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2018 High-Resolution Melt Curve PCR Assay for Specific Detection of E. coli O157:H7 in Beef Yuejiao Liu, Prashant Singh and Azlin Mustapha
This is the accepted manuscript, and the version of record can be found at https://doi.org/10.1016/j.foodcont.2017.11.025.
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8 High-Resolution Melt Curve PCR Assay for Specific Detection of E. coli O157:H7 in Beef
9 Yuejiao Liu1, Prashant Singh2, Azlin Mustapha1*
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11 Food Science Program, University of Missouri, Columbia1
12 Department of Nutrition, Food and Exercise Sciences, Florida State University2
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21 *Corresponding author: Food Science Program, 246 William Stringer Wing, Eckles Hall,
22 University of Missouri, Columbia, MO 65211; Tel: (573) 882-2649, Fax: (573) 884-7964, E-
23 mail: [email protected]
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24 Abstract
25 Among the disease-causing Shiga toxin-producing Escherichia coli (STEC), E. coli O157:H7 is
26 estimated to cause one-third of the total STEC illnesses and the most cases of hemolytic uremic
27 syndrome (HUS) in the U.S. The uidA gene which is present in the majority of E. coli strains,
28 codes for the synthesis of β-D-glucuronidase (GUD). In E. coli O157:H7, the uidA gene has a
29 single point mutation at the +93 position that leads to an alteration in the amino acid sequence
30 encoding the GUD enzyme. The aim of this study was to distinguish E. coli O157:H7 from other
31 E. coli using a high-resolution melt curve (HRM) real-time PCR assay. Based on the uidA
32 mutation in E. coli O157:H7, a reliable PCR assay targeting the uidA gene was developed to
33 differentiate E. coli O157:H7 from other E. coli serotypes and the closely related Shigella spp.
34 The assay was validated using a set of 129 pure bacterial DNA samples and spiked ground beef
35 and beef trim. Isolates of E. coli O157:H7 formed distinctive melt peaks that were easily
36 distinguishable from those of other E. coli serogroups and Shigella isolates in the PCR plot.
37 Therefore, this assay was able to clearly discriminate E. coli O157:H7 strains from other E. coli
38 and Shigella. With a 5-8 h enrichment time, 10 CFU of E. coli O157:H7 were detectable in 325 g
39 spiked beef samples. The HRM E. coli O157:H7 detection assay standardized in this study will
40 allow for accurate identification of contaminated food samples and help in preventing foodborne
41 outbreaks caused by this pathogen.
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45 Keywords: E. coli O157:H7, ground beef, high resolution melt curve (HRM) PCR,
46 immunomagnetic separation, uidA
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47 1. Introduction
48 Escherichia coli is a bacterium that is widely found in the intestinal tract of humans and
49 animals. Most E. coli strains are harmless, however some E. coli are pathogenic, among which,
50 Shiga toxin-producing E. coli (STEC) are of the most concern because of their strong association
51 with foodborne disease outbreaks. STEC typically cause mild diarrhea but can sometimes result
52 in severe illnesses, including hemolytic uremic syndrome (HUS) and bloody diarrhea (Tarr,
53 Gordon, & Chandler, 2005). STEC are reported to lead to numerous infections caused by
54 consumption of contaminated foods annually.
55 According to the CDC (2011), E. coli O157:H7 is responsible for over 73,000 illnesses in
56 the U.S. annually, resulting in around 2,000 hospitalizations and 60 deaths. Among disease-
57 causing STEC, E. coli O157:H7 is the predominant serotype that causes HUS in human. HUS is
58 a life-threatening disease characterized by acute renal failure and microvascular thrombi (Tarr, et
59 al., 2005). The management of HUS remains challenging since no specific treatments have been
60 developed. Therefore, the most effective way to prevent HUS thus far is to prevent the initial
61 infection with E. coli O157:H7. Because more than one-third of the total STEC illnesses and
62 outbreaks in the United States are caused by E. coli O157:H7 (Scallan, et al., 2011),
63 distinguishing this strain from other STEC is of critical importance for treating HUS. Further,
64 accurate detection of E. coli O157:H7 will help in pin-pointing its food source with the intention
65 of controlling foodborne outbreaks related to this dangerous pathogen.
