Visual Detection of ACE I/D Polymorphism Using T-ARMS-PCR Combined with a Lateral Flow Assay and Its Clinical Application.

Abstract

Insertions/deletions (indels) variations have been recognized as a promising marker for the development of various diseases. However, methods used for the genotyping of indels in studies were tedious, complicated, and required sophisticated or expensive instruments, as well as complex data analysis, which makes it difficult to meet the demand of point of care testing. Herein, we presented a fast and accurate biosensor (T-ARMS-PCR-LFA) by the combination of tetra-primer amplification refractory mutation system polymerase chain reaction (T-ARMS-PCR) and GoldMag lateral flow assay (LFA) for visual genotyping of ACE I/D polymorphism. ACE I/D can be distinguished by employing four primers in one PCR reaction, and genotyping results were presented by the visual inspection of colors on the nitrocellulose membrane of LFA strips within 5 min. And 50 of the human genomic DNA samples were used for the detection of ACE I/D to further validate the accuracy of the T-ARMS-PCR-LFA system. As a demonstration, we showed that ACE I/D could be genotyped using a low amount of DNA sample (25 ng) with an accuracy of 100%, without complicated operation steps and data analysis, which is better than that of the conventional method (agarose gel electrophoresis analysis after common PCR). In conclusion, the biosensor is highly applicable for genotyping specific large indel variants in clinical practices, which enables rapid clinical decision-making, improves the management of disease diagnosis, and facilitates personalized medicine.