Fluorescence biosensor for HLA-B*1502 detection based on double primer pairs enzymatic recombinase amplification (D-ERA) coupled with optimized CRISPR/Cas12a system

The single-nucleotide polymorphism (SNP) rs144012689 has been validated to exhibit high sensitivity and specificity for the detection of the HLA-B*1502 allele in both American and Chinese populations. Therefore, we designed a detection system for rs144012689 single base mutation detection based on double primer pairs enzymatic recombinase amplification (D-ERA) isothermal amplification and the optimized CRISPR/Cas12a system. First, we screened candidate crRNAs and optimized the D-ERA and CRISPR-Cas12a reaction conditions. Second, we determined the limit of detection (LOD) of the assay to be 0.1 ng/μL (equivalent to approximately 66 target copies per reaction) and established a fluorescence threshold of 500 arbitrary units (a.u.). Finally, we implemented a detection strategy that enables HLA-B*1502 genotyping within 30 min. Compared with the results of sanger sequencing, this method provided completely consistent results for clinical samples testing. So, this method provides a new pathway for rapid detection of single base mutation.