66 Beef products and carcass were implicated to be the primary vehicle for enterohemorrhagic
67 E. coli O157:H7, and the transmission of E. coli O157:H7 usually happens during slaughter
68 processes. More than half of the E. coli O157:H7 infection cases were linked to foods derived
69 from cattle (Rangel, Sparling, Crowe, Griffin, & Swerdlow, 2005). In the U.S., E. coli O157:H7
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70 is declared as an adulterant in ground beef. Therefore, mitigating and controlling this pathogen at
71 the initial stages of processing are crucial to decreasing the risk of E. coli O157:H7 infections.
72 More importantly, the minimum level of E. coli O157:H7 that can cause illness is very low,
73 usually less than several hundred CFU/g, necessitating a reliably selective detection method with
74 a high sensitivity (Karmali, 2004).
75 The traditional serotyping method is based on the immunological reaction of antigens
76 located on E. coli with specific antibodies. However, this method is time-consuming because it
77 involves sample enrichment, isolation of pure cultures using selective agar, biochemical testing
78 and serotyping. Further, culture-based methods have a low specificity since other bacteria, such
79 as Citrobacter freundii share the same antigens with E. coli O157:H7, resulting in cross-
80 reactions and false positive results (Bellisle, 1999; Vinogradov, Conlan, & Perry, 2000).
81 The 4-methylumbelliferyl-β-D-glucuronide (MUG) test is frequently used in identifying E.
82 coli (Feng & Hartman, 1982) since the majority of E. coli strains produces the enzyme, β-D-
83 glucuronidase (GUD). When MUG is hydrolyzed by GUD, a fluorogenic compound, 4-
84 methylumbelliferyl, is released which fluoresces when exposed to long-wave UV light (Feng,
85 1993). More than 90% of E. coli strains are positive in the MUG assay, while E. coli O157:H7 is
86 consistently negative for this test. The uidA gene controls the synthesis of GUD. Although E.
87 coli O157:H7 is MUG-negative, it contains the uidA gene that is highly identical to that of other
88 serotypes. The only difference is a base substitution in the sequence of the uidA gene. At the +93
89 position of its uidA gene sequence, E. coli O157 harbors a thiamine, while in other E. coli
90 serotypes, it is a guanine Feng, 1993). This leads to an alteration in the amino acid sequence that
91 encodes GUD enzyme. Therefore, this mutation is used as one of the most common targets for
92 PCR-based identification of the E. coli O157:H7 serotype.
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93 Compared with culture-based methods, the Polymerase Chain Reaction (PCR) technique
94 which allows for rapid amplification of target genes in DNA isolates is widely used for
95 distinguishing bacterial species. The HRM-based PCR assays are highly sensitive to any single-
96 base change in amplified targets. HRM dyes are able to completely saturate amplicons, thus
97 yielding a melt curve with higher resolution. Owing to the simplicity, relatively low cost and
98 ease of use, HRM application in the areas of clinical diagnostics and food safety has also
99 increased.
100 Therefore, the objective of this study was to develop a HRM real-time PCR assay for
101 identification of E. coli O157:H7. This study will also provide a useful tool to help in
102 epidemiological analyses and control of foodborne outbreaks associated with this pathogen.
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104 2. Materials and Methods
105 2.1 Bacterial DNA extraction
106 E. coli strains were obtained from the University of Missouri, Food Microbiology Lab
107 culture collection. Cultures were grown overnight at 37 oC in Tryptic Soy broth (TSB) (Difco
108 Labs, Sparks, MD, USA). Genomic DNA of pure E. coli strains was isolated from overnight
109 cultures in TSB broth. One milliliter of the overnight cultures in TSB broth was centrifuged at
110 6,000 ×g for 2 min. DNA from the obtained cell pellet was extracted by using PrepMan™ Ultra
111 Sample Preparation Reagent (Applied Biosystems, Foster City, CA, USA) following the
112 manufacturer’s instructions. The concentration and purity of the obtained DNA samples were
113 measured by a Nanodrop Lite Spectrophotometer (Thermo Fisher, Wilmington, DE, USA).
