Development of Polymerase Chain Reaction and Dot Blot Hybridization to Detect Escherichia coli Isolates from Various Sources
N. Al Haj,
Diarrhea is one of the leading causes of illnesses and death among children in developing countries, where an estimated 1.3 billion episodes and 4-10 million deaths occur each year in children below 5 years of age. Escherichia coli strains are among the major bacterial causes of diarrheal illness. There are now 7 classes of diarrheagenic E. coli, namely enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), diarrhea-associated hemolytic E. coli (DHEC) and cytolethal distending toxin (CDT)-producing E. coli. Due to the need for costly and labor-intensive diagnostic procedures, identification of diarrheagenic E. coli (DEC) is difficult at standard laboratories. Therefore, the epidemiology of DEC infections remains an important issues particularly developing country. Recently, Polymerase Chain Reaction (PCR) or dot blot has been used for genetic detection of DEC. In this study, we analyzed 25 E. coli isolates from different sources in Malaysia. Using primers for 671 bp gad gene successfully amplified by PCR. Dot blot analysis for high-throughput, rapid, simple and inexpensive quantification of specific microbial populations was evaluated and used to confirm the results of PCR. The protocol of the assays is readily applicable for implementation in the food processing, water quality control and clinical diagnosis.
to cite this article:
N. Al Haj, N.S. Mariana, A.R. Raha and Z. Ishak, 2007. Development of Polymerase Chain Reaction and Dot Blot Hybridization to Detect Escherichia coli Isolates from Various Sources. Research Journal of Microbiology, 2: 926-932.
Conventional microbiological methods for detection of pathogenic E. coli,
however, usually include multiple subcultures and biotype or serotype identification
steps and thus are laborious and time-consuming (Blackburn, 1993; Naravaneni
and Jamil, 2005; Swaminathan and Feng, 1994). One of the inherent difficulties
in the detection of water-food borne pathogens is that they are generally present
in very low numbers (<100 cfu g-1) in the midst of up to a million
or more other bacteria. These microbes may be lost among a background of indigenous
microflora and substances in the foods themselves may hinder recovery or injured
cells and or hide in sediment. There is also the difficulty of demonstrating
that the strains recovered from any sources particularly clinical and water-food
sample are, indeed, pathogenic to human beings (Sockett, 1991). Existing methods
for detection of pathogenic E. coli first require time-consuming biochemical
procedures to determine if an isolate is E. coli, followed by molecular
and cell assays to determine whether specific virulence markers are present
(Grant et al., 2001). The task would be enormous if one contemplates
the monitoring of 100 of pathogens on a routine basis in water and environmental
samples. E. coli is an indicator of recent fecal contamination recommended
by the US Environmental Protection Agency universally to be used for monitoring
the microbiological quality of water, a sensitive measure of fecal pollution
since it is common to almost all warm-blooded animals, including humans (USEPA,
1986, 2005). Therefore, indicators of fecal pollution were much needed (Naravaneni
and Jamil, 2005).The use of E. coli as an indicator of fecal contamination
relies on the assumption that its presence in water is a direct evidence of
fecal contamination and indicates the possible presence of pathogens (McLellan,
2004). There is currently no procedure for isolation of E. coli from
food or water based on a characteristic unique to the species and found in all
virulence subgroups. In order to control outbreaks caused by food or water-borne
E. coli it is important that this process be conducted rapidly and identification
of a diagnostic marker to the species which can be get in all groups would be
valuable. The enzyme glutamate decarboxylase (GAD) reported to be limited to
E. coli and is encoded by two virtually identical genes, gadA
and gadB (McDaniels et al., 1996; Grant et al., 2001; Smith
et al., 1992). Objective of this study is to develop PCR and probe to detect
the E. coli from different sources simultaneously.
MATERIALS AND METHODS
Sources of Sampling
Twenty five E. coli isolates as clinical, marine, river, food and
animal were studied from 5 different sources in Malaysia 2003-2004. The clinical
samples were stock culture of Microbiology Laboratory there were collected from
Kula Lumpur Hospital (HKL), where marine and river isolates were collected from
Costrica beach, Sunggi linggi river Nergeri Semblan State. The food sample was
selected randomly from different restaurant in Seri Serdange area, Selangor
state. The last samples of animal source were provided by bacteriology department,
Faculty of Veterinary University Putra Malaysia (UPM). Water samples were collected
in 500 mL sterile containers, refrigerated at 4°C and proceeds applicant
within 8 h of collection in adherence to methods for E. coli enumeration
(USEPA, 1986). All samples were grown on selective and differential Chromocult
Coliform Agar (Merek, Germany). Confirmation was achieved by monitoring acidification
and gas production during growth in Kligler Iron Agar (KIA) and Sulfide Indole
Motility (SIM) media (Oxoid. UK). The presence of E. coli was confirmed
by demonstration of indole production from SIM.
