Rapid and ultrasensitive point of care test for SLCO1B1 rs4149056 SNP detection using RNase H2 assisted Tetra-ARMS...

Single nucleotide polymorphisms (SNPs) are critical genetic variations that play an essential role in disease diagnosis, treatment, and personalized medicine. Traditional SNPs detection methods, including DNA sequencing and TaqMan real-time PCR, offer reliable results but are often limited by complexity, time requirements, and the need for expensive equipment. These constraints make them less suitable for point-of-care testing (POCT) in resource-limited environments. In this study, we utilized the precise recognition capabilities of RNase H2 for SNP sites. Based on this specificity, we developed an ultrasensitive (10 copies/reaction) and rapid (within 30 min) innovative method combining RNase H2-assisted Tetra-ARMS RPA (RTAR) with lateral flow strip (LFS) technology for the detection of rs4149056 SNP in the SLCO1B1 gene. SNP sites were amplified isothermally in distinct orientations, with each site assigned unique labels for precise identification. The clinical sample genotypes (TT, TC or CC) were then determined by observing the color changes on the T1 and T2 lines of the lateral flow assay, providing a clear and reliable method for genotype differentiation. We validated RTAR-LFS by detecting 30 clinical blood samples in assessing the lipid-lowering efficacy of statin therapy, demonstrating a sensitivity of 92.5 % and a specificity of 100 %. In addition, we further confirmed its applicability across multiple specimen types, including dried blood spots, buccal swabs, and saliva samples. These results highlight the great potential of RTAR-LFS for specific and sensitive SNP detection at low cost, particularly in resource-limited regions.