Journal of Dairy Science and Biotechnology
Korean Society of Dairy Science and Biotechnology

Status and Prospects of PCR Detection Methods for Diagnosing Pathogenic Escherichia coli : A Review

Jin-Hyeok Yim1,, Kun-Ho Seo1,, Jung-Whan Chon1, Dongkwan Jeong2, Kwang-Young Song1,3,4,*
1Center for One Health and College of Veterinary Medicine, Konkuk University, Seoul, Korea
2Department of Food & Nutrition, Kosin University, Busan, Korea
3Institue for Dairy Foods, Gangneung, Korea
4Department of Biological Engineering, Yanbian University of Science and Technology, Yanji, JL, China
*Corresponding author : Kwang-Young Song, Center for One Health and College of Veterinary Medicine, Konkuk University, Seoul, Korea, Tel : +82-2-450-4121, Fax : +82-2-3436-4128, E-mail :

† These authors contributed equally to this study.

© Copyright 2021, Korean Society of Dairy Science and Biotechnology. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received: May 26, 2021; Revised: Jun 16, 2021; Accepted: Jun 17, 2021

Published Online: Jun 30, 2021


Escherichia coli are the predominant facultative bacteria found in the gastrointestinal tract of animals and humans. Some strains of E. coli that acquire virulence factors and cause foodborne and waterborne diseases in humans are called pathogenic E. coli and can be divided into five pathotypes according to the virulence mechanism: EAEC, EHEC, EIEC, EPEC, and ETEC. Although selective media have been developed to detect E. coli, distinguishing pathogenic strains from non-pathogenic ones is difficult because of their similar biochemical properties. Therefore, it is very important to find a new and effective diagnostic method to identify pathogenic E. coli. With recent advances in molecular biology and whole genome sequencing, the use of polymerase chain reaction (PCR) is increasing rapidly. In this review paper, we provide an overview of pathogenic E. coli and present a review on PCR detection methods that can be used to diagnose pathogenic E. coli. In addition, the possibility of real-time PCR incorporating IAC is introduced. Consequently, this review paper will contribute to solving the current challenges related to the detection of pathogenic E. coli.

Keywords: pathogenic Escherichia coli; polymerase chain reaction (PCR); real-time PCR; internal amplification control (IAC); virulence genes


Pathogenic Escherichia coli are one of the major causative agents of food poisoning accidents occurring in Korea and abroad. Pathogenic Escherichia coli infect human through contaminated food and drinking water [13] Pathogenic Escherichia coli can be divided into five types according to the pathological mechanism, and some Escherichia coli have high pathogenicity [2]. In 2011, numerous food poisoning accidents caused by Escherichia coli O104 were reported in Europe, most of which were fatal [3,4]. Therefore, detecting and discriminating pathogenic Escherichia coli in food are necessary. However, the biochemical properties of pathogenic Escherichia coli are similar to those of normal Escherichia coli conventional medium except for Escherichia coli O157:H7, rendering it difficult to discriminate them [5].

The currently available method for discriminating pathogenic Escherichia coli according to the pathological mechanism requires skilled technicians. Nonetheless, pathogenic Escherichia coli can be detected using polymerase chain reaction (PCR) [6]. Although PCR has the advantage of rapid detection, it requires considerable time and resources to discriminate the five kinds of Escherichia coli. In addition, when PCR is used, the test results can be considered valid only when false-positive or false-negative results can be discriminated. Therefore, in this review paper, we tried to present the possibility of developing multiplex PCR that can simultaneously distinguish 5 types of pathogenic Escherichia coli using an internal amplification control (IAC).

Therefore, this review paper was organized to provide general information about (1) tan summary of PCR detection methods that could be used to confirm pathogenic Escherichia coli and also (2) the possibility of real-time PCR incorporating IAC would be introduced.

Pathogenic Escherichia coli

Escherichia coli are the part of the normal flora found in the intestine of human beings and animals [7]. However, several strains of Escherichia coli are identified as pathogenic and cause severe diseases in their host [8]. Pathogenic Escherichia coli have different virulence strategies, and the symptoms vary according to pathogenicity [9]. Pathogenic Escherichia coli can be classified according to their pathogenicity into five types: enteroaggregative Escherichia coli (EAEC), enterohemorrhagic Escherichia coli (EHEC), enteroinvasive Escherichia coli (EIEC) enteropathogenic Escherichia coli (EPEC), and enterotoxigenic Escherichia coli (ETEC) [10].

