Rapid MPN-Qpcr Screening for Pathogens in Air, Soil, Water, and Agricultural Produce

Abstract

A sensitive, high-throughput, and costeffective method for screening bacterial pathogens in the environment was developed. A variety of environmental samples, including aerosols, soil of various types (sand, sand/clay mix, and clay), wastewater, and vegetable surface (modeled by tomato), were concomitantly spiked with Salmonella enterica and/or Pseudomonas aeruginosa to determine recovery rates and limits of detection. The various matrices were first enriched with a general pre-enrichment broth in a dilution series and then enumerated by most probable number (MPN) estimation using quantitative PCR for rapid screening of amplicon presence. Soil and aerosols were then tested in non-spiked environmental samples, as these matrices are prone to large experimental variation. Limit of detection in the various soil types was 1-3 colony-forming units (CFU) g −1 ; on vegetable surface, 5 CFU per tomato; in treated wastewater, 5 CFU L −1 ; and in aerosols, >300 CFU mL −1 . Our method accurately identified S. enterica in non-spiked environmental soil samples within a day, while traditional methods took 4 to 5 days and required sorting through biochemically and morphologically similar species. Likewise, our method successfully identified P. aeruginosa in non-spiked aerosols generated by a domestic wastewater treatment system. The obtained results suggest that the developed method presents a broad approach for the rapid, efficient, and reliable detection of relatively low densities of pathogenic organisms in challenging environmental samples.

Optimized Co-extraction and Quantification of DNA From Enteric Pathogens in Surface Water Samples Near Produce Fields in California

“…Typical resuscitation (enrichment) will produce an unknown number of cell divisions depending on the cell physiology, making direct plating unsuitable for quantification. Quantification methods are available which utilize enrichment and Most Probable Number (MPN) determination by either culture methods or PCR methods (Mcegan et al, 2013 ; Orlofsky et al, 2015 ; Benami et al, 2016 ). However these methods are labor intensive, especially if a large number of samples is involved.…”

Genome-Enhanced Detection and Identification (GEDI) of plant pathogens

“…The conceptual and practical simplicity of qPCR, in combination with its speed and sensitivity, have made it the technology of choice in many diagnostic applications, including microbial quantification ( Yu et al, 2005 ; Narihiro & Sekiguchi, 2011 ; Thonar, Erb & Jansa, 2012 ) and pathogen detection ( Orlofsky et al, 2015 ). Multiple qPCR assays have become extremely powerful to detect fungi, bacteria and parasites ( Kamau et al, 2014 ; Gosiewski et al, 2014 ; Bilodeau et al, 2009 ) and we believe that this pipeline should enable development of qPCR assays in crop and tree pathogens.…”

Peer Review #1 of "Genome-Enhanced Detection and Identification (GEDI) of plant pathogens (v0.1)"

“…The conceptual and practical simplicity of qPCR, in combination with its speed and sensitivity, have made it the technology of choice in many diagnostic applications, including microbial quantification (Yu et al, 2005;Narihiro and Sekiguchi, 2011;Thonar et al, 2012) and pathogen detection (Orlofsky et al, 2015). Multiple qPCR assays have become extremely powerful to detect fungi, bacteria and parasites (Kamau et al, 2014;Gosiewski et al, 2014 and we believe that this pipeline should enable development of qPCR assays in crop and tree pathogens.…”