Visual endpoint detection of Escherichia coli O157:H7 using isothermal Genome Exponential Amplification Reaction (GEAR) assay and malachite green
Introduction
Escherichia coli O157:H7 is a bacterial pathogen that can be transmitted through food and water (Gould et al., 2013, Hlavsa et al., 2011). E. coli O157:H7 infection is typically associated with bloody diarrhea and can cause hemolytic uremic syndrome (HUS), especially in the young and immunocompromised individuals (Sanchez et al., 2010). E. coli O157:H7 are gram negative bacteria that produce shiga toxin (stx) and are often referred as shiga-toxin E. coli (STEC) (Dean-Nystrom et al., 1998) or enterohemorrhagic E. coli (EHEC) (Cebula et al., 1995). A small percentage of other shiga toxin producing serotypes of E. coli, including O26 (stx1 and stx2), O45 (stx1), O103 (stx2 and eaeA), O111 (stx1, stx2, and eaeA), O121 (stx1 and eaeA) and O145 (stx2) also cause similar illness (Beutin et al., 2009, Couturier et al., 2011, Fratamico et al., 2011, Madic et al., 2010, Madic et al., 2011, O'Hanlon et al., 2004). Other shiga-toxin producing serotypes of E. coli O91, O113 and O128 also cause HUS and bloody diarrhea (Friedrich et al., 2003, Kappeli et al., 2011, Orden et al., 1998). With such a diversity of serotypes potentially present in food and environmental samples, it is important to distinguish E. coli O157:H7 from other bacteria using appropriate virulence markers. The molecular identification of E. coli O157:H7 often targets the marker genes of stx1 (shiga toxin 1), stx2 (shiga toxin 2), eaeA (intimin gene — A/E lesions widely present in enteropathogenic E. coli), hly (60 mDa plasmid pO157 encoding enterohemolysin gene), rfbE (O antigen cluster — locus containing lipopolysaccharide gene present in E. coli O157 serogroup) and fliC (H7 flagellin gene present in all serotypes of H7 serogroup) (Prendergast et al., 2011, Wang et al., 2002). Assays for specific detection of E. coli O157:H7 often require screening for virulence markers of stx1, stx2, hlyA and eaeA, and serotype specific markers of rfbE and fliC genes (Bai et al., 2010).
Real-time PCR based rapid detection of E. coli O157:H7 has been reported for several marker genes individually or in multiplex format for confirmation (Carey et al., 2009, Jothikumar and Griffiths, 2002, Oberst et al., 1998, Singh et al., 2009, Suo et al., 2010). Real-time PCR machines, and associated reagents, are relatively expensive, which limits the implementation of molecular testing in resource-limited laboratories. Other molecular techniques have been reported for the rapid identification of E. coli O157:H7 that are based on isothermal amplifications such as nucleic acid based amplification (NASBA) (Won and Min, 2010), ramification amplification (Li et al., 2005) and loop mediated isothermal amplification (LAMP) (Ohtsuka et al., 2010, Zhao et al., 2010). The GEAR technique offers advantages over established isothermal amplification methods, such as the widely used loop mediated isothermal amplification (LAMP) assay. The GEAR technique differs from the LAMP technique in that the pair of core GEAR primers (FT and BT) targets three regions (Prithiviraj et al., 2012), while the pair of core LAMP primers (FIP and BIP) targets four regions (Notomi et al., 2000). Additionally, the reagent, malachite green, can be used in conjunction with GEAR to enable visible observation of positive GEAR assays. Isothermal amplification of target nucleic acids can be performed in a water bath or a heat-block that maintains a constant temperature at 65 °C and the amplified products result in color change that can be visually observed without the need for any reader.
The objective of the present study was to develop a rapid isothermal molecular assay method using the GEAR technique for rapid detection of E. coli O157:H7. The GEAR assay was applied to the detection of E. coli O157:H7 in 100 L drinking water samples concentrated by tangential flow ultrafiltration (UF). Previous studies have investigated molecular detection of E. coli O157:H7 in water (Mull and Hill, 2009), but no previous study has investigated the use of isothermal amplification methods for detection of this pathogen in water. The study incorporated experiments designed to determine the method detection limit for recovery of E. coli O157:H7 in 100-L drinking water samples using hollow-fiber ultrafiltration (HFUF) and broth culture, followed by application of the GEAR assay for E. coli O157:H7.
Section snippets
Bacterial strains
The specificity of the E. coli O157:H7 assay was determined using a panel of 86 bacterial isolates, including 56 isolates representing a diverse array of pathogenic serotypes. Pathogenic E. coli isolates used for the study were previously characterized by the CDC's National Escherichia coli Reference Laboratory, as follows: EHEC E. coli O157:H7 (10 isolates), EHEC non-E. coli O157:H7 [O26:H11 (3 isolates); O103:H2 (1 isolate), O121:H19 (2 isolates), O45:H2 (3 isolates), O60:H8 (1 isolate),
Specificity of GEAR assay
The primers for the GEAR assay were designed to target the rfbE gene, which codes for an O-antigen biosynthesis enzyme that is highly conserved among E. coli O157:H7. Specificity testing indicated that the GEAR assay was 100% specific for detection of E. coli O157:H7. DNA from all 10 E. coli O157:H7 isolates was amplified, but the GEAR assay did not amplify DNA from the 45 isolates of non-O157:H7 pathogenic E. coli, the 8 isolates of non-pathogenic E. coli isolates, or the 22 non-E. coli
Discussion
The present study reports the development of a novel real-time isothermal amplification based molecular assay (GEAR) targeting the rfbE gene for specific and sensitive detection of E. coli O157:H7. The GEAR assay was found to be 100% specific to E. coli O157:H7 using a panel of 86 bacterial isolates. The detection limit of the GEAR assay was found to be 20 CFU/reaction when SYTO 9 was used and fluorescence detected using a real-time PCR instrument. The same detection limit was determined for the
Competing interests
The authors are inventors on a pending US patent application and international application covering GEAR assay.
Acknowledgments
The findings and conclusions in this report are those of the authors and do not necessarily represent those of the CDC. Use of trade names and commercial sources is for identification only and does not imply endorsement by CDC or the U.S. Department of Health and Human Services.
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