The most common virulence factor of pathogenic Escherichia coli is the production of various toxins within the host [11]. The following toxins are produced by pathogenic Escherichia coli, Shiga toxins (Stx1 and/or Stx2), heat-labile enterotoxins (LT), and heat-stable enterotoxins (ST) [12]. Moreover, specific invasion plasmids, colonization factors, fimbriae, and adhesions are known to affect the pathogenic properties of Escherichia coli isolates [13].

Virulence factors are determined by the genetic properties acquired through plasmids, phages, or other gene transfer events [14]. The common symptoms due to pathogenic Escherichia coli are diarrhea, acute inflammation, hemorrhagic colitis, urinary tract infections, and septicemia [15].

1. Enteroaggregative Escherichia coli

EAEC have a plasmid of 60–65 MDa, which encodes the aggregative adherence fimbriae AAFI or AAFII [16]. In addition, EAEC produce several toxins, of which Pic and Shigella enterotoxin 1 (ShET1) share the same chromosomal locus on opposite strands [17]. EAEC have a unique LT plasmid that encodes the entero-aggregative toxin EAST1 [18]. The virulence factors of EAEC are regulated by a single transcriptional activator called AggR, a member of the AraC family of transcriptional activators [19].

2. Enterohemorrhagic Escherichia coli

EHEC is characterized by the production of Shiga toxins (Stx) [20]. The Stx causes hemorrhagic colitis and hemolytic uremic syndrome (HUS) [21]. EHEC also has the locus of enterocyte effacement (LEE), which is characterized by the ability to attach to the enterocyte [22]. Although more than 200 serotypes produce Stxs, most serotypes do not have the LEE [23]. Stx-producing Escherichia coli (STEC) or verotoxin-producing Escherichia coli (VTEC) produce Stx, but do not have the LEE, whereas EHEC produce Stx and have the LEE [23,24].

3. Enteroinvasive Escherichia coli

The pathogenic mechanism and clinical symptoms (dysentery-like diarrhea with fever of EIEC) are similar to those of Shigella spp. EIEC invade and proliferate within the epithelial cells of the colon, causing extensive cell destruction [25]. EIEC pathogenesis occurs via a plasmid-borne type III secretion system that secretes several proteins such as IpaA, IpaB, IpaC, and IpgD [26]. Among them, IpaH, which encodes the invasive plasmid antigen H, is present on both the chromosome and invasion plasmid [27].

4. Enteropathogenic Escherichia coli

EPEC are characterized by attaching and effacing (A/E) lesions on the intestinal epithelium [28]. The genetic element responsible for the A/E lesions is located on a 35 kb pathogenicity island called the LEE, which encodes an intimin, a type III secretion system, many secreted (Esp) proteins, and the translocated intimin receptor named Tir [29]. A typical EPEC has 70–100 kb of EPEC adherence factor (EAF) plasmid, and this plasmid encodes a type IV pilus called the bundle-forming pilus (BFP) [30]. In a typical EPEC, BFP mediates interbacterial adherence and epithelial cell adhesion [31]. Atypical EPEC has only the LEE plasmid, but not the EAF plasmid [32].

5. Enterotoxigenic Escherichia coli

ETEC produce enterotoxins and cause fever-free diarrhea [33]. ETEC can produce LT and/or ST enterotoxins; they can produce one or two toxins simultaneously, each with one or more colonization factors [34]. LT toxins are structurally and functionally similar to cholera enterotoxin and are classified as LT I (associated with humans and animals) and LT II (associated primarily with animals) [35]. ST toxin variants include ST1a and STb [34].

Serotyping of Escherichia coli

Serotyping by using somatic (O) and flagellar (H) antigens is the most basic method of classifying Escherichia coli [36]. However, serology is not always sufficient to identify the pathotypes because it does not involve checking for the presence of virulence factors [36]. Better strain identification requires specialized knowledge and the use of various detection methods, but these methods are difficult to perform and to apply to routine investigation [36].

Occurrence of Pathogenic Escherichia coli

1. Pathogenic Escherichia coli in world

Over the past 10 years, food poisoning has been mainly caused by EPEC, STEC/EHEC, EIEC, ETEC, and EAEC. Vegetables, fruits, meat products, and cooked foods were mainly contaminated by bacteria from food handlers. Pathogenic Escherichia coli originate from contaminated environments (water and soil), animals, and humans. Food poisoning due to pathogenic Escherichia coli is attributed to the consumption of less cooked and contaminated food and by contamination from food workers [36]. STEC is more commonly responsible for food poisoning, and contamination by STEC strains O104:H4, O157 PT8, and O111:NM leads to death [35].

