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

Michael Erlichster; Jason A Tye-Din; Michael D Varney; Efstratios Skafidas; Patrick Kwan

doi:10.1016/j.jmoldx.2018.01.005

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

J Mol Diagn. 2018 May;20(3):307-315. doi: 10.1016/j.jmoldx.2018.01.005. Epub 2018 Feb 17.

Authors

Michael Erlichster¹, Jason A Tye-Din², Michael D Varney³, Efstratios Skafidas⁴, Patrick Kwan⁵

Affiliations

¹ Centre for Neural Engineering, University of Melbourne, Melbourne, Victoria, Australia; Department of Medicine, University of Melbourne, Melbourne, Victoria, Australia.
² Immunology Division, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia; Department of Medical Biology, University of Melbourne, Melbourne, Victoria, Australia; Murdoch Children's Research Institute, Melbourne, Victoria, Australia; Department of Gastroenterology, The Royal Melbourne Hospital, Melbourne, Victoria, Australia.
³ Victorian Transplantation and Immunogenetics Service, Australian Red Cross Blood Service, Melbourne, Victoria, Australia.
⁴ Centre for Neural Engineering, University of Melbourne, Melbourne, Victoria, Australia; Department of Electrical and Electronic Engineering, University of Melbourne, Melbourne, Victoria, Australia; The Florey Institute of Neuroscience and Mental Health, Melbourne, Victoria, Australia.
⁵ Department of Medicine, University of Melbourne, Melbourne, Victoria, Australia; Department of Electrical and Electronic Engineering, University of Melbourne, Melbourne, Victoria, Australia; Department of Neurology, The Royal Melbourne Hospital, Melbourne, Victoria, Australia. Electronic address: patrick.kwan@unimelb.edu.au.

PMID: 29458095
DOI: 10.1016/j.jmoldx.2018.01.005

Abstract

Human leukocyte antigen (HLA) genotyping has become a useful investigation in the diagnostic work-up of celiac 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 (95.1%) genotypes. CD-LAMP results are easily visualized and instrument free through the addition of a DNA intercalating dye after amplification. Combined with point-of-care antibody testing, CD-LAMP may enable immediate, confident CD diagnosis at a low cost in the clinical setting.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Alleles*
Base Sequence
Celiac Disease / genetics*
DNA / genetics
Genetic Predisposition to Disease*
Humans
Nucleic Acid Amplification Techniques / methods*
Reproducibility of Results
Risk Factors
Sensitivity and Specificity

Substances

DNA