Visual screening of Toxigenic Alternaria Fungi in citrus using CRISPR/Cas12a and quantum dot

Abstract

Citrus infected by Alternaria not only manifests as brown spot, one of the most detrimental diseases affecting citrus crops, but also produces highly toxic mycotoxins. Large-scale screening effectively controls its widespread, thereby ensuring consumer safety. This study presents a high-throughput visual fluorescent assay for Alternaria screening in citrus, which integrates the signal amplification capabilities of CRISPR/Cas12a with hybridization chain reaction (HCR) and the bright fluorescence of quantum dots (QDs). A biotin-labeled single-stranded DNA (biotin-ssDNA) served as both a trans-cleavage substrate for the CRISPR/Cas12a system and the initiator of HCR. When Alternaria is not present, the CRISPR/Cas12a system remains inactive, allowing intact biotin-ssDNA to bind to the streptavidin (SA) coated microplate and trigger HCR in the presence of two biotin-labeled hairpin nucleic acids. The subsequent biotin-SA interaction facilitates the accumulation of SA-QDs on the microplate. Upon excitation the collected supernatant with a portable ultraviolet light source, samples devoid of Alternaria exhibit weak red fluorescence, whereas Alternaria-infected samples exhibit pronounced red fluorescence. The detection of synthetic target sequences was achieved with a detection limit of 10 pM. Furthermore, the high specificity of the assay was demonstrated by its ability to effectively differentiate Alternaria from other citrus pathogens. The practical applicability was validated through the analysis of cultured Alternaria and 36 field samples collected from citrus leaves and fruits, achieving a 100 % accuracy rate in comparison to quantitative PCR. This innovative approach does not rely on sophisticated instrumentation, making it potentially valuable for Alternaria screening in resource-limited conditions.