Rapid, Loop-Mediated Isothermal Amplification Detection of Coeliac Disease Risk Alleles

Publication date: Available online 17 February 2018 Source:The Journal of Molecular Diagnostics Author(s): Michael Erlichster, Jason A. Tye-Din, Michael D. Varney, Efstratios Skafidas, Patrick Kwan Human leukocyte antigen (HLA) genotyping has become a useful investigation in the diagnostic work-up of coeliac disease (CD), with utility in risk stratification and screening. However, broad application of this technology has been hindered by the cost and time burden of conventional laboratory-based assays. We have developed and validated CD–loop-mediated isothermal amplification (CD-LAMP), a LAMP assay, which enables rapid identification of the signature CD risk genotypes, HLA-DQ2.5, HLA-DQ8, HLA-DQ2.2, and HLA-DQA1*05. Sample-to-answer is achieved in approximately 65 minutes without DNA purification, thermal cycling, or specialized analytical equipment. CD-LAMP genotyping of samples was 100% concordant with accredited pathology genotyping on a panel of 40 blood and 20 saliva samples. In a panel of 100 purified DNA samples, genotyping of the high risk DQ2.5 genotype was 100% concordant with accredited pathology genotyping, with slightly reduced sensitivity for the DQ8 genotype (97.1%), and reduced specificity for the DQ8 (93.9%) and DQ2.2 genotypes (95.1%). CD-LAMP results are easily visualized, instrument free, through the addition of a DNA intercalating dye following amplification. Combined with point-of-care antibody testing, CD-LAMP may enable immediate, confident CD...
Source: The Journal of Molecular Diagnostics - Category: Pathology Source Type: research