**2. Polymerase chain reaction (PCR) method**

PCR being a mighty and handy tool with molecular biologists showed enormous potential in various forms including multiplex PCR and quantitative real-time PCR. The advantage of PCR is that despite its inability to distinguish between live and dead cells, nonculturable cells may be detected rapidly. In the recent two decades, various PCR-based strategies have been introduced to improve the detection of indicator organisms [23, 24]. Genetic markers such as 23S rRNA and lacZ are often used to establish PCR tests for detecting *E. coli* in environmental samples [25, 26]. The uidA and tuf genes have been identified as potential targets for *E. coli*/Shigella detection using PCR [27, 28]. Most of the PCR assays were reported to amplify the virulence genes, such as *eaeA*, and *stx1, stx2* [29–33] or phenotypic genes, such as *rfbE* (O antigen), and *fliC* (H antigen), *uidA* and *lacZ* which are commonly shared [26, 28, 32]. The ability to generate these lesions is restricted to 43-kb loci of the *E. coli* O157:H7 chromosome [17]. Intimin encoded by eae locus is necessary for early bacterial cell attachment to host cells and the creation of A/E lesions [34, 35]. In a couple of studies, virulence genes like *stx1* and *stx2* were unable to accurately identify a species, owing to the fact that they are widely shared by different species or strains [33]. *Shigella dysenteriae* and *Aeromonas* spp. have been described as the two outliers as non-*E. coli* bacteria bearing Shiga toxin genes [36, 37]. Real-time PCR techniques targeting *Shigella* spp. in food or water utilizing *ipaH* as a target have also been developed to detect enteroinvasive *E. coli* (EIEC) that carries ipaH [36]. Therefore, phenotypic genes such as *rfbE* and *fliC* have been utilized as targets for confirmed identification of *E. coli* in PCR [30].

The *E. coli* genes such as uidA and tuf were used for the detection of *E. coli* and Shigella strains [27, 38, 39]. However, the uidA gene used as a marker was not reported in 3.4% of 116 *E. coli* strains [37]. In another work, Maheux et al. [27] detected Escherichia fergusonii in a PCR targeting the tuf gene. Albeit, it has been extensively reported, neither β-D-glucuronidase activity nor *uidA* gene amplification is the full proof for the accurate molecular detection *E. coli* in the presence of this enzyme or gene has been reported in Flavobacteria and to a great extent in *Shigella, Salmonella* and *Yersinia* [38, 40, 41]. Contrarily, Fricker & Fricker [42] using uidA primer pair detected five non-*E. coli* coliforms in water samples. Recently, Molina et al. [40] designed a set of primers targeting the *E. coli* orphan gene *yaiO* that encodes an outer membrane protein and succeeded in obtaining the *yaiO* amplicon of 115 bp size from unfermented and fermented dairy samples. These workers in terms of specificity claimed superiority of *yaiO* gene-based primers to uidA primers though the study was limited by small sample size. In another recent study, the xanQ-PCR using novel primer set for amplification of *xanQ* gene was demonstrated for specific detection of a large number of *E. coli* strains [41].

Li et al. [43] established a multiplex real-time PCR test that targets the *z3276* and Shiga toxin genes to specifically detect *E. coli* O157:H7 and screen for non-O157 STEC (*stx1* and *stx2*). The reaction mixture contained a primer set; four probes (*z3276, stx1, stx2,* and IAC), and the template DNA of appropriate concentrations. The optimized multiplex assay achieved the limit of detection (LOD) as low as 200 femto grams of bacterial DNA from beef and fresh spinach samples (40 CFU/ reaction). In a separate study, a multiplex fluorogenic PCR assay was developed to

quantify *E. coli* O157:H7 in manure, soil, dairy wastewater, and cow and calf feces in an artificial wetland. Oligonucleotides were designed to amplify the *stx1* and *stx2* and the *eae* genes of *E. coli* O157:H7 in a simplex reaction [44].

Being a rapid, sensitive, and specific method enabling the detection of multiple pathogens simultaneously this method finds applications in different types of foods and poultry industries. Nguyen et al. [45] developed a multiplex PCR for the rapid and simultaneous detection of three epidemic food-borne pathogens: *E. coli* O157:H7, *Salmonella* spp., and *Listeria monocytogenes* in food samples.