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114 Genomic DNA of E. coli from spiked beef samples was isolated using DNeasy® Blood and
115 Tissue Kit (Qiagen, Hilden, Germany), according to the instructions of the Purification of Total
116 DNA from Animal Tissues.
117 118 2.2 Primer and HRM-PCR assay design
119 The primer pair, HRM-F and HRM-R, was designed using the Primer3 software
120 (Untergasser et al., 2012). The specificity of the designed PCR primers was tested using
121 NCBI/Primer-BLAST, and all oligonucleotides were commercially synthesized (IDT, Coraville,
122 IA, USA) as shown in Table 1.
123 Real-time PCR was performed using 2× LightCycler® 480 High Resolution Melting Master
124 (Roche Diagnostics Corp., Indianapolis, USA), and the HRM assay was standardized on a
125 LightCycler® 96 real-time PCR instrument (Roche Diagnostics Corp., Indianapolis, USA). The
126 PCR reaction was performed in a 10 µL reaction volume in duplicate, with 30 ng of genomic
127 DNA, 0.5 µM primers and 2.5 mM MgCl2. A two-step amplification protocol included an initial
128 denaturation step at 94 oC for 10 min, followed by 40 cycles of 95 oC for 15 s and 64 oC for 30 s.
129 A HRM step was added at the end of the PCR amplification (from 60 oC to 95 oC, with gradual
130 temperature increments of 0.04 oC/s) A HRM analysis was performed with a pre-melt region
131 76.9 -77.9 oC and a post-melt region 87.7-88.7 oC.
132 E. coli O157:H7 and other E. coli DNA isolates were tested to validate this assay.
133 134 2.3 Food sample preparation
135 Ground beef (20% fat) and beef trim were purchased from the University of Missouri Meat
136 Lab. Three hundred and twenty-five grams of ground beef were placed in filter stomacher bags
137 and spiked with 10 CFU E. coli O157:H7. After 15 min at room temperature, the spiked ground
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138 beef was placed in 4 oC for 72 h, in order to cold-stress the E. coli O157:H7, mimicking normal
139 industry scenarios. After that, 975 mL of buffered peptone water (BPW), pre-heated at 37 oC and
140 supplemented with 8 mg/L vancomycin, was added into the bag and the sample was
141 homogenized by hand massaging (Singh and Mustapha, 2015). Samples were enriched by
142 incubating at 37 oC for 5 h, 6 h, 8 h, 10 h and 12 h. Two milliliters of homogenate were
143 transferred from the enriched sample into a centrifuge tube. After centrifuging at 3800 ×g for 1.5
144 min, 1 mL of the supernatant (without withdrawing the fat layer on top) was removed and
145 transferred into a new centrifuge tube.
146 147 2.4 Preparation of E. coli O157:H7 and non-O157 STEC cocktail
148 Seven strains of non-O157 STEC and one strain of E. coli O157: H7 were chosen as the
149 inocula for food samples. E. coli strains used in this study are shown in Table 3. Each strain was
150 separately grown overnight in TSB at 37 oC. One hundred microliters of each non-O157 STEC
151 culture were then mixed and sub-cultured in a fresh tube containing 9 mL TSB. E. coli O157:H7
152 was sub-cultured in a separate tube of 9 mL TSB. After incubating both tubes at 37 oC overnight,
153 both samples were enumerated after serial dilutions and pour plating on TSA. Colonies were
154 counted after incubation at 37 oC for 24 h. The non-O157 E. coli cocktail and E. coli O157:H7
155 cultures were stored at 4 oC while awaiting the colony counts. The obtained bacterial counts
156 were used to precisely calculate appropriate dilutions and inoculum volumes to achieve 10
157 CFU/325 g. Beef samples were spiked with the calculated amounts of inocula and stored at 4 oC
158 for 72 h to cold-stress the inoculated bacterial cells. Inoculated beef samples, after the cold-stress
159 step, were enriched for 5-8 h and DNA was isolated from samples as described in the previous
160 section.