The most serious food poisoning accident in Germany in 2011 was caused by STEC O104:H4 [37]. A large-scale food poisoning outbreak resulted in 3816 STEC infections and 54 deaths, of which 32 died from HUS, which is known to mainly affect children, but 89% of all patients with HUS were adults. The source of infection was found to be raw sprouts. In addition to Germany, STEC O104:H4 infection incidents have been reported in Europe and North America (Table 1). Six cases of STEC O104:H4 infection were confirmed in the United States, and five of them had traveled to Germany during the outbreaks. Of the 6 patients, 4 developed HUS, and 1 died. In France, 24 cases of STEC O104:H4 infection were reported, of which 22 (92%) were reported in adults: 7 cases (29%) developed HUS; 5 cases (21%), bloody diarrhea; and 12 cases (50%), diarrhea [38].

Table 1. Foodborne outbreaks caused by pathogenic Escherichia coli during 2010–2019
Country Year Pathotype Serotypes Source Incidence
USA 2010 EHEC O157:H7 Cheese and beef 59
EHEC O145 Romaine lettuce 26
2011 EHEC O104 Sprouts 6
EHEC O157:H7 Romaine lettuce 58
2014 EHEC O121 Raw clover sprouts 19
EHEC O157:H7 Ground beef 12
2015 EHEC O26 Restaurant 55
EHEC O157:H7 Chicken salad 19
2016 EHEC O157:H7 Beef product 11
EHEC O121, O26 Flour 63
EHEC O157 Alfalfa sprouts 11
2017 EHEC O157:H7 Butter, leafy greens 57
2018 EHEC O157:H7 Romain lettuce 210
EHEC O157:H7 Ground beef 18
2019 EHEC O157:H7 Salad kit and romaine lettuce 177
EHEC O103 Ground beef 209
EHEC O26 Flour 21
EHEC O103 and O121 Bison 33
Korea 2012 EPEC O169 Kimchi 230
2013 ETEC O157:H45 Egg soup and tuna bibimbap 33
Japan 2011 EHEC O111 and O157 Raw beef dishes 181
2012 ETEC O169:H41 Japanese restaurant 102
China 2010 EPEC O127a:K63 Dining room 112
Italy 2012 EIEC O96H19 Cooked vegetables 109
German 2011 EHEC O104:H4 Sprouts 3,816
France 2011 EHEC O104:H4 Fenugreek seeds 24
EHEC Sorbitol-fermenting O157:H7 Frozen ground beef products 18
England 2010 EHEC O157 PT8 Raw leeks and potatoes 252
2013 EAEC O131:H27, O104:H4, O20:H19 Food festival 592
Denmark 2010 ETEC O6:K15:H16 Lettuce 264
Norway 2012 ETEC O78 Imported chives and scrambled eggs >300

Adapted from Yim with permission of author [7].

EHEC, enterohemorrhagic Escherichia coli; EPEC, enteropathogenic Escherichia coli; ETEC, enterotoxigenic Escherichia coli; EIEC, enteroinvasive Escherichia coli; EAEC, enteroaggregative Escherichia coli.

Download Excel Table
2. Pathogenic Escherichia coli in South Korea

The Korea Centers for Disease Control and Prevention (KCDC) analyzed the epidemic pattern and pathotype of pathogenic Escherichia coli between 2010 and 2019 and isolated 6,485 pathogenic Escherichia coli, of which 5,785 (89.2%) and 700 (10.8%) were isolated from domestic and foreign samples, respectively [39]. By pathotype, EPEC were the highest (3,921 [60.5%]), followed by ETEC (2,025 [31.2%]), EIEC (101 [1.5%]), and EHEC (438 [6.8%]). Of the ETEC isolated, 556 (27.5%) were of foreign origin, which required continuous monitoring and quarantine (Table 2).

