Construction of internal and external amplification controls for HLA-B27 diagnostic by PCR

BARCA, Y; LAPADULA W.J.; CARGNELUTTI E.; JURI AYUB M; DI GENARO M.S.

Congreso; XXXI Reunión científica anual de la sociedad de Biología de Cuyo; 2013

Spondyloarthropathies (SpAs) are a group of interrelated inflammatory arthritic diseases that share clinical, radiological features and genetic predisposing factors. According to the diagnostic criteria proposed by the European Study Group Spondylarthritis (ESGS) has recognized six entities within the group of SpA: Ankylosing spondylitis (AS), reactive arthritis (ReA), psoriatic arthritis (PsA), SpA associated with inflammatory bowel disease (IBD-SpA), juvenile - onset SpA and undifferentiated spondyloarthritis (uSpA). SpAs are characterized by axial and or peripheral arthritis, associated with enthesitis, dactylitis and potential extra-articular manifestation such us uveitis and skinrash. While the major genetic risk factor in SpA is the presence of human leukocyte antigen B27 (HLA-B27) is a molecule of the major histocompatibility complex type I (MHC I). Actually we are determining the presence of this molecule by amplification of its 3 exon by qPCR, which cause an amplification product of 135 base pairs (bp). In this work we desing a synthetic construct of 423 pb using bioinformatics tools, including search of sequence in databases, multiple sequence alignment and search of restriction site between others. Finally we cloned it in the commercial vector pGEM-T easy. This construction can be amplified using the same primer pairs above mentioned, to amplify the exon 3 in a competitive qPCR. Moreover can be used as postive control both conventional PCR and qPCR.