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162 2.5 Biochemical confirmation tests
163 Immunomagnetic separation (IMS) was performed with 1 mL of enriched samples using
164 Dynabeads® E. coli anti-O157 (Thermo Fisher, Wilmington, DE, USA), followed by DNA
165 isolation with the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). Another 1.0 mL
166 supernatant of the enriched broth sample was processed using IMS and the collected beads were
167 diluted to 10-4 using peptone water. One hundred microliters of the diluted sample were
168 transferred and spread-plated on Rainbow Agar (Biolog, Hayward, CA, USA). Plates were
169 incubated at 37 oC for 24 h. The distinctive black or gray colonies on Rainbow Agar were
170 suspected to be E. coli O157:H7.
171 A single colony of suspect E. coli O157:H7 from the Rainbow Agar plate was picked and
172 confirmed with DrySpot™ E. coli O157 Latex Agglutination Test (Oxoid Diagnostic Reagents,
173 Hampshire, England) according to the manufacturer’s instructions. A positive result was
174 interpreted as large clumps of agglutination with partial or complete clearing of the background
175 latex within 1 to 2 min.
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177 Results and Discussion
178 Conventional culture-dependent methods remain the gold standard and have been
179 extensively used for foodborne pathogen detection worldwide (Gracias & McKillip, 2004;
180 Mandal, Biswas, Choi, & Pal, 2011). However, conventional methods are tedious and require at
181 least 4-5 working days to complete the whole process. In this study, a HRM real-time PCR
182 method was developed to distinguish E. coli O157:H7 from other E. coli in ground beef and beef
183 trim. The obvious advantage of this assay over phenotypic assays is the shortening of the whole
184 process to less than 24 h. Moreover, the HRM assay does not require special equipment other
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185 than a real-time PCR instrument, thereby making it accessible and affordable for most
186 laboratories.
187 The HRM assay was standardized using pure DNA samples of E. coli O157:H7 and other
188 E. coli. E. coli O157:H7 isolates formed a specific melt profile in normalized and differential
189 plots after HRM analysis, as shown on Figs. 1 and 2, making this serotype distinguishable from
190 other E. coli. Shigella, which can sometimes be difficult to distinguish from E. coli O157, was
191 found to form melt peaks which were similar to melt peaks obtained from other E. coli serotypes,
192 thus avoiding false positives from this organism. HRM-based methods have previously been
193 used for detection of pathogens in foods. It is simple to perform and can be accomplished in a
194 single closed-tube, thus avoiding cross-contamination from sample handling errors (Jeng, et al.,
195 2012).
196 The optimal concentration of MgCl2 for the PCR was determined to be 2.5 mM. A proper
197 MgCl2 concentration in a PCR reaction mix is one of the most important factors for
198 standardizing any HRM assay because it increases the PCR amplification efficiency, influences
199 the Tm of amplicons and, most importantly, affects genotype separation.
200 The optimized assay was validated using 129 bacterial stains, comprising 21 strains of E.
201 coli O157, 9 of E. coli O26, 10 of E. coli O45, 10 of E. coli O103, 6 of E. coli O104, 10 of E.
202 coli O111, 10 of E. coli O121, 9 of E. coli O145, 3 Shigella strains including S. dysenteriae, S.
203 flexneri and S. sonnei, and 40 non-STEC E. coli strains isolated from the feces of eight different
204 animals (chicken, cattle, duck, dog, goose, goat, human, pig). An equal concentration mixture of
205 E. coli O157:H7 and E. coli DNA was used to create a heterozygous genotype. This HRM assay
206 was able to clearly discriminate E. coli O157:H7 strains from other E. coli and Shigella.
