The rapid detection of human HLA-B*27 gene based on BASIC isothermal detection

Abstract

Abstract Ankylosing spondylitis (AS) is a chronic, inflammatory arthritis of the spine and peripheral joints which is known to have a strong association with the human leukocyte antigen B27 (HLA-B27). Quantitative real-time PCR and flow cytometry are the predominant methods for HLA-B27 gene and antigen, respectively, which are too time-consuming and labor-intensive to realize rapid analysis. Therefore, a rapid diagnostic tool is highly required. In this study, we developed a rapid HLA-B*27 detection platform (namely BASIC) by combining our previously invented BASIS isothermal amplification method with the widely used CRISPR/Cas12a signal output tool. The BASIS can efficiently amplify all HLA-B*27 genotypes by using a set of universal primers, which target the conserved regions. The amplicons are subsequently applied to CRISPR/Cas12a analysis. The CRISPR/Cas12a recognizes the pathogenic HLA-B*27 amplicons specifically by using a well-designed gRNA, thereby achieving fluorescence signal output. Our results showed that the BASIC can be completed in 1 h with analytical sensitivity up to 100 aM. It could resist interference of homologous genes, hemoglobin, bilirubin, and triglyceride. For clinical sample detection, the BASIC offered completely consistent results with qPCR. Given the advantages of sensitivity, specificity, simplicity and rapidity, the BASIC was demonstrated a promising HLA-B*27 gene rapid detection tool for the early screening and diagnosis of AS.

Keywords

• Ankylosing spondylitis
• HLA-B*27 gene
• Rapid detection
• BASIS isothermal amplification
• CRISPR/Cas12a