**3. Loop mediated isothermal amplification (LAMP) assay**

In order to circumvent the use of thermocyclers that entail the time-consuming thermal cycling, an innovative method such as isothermal DNA amplification has been introduced which finds its application in the advanced Research & Development (R & D) unit of the food industry. The LAMP reaction that involves isothermal amplification chemistry has a good range of possible applications, including point-of-care testing with the potential of getting developed into portable diagnostic systems, and quick testing of food products, clinical and environmental samples.

The isothermal characteristics of LAMP enable the simplification of the detection process without involving any costly and complex instrumentation wherein a simple heating block or a precise digital water bath would work. Though conventional PCR and LAMP techniques were reported to be vulnerable to several inhibitors while testing various biological (for example urinary and plant materials) matrices [64], yet LAMP is much less sensitive to amplification inhibitors [64], potentially permitting its application bypassing the general requirement for cultural enrichment or DNA purification.

Despite some disadvantages like its qualitative nature of detection, the LAMP offers several advantages over PCR. LAMP assay emphasizes the requirement of a heating block and obviates the need for a thermal cycler. Unlike PCR that requires DNA extraction from samples for amplification, LAMP assay does not require DNA extraction step. The difficulties in amplifying DNA in PCR from unprocessed urinary samples in the presence of a high concentration of urea were reported by Khan et al. [65]. Therefore the LAMP assay, by rendering the DNA extraction step redundant, has made the process more rapid and facile [67]. The implementation of LAMP does not require any denatured template as due to the

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

use of Bst DNA polymerase from *Geobacillus stearothermophilus* with auto-cycling strand-displacement activity denatured template use has been eliminated. In the LAMP reaction, the nucleic acid amplification takes place at a fixed temperature (isothermal) through repetition of two types of elongation reactions occurring at the loop regions: self-elongation of templates from the stem-loop structure formed at the 3′-terminal and the binding and elongation of new primers to the loop region (**Figure 2**) [68]. LAMP reaction time is merely 60–65 min at 60–65°C involving four

#### **Figure 2.**

*Schematic representation of the principle steps in a LAMP assay and localization of the eight LAMP primers for specific amplification of target DNA. Adapted from Gallas-Lindemann et al. [68]. Copyright (2017). IntechOpen. Inner primer: FIP (consisting of F1c and F2), BIP (consisting of B1c and B2), typical length ~ 40 bp; outer primers: F3, B3 typical length ~ 20 bp; loop primers: LF and LB, typical length ~ 20 bp.*

to six precisely designed primers to amplify DNA targets at the specific amplification temperature [68].

The very purpose of inner primers that consisted of two different sequences was to recognize a sense and antisense sequences of the target viral DNA, and the outer primers were designed to recognize an external sequence of the target viral DNA [69]. Additionally, in the LAMP assay, as an advantage, the identification of a positive reaction does not involve any special processing or electrophoresis. Only the visual observation of color change of the reaction mix in normal light is enabled when the appropriate DNA-binding dye is used. Thus, LAMP positive results could be better detected through visual observation of turbidity changes [70]. This visualization process can be improved by a UV transilluminator. Hill et al. [67] had demonstrated the use of propidium iodide for detecting the LAMP products.

In order to detect generic *E. coli*, *E. coli* O157, or different VTEC virulence genes a number of LAMP assays were developed and discussed in several previous studies [71–74]. In order to develop the LAMP assays for the simultaneous detection of an *E. coli*-specific gene and verotoxin-elaborating genes, and capable of distinguishing between generic *E. coli* and VTEC that would serve the purpose of simultaneous detection both *E. coli* and VTEC simultaneously in beef would allow the simultaneous monitoring of hygienic status/quality of beef. Therefore, the development of multiplex LAMP assay was of paramount importance. In the study, the LAMP assay was designed to detect nonpathogenic *E. coli* targeting the phoA and VTEC targeting the *stx1* and *stx2* without the need for a cumbersome culture enrichment process. The specificity of the *phoA* LAMP-based detection assay for *E. coli* showed 100% specificity (when a total of 58 bacterial strains were used for detection purposes) to determine with no false-positive or false-negative results with strains of any of the other bacterial genera tested. Interestingly only phoA gene-positive *E. coli* strains showed detectable amplification and non-*E. coli* showed no amplification. LAMP-false negative tests were reported by Stratakos et al. [75] while determining non-pathogenic *E. coli* and verocytotoxigenic *E. coli* (VTEC) in beef and bovine feces. Of note for the improvement of LAMP detection sensitivity, an enrichment step (which would not allow the post-enrichment quantification of *E. coli* or VTEC) prior to LAMP was suggested following the demonstration in previous studies by Wang et al. [76], and also a touchdown LAMP approach was suggested by Wang et al. [73].