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207 Based on the single point mutation at position +93 of the uidA in E. coli O157, Cebula et
208 al. (1995) designed a mismatch amplification mutation assay (MAMA) for the detection of this
209 serotype. It was one of the most commonly used methods for the identification of E. coli
210 O157:H7. It was found that under relaxed PCR conditions, the assay amplified the uidA gene of
211 other E. coli and Shigella, generating a false positive result. Hence, the HRM assay developed in
212 this study was further validated using Shigella strains. The melt profile generated by the Shigella
213 uidA gene amplicon grouped with other E. coli, proving the specificity of the assay towards the
214 E. coli O157:H7 serotype.
215 In this study, out of 40 E. coli strains isolated from the feces of eight different animals, the
216 melt curve profile of 38 strains aligned with one another but two strains, E. coli Dg2 and E. coli
217 H2, generated a separate profile in the differential plot (Fig. 2). The formation of a separate melt
218 curve profile in the differential plot indicates the presence of mutations in the amplicon region of
219 targeted uidA gene sequences. GenBank nucleotide data search on the Shigella uidA gene
220 revealed the presence of two single nucleotide polymorphism (SNP) in the amplicon region. If
221 Shigella strains with these SNP are tested using our assay, these SNPs are expected to form a
222 separate melt curve profile other than E. coli O157:H7 on the differential plot due to the high
223 sensitivity of the HRM assay.
224 Raw ground beef is a type of food product that has a high level of background microflora,
225 which makes the isolation of target bacteria difficult, especially if the target bacteria is cold-
226 stressed and present in low numbers. In this study, artificially inoculated ground beef was found
227 to be positive for E. coli O157:H7 after an 8-h enrichment. However, spiked beef trim required a
228 shorter enrichment period of only 6 h (Table 4). This was probably because the beef trim carried
229 less amounts of initial microflora than the ground beef. Beef samples inoculated with a cocktail
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230 of E. coli O157:H7 and non-O157 showed similar results. A 5-8 h enrichment time was required
231 for the identification of E. coli O157:H7 (Table 4). Therefore, this assay was proved to be
232 specific for E. coli O157:H7 in a single pure culture, pure culture mix and food samples. The
233 black colonies on Rainbow Agar, suspected to be E coli O157:H7, were positively confirmed by
234 DrySpot™ E. coli O157 Latex Agglutination Test.
235 IMS is a useful method for isolating target organisms from a wide range of samples. In our
236 previous studies (Wang, Li & Mustapha, 2007), IMS showed its high sensitivity and specificity
237 at capturing E. coli O157:H7 from enriched food samples. It is reported that IMS can reach a
238 detection limit of less than 1 CFU/g when applied to spiked animal feces and meat samples
239 (Islam, Heuvelink, Talukder, & De Boer, 2006). Other studies also proved IMS as a sensitive
240 tool for isolating E. coli O157:H7 from diverse bacterial microflora, pathogen cocktails, food
241 samples and bovine feces (Chapman, Wright, & Siddons, 1994). Therefore, conducting IMS
242 prior to a PCR increases the target isolation efficiency and removal of PCR inhibitors from food
243 samples. In Weagant’s (2001) study, the recovery of E. coli O157:H7 after a 5-h enrichment
244 period was significantly improved after the addition of an IMS step. Likewise, Fedio, et al.
245 (2011) found that the introduction of IMS for food samples could prompt culture recovery from
246 56% without IMS to 100% after IMS within a 24-h enrichment.
247 Beef products, after fabricating and packing, are typically stored at refrigerated
248 temperatures to extend their shelf life before shipping to markets. In this study, beef samples
249 after inoculation were cold-stressed for 72 h at 4 oC to simulate a typical storage procedure.
250 Previous studies have shown that a cold shock of food samples could affect the enrichment
251 period required for pathogen detection. Uyttendaele et al. (1998) claimed in their findings that
252 ground beef inoculated with E. coli O157:H7 after a cold stress at 4 oC for 14 days required a 3.5
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253 h longer enrichment time. In another study (Fratamico, Bagi, & Pepe, 2000), when the cold stress
254 period was shortened to 48 h, the enrichment period required to reach the detectable level was
255 the same as that for samples without a cold-stress treatment.
256 Buffered peptone water (BPW) is a common pre-enrichment broth used for pathogen
257 recovery in a various range of foods (Baylis, MacPhee, & Betts, 2000). A previous study verified
258 that BPW provided appropriate conditions for bacterial resuscitation and recovered suppressed
259 cells during enrichment (Thomason, Dodd, & Cherry, 1977). Vancomycin could suppress the
260 growth of Gram-positive bacteria, especially Staphylococcus aureus, which is a prevalent
261 pathogen associated with ready-to-eat meat products (Williams and Bardsley 1999). Thus, the
262 addition of vancomycin in BPW inhibited S. aureus without interfering with E. coli O157:H7
263 growth.
