Real-Time PCR High-Resolution Melting Assays for the Detection of Foodborne Pathogens

Prashant Singh; Frank J Velez

doi:10.1007/978-1-0716-3358-8_4

Real-Time PCR High-Resolution Melting Assays for the Detection of Foodborne Pathogens

Methods Mol Biol. 2023:2967:41-52. doi: 10.1007/978-1-0716-3358-8_4.

Authors

Prashant Singh¹, Frank J Velez²

Affiliations

¹ Department of Nutrition, and Integrative Physiology, Florida State University, Tallahassee, FL, USA. [email protected].
² Department of Nutrition, and Integrative Physiology, Florida State University, Tallahassee, FL, USA.

PMID: 37608101
DOI: 10.1007/978-1-0716-3358-8_4

Abstract

Real-time PCR high-resolution melting assays are a method for the identification of single nucleotide polymorphisms (SNPs). The assay is performed by amplifying a short DNA fragment using a specific primer pair flanking a target SNP in the presence of a high-resolution melting dye. The HRM analysis of amplicons groups the samples based on the differences in the melting temperature and the shape of the melt curves, facilitating a convenient genotyping of samples. This chapter describes the steps and considerations of real-time PCR HRM assay standardization.

Keywords: Genotyping; Melting; Post-melt; Pre-melt; Protocol; SNP; Saturating dye.

MeSH terms

Biological Assay*
Polymorphism, Single Nucleotide*
Real-Time Polymerase Chain Reaction
Temperature