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T10-04 The Escherichia coli Common Pilus: A Diagnostic Target for Point-of-Need LAMP Assays Detecting the Fecal Indicator E. coli

Wednesday, July 25, 2012: 9:15 AM
Ballroom E (Rhode Island Convention Center)
Jeffrey Chandler, Colorado State University, Fort Collins, CO
Alma Perez-Mendez, Colorado State University, Fort Collins, CO
Bledar Bisha, Colorado State University, Fort Collins, CO
Shannon Coleman, Colorado State University, Fort Collins, CO
Lawrence Goodridge, Colorado State University, Fort Collins, CO
Introduction:  Pathogens derived from fecal contamination of food and water pose significant public health concerns, yet assays to identify fecal indicator bacteria (FIB) at the point-of-need are typically limited to slow culture based techniques.  Molecular assays can accelerate the identification of FIB, but currently these assays are not conducive to point-of-need detection because of their expense, need for specialized equipment, and need for highly trained personnel.  Thus, new molecular detection technologies are needed for FIB identification.

Purpose:  Here, we describe a field-capable molecular detection platform to rapidly identify the FIB Escherichia coli in irrigation water and seawater.  This platform relies on an advanced nucleic acid amplification strategy, loop-mediated isothermal amplification (LAMP), and field-ready instrumentation to monitor the progression of LAMP reactions.

Methods:  A LAMP assay was developed targeting a portion of ecpA, the major pilus subunit of the E. coli common pilus (ECP), and evaluated with genomic DNA from 15 isolates of pathogenic E. coli (representing six common Shiga toxin-producing E. coli serotypes, as well as O104:H4, and O157:H7).  Efficacy of the LAMP reaction was also tested by detecting naturally present E. colifiltered from seawater (obtained from oyster beds) and spiked irrigation water.

Results:  LAMP reactions targeting ecpA reliably detected 1 pg of genomic DNA from all isolates tested, the equivalent of approximately 2.5 × 102 bacteria, in less than 25 minutes.  Detection times were dependent on the concentration of template DNA (100 ng of DNA was detectable in less than 13 minutes), thus allowing for semi-quantitative estimates of bacterial contamination.  Further, LAMP detection of E. coliwas successful in filtrates of seawater and spiked irrigation water.

Significance:  These results demonstrate the feasibility of utilizing the ECP of E. coli as a diagnostic target, and the studies presented here provide the foundation for the development of rapid, point-of-need, LAMP assays to evaluate fecal contamination of food and water.

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