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

BARCA YA; LAPADULA WJ,; CARGNELUTTI E,; JURI AYUB M; DI GENARO MS

Mendoza

Congreso; XXXI Reunión Anual de la Sociedad de Biología de Cuyo; 2013

Spondyloarthritis (SpAs) are a group of interrelated inflammatory arthritis which share multiple clinical features and genetic predisposing factors. The major genetic risk factor is the presence of human leukocyte antigen B27 (HLA-B27). In our laboratory we determinate HLA-B27 by real-time polymerase chain reaction (qPCR) using whole blood as sample. The amplification of the exon 3 of HLA-B27 was carried out using specific primers, yielding a 135 pair bases (pb) amplicon. In this work we constructed a 423 pb fragment to be used as Internal Amplification Control (IAC) in order to discard false negative results. For this purpose, an exogenous sequence of 288 pb was inserted into the 135 pb amplicon sequence, flanked by HindIII restriction sites.The construct was amplified by PCR and cloned into a conventional vector. The identity of clones was confirmed by restriction enzyme mapping and sequencing. This clon was digested with HindIII and religated, generating a fragment of 135 pb to be used as external control. Theses constructions can be amplified using the primers specific to the exon 3 of B27 allele in a competitive qPCR and furthermore used as positive control both in conventional PCR and qPCR.