Centre for Neural Engineering, University of Melbourne, Melbourne, Victoria, Australia; Department of Medicine, University of Melbourne, Melbourne, Victoria, Australia.
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.
J Mol Diagn. 2018 May;20(3):307-315. doi: 10.1016/j.jmoldx.2018.01.005. Epub 2018 Feb 17.
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.
人类白细胞抗原 (HLA) 基因分型已成为乳糜泻 (CD) 诊断工作中的一项有用的检查，可用于风险分层和筛查。然而，由于传统实验室检测方法的成本和时间负担，这项技术的广泛应用受到了阻碍。我们开发并验证了 CD 环介导等温扩增 (CD-LAMP)，这是一种 LAMP 检测方法，可快速鉴定 CD 风险基因型 HLA-DQ2.5、HLA-DQ8、HLA-DQ2.2 和 HLA-DQA1*05。无需 DNA 纯化、热循环或专用分析设备，即可在大约 65 分钟内实现样本到答案。对 40 个血液和 20 个唾液样本的面板进行 CD-LAMP 基因分型，与认证病理基因分型的结果完全一致。在 100 个纯化 DNA 样本的面板中，高风险 DQ2.5 基因型的基因分型与认证病理基因分型完全一致，DQ8 基因型的敏感性略低（97.1%），DQ8（93.9%）和 DQ2.2（95.1%）基因型的特异性降低。通过在扩增后添加 DNA 嵌入染料，可轻松可视化和无仪器查看 CD-LAMP 结果。结合即时抗体检测，CD-LAMP 可能会使临床环境中的即时、自信的 CD 诊断得以实现，且成本低廉。
