Medium-high resolution electrochemical genotyping of HLA-DQ2/DQ8 for detection of predisposition to coeliac disease

Abstract

Coeliac disease is a small intestinal disorder, induced by ingestion of gluten in genetically predisposed individuals. Coeliac disease has been strongly linked to human leukocyte antigens (HLA) located on chromosome 6, with almost 100 % of coeliac disease sufferers carrying either a HLA-DQ2 or HLA-DQ8 heterodimer, with the majority carrying HLA-DQ2 encoded by the DQA1*05:01/05:05, DQB1*02:01/02:02 alleles, whereas the remaining carry the HLA-DQ8 encoded by the DQA1*03:01, DQB1*03:02 alleles. In this work, we present the development of a multiplex electrochemical genosensor array of 36 electrodes, housed within a dedicated microfluidic platform and using a total of 10 sequence-specific probes for rapid medium-high resolution HLA-DQ2/DQ8 genotyping. An evaluation of the selectivity of the designed probes was carried out with the target sequences and 44 potentially interfering alleles, including single base mismatch differentiations; good selectivity was demonstrated. The performance of the electrochemical genosensor array was validated, analyzing real human samples for the presence of HLA-DQ2/DQ8 alleles, and compared with those obtained using laboratory-based HLA typing, and an excellent correlation was obtained.

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

“…The previous reports of direct electrochemical detection of PCR product (Bonanni et al, 2009;Brasil de Oliveira Marques et al, 2009;de Lumley-Woodyear et al, 1999) using labelled primers were not suitable for multiplexing and required considerably longer assay times (10-60 min). A recent report for the electrochemical microfluidic medium-high genotyping of HLA-DQ2/DQ8 using ten different sequence specific probes has been detailed and whilst very useful an increased assay time was required, as ssDNA had to be generated and the solidphase hybridisation of the target needed longer times to hybridise to their cognate targets (Joda et al, 2014).…”

“…Following cleaning the electrodes were functionalised, via coself-assembling (Acero Joda et al, 2012;Henry et al, 2010;Joda et al, 2014) of the DNA thiolated probes (1 mM) and a spacer (Dithiol 16-(3,5-bis((6-mercaptohexyl)oxy) phenyl)-3,6,9,12,15-pentaoxahexa-decane), DT1, CAS# 936115-52-5, where the DT1 also serves to prevent non-specific binding (Civit et al, 2010), SensoPath Technologies, Bozeman, MT, USA) (100 mM) in 1 M KH 2 PO 4 and left to incubate for 3 h at room temperature within a humidified chamber. Subsequently, the electrode array was rinsed with Milli-Q water and dried with nitrogen gas.…”

DNA biosensor based on hybridization refractory mutation system approach for single mismatch detection

“…The proposed system consisted 58 of the deliberate insertion of an additional mismatch near the 3 0 end 59 of a primer to prevent primer extension with non-completely com-60 plementary sequences, hence increasing primer specificity. 61 Guo and coworkers [13] and Burgner and coworkers [14] reported [15,16] and can be found in the online supplementary material. 92 A detailed description of the optimized design of the probes for the 93 HLA typing of DQ2 and DQ8 genes, together with the description of 94 relevant interfering alleles, has also been detailed previously [15,16].…”

“…61 Guo and coworkers [13] and Burgner and coworkers [14] reported [15,16] and can be found in the online supplementary material. 92 A detailed description of the optimized design of the probes for the 93 HLA typing of DQ2 and DQ8 genes, together with the description of 94 relevant interfering alleles, has also been detailed previously [15,16]. 95 Because DQA1*05:01 and DQA1*05:05 alleles are involved in 96 the genetic predisposition to celiac disease detection, clear dis-97 crimination between them is required.…”

“…95 Because DQA1*05:01 and DQA1*05:05 alleles are involved in 96 the genetic predisposition to celiac disease detection, clear dis-97 crimination between them is required. First, a probe able to detect 98 both alleles, namely DQA05, was designed and investigated 99 [15,16]. Subsequently, discrimination between the DQA1*05:01 (Fig.…”