Gold nanoparticle-based electrophoresis-free colorimetric detection method for allele-specific PCR-SNP genotyping

Conventional allele specific PCR (AS-PCR) genotyping using gel electrophoresis and ethidium bromide (EtBr) is costly, particularly in developing countries. It also poses health risks to working personnel as it requires specialized equipment and toxic dyes like EtBr. Hence, the present study developed a simple and cost-effective colorimetric genotyping method using gold nanoparticles solution (AuNPs) and unmodified primers. Specifically, 15 μl of AuNPs solution was found sufficient for detecting an amplicon in 5 μl of PCR product. In this approach, the amplified PCR products appear red while the non-amplified PCR products appear blue with a PCR mastermix without a dye. Transmission Electron Microscopy (TEM) revealed the sequestration of AuNPs in amplified PCR products and the aggregation of AuNPs in non-amplified PCR products, resulting in red and blue colors, respectively. The method was tested on genotyping of six SNPs from six genes (Akr1c3, Plg, Myf5, Sec14l2, Tpm1, and Lama2) in buffaloes, and the results were perfectly matched with those obtained using agarose gel electrophoresis analysis. Therefore, the AS-PCR combined with AuNPs provides an easy visual detection method for the amplified and non-amplified PCR products of single-nucleotide polymorphisms (SNPs). In addition, the presented method has the potential to replace agarose gel electrophoresis, the use of EtBr, and UV-transilluminator.