Most probable number - loop mediated isothermal amplification (MPN-LAMP) for quantifying waterborne pathogens in < 25 min

Abstract

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 5 min), 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 < 10 CFU 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 < 10 CFU, and time to positivity of about 20 min. MPN-LAMP assays were performed for cell concentrations in the range of 105 CFU to < 10 CFU. MPN values from LAMP assays confirmed that the amplifications were from < 10 CFU. 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.

Loop-Mediated Isothermal Amplification Assay for Identification of Clinical Enterococcus Species

“…In our study, the optimal temperatures were 60 and 61ºC for the detection of E. faecalis and E. faecium, respectively, in the LAMP assay. However, a recent study showed no difference in gene amplification conditions between Gram-negative and Gram-positive bacteria in the LAMP assay (19). In another study, the optimum temperature was 65ºC for gene amplification in the LAMP test for the detection of E. faecalis (6).…”

Challenges and perspectives in the application of isothermal DNA amplification methods for food and water analysis

“…Researchers also attempted to completely avoid nucleic acid isolation and instead apply the sample directly into the isothermal amplification mix. This has been demonstrated for plants using the example of papaya [ 15 ] and for the fecal indicator bacteria E. coli and E. faecalis [ 16 ]. The authors concluded that the elevated temperature of 63 °C could lead to increased cell permeability, allowing direct amplification of the target DNA.…”

Loop‐Mediated Isothermal Amplification (LAMP): A Rapid and Sensitive Tool for Quality Assessment of Meat Products

“…Ahmad and others () used the most probable number approach with LAMP (MPN–LAMP) for detection of Gram‐negative E. coli and Gram‐positive Enterococcus faecalis without NA extraction. This method can be applied directly to the bacterial cells, which reduces the chances of NA loss during upstream sample processing, thus it is more effective in detecting target NA.…”