Development of a prototype rapid method for the detection of Escherichia coli and Salmonella enterica subspecies enterica serovar Newport based on phage amplification and detection of progeny phage by Loop Mediated Isothermal Amplification
The goal of this project was to develop a rapid detection method for Escherichia coli B and Salmonella enterica subspecies enterica serovar Newport. T4 was used for the detection of E. coli B. For the purpose of detection of S. Newport, phage isolation and characterization were done. Four different phage isolates were selected and characterized by host-range analysis, transmission electron microscopy, restriction endonuclease fingerprinting, and stability under different pH and temperature conditions. A broad host-range phage called vB_SN_CGG41 that was able to infect all of the Salmonella serovars against which it was screened was selected for genome sequencing. Loop mediated isothermal amplification (LAMP) primers targeting respective unique sequences in the genomes of phages T4 and CGG41 were designed and used to detect phage nucleic acid released by boiling. Two different LAMP methods were used, which were Bioluminescent Assay in Real-Time (BART)-LAMP and endpoint colourimetric detection of pyrophosphate following conventional LAMP. Phage amplification assays (PAA) were performed with E. coli B and S. Newport to allow LAMP-based detection of phage following phage-host co-incubation.