title: Most probable number - loop mediated isothermal amplification (MPN-LAMP) for quantifying waterborne pathogens in <25min.
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 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.
authors.affiliation: Department of Civil and Environmental Engineering, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Farhan
authors.initials: F
authors.lastname: Ahmad
authors.affiliation: Department of Civil and Environmental Engineering, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Robert D
authors.initials: RD
authors.lastname: Stedtfeld
authors.affiliation: Department of Civil and Environmental Engineering, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Hassan
authors.initials: H
authors.lastname: Waseem
authors.affiliation: Department of Civil and Environmental Engineering, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Maggie R
authors.initials: MR
authors.lastname: Williams
authors.affiliation: Department of Civil and Environmental Engineering, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Alison M
authors.initials: AM
authors.lastname: Cupples
authors.affiliation: The Center for Microbial Ecology, Michigan State University, East Lansing, MI 48824, USA; Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: James M
authors.initials: JM
authors.lastname: Tiedje
authors.affiliation: Department of Civil and Environmental Engineering, Michigan State University, East Lansing, MI 48824, USA; The Center for Microbial Ecology, Michigan State University, East Lansing, MI 48824, USA. Electronic address: hashsham@egr.msu.edu.
authors.forename: Syed A
authors.initials: SA
authors.lastname: Hashsham
mesh_terms: D001386:Azides / Q000737:chemistry; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000302:isolation & purification*; D013293:Enterococcus faecalis / Q000302:isolation & purification*; D004926:Escherichia coli / Q000302:isolation & purification*; D005506:Food Contamination; D005516:Food Microbiology; D005798:Genes, Bacterial; D050296:Microbial Viability; D046210:Microfluidic Analytical Techniques; D011419:Propidium / Q000031:analogs & derivatives / Q000737:chemistry; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D059629:Signal-To-Noise Ratio; D014871:Water Microbiology
chemical_list: D001386:Azides; D004269:DNA, Bacterial; C533957:propidium monoazide; D011419:Propidium
keywords: Detection limit; Loop-mediated isothermal amplification; Most probable number; Point-of-care diagnostics; Propidium monoazide