Different molecular approaches used for detection of Escherichia coli O 157 : H 7 from different sources

Escherichia coli (E. coli) O157:H7 was first recognized in 1982 after investigation of two cases of hemorrhagic colitis and hemolytic uremic syndrome. E. coli O157:H7 the shiga toxin producing strain of E. coli are most commonly involved in many outbreaks of human and animal diseases with low infectious dose. Human infections caused by E. coli O157:H7 are due to consumption of undercooked ground beef, raw milk and raw milk products, water, cold sandwiches, vegetables, raw apple cider and person to person contact. Due to low infectious dose and different mode of transmission it is essential to develop more rapid and sensitive methods for detection of shiga toxin producing E. coli. In this review different molecular based detection methods are discussed. PCR speeds up the analysis time and amplify small number of microbes are described along different variations of PCR following DPCR, omitting the tedious step of DNA extraction. Genomic techniques are reviewed along further epidemiological typing like phage typing and biotyping. Biosensors provide real time detection described along with use of fluorescence and microscopy. Emerging technologies including microarray, molecular beacons and lab-on-a-chip are described along with new improvements in molecular beacons. Merits and demerits of each approach discussed are examined throughout the article.


Introduction Enterohemorrhagic
Escherichia coli (EHEC) contains numerous serotypes, among which E. coli O157:H7 is the most common serotype causing epidemic and sporadic diseases in humans throughout North America, Japan, Scotland, United State, Canada and many parts of Europe [1-4].From investigation of two outbreaks of Hemorrhagic colitis (HC), a leading cause of Hemolytic uremic syndrome (HUS) in 1982 the E. coli O157:H7, a shiga toxin (stx) producing E. coli (STEC) was confirmed a pathogenic bacteria [5].Other shiga toxin producing serotypes e.g.O103, O111, O145 and O26 occur, increasing human pathogens in Australia, South America and Europe [6, 7].Serotype O157:H7 are the most virulent serotype because of production of stx genes also known as Vero toxins (VT).The stx produced by E. coli O157:H7 in the intestine [8, 9], begin illness with watery diarrhea followed by bloody diarrhea called HC [10].Sometime HC results into life threatening disease such as HUS following thrombotic thrombocytopenic purpura (TTP), renal failure and even death [11-13].There are other strains of EHEC that are E. coli O157, yet do not show flagellar H antigen and named E. coli O157:NM.Most  A verity of methods developed includes culturing on specific media, assessing of biochemical characteristics, screening and serotyping for existence of virulence factors.However these methods are not rapid and reliable to differentiate such pathogenic strain from other stx producing E. coli.Therefore this paper describes a number of sensitive molecular techniques used for isolation and identification of STEC O157:H7 from different sources.

Molecular detection of E. coli O157:H7
There are several ways for detection of E. coli O157:H7, among which the below diagram demonstrates the main methods discussed in present review (Figure 1)  describe mPCR for recognition of pathogenic bacteria from soil and water samples.Samples spiked with serotype O157:H7 were enriched before PCR.The recognition limit found was 1 cfu ml-1 water and 2 cfu g-1 soil.An important feature occur was starvation serotype O157: H7 for about 35 days hitherto added to soil could not infect the identification of primary cell numbers as below 10 cfu g -1 soil.Real time PCR is developed for both identification and determination of solitary bacterial species from multiplex microbial community along from complex samples.studied cattle and dairy wastewater for identification of O157:H7 by using real-time PCR.Samples were artificially inoculated 10 4 , 10 7 and 10 8 cells ml -1 of serotype O157:H7.Extracted DNA from strain was used first to test PCR protocol to modify the amplification condition prior to real-time PCR experiments.Exact fragments of ca 106, 150 and 200 bp toward eae, stx1 and stx2 genes subsequently were acquired.Real-time PCR protocols were conducted with DNA (extracted) from inoculated wastewater samples.The susceptibility limit obtained occur 10 -1 pg µl -1 for eae gene, stx2 and 16S rRNA and 1 pg µl -1 for stx1 subsequently.The reverse transcriptase PCR (RT-PCR) assay has been progressed for detection of E. coli O157:H7.Reverse transcriptase is an enzyme able to synthesize DNA (single standard) from RNA (especially mRNA) within 5'-3' direction

Multilocus variable number tandem repeat analysis (MVTR)
This is DNA construct molecular typing approaches that can identify several repeats at multiple-variable number tandem repeat loci [63].MVTR assay identify isolates from different outbreaks caused by serotype O157:H7.This method is easy and sensitive as PFGE for detection of serotype O157:H7 [69].The approach introduces new, fast and safe methodologies for typing, so impact the health field of public.It has high resolution and reduced typing time so it is important in resolving complex outbreaks.The method is beneficial too for extensive automation.However the limitation of the assay is its high particularity, due to which lack of standardization for published assays [68].