264 The initial beef microflora count is another factor that influences the recovery of E. coli
265 O157:H7 from foods. The complexity of food samples (e.g. fat and protein content) and high
266 background microflora count were found to be inhibitory for the detection of STEC (Singh and
267 Mustapha, 2015; Johnson, Brooke, & Fritschel, 1998). When testing foods with high levels of
268 non-target microflora, a shorter enrichment period is not sufficient to bring target bacteria to a
269 detectable level. In such cases, the application of IMS has been found to be very useful. In a
270 previous study, samples enriched for 5 h and 24 h and tested using real-time PCR after an IMS
271 step, showed no significant difference in the E. coli O157 isolation efficiency following two
272 different enrichment periods (Fedio, et al., 2011). In this study, the application of IMS before the
273 HRM assay allowed us to achieve a similar enrichment time, even after the cold-stressing period.
274 Choosing a proper DNA purification kit is critical for obtaining stable and pure DNA
275 extracts with less inhibitors. This is crucial for generating reproducible results using a HRM
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276 assay. DNA isolated from enriched food samples using different DNA isolation kits has varying
277 purity levels. Variations in the DNA purity level can lead to variations in melt curve profiles and
278 eventually affect genotyping during the analysis step. Therefore, PrepMan™ Ultra was employed
279 for isolating DNA from pure broth cultures, while DNA from enriched food samples were
280 isolated with the DNeasy Blood & Tissue Kit.
281 Due to the complex and high counts of microflora in ground beef, distinguishing E. coli
282 O157:H7 from the background microflora is challenging. This assay involves IMS in
283 combination with HRM-PCR, making the rapid and reliable detection method feasible. However,
284 further improvements on rapid capture of target bacteria are necessary to lessen the enrichment
285 time and further improve the detection limit.
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287 Conclusions
288 The HRM-PCR assay was able to distinguish E. coli O157:H7 from other E. coli and
289 Shigella and obtain a low detection limit of 10 CFU/325 g with a 5-8 h enrichment period when
290 applied to beef samples. The advantages of low cost, easiness, rapidness and non-destructive
291 nature make this HRM assay a reliable method for distinguishing E. coli O157:H7 from other E.
292 coli serotypes and background microflora. This assay may also aid in epidemiological studies
293 and infection control.
294
295 Acknowledgments
296 This research was supported by the USDA NIFA Hatch/Multistate Grant No. S1056.
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299 References
300 Baylis, C., MacPhee, S., & Betts, R. (2000). Comparison of two commercial preparations of
301 buffered peptone water for the recovery and growth of Salmonella bacteria from foods. J.
302 Appl. Microbiol., 89(3), 501-510.
303 Bellisle, F. (1999). Glutamate and the UMAMI taste: sensory, metabolic, nutritional and
304 behavioural considerations. A review of the literature published in the last 10 years. Neurosci.
305 Biobehav. Rev., 23(3), 423-438.
306 Chapman, P., Wright, D., & Siddons, C. (1994). A comparison of immunomagnetic separation
307 and direct culture for the isolation of verocytotoxin-producing Escherichia coli O157 from
308 bovine faeces. J. Medical Microbiol., 40(6), 424-427.
309 Control, C. f. D., & Prevention. (2011). Vital signs: incidence and trends of infection with
310 pathogens transmitted commonly through food--foodborne diseases active surveillance
311 network, 10 US sites, 1996-2010. MMWR, 60(22), 749.
312 Fedio, W. M., Jinneman, K. C., Yoshitomi, K. J., Zapata, R., Wendakoon, C. N., Browning, P.,
313 & Weagant, S. D. (2011). Detection of E. coli O157: H7 in raw ground beef by Pathatrix™
314 immunomagnetic-separation, real-time PCR and cultural methods. Int. J. Food Microbiol.,
315 148(2), 87-92.
