Discrimination of a single nucleotide polymorphism in the haptoglobin promoter region, rs5472, using a competitive fluorophore-labeled probe hybridization assay following loop-mediated isothermal amplification

Abstract

Personalized peptide vaccination, which involves activation of the host immune system against cancer cells using personalized peptide vaccines (PPVs), can improve overall survival in multiple cancer types. However, the clinical efficacies of PPVs vary for unknown reasons. Recently, a single nucleotide polymorphism (NG_012651.1:g.4461_5460[4960A>G]) in the haptoglobin promoter region, rs5472, was significantly associated with clinical response of PPV. Therefore, rs5472 is expected to be a predictive biomarker for PPV therapy. Here, we described a single nucleotide discrimination method for rs5472 analysis by combining the loop-mediated isothermal amplification and quenching probe methods. In evaluation of saliva samples, this method showed high concordance with the results of Sanger sequencing (100%, n = 36). Importantly, this method did not require calculation of melting temperature for single nucleotide discrimination and could therefore be carried out on a simple instrument. Accordingly, this method may be more robust and applicable to near-patient testing.

Generic Platform for the Multiplexed Targeted Electrochemical Detection of Osteoporosis-Associated Single Nucleotide Polymorphisms Using Recombinase Polymerase Solid-Phase Primer Elongation and Ferrocene-Modified Nucleoside Triphosphates

“…Isothermal amplification has been exploited for the detection of SNPs, including approaches exploiting loop-mediated isothermal amplification (LAMP) for the genotyping of blood and buccal cells as well as an approach using loop-primer endonuclease cleavage (LEC)-LAMP for the detection of SNPS in Neisseria meningitidis . Further examples include a competitive fluorophore-labeled probe hybridization assay following LAMP, which was applied to the detection of an SNP associated with the clinical response of personalized peptide vaccination in saliva samples . In another report, the use of loop-primer LAMP was used for the detection of SNPs in Salmonella enterica serovar Gallinarum biovars Pullorum and successfully applied in real sample testing of embryos, livers, and anal swabs from chickens in poultry farms .…”

“… 35 Further examples include a competitive fluorophore-labeled probe hybridization assay following LAMP, which was applied to the detection of an SNP associated with the clinical response of personalized peptide vaccination in saliva samples. 36 In another report, the use of loop-primer LAMP was used for the detection of SNPs in Salmonella enterica serovar Gallinarum biovars Pullorum and successfully applied in real sample testing of embryos, livers, and anal swabs from chickens in poultry farms. 37 LAMP has also been used for the duplex detection of Factor V Leiden and Factor II G20210A variants in whole blood samples 38 in a molecular beacon LAMP format for the detection of BRAF V600E, 39 and further examples of the use of LAMP for the detection of SNPs has been comprehensively reviewed.…”