Phenotypic tests for E. coli O157:H7 3.1. Serotyping
Full serotyping of stx producing E. coli is necessary because stx production has been investigated in strain of distinct serotypes.For full serotyping stx could be sent to reference laboratories for investigation of antisera in contrast to O antigens and H antigens.Mostly O157 strains are usual with flagellar H7 antigen, but non motile strain occur also been investigated lacking flagellar H antigen, a useful feature to distinguish between E. coli O157:H7 and non O157.Some stx producing E. coli belongs to enteropathogenic serogroups e.g.O26, O111, O55 and O128.Hence strains from these serogroups isolated from HUS or HC cases or outbreaks of diarrhea should be fully serotype or address to reference laboratories for additional tests to distinguish between VTEC [75].

Biosensor
The problem with rapid developed method is number of steps involve, including time consuming enrichment procedures.Biosensor are used ordinary but with many issues still remaining to be solved.An evanescent wave fiber optic biosensor has been used for identification of bacteria from ground beef samples [82].Biosensor directs the light onto optical fiber probe by using a 635-nm laser diode.The optical fiber probes generate the evanescent wave.Fluorescent molecules are elevated and a part of emission re-couples in fiber probe.The fluorescent signals are detected and quantified by photodiode.A sandwich immunoassay can detect 9.0*10 3 cfu g -1 for 25 g sample and 5.2*10 2 cfu g -1 for 10 g samples.No false positive were obtained from results.Fiber optic biosensor has been used for detection of genomic DNA from E. coli and other coli forms [83].A combination of PCR-acoustic wave sensor was added for detecting sequences of E. coli O157:H7 [84].PCR was used for amplification of a unique sequence (509 bases) of pathogen and biotinylated probe to attach on sensor surface.This approach was suggested by author to be suitable for detection of bacterium from different sources e.g.water, food and clinical samples.Additionally it was suggested that this approach may assimilate for PCR based analysis of DNA.But achieving of this aim requires much more research.Leach et al.
[85] studied spinach for presence of E. coli O157:H7 by using electro chemiluminescent (ECL) assay and fluorescence based cytometric bead assay.The ECL system detected >0.1 cfu of O157:H7 from per gram of spinach after enrichment of 5 hour, with total assay time of 6.5 hour.Cytometric bead assay detected >0.1 cfu per gram after enrichment of 7 hour, with total assay time of >10 hrs.The author demonstrates both biosensor based assays are sensitive for rapid identification of EHEC O157:H7 on produce in time frames as compare to other testing formats.Biosensor is also used with surface plasmon resonance (SPR) for rapid detection of O157:H7.BIAcore, as shown in Figure 3 are most widely used SPR based systems using antibodies against O157:H7 [86-88].GAI, L., 2011 used SPR biosensor for identification of O157:H7.Dextran modified sensor chip (CM5) and BIAcore 3000 (a SPR instrument) was used in this method.Following activation with use of EDC/NHS antibody for O157:H7 was immobilized on gold surface of SPR sensor, after that ethanolamine was injected and chip was organized for E. coli O157:H7.The detection limit found was 3*10 5 cfu X ml -1 for E. coli O157:H7 with time range of 5 to 7 min.Regeneration results were effective and allowed the chip to be reused for more than 50 samples [89].In another study E. coli O157:H7 was modified using plasmid vector red-shifted green fluorescence protein (pEGFP) to show red shifted green fluorescence protein (EGFP) from aequorea victoria.Total cells and nonviable cells were used to determine the expression of EGFP at cellular by microscopic study of immunestained and membrane impermeable, dye stained cultures.The expression of EGFP was improved by sub culturing; reaching 83.4+/-0.1%,while the intensity of fluorescence varied among cells.EGFP show nonviable cells at low percentage.The percentage of EGFP expression was decreased by keeping culture plates at 4 0 C, suggesting that during refrigeration same cells loss pEGFP expression.The culture suspensions were stored in deionized water at 4 0 C for 24 hours that decrease the percentage of EGFP expression and indicate some EGFP has been denatured.The application of EGFP was evaluated by using it as a marker for E. coli O157:H7 in cauliflower, green leaf lettuce and tomato by using confocal scanning laser microscope.The number of O157:H7 cells identified by EGFP were equal to green leaf lettuce and cauliflower detected by immunostaining but found less for tomato.The study suggests EGFP can be used as a marker to detect attachment of E. coli O157:H7 on cauliflower, green leaf lettuce but found unsuitable on tomato For detection of O157:H7, rabbit anti E. coli O157:H7 antibody was used as a primary antibody, afterward protein A with FITCdoped silica NPs (FSiNPs) was used for fluorescent signal generation.E. coli O157:H7 was detected in various beef samples with adequate results.FSiNPs are useful for pathogens in single amplified bioassay because of brighter fluorescence and higher photo stability.

