Most probable number - loop mediated isothermal amplification (MPN-LAMP) for quantifying waterborne pathogens in...

We are reporting a most probable number approach integrated to loop mediated isothermal technique (MPN-LAMP) focusing on Gram-negative Escherichia coli and Gram-positive Enterococcus faecalis bacterial cells without nucleic acids extraction. LAMP assays for uidA from E. coli and gelE from E. faecalis were successfully performed directly on cells up to single digit concentration using a commercial real time PCR instrument. Threshold time values of LAMP assays of bacterial cells, heat treated bacterial cells (95°C for 5min), and their purified genomic DNA templates were similar, implying that amplification could be achieved directly from bacterial cells at 63°C. Viability of bacterial cells was confirmed by using propidium monoazide in a LAMP assay with E. faecalis. To check its functionality on a microfluidic platform, MPN-LAMP assays targeting <10CFU of bacteria were also translated onto polymeric microchips and monitored by a low-cost fluorescence imaging system. The overall system provided signal-to-noise (SNR) ratios up to 800, analytical sensitivity of <10CFU, and time to positivity of about 20min. MPN-LAMP assays were performed for cell concentrations in the range of 10⁵CFU to <10CFU. MPN values from LAMP assays confirmed that the amplifications were from <10CFU. The method described here, applicable directly on cells at 63°C, eliminates the requirement of complex nucleic acids extraction steps, facilitating the development of sensitive, rapid, low-cost, and field-deployable systems. This rapid MPN-LAMP approach has the potential to replace conventional MPN method for waterborne pathogens.