316 Feng, P. (1993). Identification of Escherichia coli serotype O157: H7 by DNA probe specific for
317 an allele of uidA gene. Mol. Cell. Probes, 7(2), 151-154.
318 Feng, P., & Hartman, P. A. (1982). Fluorogenic assays for immediate confirmation of
319 Escherichia coli. Appl. Environ. Microbiol., 43(6), 1320-1329.
14
320 Fratamico, P. M., Bagi, L. K., & Pepe, T. (2000). A multiplex polymerase chain reaction assay
321 for rapid detection and identification of Escherichia coli O157:H7 in foods and bovine feces.
322 J. Food Prot., 63(8), 1032-1037.
323 Gracias, K. S., & McKillip, J. L. (2004). A review of conventional detection and enumeration
324 methods for pathogenic bacteria in food. Canadian J. Microbiol., 50(11), 883-890.
325 Islam, M. A., Heuvelink, A. E., Talukder, K. A., & De Boer, E. (2006). Immunoconcentration of
326 Shiga toxin-producing Escherichia coli O157 from animal faeces and raw meats by using
327 Dynabeads anti-E. coli O157 and the VIDAS system. Int. J. Food Microbiol., 109(1), 151-
328 156.
329 Jeng, K., Gaydos, C. A., Blyn, L. B., Yang, S., Won, H., Matthews, H., Toleno, D., Hsieh, Y.-H.,
330 Carroll, K. C., & Hardick, J. (2012). Comparative analysis of two broad-range PCR assays for
331 pathogen detection in positive-blood-culture bottles: PCR–high-resolution melting analysis
332 versus PCR-mass spectrometry. J. Clin. Microbiol., 50(10), 3287-3292.
333 Johnson, J. L., Brooke, C. L., & Fritschel, S. J. (1998). Comparison of the BAX for screening/E.
334 coli O157: H7 method with conventional methods for detection of extremely low levels of
335 Escherichia coli O157:H7 in ground beef. Appl. Environ. Microbiol., 64(11), 4390-4395.
336 Karmali, M. A. (2004). Prospects for preventing serious systemic toxemic complications of
337 Shiga toxin–producing Escherichia coli infections using Shiga toxin receptor analogues. J.
338 Infect. Dis., 189(3), 355-359.
339 Mandal, P., Biswas, A., Choi, K., & Pal, U. (2011). Methods for rapid detection of foodborne
340 pathogens: an overview. Am. J. Food Technol., 6(2), 87-102.
15
341 Rangel, J. M., Sparling, P. H., Crowe, C., Griffin, P. M., & Swerdlow, D. L. (2005).
342 Epidemiology of Escherichia coli O157:H7 outbreaks, United States, 1982–2002. Emerg.
343 Infect. Dis., 11, 603-609.
344 Scallan, E., Hoekstra, R. M., Angulo, F. J., Tauxe, R. V., Widdowson, M.-A., Roy, S. L., Jones,
345 J. L., & Griffin, P. M. (2011). Foodborne illness acquired in the United States—major
346 pathogens. Emerg. Infect. Dis., 17(1), 7-15.
347 Singh, P., & Mustapha, A. (2015). Multiplex real-time PCR assays for detection of eight Shiga
348 toxin-producing Escherichia coli in food samples by melting curve analysis. Int.l J. Food
349 Microbiol., 215, 101-108.
350 Tarr, P. I., Gordon, C. A., & Chandler, W. L. (2005). Shiga-toxin-producing Escherichia coli and
351 haemolytic uraemic syndrome. The Lancet, 365(9464), 1073-1086.
352 Thomason, B. M., Dodd, D. J., & Cherry, W. B. (1977). Increased recovery of salmonellae from
353 environmental samples enriched with buffered peptone water. Appl. Environ. Microbiol.,
354 34(3), 270-273.
355 Vinogradov, E., Conlan, J. W., & Perry, M. B. (2000). Serological cross-reaction between the
356 lipopolysaccharide O-polysaccharide antigens of Escherichia coli O157:H7 and strains of
357 Citrobacter freundii and Citrobacter sedlakii. FEMS Microbiol. Lett., 190(1), 157-161.
358 Wang, L., Li, Y., & Mustapha, A. (2007). Rapid and simultaneous quantitation of
359 Escherichia coli O157:H7, Salmonella, and Shigella in ground beef by multiplex real-time PCR
360 and immunomagnetic separation. J. Food Prot., 70(6) 1366-1372.