Emerging technologies 6.1. The microarray based assays
Microarray, a device authorizes for detection of thousands of specific DNA/ RNA sequences on a small slide.These are called fast and rapid analyze to take place [84].Nucleic acid microarrays developed for rapid recognition and genotyping of pathogenic bacteria [96].Microarrays are also used for identification of bacterial gene also for pathogenic surveillance.For this requisition required labeled nucleic acid (Genomic DNA, rRNA etc, PCR products) are combined to clip of microarray, through which few types of direct or indirect fluorescent signal systems the target probe duplexes are detected.The comparative signal of each probe can be identified and quantified via confocal laser or by filtered light scanner [97] (Figure 4).Call and coworkers (2001) used mPCR and nucleic acid microarray for specific and sensitive detection of bacteria.The array contained 25-30-mer oligonucleotide probes interdependent to four objectives (intimin, stx1, stx2 and heamolysin A). mPCR amplified the target DNA and amplicons were hybridized without any purification.The assay was 32 times more sensitive then gel electrophorases.The author suggested the combined use of immunomagnetic capture with PCR and microarray detected 55 cfu ml-1 of O157:H7 from chicken corpse wash water lacking pre-enrichment [98].Viable but non culturable (VBNC) E. coli additionally act as a big warning, basically in potable water.Microarray has become advanced for sensitive detection of VBNC O157:H7 from water.The rfbE gene and fliC-H7 gene were select as viable marker and identified by electronic microarray.From 1 liter (L) of river water 50 VBNC O157:H7 were detected [99].The advantage of microarray is that the assay could not lie on length of identified PCR product.On need when microarray assay combined with PCR, improve the sensitivity of analysis, diagnostic and diagnostic specificity of O157:H7 [100].But microarray approach has the limitation of expensive instruments and also requirements of special knowledge and instruction to remove useful information from enormous data generated [84].Some technical issues included high framework interference mostly at short signal product and difference in organization of labeling nucleic acid with variation in experiments results from different laboratories can infect explanation of results.There for more researches needed on microarray based approaches before making this technology most able and sensitive for routinely used tests for identification of serotype O157:H7.

