Electrochemical monitoring of single nucleotide polymorphisms of rice varieties related to blast resistance based on PCR product and T4 DNA polymerase

Rice blast is one of the most destructive disease, which is estimated to cause 10-30% loss of global rice output annually. Single nucleotide change in bsr-d1 promoter (SNP-33 G) is conferred broad-spectrum resistance to rice blast. In this paper, we reported the construction of a relatively simple, inexpensive and ultrasensitive electrochemical DNA biosensor to detect SNP-33 G for the first time. PCR product with a 19-nt-long sticky end was generated in the presence of T4 DNA polymerase and dGTP, which was used as a powerful signal amplifying tool in the assay. The biosensor was immersed in RuHex buffer as a redox, and a high current signal could be observed due to the electrostatically bound between RuHex and the phosphate backbone of PCR product. Under optimal conditions, the electrochemical DNA biosensor exhibited satisfied performance for the determination of mutant-type of bsr-d1 promoter with a linearity ranging from 0.1 fM to 0.1 pM and a relatively low detection limit of 0.1 fM. The proposed biosensor showed excellent selectivity of SNP-33 G compared to that of wild type bsr-d1 promoter at a ratio of 1:10,000. The results provided a novel method for potential applications in variety screen and breeding for rice blast resistance.