In developing countries, the identification of enteric pathogens in food and other edible items are time-consuming process and often results in wrong and delayed diagnosis. Enteropathogenic *E. coli* (EPEC) has been reported to be frequently associated with outbreaks of infantile diarrhea and recognized as a causative agent for diarrheagenic ailments [46]. In order to detect and identify the Shiga toxin producing *E. coli*, enterohemorrhagic *E. coli* (EHEC), and EPEC primers were designed to amplify *eae* gene and long polar fimbriae (*lpfA*) variants, the bundleforming pilus gene *bfpA*, and the Shiga toxin-encoding genes *stx1* and *stx2* [47]. This group demonstrated consistent amplification of genes specific to the prototype EHEC O157:H7 EDL933 (*lpfA1–3, lpfA2–2, stx1, stx2*, and *eae-γ*) and EPEC O127:H6 E2348/69 (*eae-α, lpfA1–1*, and *bfpA*) strains using the optimized mPCR protocol with purified genomic DNA (gDNA). A screen of gDNA from isolates in a diarrheagenic *E. coli* collection revealed that the mPCR assay was successful in predicting the correct pathotype of EPEC and EHEC clones grouped in the distinctive phylogenetic disease clusters EPEC1 and EHEC1, and was able to differentiate EHEC1 from EHEC2 clusters. The mPCR assay detection threshold was 2 × 104 CFU per PCR reaction for EHEC and EPEC. Thus, mPCR methodology permitted differentiation of EPEC, STEC, and EHEC strains from other pathogenic *E. coli* and the developed assay has the potential tool for rapid diagnosis of these pathogens. Wang et al. [48] demonstrated the ability of the mPCR assay to detect six bacterial pathogens viz., *E. coli*, *Pasteurella multocida, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella* spp. and *Staphylococcus aureus* in liver, spleen, and blood samples from experimentally infected chicks without cross-amplification with viruses or parasites. In the mPCR assay, gene targets were *phoA, KMT1, ureR, toxA, invA*, and *nuc* of these six pathogens, and six sets of specific primers were designed.

Toma et al. [49] used a single-tube mPCR for the identification of enteropathogenic *E. coli* (EPEC), enteroinvasive *E. coli* (EIEC), enterotoxigenic *E. coli* (ETEC), enteroaggregative *E. coli* (EAEC), and Shiga toxin-producing *E. coli* (STEC). In total six targets were chosen for (*eae*) enteropathogenic *E. coli*, (*stx*) Shiga toxinproducing *E. coli* enterotoxigenic *E. coli*, elt, and est. for enterotoxigenic *E. coli*, (*ipaH*) for enteroinvasive *E. coli* for, and *aggR* for enteroaggregative *E. coli*.

Chen et al. [50] developed a multiplex rtPCR assay for the identification of diarrheagenic *E. coli* (DEC) and claimed it to be a highly sensitive and specific and suggested the rapid identification of DEC in clinical and public health laboratories. Specific virulence genes were selected to identify specific pathogens: *ipaH* for EIEC, *stp/sth/lt* for ETEC, *eaeA/escV* for EPEC, *stx1/stx2* for EHEC, *aggR* for EAEC. The 5′ end of primers were added with a homo tail sequence to reduce the primer dimer formation and the addition of homo tail to 5′ end of primer sequences allowed proper annealing temperature that would fall into broad range in each individual PCR reaction. Molecular beacons were modified and designed using DNA folding form website (http://mfold.rit.albany.edu/?q=mfold/DNA-Folding-Form) [50]. Five categories of DEC were split into two tubes. For tube number one, *stp/sth/lt* for ETEC, *aggR* for EAEC and IAC were included, while *ipaH* for EIEC, *eaeA/escV* for EPEC, *stx1/stx2* for EHEC and IAC were included in tube number two. Carboxy fluorescein (FAM), Hexachloro fluorescein (HEX), Carboxy-X-rhodamine (ROX),

*Molecular Diagnostic Platforms for Specific Detection of* Escherichia coli *DOI: http://dx.doi.org/10.5772/intechopen.101554*

**Figure 1.**

*Schematic depicting the steps in culture-independent detection of* E. coli *in a sample using qPCR method. Bacillus atrophaeus Spores are used as an internal control for monitoring of possible PCR inhibition [52].*

Quasar 705, and indodicarbocyanine5 (Cy5) fluorescence were collected and recorded at the end of the annealing step during the third stage.

