Journal of Microbiological Methods 98 (2014) 122–127

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Visual endpoint detection of Escherichia coli O157:H7 using isothermal Genome Exponential Amplification Reaction (GEAR) assay and malachite green Prithiviraj Jothikumar a, Jothikumar Narayanan b,⁎, Vincent R. Hill b a b

Georgia Tech, Institute of Bioengineering and Biosciences, 315 Ferst Drive, Atlanta, GA 30332, USA Centers for Disease Control and Prevention, National Center for Emerging and Zoonotic Infectious Diseases, Waterborne Disease Prevention Branch, 1600 Clifton Road, Atlanta, GA 30329, USA

a r t i c l e

i n f o

Article history: Received 28 October 2013 Received in revised form 2 January 2014 Accepted 2 January 2014 Available online 11 January 2014 Keywords: E. coli O157:H7 Molecular assay Isothermal amplification Malachite green Water microbiology

a b s t r a c t Rapid and specific detection methods for bacterial agents in drinking water are important for disease prevention and responding to suspected contamination events. In this study, an isothermal Genome Exponential Amplification Reaction (GEAR) assay for Escherichia coli O157:H7 was designed specifically to recognize a 199-bp fragment of the lipopolysaccharide gene (rfbE) for rapid testing of water samples. The GEAR assay was found to be specific for E. coli O157:H7 using 10 isolates of E. coli O157:H7 and a panel of 86 bacterial controls. The GEAR assay was performed at a constant temperature of 65 °C using SYTO 9 intercalating dye. Detection limits were determined to be 20 CFU for the GEAR assay. When SYTO 9 fluorescence was measured using a real-time PCR instrument, the assay had the same detection limit as when malachite green was added to the reaction mix and a characteristic blue color was visually observed in positive reactions. The study also found that 50 and 20 CFU of E. coli O157:H7 seeded into 100-liter of tap water could be detected by the GEAR assays after the sample was concentrated by hollow-fiber ultrafiltration (HFUF) and approximately 10% of HFUF concentrate was cultured using trypticase soy broth–novobiocin. When applied to 19 surface water samples collected from Tennessee and Kentucky, the GEAR assay and a published real-time PCR assay both detected E. coli O157:H7 in two of the samples. The results of this study indicate that the GEAR assay can be sensitive for rapid detection of E. coli O157:H7 in water samples using fluorometric instruments and visual endpoint determination. Published by Elsevier B.V.

1. 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) (DeanNystrom 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

⁎ Corresponding author at: Centers for Disease Control and Prevention, National Center for Emerging and Zoonotic Infectious Diseases, Waterborne Disease Prevention Branch, 1600 Clifton Road NE, Mail Stop D-66, Atlanta, GA 30329, USA. E-mail address: [email protected] (J. Narayanan). 0167-7012/$ – see front matter. Published by Elsevier B.V. http://dx.doi.org/10.1016/j.mimet.2014.01.002

(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

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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.

2. Materials and methods 2.1. 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), O145:NM (1 isolate)], DAEC E. coli (1 isolate), EAEC E. coli [O126:H27 (1 isolate), O111:H2 (2 isolates) and O44:H18 (1 isolate)], EIEC E. coli (4 isolates), O22:NM (1 isolate), O28ac:NM (1 isolate), EPEC E. coli [O55:NM (1 isolate), O128:H1 (1 isolate), O111:NM (1 isolate), O127:NM (1 isolate), O55:H6 (1 isolate), O119:H6 (1 isolate), O86:H34 (1 isolate)], ETEC E. coli (4 isolates), other pathogenic E. coli [ExPEC/NMEC/UPEC; O1:H7 (1 isolate), O4-H5 (1 isolate), O6:H1 (3 isolates), O6:H31 (1 isolate), O7:NM (2 isolates), O157:H18 (1 isolate), O18:H7 (1 isolate)], and STEC E. coli [O111:H8 (2 isolates), O11:NM (1 isolate)]. Non-pathogenic E. coli used in this study were obtained from ATCC (8 isolates). Non-E. coli used in this study were: Escherichia albertii (1 isolate), Escherichia vulneris (2 isolates), Escherichia hermannii (2 isolates), Escherichia fergusonii (3 isolates), Escherichia blattae (1 isolate), Salmonella enterica subsp. enteric serovar Typhimurium (2 isolates), Yersinia pestis (1 isolate), Shigella boydii (2 isolates), Shigella dysenteriae (3 isolates), Shigella sonnei (2 isolates) and Shigella flexneri (3 isolates). After overnight culturing of bacterial isolates, 200 μL of each sample was subjected to nucleic acid extraction as described previously (Hill et al., 2007), and DNA was eluted from the silica column with 200 μL of TE buffer (pH 8.0).

