Optimization of an in-house PCR method for the detection of HLA-B*27 alleles

Abstract

The association of spondyloarthropathies with different alleles of human leukocyte antigen (HLA) B*27 is well established. Different subtypes of HLA-B*27 may be linked with different ethnic groups, distinct clinical manifestations, specific age of onset and different prognoses. Polymerase chain reaction with sequence specific primers (PCR-SSP) is the most frequently adapted molecular method used for the recognition of HLA-B*27-specific DNA sequences. The aim of the present study was to standarise an in-house protocol of PCR-SSP for HLA-B*27 allele detection for use in the Armed Forces Institute of Pathology (AFIP), Pakistan, with consideration of its cost effectiveness. A total of 49 individual samples were included, comprising 10 transplant samples determined to be HLA-B*27-negative by PCR-SSP and 39 HLA-B*27-positive samples determined by flow cytometry, obtained from patients who were symptomatic and referred for HLA-B*27 testing. By altering each variable individually, an in-house PCR-SSP protocol was optimized to amplify common HLA-B*27 alleles (2701-2721, 2723-2730). To discriminate B*27 from all other HLA-B alleles, a low-resolution HLA-B typing set with a 96 PCR-SSP primer mixture was used in conjunction. Among the 39 HLA-B*27-positive specimens, 31 (79%) were detected as positive by PCR-SSP, with the remaining samples failing due to a sub-optimized protocol and/or low DNA concentration. Additionally, there was complete concordance between flow cytometry and in-house PCR, and the sensitivity and specificity of the PCR-SSP were determined to be 100%. In conclusion, in-house SSP-PCR is, standard method for the detection of HLA-B*27 alleles. The determination of associations between specific HLA-B*27 alleles and AS may aid to identify individuals at higher risk of developing the disease. Furthermore, the identification of individuals at risk may aid to adapt preventive strategies.