Lab-on-a-chip assays
A lab-on-a-chip method has been newly recommended as best medium for real-time and portable diagnostics of medical.It is a tool that combines different laboratory purpose on one little platform.It involves handling of small volumes (Fluid), which initiate the area of micro fluidics.The technology employ a network of wells and channels engrave onto glass, polymer chips or silicon for building mini laboratories.The small volumes move through channels by electro kinetic forces.The merits of lab-ona-chip method are: speed of analysis, ease of use, high reproducibility and low sample and low reagent utilization [103].Woolley et al.
[104] reported the integration of PCR and capillary electrophoresis in a micro fabricated DNA analysis device.The method integrates thermal cycling with high-speed DNA separation by the CE chips.This system gives an assay of genomic Salmonella DNA in about 45 min.Several types of immunoassay could be implemented in Lab-on-a-chip platforms [105, 106].Here we will report few kinds of immunoassay lab-on-a-chip and individual function for identification of pathogenic bacteria.As generally ELISA is performed on microplate (96 microwells) and assess by microplate reader for results.In ELISA Labon-a-chip the wells are replaced with micro channel through which the target solution flows continuously.The antibodies are crippled on to inner surface of microchannel.The constant flow results in affective rinsing.Light is brightening to immobilized antibodies, while the signals from different side of micro channel are read by light sensor (Figure 6).Recently various unusual detection strategy have been determined for detection of food borne pathogens employing ELISA type lab-on-achip, which is most sensitive as to real time methods [103].Guan et al.
[107] use anti E. coli for bioluminescence based recognition of EHEC O157:H7.The detection limit found was 3.2*10 1 cfu per ml with examination time of 20 min.ELISA lab-ona-chip requires "labels" secondary antibodies, e.g.fluorescent dye, gold nanoparticles and chemiluminescent dye, with a couple of steps of rinsing.Many options advanced that don't require "labels" e.g.label unfastened immunoassay lab-on-achip.SPR in lab-on-a-chip is a well-known label-unfastened immunoassay lab-on-achip.In SPR lab-on-a-chip the gold surface is immobilized with antibodies.The chip of sensor is discovered to flow channel to ease the goal solutions and successive rinsing [103] (Figure 7).Fernández et al. [108] decided the antibiotic detection from milk using SPR lab-on-a-chip approach.The detection limit found becomes 1.1-2.1 ng per mL in diluted (five-fold) milk.Carbon nanotube (CNT) immunoassay lab-on-a-chip is popular "label-free" immunoassay lab-ona-chip with advantage of increased electronic properties and fast electrode kinetics.There are two kinds of CNTs, single walled CNT (SWCNT) and multi walled CNTs (MECNT)

Conclusion
In this review we have examined different molecular approaches antiquated for detection of E. coli O157:H7.As a result of low infectious dose and ability of causing more and more outbreaks, rapid and sensitive approaches are needed for diagnosis of serotype O157:H7.A summary of all the assays is given in Table 1.
Conventional assays are tedious and labor intensive.However application of molecular techniques rapidly and accurately full fills such limitations.PCR based detection methods (molecular methods) show notable advances and made detection easier.However the process could be reticent by food matrix and non-target DNA obtained from food origin.These complexes result in reaction frustration and false negative outcomes.DNA extraction was also famed to be a time consuming process.Finally it was noted that IMS or enrichment procedures are still needed to increase analysis of time.Among many variations of PCR, despite their drawbacks RT-PCR and mPCR made detection much easier for all researchers.DPCR omit the time consuming process of DNA extraction.Genomic techniques like PFGE and restricted fragment length polymorphism are described along with phenotyping (Table 1).As described above, Genomic techniques have many limitations e.g.require high professional experienced personals.Biosensor assays allot fast analysis time and real time measurement.As compared to other described methods, this method require less preparation time.Ordinarily enrichment procedures are not necessary.Biosensor is used with SPR for rapid detection.Fluorescent and microscopy have been described too for detection methods.Emerging technologies like microarray along with molecular beacons and Lab-on-achip assays described, allot rapid analysis and real time measurement without preenrichment.According to researchers, new designed improvements in molecular beacons are applicable for simultaneous identification of target DNA sequences by single run.However each method has its own advantages and limitations, decision for choice of detection method will require noticeable balance between some factors e.g.speed, specificity, sensitivity, discrimination power and accessibility of skilled personnel's.Consequently depend on the aloft conclusion; more research is needed to progress more specific, rapid and systematic techniques for diagnosing pathogenic bacteria including E. coli O157:H7.

Figure 1 .
Figure 1.Molecular approaches for identification and genotyping of E. coli O157:H7

62]. 2.2. Partial genome genotyping 2.2.1. Restriction fragment length polymorphism (RFLP)
The advantage of the approach is that it does not utilize probe and restriction enzyme of any kind, but utilize a DNA polymerase in PCR, that can tolerate high temperature.
A phage typing strategy was developed for investigation of epidemiology's caused by VT producing strains of EHCE O157:H7 [76].The strategy employs 16 phages, recognized to use above 80 types [77-79].In UK 24 phage types have been identified, while mostly strains belongs to phage type 1, 2, 4 and 49.This assay is beneficial for E.