2.1.1. Sensitivity of the GEAR assay A standard curve for the GEAR assay was performed using a 10-fold dilution series of DNA extracted from a stock of 8 × 108 CFU/mL of E. coli O157:H7 in nuclease free-water. The standard curve was generated using triplicate reactions of 100 to 105 CFU per reaction.

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2.1.2. Genome Exponential Amplification Reaction (GEAR) assay The GEAR assay developed in the present study was used for detecting the rfbE gene (coding for the O antigen for O157). The GEAR assay was designed using Tab primers (FT and BT) that are complementary at their 5′ end (Fig. 1A). The primer sequences for the GEAR assay and its locations are shown in (Fig. 1B and C). In a previously reported GEAR assay (Prithiviraj et al., 2012), the primers DF and DR were not included since the GEAR assay targeted a high copy number gene. In the present study, additional primers (DF and DR) were included to improve the sensitivity and speed of the reaction (Fig. 1B and C). Amplification of DNA targets was performed using an ABI 7500 instrument, but the instrument was programmed to maintain a constant temperature at 65 °C for 90 min. Fluorescence was acquired at the end of every minute and collected up to 90 min. An amount of 2 μL of DNA template was tested in 20 μL reactions. Each 20 μL reaction mix contained 10 μL of 2 × Loopamp DNA Amplification mastermix (Eiken Chemical Co., Ltd., Japan), 0.8 μL of Bst DNA polymerase (New England Biolabs, Ipswich, MA, USA), 0.8 μL of 25 μM SYTO 9 (Invitrogen, Carlsbad, CA, USA), 2 μL of DNA template, and six primers [outer displacement primers DF and DR (each at 0.2 μM), GEAR primers FT and BT (each at 1.2 μM), inner primers IR and IF (each at 1.2 μM)]. Positive and negative controls were included in each run.

2.2. Malachite green Experiments were also performed to determine the detection limit of the E. coli O157:H7 GEAR assay when malachite green was used to enable observation of positive reactions using a visual color change instead of using SYTO 9 fluorescence detection by a real-time PCR instrument. Malachite green was obtained as a 5% stock solution and further diluted with nuclease free water to obtain a stock solution of 0.2%, then stored at room temperature. The reaction mixture (20 μL reaction) was overlaid with 20 μL mineral oil. The malachite green (4 μL) was added on top of the mineral oil. Mineral oil was used to initially separate malachite green from the GEAR reaction mixture because direct addition of malachite green was associated with inhibition of the GEAR assay (data not shown). At the end of the 90 min reaction time, amplification of target DNA was determined by observation of a blue color in the reaction mix. Negative GEAR assays were associated with a colorless reaction mix.

2.3. Detection of E. coli O157:H7 in seeded tap water samples The GEAR assay was evaluated for the detection of E. coli O157:H7 seeded into 100 L volumes of tap water. The tap water samples were obtained from the study laboratory and were dechlorinated prior to seeding. The 100 L tap water samples were concentrated using UF according to the method of Hill et al. (2007). E. coli O157:H7 (ATCC 43895) was seeded into tap water samples at two different levels (50 and 20 CFU). Non-seeded 100 L control samples were also processed by UF and assayed for background E. coli O157:H7. Three replicate experiments were performed at each seeding level (including nonseeded controls). The UF procedure resulted in concentrated samples with average volumes of approximately 450 mL. To assay UF concentrates for E. coli O157:H7, 10% of the volume of the UF concentrate was filtered through a 0.45-μm pore size mixed cellulose ester membrane filter. Membrane filters were incubated separately at 37 °C in 100 mL of modified tryptic soy broth with novobiocin (Merck, Darmstadt, Germany) and agitation for 18–24 h (Mull and Hill, 2009). Following culture, the genomic DNA was obtained by direct heating. Two hundred microliters of each log-phase bacterial broth culture was subjected to heating at 95 °C for 5 min in a heating block and cell debris was removed by centrifugation at 10,000 ×g for 1 min. Two microliters of the supernatant containing DNA was added to reaction tubes.