Table 2. The number of pathogenic Escherichia coli collected by the Enteric Pathogens Active Surveillance Network (Enter-Net), 2010–2019
Pathotype No. of isolates (%)
Domestic strains Imported strains Total
Enteropathogenic Escherichia coli (EPEC) 3,855 (66.6) 66 ( 9.4) 3,921 (60.5)
Enterotoxigenic Escherichia coli (ETEC) 1,469 (25.4) 556 (79.4) 2,025 (31.2)
Enteroinvasive Escherichia coli (EIEC) 47 ( 0.8) 54 ( 7.7) 101 ( 1.5)
Enterohemorrhagic Escherichia coli (EHEC) 414 ( 7.2) 24 ( 3.4) 438 ( 6.8)
Total 5,785 (100) 700 (100) 6,485 (100)

Rearranged by referring to the Table in Yun et al. [39] with public domain.

Download Excel Table

Pathogenic Escherichia coli were mostly isolated in summer from June to September, accounting for 61.7% of the total, and were more frequent in children under 9 years of age (37.9%). In children under the age of 9 years, EHEC was more common (51.7%) than other pathogenic Escherichia coli. The major virulence genes for each pathogenic Escherichia coli were detected in the following order (Table 3): EIEC ipaH (100%), EPEC eaeA (97.4%), ETEC st (53.4%), EHEC stx1 (45.7%), and EHEC with both Stx gene and eaeA (57.5%).

Table 3. Virulence gene profiles of pathogenic Escherichia coli collected by the Enteric Pathogens Active Surveillance Network (Enter-Net), 2010–2019
Pathotype No. of isolates (%) according to virulence genes
stx1 stx2 stx1+stx2 eaeA bfpA eaeA+bfpA lt st lt+st ipaH
Enterohemorrhagic 200 123 115
Escherichia coli (EHEC) (45.7) (28.1) (26.3)
Enteropathogenic 3,818 8 95
Escherichia coli (EPEC) (97.4) (0.2) (2.4)
Enterotoxigenic 407 1,081 537
Escherichia coli (EIEC) (20.1) (53.4) (26.5)
Enteroinvasive 101
Escherichia coli (EIEC) (100.0)
Total 438 (100) 3,921 (100) 2,025 (100) 101 (100)

Rearranged by referring to the Table in Yun et al. [39] with public domain.

Stx, Shiga toxins.

Download Excel Table

Polymerase Chain Reaction and Internal Amplification Control for diagnosing Pathogenic Escherichia coli

1. Conventional PCR and real-time PCR for pathogenic Escherichia coli

PCR is an easy alternative tool for the identification of Escherichia coli that can be used for diagnosis by amplifying specific genes of interest present in the target pathotype [40]. Multiplex PCR simultaneously amplifies more than one target sequence in the same reaction mixture [41]. Multiplex PCR can be applied to various virulence-associated genes to differentiate between different pathotypes.

Until now, the various methods that are explored to diagnose Escherichia coli and diarrheagenic Escherichia coli in water samples using multiplex PCR [42], multiplex real-time PCR [43], nucleic acid based sequence amplification real-time PCR [44], propidium monoazide real-time PCR [45], real-time PCR and quantitative real-time PCR [46], reverse transcriptase PCR [47], and so on.

The main advantages and disadvantages (limitations) of each method are as follows.

The advantages of standard PCR are (a) Higher sensitivity and specificity than culture-based methods, (b) Possibility of multiplex PCR for multiple pathogen detection, (c) Detects viable but nonculturable cells, (d) Simultaneous detection of different targets within the same species is possible (multiplex PCR), and the disadvantages are (a) Post-PCR confirmation step needed (for example, electrophoresis), (b) Non-quantitative, (c) No distinction between viable and dead cells (detects both), (d) Inhibition of the amplification when environmental samples are analyzed due to the presence of contaminants (for example, organic, inorganic and biomass content), (e) Low nucleic acid concentration causes frequent variability on the results, which leads to tube-to-tube variability [42,48].

The advantages of real-time PCR are (a) Faster than conventional PCR, (b) High level of sensitivity and specificity, (c) Real-time detection, (d) Quantification of the target in the sample is possible (quantitative real-time PCR), and the disadvantages are (a) Inhibition of the amplification when environmental samples are analyzed due to the presence of contaminants, (b) No distinction between viable and dead cells (detects both) [43,48].

The advantages of nucleic acid based sequence amplification real-time PCR are (a) Distinguishes viable from dead cells, (b) No interference from background DNA, and the disadvantage is (a) The same as in RT-PCR [44,48].

The advantages of propidium monoazide real-time PCR are (a) Distinguishes live from dead cells and from free DNA, (b) Simple to perform, and the disadvantages are (a) Possible inhibition from high solid content samples, (b) Use of an extremely toxic compound [45,48].

