Abstract
Conventional methods of detecting economically essential mutations have several disadvantages. Even though fluorescence-based methods are still the best option, Surface-Enhanced Raman Spectroscopy (SERS) may soon emerge as an alternative to the current techniques for detecting these mutations, because of its ability to detect molecular vibrational signatures. We were able to identify and develop a PCR-based SERS assay that can relentlessly differentiate between different types of indels, and SNPs as demonstrated in the case of tomato genome related to tomato yellow leaf curl virus and root-knot nematodes, diseases that are economically significant to the global agriculture industry and where the selection of resistant crops is the best solution. This tri-primer assay utilizes mutation-specific forward primers and SERS probes tagged with FAM and Cy3 dyes, specific for each allele of a particular gene (Ty-3 and Mi-1). The unique Raman spectral features of these dyes enabled to perform of multiplexing, which made it possible to detect not only the indel type but also the zygosity in a single experiment. Moreover, this technique successfully differentiated between two different SNP-based alleles. Therefore, due to its efficient multiplexing capability and lack of the need for quenchers, it has the potential to become a powerful onsite and offsite screening tool in the not-too-distant future.