In silico Comparison of Real-Time PCR Probes for Detection of Pathogens

Abstract

Rapid diagnostics and risk assessment of the pathogens is possible by Real-Time Polymerase Chain Reaction (PCR) probes like TaqMan, Molecular Beacon (MB) and FRET. However, validation of such probes for real-life samples is an expensive and time consuming proposition. Hence, development and comparison of real-time probes in silico can be the first step in selection of most appropriate probe chemistry. The virulence genes specific for a model pathogen, Escherichia coli O157:H7, transmitted worldwide by contaminated water and food, were chosen to compare probe chemistries. MB was observed to be the best probe chemistry for virulence genes stx1, stx2 and eae, while FRET was preferred for hlyA gene, based on T $_{m}$ and free energy values for self-dimer, hairpin and cross-dimer. Secondary structure analysis indicated that MB design was flexible and less dependent on nucleotide arrangement and repetitive sequences in the genes compared to TaqMan and FRET probes. In addition, multiplexed MB probes could be a feasible option using a single non-fluorescent quencher for high throughput diagnostics.