It is to be noted that the LAMP assay reported by Hill et al. [67] was able to detect a large number of strains with very high sensitivity. Since biological samples such as cerebrospinal fluid and blood require very high sensitivity as compared to urine samples LAMP can be suitably modified for its clinical uses. LAMP has also been proposed to detect a lower copy number in partially treated infections (postempirical antibiotic doses) [67].

## **4. DNA-based biosensors**

A biosensor typically consists of a bioreceptor element with a transducer. The bioreceptor, interacts specifically with the analyte, whereas the transducer converts the biomolecular interaction into an electronic signal. Three basic parts of a biosensor are recognition material, transducer or detector system, and signal processor [74]. Monitoring the molecular interaction between the DNA-based bioreceptor and the analyte is an essential element of various DNA-based sensing strategies. The measurement methods of DNA–DNA interactions that take place on the various sensor surfaces are gaining much interest to improve sensor performance. The assays are applicable to the determination of low numbers of *E. coli* cells in various

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

matrices. In addition, the molecular detection of *E. coli* using single-stranded nucleic acids or aptamers coupled with the electrochemical impedance spectroscopy for sensing of DNA is a growing field of research and proving to be an alternative method of detection to traditional techniques [74].

Arora et al. [77] reported an electrochemical DNA biosensor for the detection of *E. coli*. In this study, avidin was modified with –COOH and then attached to the polyaniline (PANI)-modified platinum disk by the covalent binding between –COOH and –NH/NH2 of PANI. Subsequently, the biotin-labeled DNA probe was functionalized on the electrode surface to achieve a LOD of 0.01 ng/uL for *E. coli* genomic DNA. Few studies reported the use of nanomaterials with graphene oxide (GO) to enhance the sensitivity of the DNA biosensor for *E. coli* detection. For example, a DNA biosensor for the detection of *E. coli* O157:H7 eaeA gene based on a novel sensing tag of GOx-Thi-Au@SiO2 nanocomposites is reported [78]. The combined use of GO and Au@SiO2 creates an environment for maintaining the appropriate conformation of DNA. These biosensor modalities led to wide linear response for *E. coli* O157:H7 eaeA gene in the range of 0.02 to 50.0 nM with LoD of 0.01 nM. In addition, Tiwari et al. [79] reported a DNA biosensor for *E. coli* O157:H7 using a DNA probe sequence. The DNA probe was immobilized onto GO modified iron oxide-chitosan hybrid nanocomposite (GIOCh) film. The DNA biosensor resulted in linear response to *E. coli* DNA in the range of 10−6 to 10−14 M with a LoD of 10−14 M.

Since its discovery in the 1980s, the system has demonstrated widespread applications in basic biotechnology research and disease treatment [80, 81]. A pressing need of the hour is the availability of a cost-efficient, rapid and selective molecular diagnostic platform to detect different pathogens and lethal diseases in the early stage of the infection. Quantitative PCR and metagenomic next-generation sequencing (mNGS) are the most commonly explored molecular platforms for the same; however, these methods have their disadvantages and limitations. Clustered Regularly Interspaced Short Palindromic Repeat/associated protein (CRISPR/ Cas)-based diagnostic platform for the detection of nucleic acids has progressively demonstrated its potential as an ideal diagnostic approach for pathogens, cancer biomarker, and single-nucleotide polymorphisms (SNPs) detection. CRISPR systems have evolved in prokaryotes as a defensive mechanism against foreign viruses by cleaving their nucleic acids [82–84].

Additionally, the unique cleavage activity of Cas9 is often utilized for the development of ultra-low abundance DNA biosensors. A highly innovative and sensitive CRISPR/Cas9 system was developed by Huang et al. [84] that triggered isothermal exponential amplification reaction (CAS-EXPAR) strategy to detect DNA targets with attomolar (aM) sensitivity and single-base specificity [84]. CAS-EXPAR was primed by the target DNA fragment produced by cleavage of CRISPR/Cas9, and associated with the cyclical amplification reaction to produce numerous DNA replicates capable of getting detected by a real-time SYBR Green fluorescence signal [83].

Recently, Sun et al. [84] reported the detection of *E. coli* O157:H7 based on the CRISPR/Cas9 coupled with metal–organic framework platform (MoF) (**Figure 3**). In this approach, the virulence gene sequences of *E. coli* O157:H7 were identified and spliced by the CRISPR/Cas9 system leading to strand displacement and rolling circle amplification. Subsequently, amplified products were hybridized with the target-specific probes. The virulence genes were detected by the fluorescence quenching caused due to MoF platform. The method showed high sensitivity with LoD of 4.0 × 101 CFU mL−1 [84]. Although there is only one reported work available for CRISPR/Cas-based detection of *E. coli*, however, the CRISPR/Cas system can be exploited further for the detection of *E. coli* and other waterborne pathogens using novel strategies.

**Figure 3.**

*CRISPR/Cas9 platform coupled with two-step isothermal amplification for detection of* E. coli *O157:H7. Reprinted with permission from Sun et al. [84]. Copyright (2020) American Chemical Society.*