Detection of harmful bacteria with higher specificity, sensitivity, and reliability is the focus of nucleic acid-based approaches. The desired nucleic acid sequence is hybridized to a synthetic oligonucleotide for specific detection of the pathogen [51]. Nucleic acid-based approaches are routinely used to detect bacterial infections and their toxin-producing genes [51]. Nucleic acid-based methods are rapid and easy to use, and they do not require the pathogens to be cultured (**Figure 1**).

Even a decade ago, the identification and measurement of specific target genes with absolute accuracy and as little as a few copies in a matter of hours was a dream. In the area of water quality assessment, however, qPCR technology has proven to be a powerful technique [53]. Unlike the classical PCR, which needs agarose-gel electrophoresis to identify the end-point PCR products, the qPCR enables assessing PCR product amplification by measuring fluorescence signals released by specialized dual-labeled probes or the intercalating dyes. The fluorescence intensity generated during the qPCR is directly related to the quantity of PCR products produced [12, 54, 55]. The most often used fluorescent systems for qPCR include SYBR green, TaqMan probes, and molecular beacons [56]. The qPCR techniques, which have higher specificity, sensitivity, and reliability than classic culture methods and mPCR [57], allow for the time-efficient detection of harmful bacteria with higher specificity, sensitivity, and reliability [12, 56, 58]. Although the qPCR has been used to detect and quantify *E. coli* O157:H7 in food and clinical samples, it has not been thoroughly evaluated with environmental samples [57, 59, 60].

Utilizing TaqMan probes labeled with different fluorophores, microfluidic qPCR was shown to identify pathogens such as *Listeria monocytogenes, Vibrio cholerae, Vibrio parahaemolyticus, Pseudogulbenkiana* spp., *Salmonella typhimurium, Shigella flexneri, Clostridium perfringens*, and *E. coli* at a limit of detection of 100 CFU/L [56, 61]. Despite its high sensitivity, qPCR has significant drawbacks, such as the inability to provide information on the physiological status of target cells in environmental samples. Humic substances found in environmental samples such as water hinder DNA polymerase activity, and colloidal debris has been reported to have a DNA affinity [62, 63]. There is no universal answer to avert such problems. As a result, the existence of these compounds in environmental samples has the potential to adversely affect the amplification effectiveness of qPCR, which is used to detect small quantities of bacteria [60]. To overcome these issues in qPCR, several compounds such as bovine serum albumin, methoxsalen, dimethyl sulfoxide, and internal amplification controls have been proposed. However, these approaches may have certain drawbacks as well as benefits [64, 65]. Walker et al. [63] established a new qPCR technique for detecting and quantifying *E. coli* that targeted a segment of the ybbW gene, which encodes a potential Allantoin transporter. The *ybbW* gene

is part of the *E. coli* "core genome," which means that each gene is found in >95 percent of all sequenced strains. For this work, water samples were taken at monthly intervals from different locations in the southwest of England. The *ybbW*-qPCR was found to be 100% specific towards 87 *E. coli* strains tested. This work also reported that despite the theoretically low detection levels achievable by qPCR, the quantity of *E. coli* DNA has been the key issue in limiting the detection in real samples. This could be addressed in part by filtering greater quantities of water samples, but this is likely to be unfeasible for regular sample analysis and could result in the accumulation of higher inhibitory substance quantities.

In another study, Liu et al. [66] reported designing of the novel oligonucleotide primer set and TaqMan probes targeting the specific virulence genes of twelve common food pathogens such as *E. coli* O157:H7, *Salmonella enterica, L. monocytogenes/ ivanovii*, β-*Streptococcus hemolyticus, Enterococcus faecalis, Yersinia enterocolitica, Shigella sp., P. mirabilis, V. fluvialis, V. parahaemolyticus, S. aureus* and *Campylobacter jejuni*. Liu et al. [66] reported the use of TaqMan in artificially spiked dilution series of each pathogen into meat to detect 12 strains. The TaqMan assays demonstrated expected amplification with no amplification inhibition. In spiked food samples, *V. parahaemolyticus* was found in concentrations ranging from 103 to 107 CFU/g, while the remaining 11 strains were from 104 to 107 CFU/g. The qPCR has been touted as a specific and sensitive method with high throughput sample analysis. Smati et al. [66] reported a rapid, sensitive, and reliable qPCR method to quantify *E. coli* phylogroup from 100 healthy human stool specimens and demonstrated the existence of subdominant clones. The new 16S-rRNA-qPCR assay was highly repeatable, with a detection limit of 105 CFU/g of feces.