The advantage of reverse transcriptase PCR is (a) Distinguishes viable from dead cells, and the disadvantages are (a) Complexity of the procedures, (b) Short half-life of RNA, (c) Technical expertize is necessary, (d) Environmental samples can inhibit the detection [47,48].

Mendes Silva and Domingues [48] reported in detail the target gene and the method used to detect pathogenic Escherichia coli. It is summarized in detail in Table 4.

Table 4. PCR methods used to detect pathogenic Escherichia coli in samples
Type of pathogenic Escherichia coli Target gene Detection method used
Escherichia coli clpB-mRNA Nucleic acid based sequence amplification real-time PCR (Molecular beacon probe)
Escherichia coli Internal transcribed spacer (ITS) region between 16S-23S rRNA subunit genes Quantitative real-time PCR (SYBR Green)
Escherichia coli, Helicobacter pylori lacZ Quantitative real-time PCR (TaqMan probe)
Enterococcus spp., Enterococcus faecalis/faecium, Escherichia coli, and Shigella spp. 23S rRNA, mtlf, ddl, and atpD Reverse transcriptase PCR (TaqMan probe)
Escherichia coli O157:H7 Normal rfbE PCR
rfbE and fliC Real-Time PCR and electronic microarray
stx1 and/or stx2 Multiplex-Reverse transcriptase PCR (SYBR Green)
stx1, stx2, and rfbE Reverse transcriptase PCR (TaqMan probe)
Stressed eae, stx1, and stx2 Multiplex-Quantitative real-time PCR (Minor groove binding probes)
Enterohemorrhagic Escherichia coli (EHEC) stx1, stx2, and eae Multiplex-Reverse transcriptase PCR (SYBR Green)
Enterotoxigenic Escherichia coli (ETEC) LT1 Quantitative real-time PCR (Molecular beacon probe)
LT1 and ST1 Quantitative real-time PCR (SYBR Green)
Shiga toxin-producing Escherichia coli (STEC) stx2 Quantitative real-time PCR (Molecular beacon probe)

Rearranged by referring to the Table in Mendes Silva and Domingues [48] with permission of Elsevier.

PCR, polymerase chain reaction; Stx, Shiga toxins; LT, heat-labile enterotoxins; ST, heat-stable enterotoxins.

Download Excel Table

Waturangi et al [49] reported that prevalence of pathogenic Escherichia coli from salad vegetable and fruits sold in Jakarta. Fruits and Vegetables were analyzed by multiplex conventional PCR which consisted of six sets of primer encoding virulence genes were used such as aggr (EAEC), stx (EHEC), ipah (EIEC), eae (EPEC), and elt & est (ETEC) [49].

And Rani et al [50] demonstrated that trends in point-of-care diagnosis for Escherichia coli O157:H7 in food and water. Various strategies could be applied to manage the outbreak of infection from Escherichia coli O157:H7. However, since early diagnosis of Escherichia coli O157:H7 was not easy, prevention strategies to minimize infection were difficult. Unfortunately, the gold standard method currently used to detect Escherichia coli O157:H7 was the culture methods. For the purpose of overcoming the limitations of Escherichia coli O157 diagnosis, mobile PCR and CRISPR-Cas diagnosis platforms have been recently developed [50].

Furthermore, various methods are currently being used for the diagnosis of Escherichia coli O157, for example, isothermal amplification method, biosensor, surface-enhanced Raman spectroscopy, paper-based diagnosis, and smart phone-based digital method [50].

2. Internal amplification control for effectively eliminating false-negative results in PCR

Although PCR is a routinely used method, it may be difficult to reproduce the results owing to the differences in the performance of PCR thermal cyclers and the efficiency of DNA polymerase and presence of various PCR inhibitors in the environment [51].

IAC is a nontarget DNA sequence that can be added to the sample and is amplified simultaneously with the target sequence [52]. IAC can prevent false-negative results that may be caused by PCR inhibitors [53]. The European standardization committee, in cooperation with the International Standard Organization, proposed the guidelines for testing pathogens by using PCR, including IAC [54].

The approach used for developing an IAC largely depends on whether it will act competitively or non-competitively with the target sequence. In a competitive strategy, the target sequence and IAC are amplified using a common primer set under the same conditions [55]. In this strategy, the amount of IAC used is very important because it affects the limit of detection of the target sequence [56]. In a noncompetitive strategy, target sequence and IAC are amplified using different primer sets [57].

