Comparative Analysis of a Developed Probe-Based Real-Time PCR and PCR-SSP for HLA-B*27 Detection among Thai Blood Donors

Abstract

The human leukocyte antigen (HLA) class I gene, the B locus, allele 27, HLA-B*27 is one of the most fascinating risk factors that is strongly associated with developing spondyloarthropathies (SpA). HLA-B27 testing has been routinely available in the diagnosis of those diseases. This study aimed to develop a fluorogenic real-time PCR and to compare it with PCR-SSP to detect the HLA-B*27 allele among Thai blood donors. A total of 391 DNA samples were obtained from Thai blood donors at Thammasat University Hospital and tested for HLA-B*27 allele detection. A new real-time PCR was developed and validated to identify this allele and subsequently compared with those results tested with PCR-SSP. The sensitivity of detection was performed using known HLA-B*27-positive and -negative samples with concentrations ranging from 0.001 to 100 ng/µL. Additionally, HLA-B27 subtyping was performed by DNA sequencing containing second and third exons of this gene among all the HLA-B*27-positive donors. The validity of real-time PCR using known DNA controls and the results obtained by PCR-SSP techniques were in 100% concordance. The method was sensitive even at low DNA concentrations (1 ng/µL). Of 391 donors, 24 (6.14%; 95% CI, 3.97 - 9.00) were found to have the HLA-B*27 allele, while the remaining 367 (93.86%; 95% CI, 91.00 - 96.03) did not have this allele. Donors presented HLA-B*27-positive, HLA-B*27:06, the most common allele, followed by HLA-B*27:04, -B*27:05, and -B*27:07. HLA-B*27 using fluorogenic real-time qualitative PCR was found to be superior compared with that of PCR-SSP. The method is rapid, accurate, reliable, and sensitive for detection. In addition, this method provides convenience in the early treatment of SpA patients and relieves their suffering.