A cost-effective and labor-saving method for detecting HLA-B27 status via sequence-encoded fluorescence amplification assay

Identification of Human leukocyte antigen B27 (HLA-B27) by flow cytometry (FCM) has been widely applied in clinical practice for auxiliary diagnosis of ankylosing spondylitis (AS). However, FCM requires fresh-prepared samples and relies on expensive equipment, reagents, and experienced operator. To provide a cheaper and more convenient method for HLA-B27 detection, we proposed a new method termed sequence-encoded fluorescence amplification assay (SEFA), which specially recognized sequences of HLA-B27 gene (HLA-B*27) covering current common subtypes in a single closed tube. SEFA could detect as low as 10 pg (equals to 3 copies) genomic DNA (gDNA) per reaction and distinguish HLA-B*27 from other HLA-B alleles with highly similar sequences. A total of 288 clinical samples were tested by SEFA, including 181 AS patients and 107 healthy controls. Compared with the detection results from FCM, two controversial samples of AS patients were obtained and further confirmed to be consistent with SEFA by sanger sequencing, indicating that our method was more accurate than FCM. Moreover, SEFA could detect HLA-B27 status by using supernatant from crude extract of 10-μL blood without commercial reagents. Overall, SEFA has the potential to be an alternative for HLA-B27 identification with the advantage of convenience and low-cost, especially suitable for early diagnosis of AS in areas with limited medical resources.