Conflict of Interest

The authors declare no potential conflict of interest.


This paper was supported by the Konkuk University Researcher Fund, 2020. Also, the authors thank the support of all members of Center for One Health in Konkuk University.



Jang J, Hur HG, Sadowsky MJ, Byappanahalli MN, Yan T, Ishii S. Environmental Escherichia coli: ecology and public health implications: a review. J Appl Microbiol. 2017;123:570-581.


Zarringhalam M, Goudarzi H, Nahaei MR, Bandehpour M, Shahbazi G. Detection of Escherichia coli pathotypes from the cases of diarrhea. Biosci Biotechnol Res Asia. 2016;13:247-255.


MacDonald E, Møller KE, Wester AL, Dahle UR, Hermansen NO, Jenum PA, et al. An outbreak of enterotoxigenic Escherichia coli (ETEC) infection in Norway, 2012: a reminder to consider uncommon pathogens in outbreaks involving imported products. Epidemiol Infect. 2015;143:486-493.


International Organization for Standardization [ISO]. Microbiology of food and animal feeding stuffs—polymerase chain reaction (PCR) for the detection of food-borne pathogens—general requirements and definitions. EN ISO 22174:2005. Geneva, Switzerland: ISO.


Cho SH, Kim J, Oh KH, Hu JK, Seo J, Oh SS, et al. Outbreak of enterotoxigenic Escherichia coli O169 enteritis in schoolchildren associated with consumption of kimchi, Republic of Korea, 2012. Epidemiol Infect. 2014;142:616-623.


Sarowska J, Futoma-Koloch B, Jama-Kmiecik A, Frej-Madrzak M, Ksiazczyk M, Bugla-Ploskonska G, et al. Virulence factors, prevalence and potential transmission of extraintestinal pathogenic Escherichia coli isolated from different sources: recent reports. Gut Pathog. 2019;11:10.


Yim. JH. Developments of novel multiplex conventional PCR and Real-time PCR with IAC for the detection of pathogenic Escherichia coli [Ph.D. dissertation] Seoul, Korea: Konkuk University; 2021.


Aijuka M, Buys EM. Persistence of foodborne diarrheagenic Escherichia coli in the agricultural and food production environment: implications for food safety and public health. Food Microbiol. 2019;82:363-370.


Belotserkovsky I, Sansonetti PJ. Shigella and enteroinvasive Escherichia coli. Curr Top Microbiol Immunol. 2018;416:1-26.


Croxen MA, Finlay BB. Molecular mechanisms of Escherichia coli pathogenicity. Nat Rev Microbiol. 2010;8:26-38.


Chandra M, Cheng P, Rondeau G, Porwollik S, McClelland M. A single step multiplex PCR for identification of six diarrheagenic E. coli pathotypes and Salmonella. Int J Med Microbiol. 2013;303:210-216.


Devi TS, Durairaj E, Lyngdoh WV, Duwarah SG, Khyriem AB, Lyngdoh CJ. Real-time multiplex polymerase chain reaction with high-resolution melting-curve analysis for the diagnosis of enteric infections associated with diarrheagenic Escherichia coli. Indian J Med Microbiol. 2018:36:547-556.


Benny E, Mesere K, Pavlin BI, Yakam L, Ford R, Yoannes M, et al. A large outbreak of shigellosis commencing in an internally displaced population, Papua New Guinea, 2013. Western Pac Surveill Response J. 2014;5:18-21.


Gomes TAT, Elias WP, Scaletsky ICA, Guth BEC, Rodrigues JF, Piazza RMF, et al. Diarrheagenic Escherichia coli. Braz J Microbiol. 2016;47:3-30.


Dallman TJ, Chattaway MA, Cowley LA, Doumith M, Tewolde R, Wooldridge DJ, et al. An investigation of the diversity of strains of enteroaggregative Escherichia coli isolated from cases associated with a large multi-pathogen foodborne outbreak in the UK. PLoS ONE 2014;9:e98103.


Escher M, Scavia G, Morabito S, Tozzoli R, Maugliani A, Cantoni S, et al. A severe foodborne outbreak of diarrhoea linked to a canteen in Italy caused by enteroinvasive Escherichia coli, an uncommon agent. Epidemiol Infect. 2014;142:2559-2566.


