Modified primers for rapid and direct electrochemical analysis of coeliac disease associated HLA alleles.

Direct detection of PCR product via hybridisation assay, would facilitate the development of rapid tools for genetic analysis. Here, a PCR primer designed to generate a PCR amplicon tagged with single stranded DNA tails at each end of the duplex, which can be used for direct hybridisation with a surface immobilised probe and an enzyme labelled reporter probe is presented. Four modified sequence specific primers (SSP) pairs were designed for the selective amplification of coeliac disease associated alleles (DQA1*05, DQB1*02, DQB1*03:02 alleles), and human growth hormone (positive control). Multiplex PCR products were electrochemically detected in less than 5 min at 37 °C via direct hybridisation to short probes immobilised on individual electrodes of a genosensor array, and subsequent hybridisation to an enzyme labelled reporter probe. The developed electrochemical genosensor array exploiting the modified primers for the direct detection of PCR products was applied to the genotyping of real patient samples.