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Fig. 1. (A) Schematic presentation of multiple GEAR primers. (B) The sequence of GEAR Tab primers (FT/BT) along with inner primers (IF and IR) and displacement primers (DF and DR) and (C) nucleotide sequences of rfbE gene and locations of primers (GenBank accession# S83460).

2.4. Application of GEAR assay to natural surface water A total of 19, 100 L water samples were collected from the Ohio River in Kentucky and from the Cheatham Reservoir in Tennessee using deadend ultrafiltration and one REXEED™-25 SX ultrafilter per sample (Mull and Hill, 2012). Fifteen water samples were collected from the Ohio River in May and July 2011, and four water samples from the Cheatham Reservoir were collected in May 2010. Ultrafilters were chilled, shipped to the analytical laboratory at CDC (Atlanta, GA), and processed within 24 h of sampling. Each ultrafilter was processed for culture and detection of E. coli O157:H7 according to the procedure of Mull and Hill (2009). Isolates from sorbitol-MacConkey agar plates, supplemented with cefixime–tellurite, were picked and DNA extracted using a noncommercial lysis buffer (Hill et al., 2007). Two microliters of DNA extract was assayed by GEAR using the SYTO 9 and malachite green methods described previously. Isolates were also tested for E. coli O157:H7 using three real-time PCR assays targeting the stx1, stx2 and rfbE genes (Mull and Hill, 2009). 3. Results 3.1. Specificity of GEAR assay

detection signals within 15–40 min, depending on the template amount tested (Fig. 2). When using malachite green, the same detection limit (80 CFU/reaction) was determined for the GEAR assay (Fig. 3). Endpoint detection assays determined that the detection limit for the GEAR assay was 20 CFU/reaction (data not shown) based on repeated positive reactions for triplicate assays. Malachite green GEAR assays of negative samples (not containing target DNA) began with a blue color but convert to colorless within 60 min. 3.3. Application of GEAR assay to seeded water samples As shown in Table 1, the GEAR assay consistently detected 20 and 50 CFU of E. coli O157:H7 in 100-L tap water samples after UF and broth enrichment. GEAR assay positive reactions were observed rapidly, within 12 to 17 min. In the three non-seeded water concentration experiments performed, no E. coli O157:H7 was detected in the water samples. The same GEAR assay detection results were obtained when malachite green was used and the characteristic color change observed. All positive reactions remained blue whereas negative samples and no template controls turned colorless. 3.4. Application of GEAR assay to natural water samples

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 nonE. coli isolates or the Shigella spp. isolates.

Of the 19 surface water samples collected, isolates from two of the samples collected in Kentucky were determined to be positive for E. coli O157:H7 using the GEAR assay when SYTO 9 was used and when malachite green was used to enable visual determination of target GEAR product formation (Table 2). The same results (2 of 19 samples positive for E. coli O157:H7) were obtained when the isolates were analyzed using real-time PCR assays targeting the stx1, stx2 and rfbE genes.

3.2. Sensitivity of GEAR assay

4. Discussion

The sensitivity of the GEAR assay was determined for a 5-log10 dilution series of E. coli O157:H7 DNA covering a template range corresponding to 8 × 100 to 8 × 105 CFU/reaction. The GEAR assay exhibited a linear standard curve for the entire dilution series (Fig. 2A). The coefficient of determination (0.9826) indicated that the GEAR assay had linear correlation between amplification time and cell numbers (Fig. 2B). For a template range corresponding to 8 × 10 1 to 8 × 105 CFU/reaction, the GEAR assay produced positive

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 GEAR assay when malachite green was used instead of

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Fig. 2. The detection sensitivity of the GEAR assay for E. coli O157:H7 (ATCC 43895). (A) Amplification plot for ten-fold serial dilutions of DNA templates for E. coli O157:H7 (800,000 to 8 CFU/reaction); (B) the log plot shows the initial CFU values plotted in X-axis and the time plotted in Y-axis.

SYTO 9 and positive reactions were determined by observation of a characteristic blue color. Colorimetric measurement of pyrophosphate and inorganic phosphate has been previously reported using malachite green (Baykov

et al., 1988; Itaya and Ui, 1966; Van Veldhoven and Mannaerts, 1987). The property of decolorization associated with malachite green has also been used to identify drug-resistant Mycobacterium tuberculosis (Farnia et al., 2008). During positive GEAR assays, the use of malachite

Fig. 3. Visual detection of GEAR assay products for detection of E. coli O157:H7 with malachite green. (A) The upper panel shows ten-fold serial dilutions of DNA for E. coli O157:H7 (800,000 to 8 CFU/reaction) before reaction commencement: label numbers 1–6 correspond to 800,000, 80,000, 8000, 800, 80 and 8 CFU/reaction, respectively; 7 and 8 represent no template control; (B) the lower panel shows color changes for positive and negative samples after isothermal amplification reaction completed.