Gutiérrez Garitano I, Naranjo M, Forier A, Hendriks R, De Schrijver K, Bertrand S, et al. Shigellosis outbreak linked to canteen-food consumption in a public institution: a matched case-control study. Epidemiol Infect. 2011;139:1956-1964.


Kaper JB, Nataro JP, Mobley HLT. Pathogenic Escherichia coli. Nat Rev Microbiol. 2004;2:123-140.


Karmali MA. Infection by Shiga toxin-producing Escherichia coli: an overview. Mol Biotechnol. 2004;26:117-122.


Soleimani M, Morovvati A, Hosseini SZ, Zolfaghari MR. Design of an improved multiplex PCR method for diagnosis of enterohaemoraghic E. coli and enteropathogic E. coli pathotypes. Gastroenterol Hepatol Bed Bench. 2012;5:106-111.


Frank C, Werber D, Cramer JP, Askar M, Faber M, an der Heiden M, et al. Epidemic profile of Shiga-toxin–producing Escherichia coli O104:H4 outbreak in Germany. N Engl J Med. 2011;365:1771-1780.


Kagambèga A, Martikainen O, Siitonen A, Traoré AS, Barro N, Haukka K. Prevalence of diarrheagenic Escherichia coli virulence genes in the feces of slaughtered cattle, chickens, and pigs in Burkina Faso. MicrobiologyOpen. 2012;1:276-284.


McDaniels AE, Rice EW, Reyes AL, Johnson CH, Haugland RA, Stelma GN. Confirmational identification of Escherichia coli, a comparison of genotypic and phenotypic assays for glutamate decarboxylase and β-D-glucuronidase. Appl Environ Microbiol. 1996;62:3350-3354.


Giron JA, Ho AS, Schoolnik GK. An inducible bundle-forming pilus of enteropathogenic Escherichia coli. Science. 1991;254:710-713.


Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988;239:487-491.


Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev. 1998;11: 142-201.


Ethelberg S, Lisby M, Böttiger B, Schultz AC, Villif A, Jensen T, et al. Outbreaks of gastroenteritis linked to lettuce, Denmark, January 2010. Euro Surveill. 2010;15: 19484.


Scheiring J, Andreoli SP, Zimmerhackl LB. Treatment and outcome of Shiga-toxin-associated hemolytic uremic syndrome (HUS). Pediatr Nephrol. 2008;23:1749-1760.


Jerse AE, Yu J, Tall BD, Kaper JB. A genetic locus of enteropathogenic Escherichia coli necessary for the production of attaching and effacing lesions on tissue culture cells. Proc Natl Acad Sci USA. 1990;87:7839-7843.


Li B, Liu H, Wang W. Multiplex real-time PCR assay for detection of Escherichia coli O157:H7 and screening for non-O157 Shiga toxin-producing E. coli. BMC Microbiol. 2017;17:215.


Nandy S, Dutta S, Ghosh S, Ganai A, Rajahamsan J, Theodore R, et al. Foodborne-associated Shigella sonnei, India, 2009 and 2010. Emerg Infect Dis. 2011;17:2072-2074.


Moyo SJ, Maselle SY, Matee MI, Langeland N, Mylvaganam H. Identification of diarrheagenic Escherichia coli isolated from infants and children in Dar es Salaam, Tanzania. BMC Infect Dis. 2007;7:92.


Hao R, Qiu S, Wang Y, Yang G, Su W, Song L, et al. Quinolone-resistant Escherichia coli O127a:K63 serotype with an extended-spectrum-beta-lactamase phenotype from a food poisoning outbreak in China. J Clin Microbiol. 2012;50:2450-2451.


Launders N, Locking ME, Hanson M, Willshaw G, Charlett A, Salmon R, et al. A large Great Britain-wide outbreak of STEC O157 phage type 8 linked to handling of raw leeks and potatoes. Epidemiol Infect. 2016;144:171-181.


Watahiki M, Isobe J, Kimata K, Shima T, Kanatani J, Shimizu M, et al. Characterization of enterohemorrhagic Escherichia coli O111 and O157 strains isolated from outbreak patients in Japan. J Clin Microbiol. 2014;52:2757-2763.


Sunabe T, Honma Y. Relationship between O-serogroup and presence of pathogenic factor genes in Escherichia coli. Microbiol Immunol. 1998;42:845-849.


Buchholz U, Bernard H, Werber D, Böhmer MM, Remschmidt C, Wilking H, et al. German outbreak of Escherichia coli O104:H4 associated with sprouts. N Engl J Med. 2011;365:1763-1770.