E. coli isolates grown on Chromoocult Coliform Agar (Merek. Germany)
overnight at 37°C. A single colony of each strain was transferred to Luria-Bertani
medium (Oxoid UK) and grown overnight in a 37°C shaking water bath. DNA
was prepared with a DNA isolation kit (Qiagen, Germany) DNA extraction according
to the manufacturers instructions. The genomic DNA was checked for the
concentrations and purities using spectrophotometer (Shimadzu 1601 Japan).
Polymerase Chain Reaction
Oligonucleotide primers used for these amplifications were identified from
GenBank listed under accession numbers M84024 (gadA). Searches for optimal
primer and probe were performed with the Primer Premier3 software program following
the general primer designing parameters and commercially synthesized (Biosynthec
In. Malaysia). The forward gad primer, corresponding to base positions
306-323, was F-GACC TGCG TTGCGTAAAT-R. The reverse gad primer, corresponding
to base positions 970-976 was F-GGGCGGGAGAAGTTGAT-R. The probe sequence F-TGCTGAACTG-TTGCTGGAAG-R.
PCR amplifications were performed with a (T-personal thermal cycler Germany)
and GeneAmp PCR reagent kits (Biosynthec Inc. Malaysia) according to the manufacturers
directions for the hot start technique. The amplification of gad gene
was performed in a final volume of 50 μL containing 1X BST buffer (Biosynthec
Inc. Malaysia), 1.8 mM MgCl2 (Biosynthec Inc. Malaysia), 200 μm
dNTPs (Fermentas Life Sciences), 5 IU taq polymerase (Biosynthec Inc. Malaysia),
10 pmoles of each primer and 200 ng μL-1 DNA template. The PCR
programmer steps performed were initial denaturation at 94°C for 2 min,
followed by 30 cycles of amplification steps consisting of denaturation at 94°C
for 1 min, annealing at 64°C for 1 min and elongation at 72°C for 2
min. The amplification was ended with final extension at 72°C for 7 min.
After amplification, an aliquot of 10 μL reaction mixture was loaded into
the wells of 1.4 % agarose gel and electrophoresed, then stained with ethidium
bromide and image was captured under UV illumination (Alpha ImagerTM
2200, Alpha Innotech Corporation).
Dot Blots Hybridization
The dot blot assay was also used as an alternative method to verify the
specificity of the above PCR assay. The chromosomal DNA was labeled with Hors
Reddish Peroxidase (HRP) using the Direct Nucleic Acid Labeling Kit ECL (Amersham
Pharmacia Biotech, UK) and then used as probe for hybridization to genomic DNA
of E. coli isolates. For each isolates, 100 ng of chromosomal DNA was
denatured by heating at 96°C for 10 min and spotted onto Hybond-N nylon
membranes (Amersham Pharmacia Biotech, UK). DNA was then fixed on to the filter
by UV cross-linking by incubating in a UV cross-linking chamber (UV-Cross linker
UVC-500, USA) for 3 min at the room temperature. The prehybridization and hybridization
temperature were both 42°C. All filters were pre-hybridized for 1 h in 5xSSC
(1.5 M sodium chloride, 0.15 M sodium citrate). Hybridization was carried out
overnight with heat-denatured probe. Detection was performed using the phototope-star
detection kit according to the manufacturers instructions (Amersham Pharmacia
Polymerase Chain Reaction
Detection of gad gene by PCR showed that all the isolates of E.
coli used in this study were gad gene positive. A single band of
approximately 671 bp was observed in all the isolates tested. The single banding
pattern observed (Fig. 1 and 2) for all
isolates was located at position slightly above the 660 bp based on the 100
bp DNA ladder marker (MBI Fermentas).
Dot Blot Detection
Gene probe method using the dot blot technique was used to confirm the presence
of E. coli specific fragment detected in PCR. The presence of E. coli
specific fragment was confirmed in the dot blot technique when a positive
signal for the spot was seen on the X-ray film. The hybridized signals on the
x-ray film showed positive signal for all E. coli isolates (Fig.
||Agarose gel of electrophoresis of products amplified 671 bp
gad gene, PCR from different sources of the E. coli. Lanes
M: markers low molecular size standards 100 bp, Lanes 1-5: clinical and
Lanes 6-10: Marine sources
||Agarose gel of electrophoresis of products amplified 671 bp
gad gene, PCR from different sources of the E. coli. Lanes
M: markers low molecular size standards 100 bp, Lanes 11-15: River, lane
16-20: food and Lane 21-25 animal sources
||Photograph of the X-ray film confirming the presence of gad
gene in E. coli isolates, indicated by a dot. E. coli isolates
from 5 sources used in this study using design probe show positive dot blot
for clinical (1-5), marine (6-10), river (11-15), food (16-20) and animal
||Four positive controls (A)ATCC (23519), (B) ATCC (12799),
(C) ATCC (23520) and (D) ATCC (12810). 1-4 that does not show any positive
signal represents (1) Vibrio cholera, (2) Salmonella sp. (3)
Klebsiella pneumonia and (4) Staphylococus aureus isolates
Specificity of E. coli Specific Probe
Specificity test by dot blot hybridization was used the Horse Radish Peroxidase
(HRP) specific DNA fragment from 4 isolates of different genera of gram-positive
and gram-negative bacteria (Fig. 4 and 5)
Vibrio cholera, Staphylococus aureus, Salmonella sp. and
Klebsiella pneumonia isolates were used as negative controls as a probe
and DNA as targets was performed with E. coli isolates.