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Table 1 Performance of GEAR assay for the rapid detection of E. coli O157:H7 seeded in 100 L water samples after enrichment. Seed level

Ct values (min)a

Malachite green

0 CFU/100 L (control) 20 CFU/100 L 50 CFU/100 L

No amplification 15.3 ± 1.5 5.7 ± 0.58

Negative Positive Positive

a Average and standard deviation for three experiments. Negative and positive represent visual inspection of blue color and colorlessness (lack of blue color), respectively, in reaction tubes.

products for the visible color change as a result of accumulation of magnesium pyrophosphate in the tube when DNA is amplified. The simplicity of the visual endpoint detection procedure, widespread and inexpensive availability of malachite green, and effectiveness when performed using low-cost incubation equipment (e.g., water bath, heat block) raise the potential for the GEAR method to be readily implemented in developed and developing countries. This GEAR assay reported in this study should be useful for monitoring water samples and other environmental samples for the presence of E. coli O157:H7. Competing interests

green is also associated with blue color; in negative reactions the GEAR product is colorless. Thus, GEAR can be used to identify positive reactions without opening reaction tubes. Several studies have reported the contamination of water samples with E. coli O157:H7 (Halabi et al., 2008; Holme, 2003; Lejeune et al., 2001; Liu et al., 2008; Schets et al., 2005). Most studies have processed 100 mL to 1 L sample volumes, but one study processed 40-L samples for E. coli O157:H7 detection. In the present study, E. coli O157:H7 levels as low as 20 CFU in 100 L could be detected using the GEAR assay after UF and enrichment of 10% of the UF-concentrated tap water sample. When applied to cultures from natural water samples, the E. coli O157:H7 GEAR assay, whether performed using SYTO 9 or malachite green, yielded the same number of detections as obtained using published real-time PCR assays. A previous study by Mull and Hill (2009) reported a detection limit of 50 CFU per 40 L surface water sample after UF, broth enrichment, immunomagnetic separation (IMS), and agar culture. The study by Mull and Hill (2009) used a suite of three real-time PCR assays to detect E. coli O157:H7, whereas the present study used an isothermal molecular amplification assay (GEAR) and applied this assay directly to the culture broth by simple heating, as opposed to suspect colonies on an agar plate. Thus, the specificity of the assays targeting the rfbE gene enabled specific detection of E. coli O157:H7 in water samples after a one-step broth culture. The present study also demonstrates that the GEAR assay can be as effective as the other published molecular assays when applied to broth cultures (Hsu et al., 2005). The GEAR technique represents a relatively simple method, performed using six primers and without the requirement of an expensive fluorophore-labeled probe. Other benefits of the GEAR technique include its ease for implementation in resourcelimited laboratories and field labs (Njiru, 2012), and its ability to be performed with simple equipment such as a heating block or water bath that can maintain a constant temperature. The GEAR method does require the use of six primers and isothermal amplification mastermix, but the costs of these reagents should be similar to other closed-tube molecular methods, such as real-time PCR, that are used for specific identification of microbes. The costs for direct analysis of samples using the GEAR method (without culture) would be similar to or less than the costs for culture identification of E. coli O157:H7, which generally requires relatively expensive immunomagnetic separation reagents and chromogenic agar. The development of a simple visual endpoint detection method using malachite green enables the GEAR technique to be considered as meeting WHO recommendations (Mabey et al., 2004) for developing and implementing diagnostic tools that are suitable for resource-limited communities because they are affordable, sensitive, specific, user-friendly, robust, rapid, equipment-free, and deliverable to end users (ASSURED). In addition to observing positive reactions using an intercalating dye in a real-time PCR instrument, positive GEAR reactions could also be determined by observing reaction