King LA, Nogareda F, Weill FX, Mariani-Kurkdjian P, Loukiadis E, Gault G, et al. Outbreak of Shiga toxin-producing Escherichia coli O104:H4 associated with organic fenugreek sprouts, France, June 2011. Clin Infect Dis. 2012;54:1588-1594.


Yun YS, Kim NO, Hong SH, Chun JH, Hwang KJ. The prevalence of pathogenic Escherichia coli isolated by the enteric pathogens active surveillance network (Enter-Net), 2010–2019. Pub Health Wkly Rep. 2020;13:2860-2870.


Mullis KB. The unusual origin of the polymerase chain reaction. Sci Am. 1990;262: 56-65.


Levine MM. Escherichia coli that cause diarrhea: enterotoxigenic, enteropathogenic, enteroinvasive, enterohemorrhagic, and enteroadherent. J Infect Dis. 1987;155:377-389.


Gómez-Duarte OG, Bai J, Newell E. Detection of Escherichia coli, Salmonella spp., Shigella spp., Yersinia enterocolitica, Vibrio cholerae, and Campylobacter spp. enteropathogens by 3-reaction multiplex polymerase chain reaction. Diagn Microbiol Infect Dis. 2009;63:1-9.


Maheux AF, Bissonnette L, Boissinot M, Bernier JLT, Huppé V, Picard FJ, et al. Rapid concentration and molecular enrichment approach for sensitive detection of Escherichia coli and Shigella species in potable water samples. Appl Environ Microbiol. 2011;77:6199-6207.


Heijnen L, Medema G. Method for rapid detection of viable Escherichia coli in water using real-time NASBA. Water Res. 2009;43:3124-3132.


Gensberger ET, Sessitsch A, Kostić T. Propidium monoazide–quantitative polymerase chain reaction for viable Escherichia coli and Pseudomonas aeruginosa detection from abundant background microflora. Anal Biochem. 2013;441:69-72.


Patel CB, Vajpayee P, Singh G, Upadhyay RS, Shanker R. Contamination of potable water by enterotoxigenic Escherichia coli: qPCR based culture-free detection and quantification. Ecotoxicol Environ Saf. 2011;74:2292-2298.


Zhang Y, Riley LK, Lin M, Hu Z. Determination of low-density Escherichia coli and Helicobacter pylori suspensions in water. Water Res. 2012;46:2140-2148.


Mendes Silva D, Domingues L. On the track for an efficient detection of Escherichia coli in water: a review on PCR-based methods. Ecotoxicol Environ Saf. 2015;113: 400-411.


Waturangi DE, Hudiono F, Aliwarga E. Prevalence of pathogenic Escherichia coli from salad vegetable and fruits sold in Jakarta. BMC Res Notes. 2019;12:247.


Rani A, Ravindran VB, Surapaneni A, Mantri N, Ball AS. Review: trends in point-of-care diagnosis for Escherichia coli O157:H7 in food and water. Int J Food Microbiol. 2021;349:109233.


Hoorfar J, Ahrens P, Rådstrom P. Automated 5’ nuclease PCR assay for identification of Salmonella enterica. J Clin Microbiol. 2000;38:3429-3435.


King LA, Loukiadis E, Mariani-Kurkdjian P, Haeghebaert S, Weill FX, Baliere C, et al. Foodborne transmission of sorbitol-fermenting Escherichia coli O157:[H7] Via ground beef: an outbreak in northern France, 2011. Clin Microbiol Infect. 2014;20: O1136-O1144.


Rådström P, Löfström C, Lövenklev M, Knutsson R, Wolffs P. Strategies for overcoming PCR inhibition. Cold Spring Harb Protoc. 2008.


He X, Shi X. Internal amplification control and its applications in PCR detection of foodborne pathogens. Wei Sheng Wu Xue Bao. 2010;50:141-147.


Siebert PD, Larrick JW. Competitive PCR. Nature. 1992;359:557-558.


Rosenstraus M, Wang Z, Chang SY, DeBonville D, Spadoro JP. An internal control for routine diagnostic PCR: design, properties, and effect on clinical performance. J Clin Microbiol. 1998;36:191-197.


Hoorfar J, Malorny B, Abdulmawjood A, Cook N, Wagner M, Fach P. Practical considerations in design of internal amplification controls for diagnostic PCR assays. J Clin Microbiol. 2004;42:1863-1868.