Rapid HLA-B27 Test with Real-Time PCR in Suspected Ankylosing Spondylitis Iraqi Patients

Dr Sadiq Al- Mukhtar; Dr. Batool Mutar Mahdi; Hyam Raouf; Haider Hashim Zalzala; Laheeb Ali Abid; Zena Nehad

Volume : II, Issue : VIII, August - 2013

Rapid HLA-B27 Test with Real-Time PCR in Suspected Ankylosing Spondylitis Iraqi Patients

Dr Sadiq Al Mukhtar, Dr. Batool Mutar Mahdi, Hyam Raouf, Haider Hashim Zalzala, Laheeb Ali Abid, Zena Nehad

Abstract :

HLA–B27 is a MHC class 1 molecule that is strongly associated with the Ankylosing Spondylitis and Seronegative Spondyloarthropathies. Various methods have been developed for the identification of the HLA–B27 allele. The PCRbased HLA–typing methods, including the standard PCR with sequence–specific primers (SSP), sequence specific oligoneuclotide (SSO) have become widely used alternatives to serology. This study is to assess a real–time TaqMan PCR assay for genotyping HLA–B*27 in the Iraqi patients with suspected Ankylosing Spondylitis. The study consisted of 50 Iraqi Arab Muslims patients suspected to have ankylosing spondylitis consulted Orthopedic and Rheumatology department in Al–Kindi Teaching Hospital in Baghdad from September 2012 to May 2013. The second control group consisted of twenty apparently healthy volunteers� age matched with first group from staff employees served as controls. Blood samples have been obtained from them and genomic DNA was extracted using using reliaprep spin column kit (Promega,USA). Real–time PCR reactions were performed using an Exicycler TM 96 Real–time Quantitative Thermal Block (Bioneer, Daejeon, Korea) and the AccuPower, HLA–B27 real–time PCR kit (Bioneer, Daejeon, Korea). The age of patients in this study were range from 16–35 years with a mean age of 28.2 � 3.5 years. Control ages range from 17–37 years with a mean of 30.28 � 2.87. All positive samples had Ct FAM values <28. All samples had Ct TAMRA values <26. A Total of 50 clinical samples who were suspected to have AS and twenty controls were used to compare the real time Taq Man PCR with traditional PCR–SSO method. Ten out of fifty (20%) of patients showed positive HLA–B27 results and did not detected in control group. This results gave concordant with PCR–SSO results.Real–time PCR methods appear to be reliable and very fast in the detection of HLA–B27 and could be potential alternatives to conventional PCR and serology methods.