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. References Bai, J., Shi, X., Nagaraja, T.G., 2010. A multiplex PCR procedure for the detection of six major virulence genes in Escherichia coli O157:H7. J. Microbiol. Methods 82, 85–89. Baykov, A.A., Evtushenko, O.A., Avaeva, S.M., 1988. A malachite green procedure for orthophosphate determination and its use in alkaline phosphatase-based enzyme immunoassay. Anal. Biochem. 171, 266–270. Beutin, L., Jahn, S., Fach, P., 2009. Evaluation of the ‘GeneDisc’ real-time PCR system for detection of enterohaemorrhagic Escherichia coli (EHEC) O26, O103, O111, O145 and O157 strains according to their virulence markers and their O- and H-antigenassociated genes. J. Appl. Microbiol. 106, 1122–1132. Carey, C.M., Kostrzynska, M., Thompson, S., 2009. Escherichia coli O157:H7 stress and virulence gene expression on Romaine lettuce using comparative real-time PCR. J. Microbiol. Methods 77, 235–242. Cebula, T.A., Payne, W.L., Feng, P., 1995. Simultaneous identification of strains of Escherichia coli serotype O157:H7 and their Shiga-like toxin type by mismatch amplification mutation assay-multiplex PCR. J. Clin. Microbiol. 33, 248–250. Couturier, M.R., Lee, B., Zelyas, N., Chui, L., 2011. Shiga-toxigenic Escherichia coli Detection in stool samples screened for viral gastroenteritis in Alberta Canada. J. Clin. Microbiol. 49, 574–578. Dean-Nystrom, E.A., Bosworth, B.T., Moon, H.W., O'Brien, A.D., 1998. Escherichia coli O157: H7 requires intimin for enteropathogenicity in calves. Infect. Immun. 66, 4560–4563. Farnia, P., Masjedi, M.R., Mohammadi, F., Tabarsei, P., Farnia, P., Mohammadzadeh, A.R., Baghei, P., Varahram, M., Hoffner, S., Velayati, A.A., 2008. Colorimetric detection of multidrug-resistant or extensively drug-resistant tuberculosis by use of malachite green indicator dye. J. Clin. Microbiol. 46, 796–799. Fratamico, P.M., Bagi, L.K., Cray, W.C., Narang, N., Yan, X., Medina, M., Liu, Y., 2011. Detection by Multiplex real-time polymerase chain reaction assays and isolation of Shiga toxin-producing Escherichia coli serogroups O26, O45, O103, O111, O121, and O145 in ground beef. Foodborne Pathog. Dis. 8, 601–607. Friedrich, A.W., Borell, J., Bielaszewska, M., Fruth, A., Tschape, H., Karch, H., 2003. Shiga toxin 1c-producing Escherichia coli strains: phenotypic and genetic characterization and association with human disease. J. Clin. Microbiol. 41, 2448–2453. Gould, L.H., Walsh, K.A., Vieira, A.R., Herman, K., Williams, I.T., Hall, A.J., Cole, D., 2013. Surveillance for foodborne disease outbreaks — United States, 1998–2008. Morbidity and mortality weekly report. Surveill. Summ. 62, 1–34. Halabi, M., Orth, D., Grif, K., Wiesholzer-Pittl, M., Kainz, M., Schoberl, J., Dierich, M.P., Allerberger, F., Wurzner, R., 2008. Prevalence of Shiga toxin-, intimin- and haemolysin genes in Escherichia coli isolates from drinking water supplies in a rural area of Austria. Int. J. Hyg. Environ. Health 211, 454–457. Hill, V.R., Kahler, A.M., Jothikumar, N., Johnson, T.B., Hahn, D., Cromeans, T.L., 2007. Multistate evaluation of an ultrafiltration-based procedure for simultaneous recovery of enteric microbes in 100-liter tap water samples. Appl. Environ. Microbiol. 73, 4218–4225. Hlavsa, M.C., Roberts, V.A., Anderson, A.R., Hill, V.R., Kahler, A.M., Orr, M., Garrison, L.E., Hicks, L.A., Newton, A., Hilborn, E.D., Wade, T.J., Beach, M.J., Yoder, J.S., 2011. Surveillance for waterborne disease outbreaks and other health events associated with

Table 2 Detection of E. coli O157:H7 in natural surface water samples using GEAR and real-time PCR assays. Sampling area

No. of samples

No. positive using real-time PCR

No. positive using GEAR (SYTO 9)

No. positive using GEAR (malachite green)

Cheatham Reservoir, Tennessee Ohio River, Kentucky

4 15

0/4 2/15

0/4 2/15

0/4 2/15

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Visual endpoint detection of Escherichia coli O157:H7 using isothermal Genome Exponential Amplification Reaction (GEAR) assay and malachite green.

Rapid and specific detection methods for bacterial agents in drinking water are important for disease prevention and responding to suspected contamina...
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