Banu M, Ng D, Zheng L, Goh LT, Bi X, Ow DS. Rapid quantification of Escherichia coli in food and media using bacteriophage T7 amplification and liquid chromatography-multiple reaction monitoring tandem mass spectrometry. J Biotechnol. 2014;192 Pt A:50‐58. doi:10.1016/j.jbiotec.2014.10.017
Conventional microbiological assays have been a valuable tool for specific enumeration of indicative bacteria of relevance to food and public health, but these culture-based methods are time-consuming and require tedious biochemical and morphological identification. In this work, we exploit the ability of bacteriophage T7 to specifically infect Escherichia coli and amplify nearly a 100-fold in 1–2 h. Bacteriophage amplification is integrated with liquid chromatography-multiple reaction monitoring tandem mass spectrometry (LC-MRM–MS/MS) for quantitation of phage-specific peptides. Heavy isotopic 15N labeled T7 is introduced as the inoculum phage and internal standard. Quantification is performed by determining the ratio of phage-specific peptides over the internal standard which value is proportional to E. coli numbers. A broad dynamic range of 6-log orders ranging from 3.0 × 10(3) to 3.0 × 10(9) CFU/ml is attained in LB, while between 4.1 × 10(4)–2.7 × 10(9) CFU/ml and 1.9 × 10(3)–3.0 × 10(7) CFU/ml was enumerated respectively in coconut water and apple juice. With this method, viable E. coli are quantified in 4 h with a detection limit of 3.0 × 10(3) CFU/ml, 4.1 × 10(4) CFU/ml and 1.9 × 10(3) CFU/ml in LB, coconut water and apple juice, respectively. This method has potential as a rapid tool for detection of fecal contamination during food bioprocessing and distribution to safeguard public health.
This research is supported by Bioprocessing Technology Institute, A*STAR