Sensitivity of E. coli Specific Probe
Probe sensitivity for E. coli was determined by hybridizing of different
concentration for E. coli genomic DNA against the HRP labeled probe (Fig.
|| Four different genus of bacteria show positive signal for
its specific probe (V) Vibrio cholera, (Sal) Salmonella sp.,
(K) Klebsiella pneumonia and (S) Staphylococus aureus and
E1-E4 that does not show any positive signal represents E. coli
||Dot 1: 50 ng of genomic DNA, dot 2-4: 100, 150, 200 and 300
ng. Dot 1 did not show any signal, dot 2 showed a faint signal, whereas
from dot 3, a strong signal was observed. The detection limit of the probe
developed is 100 ng. The probe developed was highly sensitive which detected
as low as 100 ng of genomic DNA
E. coli is primarily associated with food-borne diseases particularly pathogenic, contamination of drinking or recreational waters with some pathotypes has resulted in waterborne disease outbreaks and associated mortality. Identification of E. coli groups, isolates must first be identified as E. coli before they are tested for group-specific virulence traits. Because the groups are phenotypically diverse, existing methods for detecting E. coli are not always useful for pathogenic strains. Therefore we examined the potential of using gad gene as a marker for identifying E. coli isolated from various sources such as clinical and environments. PCR amplification of various sources showed that the gad gene was present in all local E. coli strains examined. There are other enteric bacteria that carried the gad genes particularly Shigella, a genus so closely related to E. coli that some investigators conclude they constitute a single species (Edwards, 1999). An ideal diagnostic test might differentiate between the two but from a public health perspective, the detection of Shigella by a GAD-based assay would not detract from the utility of this test, as Shigella is also a human pathogen because the important of E. coli as indicator of quality water which relies on the assumption that its presence in water is a direct evidence of fecal contamination and indicates the possible presence of pathogens such as Salmonella sp. Shigella sp. pathogenic E. coli and enteroviruses, including hepatitis A (McLellan, 2004). Currently, examples in the Great Lakes area include pathogenic E. coli O157:H7 isolates that contributed to a drinking water outbreak in Walkerton, Ontario, in 2000 that resulted in 2,300 illnesses and seven deaths (Hrudey and Hrudey, 2002) and to a recreational water outbreak in 2001 at a beach in Montreal, Quebec, that resulted in the hospitalization of 4 children (Bruneau et al., 2004). However, there have been few studies (Chern et al., 2004; Lauber et al., 2003; Martins et al., 1992; Obi et al., 2004) in which the proportion of potentially pathogenic E. coli isolates in the environment has been determined. Pathotyping of E. coli isolates present in water sources used for drinking or recreation could be an important tool in the development of strategies to better protect public health (Stoeckel and Harwood, 2007). Additionally, since gad-positive isolates we recommended further tested for virulence genes among E. coli isolates will be easily distinguished from pathogenic E. coli by these tests. In the present study, did not detect the presence of E. coli directly from samples without enrichment, because many foods known to contain inhibitors that can affect PCR assays (Lantz et al., 1994). Hence, culture enrichment often beneficial in diluting out the inhibitory effects of these components and is standard practice in attempts to isolate potential pathogens from contaminated food. Genomic DNA blotted on a membrane and can be detected by chemiluminescence of the hybridized signal, offers a rapid, simple, specific and accurate miniature system suitable in water-food borne area. Taking into account potentially serious water quality and food hygienic processing, rapid detection of the infectious agents based on the detection and identification the bioindicator is essential for achieving favorable management outcomes. The membrane-based assay was performed optimized to be simple and performed in 1-2 h to test E. coli genomic DNA or amplified product. The E. coli specific membrane assay developed in this study was very specific to E. coli as the probe sequence did not show positive signal for a variety of Gram positive and Gram negative bacterial tested. The sensitivity of dot blot allowed as few as 100 ng of E. coli probe hybridization assay was fast and sensitive and can be used in the laboratory methods be available for the accurate and the immediate detection of E. coli.
The molecular probe/primer developed and the molecular techniques optimized have led to the development of rapid molecular approach for detection E. coli isolates from various sources. We have found that the gad gene was prevalent in E. coli. DNA probe analysis showed that the gad marker was just as reliable for detecting E. coli in clinical, food and water isolates.
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