A rapid and sensitive CRISPR-Cas12a for the detection of Fusobacterium nucleatum

Abstract

ABSTRACT Fusobacterium nucleatum (Fn), as a conditional pathogen, can cause a range of oral and gastrointestinal diseases. However, existing clinical detection methods require expensive equipment and complex procedures, which are inconvenient for large-scale screening in epidemiological research. The purpose of this study was to establish a reliable, rapid, and inexpensive detection method based on CRISPR/Cas12a technology for the detection of Fn. Specific recombinase polymerase amplification (RPA) primer sequences and crRNA sequences were designed based on the nusG gene of Fn. Subsequently, a fluorescence assay and a lateral flow immunoassay were established using the RPA and CRISPR-Cas12a system (RPA-CRISPR-Cas12a). Sensitivity validation revealed a limit of detection of 5 copies/µL. This method could distinguish Fn from other pathogens with excellent specificity. Furthermore, the RPA-CRISPR-Cas12a assay was highly consistent with the classical quantitative real-time PCR method when testing periodontal pocket samples. This makes it a promising method for the detection of Fn and has the potential to play an increasingly important role in infectious disease testing. IMPORTANCE Fusobacterium nucleatum (Fn) naturally exists in the microbial communities of the oral and gastrointestinal tracts of healthy individuals and can cause inflammatory diseases in the oral and gastrointestinal tracts. Recent studies have shown that Fn is closely associated with the occurrence and development of gastrointestinal cancer. Therefore, the detection of Fn is very important. Unlike the existing clinical detection methods, this study established a fluorescence-based assay and lateral flow immunoassay based on the RPA and CRISPR-Cas12a system (RPA-CRISPR-Cas12a), which is fast, reliable, and inexpensive and can complete the detection within 30–40 minutes. This makes it a promising method for the detection of Fn and has the potential to play an increasingly important role in infectious disease testing.