A novel method for rapid and sensitive detection of viable Escherichia coli cells using UV-induced PMA-coupled quantitative PCR.

Abstract

We report a specific and sensitive method to improve the coupling of propidium monoazide (PMA) with DNA derived from killed cells of Escherichia coli using UV light of 365nm. UV light of three different intensities mainly 2.4 × 103, 4.8 × 103, and 7.2 × 103μJ/cm2 was applied to E. coli cells each for 1, 3, and 5min. PMA was found to be successfully cross-linked with the DNA from killed cells of E. coli at 4.8 × 103μJ/cm2 in 3min leading to the complete inhibition of PCR amplification of DNA derived from PMA-treated heat-killed cells. In spiked phosphate-buffered saline and potable water samples, the difference of the Cq values between PMA-treated viable cells and PMA-untreated viable cells ranged from -0.17 to 0.2, demonstrating that UV-induced PMA activation had a negligible effect on viable cells. In contrast, the difference of the Cq values between PMA-treated heat-killed cells and PMA-untreated heat-killed cells ranged from 8.9 to 9.99, indicating the ability of PMA to inhibit PCR amplification of DNA derived from killed cells to an equivalent as low as 100CFU. In conclusion, this UV-coupled PMA-qPCR assay provided a rapid and sensitive methodology to selectively detect viable E. coli cells in spiked water samples within 4h.