Development of a multiplex real-time PCR assay for phylogenetic groups analysis of Escherichia coli strains for the first time

Background and Objectives: In 2000 a simple triplex PCR method was described by Clermont that E. coli strains could fall into four main groups A, B1, B2 and D. The growing body of multi-locus sequence type (MLST) and genome data for E. coli has refined our understanding of E. coli’s phylo-group structure. In 2013 Clermont and colleagues classified E. coli strains into eight new phylogenetic groups (A, B1, B2, C, D, E, F and clade I) using a new quadruplex PCR method. This study for the first time was aimed to develop a multiplex real-time PCR assay with melting curve analysis for the detection of phylogenetic groups of E.coli. Materials and Methods: In this study, seven primer pairs were used for optimizing the multiple real-time PCR protocol. At first, optimization of each singleplex real-time PCR assay was performed. Singleplex real-time PCR assays for each seven target was performed in a 20 µL reaction volume containing 4 µL of 5X HOT FIREPol EvaGreen® qPCR Mix Plus (ROX), 0.2-1 µM of primers and 80-120 ng Genomic DNA. Results: We determined seven distinct melting peaks for each seven targets that result in the Tm identification of the amplicons. Seven targets were discriminated with Tm value of 93±0.8 for arpA, 89.3 for chuA, 86.5 for yjaA, 82.2±0.2 for TspE4C2, 87.8 for trpAgpC, 90.8±0.5 for trpBA and 85.5±0.5. We also confirmed the specific amplifications by detection of the specific bands in electrophoresis. Conclusion: Our findings showed melting curve-based multiplex real-time PCR could be a fast and easy assay for phylo-typing of E. coli strains.

Escherichia coli, Phylogenetic groups, Real-time PCR, Melting curve