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Article

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

  • Authors:
    • Noor Afshan
    • Mukarram Bashir
    • Hamid Nawaz Tipu
    • Mohammad Hussain
  • View Affiliations / Copyright

    Affiliations: Department of Immunology, Armed Forces Institute of Pathology, 46000 Rawalpindi, Pakistan
  • Pages: 385-390
    |
    Published online on: February 1, 2018
       https://doi.org/10.3892/br.2018.1055
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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.
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Copy and paste a formatted citation
Spandidos Publications style
Afshan N, Bashir M, Tipu HN and Hussain M: Optimization of an in-house PCR method for the detection of HLA-B*27 alleles. Biomed Rep 8: 385-390, 2018.
APA
Afshan, N., Bashir, M., Tipu, H.N., & Hussain, M. (2018). Optimization of an in-house PCR method for the detection of HLA-B*27 alleles. Biomedical Reports, 8, 385-390. https://doi.org/10.3892/br.2018.1055
MLA
Afshan, N., Bashir, M., Tipu, H. N., Hussain, M."Optimization of an in-house PCR method for the detection of HLA-B*27 alleles". Biomedical Reports 8.4 (2018): 385-390.
Chicago
Afshan, N., Bashir, M., Tipu, H. N., Hussain, M."Optimization of an in-house PCR method for the detection of HLA-B*27 alleles". Biomedical Reports 8, no. 4 (2018): 385-390. https://doi.org/10.3892/br.2018.1055
Copy and paste a formatted citation
x
Spandidos Publications style
Afshan N, Bashir M, Tipu HN and Hussain M: Optimization of an in-house PCR method for the detection of HLA-B*27 alleles. Biomed Rep 8: 385-390, 2018.
APA
Afshan, N., Bashir, M., Tipu, H.N., & Hussain, M. (2018). Optimization of an in-house PCR method for the detection of HLA-B*27 alleles. Biomedical Reports, 8, 385-390. https://doi.org/10.3892/br.2018.1055
MLA
Afshan, N., Bashir, M., Tipu, H. N., Hussain, M."Optimization of an in-house PCR method for the detection of HLA-B*27 alleles". Biomedical Reports 8.4 (2018): 385-390.
Chicago
Afshan, N., Bashir, M., Tipu, H. N., Hussain, M."Optimization of an in-house PCR method for the detection of HLA-B*27 alleles". Biomedical Reports 8, no. 4 (2018): 385-390. https://doi.org/10.3892/br.2018.1055
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