials and Methods

The signaling probe-based DNA microarray is composed of a “fluorescence probe” (35-mer) labeled with Cy3 at the 5’-end and a “quencher probe” (35-mer) labeled with BHQ2 at the 3’-end. The quencher probe includes a detection sequence, which is complementary to the target fragments. Cy3-labeled and BHQ2-labeled probes were immobilized in the same spots. Partial hybridization between these probes makes the fluorophore and quencher molecules in close proximity, resulting in loss of fluorescence due to fluorescence resonance energy transfer (FRET). When the target fragments are introduced on the signaling probes, the fragments form duplexes with quenching probes by the detection sequence, and fluorescence signal is detectable. In this study, three signaling probe pairs were designed to detect the 18S rDNA genes, which is widely conserved in fungi. Target DNA fragments (245-bp to 247-bp; 18S rDNA) were prepared by asymmetric PCR using the genome from A. niger, C. albicans, and C. globosum. The PCR products were subjected to the signaling probe-based DNA microarray. Hybridization was carried out for 30 min at 60°C. The entire DNA microarray images were visualized in the aqueous solution using our original wide-field imaging system, consisting of a 532 nm laser source, microlens array, dichroic mirror, bandpass filter and CCD camera. The acquired images were processed using Array Pro image analysis software to analyze the fluorescence signals.

Results & Discussion

To confirm sufficient quenching of Cy3 on the fluorescence probe by BHQ2 on the quencher probe, the FRET-based quenching of the designed probe was investigated. In the absence of target DNA, the spots of the signaling probe exhibited no fluorescence, indicating that hybridization between the signaling probe pairs induced FRET. Next, the detection performance of the signaling probe-based DNA microarray was evaluated by measuring the fluorescence signals in the presence of target DNA. Clear fluorescence signals were observed in all the tested probes after the addition of target DNA. Furthermore, little fluorescence at the background levels were detected when using a control (PCR product of Escherichia coli 16S rDNA). These results suggest that the signaling probe-based DNA microarray can be useful for various pathogens including fungi. In our proposed system, removal of the target DNA solution and washing process were not required for detection because the wide-filed imaging system allowed us to visualize the fluorescence signals from the microarray immersed in the liquid media. The total assay was completed in 95 min. Therefore, the signaling probe-based DNA microarray allows a rapid and simple determination of a wide range of microorganisms.
1) T. Taguchi, et al. Biosens. Bioelectron. 194, 113659 (2021)