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364 Figure Legends
365 Fig 1. High-resolution melting plots for the specific identification of E. coli O157:H7 and its
366 discrimination from other E. coli and Shigella strains.
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368 Fig 2. Normalized melting curves showing three different melt profiles obtained using standard
369 E. coli O157 ATCC 43894, E. coli O26:H11 DEC10B and a mixture of the two DNA
370 samples.
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386 Figure 1
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388 0.300 389 0.250 E. coli O157:H7 0.200 390 0.150 391 0.100 E. coli O157:H7 and non-O157 E. coli mix 392 0.050
393 Difference 0.000 Non-O157 E. coli -0.050 394 and Shigella -0.100 395 -0.150 396 -0.200 E. coli Dg2 and H2 397 -0.250
398 76.00 78.00 80.00 82.00 84.00 86.00 88.00 o 399 Temperature ( C)
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403 Figure 2
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405 1.000 406 0.900
407 0.800
408 0.700 E. coli O157:H7 0.600 409 E. coli O157:H7 and 0.500 O26:H11 mix 410 0.400 E. coli O26:H11 411 Fluorescence(RFU) 0.300 412 0.200
413 0.100 0.000 414 76.00 78.00 80.00 82.00 84.00 86.00 88.00 415 Temperature (oC)
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420 Table 1 The uidA primer details.
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Primer Sequence Primer Gene Product MgCl2 Reaction concentration Size concentration volume (bp) HRM-F GCCCGGCTTTCT 0.5 µM
TGTAAC uidA 62 bp 2.5 mM 10 µL
HRM-R GATCGCGAAAA 0.5 µM
CTGTGGAAT
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437 Table 2 E. coli O157:H7 strains used in spiked beef samples.
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Strain Source
E. coli O157:H7 EDL-933 Food (hamburger)
E. coli O157:H7 505B Beef (FRI)
E. coli O157:H7 3178-85 Human (CDC)
E. coli O157:H7 C7927 Human (CDC)
E. coli O157:H7 MF1847 Beef (FSIS)
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454 Table 3 E. coli O157:H7 and non-O157 STEC cocktail information.
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E. coli O157:H7 Non-O157 STEC strain strain Mix 1 EDL-933 O26:HN TB352A, O111:H8 3215-99, O104:H TW04909, O45:H2
M105-14, O103:H25 8419, O145:H[28] 4865/96, O121:H19
MT#2
Mix 2 505B O26: H11 DEC10B, O111:H2 RD8, O104:H ECOR228, O45:H2
M103-19, O103:H6 TB154A, O145:HNM GSG5578620,
O121:H19 MT#2
Mix 3 3178-85 O26: H11 DEC10B, O111:H2 RD8, O104:H ECOR228, O45:H2
M103-19, O103:H6 TB154A, O145:HNM GSG5578620,
O121:H19 MT#2
Mix 4 C7927 O26:HN TB352A, O111:H8 3215-99, O104:H TW04909, O45:H2
M105-14, O103:H25 8419, O145:H[28] 4865/96, O121:H19
MT#2
Mix 5 MF1847 O26:H1197-3250, O111:HNM 3007-85, O104:H TW04911,
O45:H2 MI01-88,O103:H N PT91-24,O145:H[28] 4865/96,
O121:H19 MT#2
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461 Table 4 Enrichment period needed to detect E. coli O157:H7 in spiked beef sample.
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Strain Enrichment time (h)
Ground beef Beef trim
E. coli O157:H7 EDL-933 8 6
E. coli O157:H7 505B 6 5
E. coli O157:H7 3178-85 8 6
E. coli O157:H7 C7927 8 5
E. coli O157:H7 MF1847 8 6
Mix 1 8 6
Mix 2 6 5
Mix 3 8 6
Mix 4 8 6
Mix 5 8 6
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