detection probabilities were 70% when a Salmonella cell suspension containing
10
3 CFU/ml was used as a template in the PCR (5 CFU per reaction) and 100%
when a suspension of 10
4 CFU/ml was used. Sharma (2006) developed a method for
detection of mRNA encoded by rfbE and eae genes of enterohemorrhagic
Escherichia coli (EHEC) O157:H7. A 129-bp and a 106-bp sequence specific to
rfbE and eae, respectively, were targeted for real-time detection. This method may
contribute to meet the enhancing demand for quality assurance laboratories as
standard diagnostic methods. Obeid et al. (2003) characterized and developed
reusable glass chip-based microfibricated monolithic microdevices using reverse
transcription (RT) and functional integration of PCR in a continuous flow mode.
This allows the selection of the number of chip amplification cycles. Samples and
reagents for PCR were pumped continuously through appropriate entry holes. After
cycles 20, 25, 30, 35, and 40, products were collected from outlet channels. Products
were collected in 0.2 ml tubes and analyzed by agarose gel electrophoresis and
ethidium bromide staining after 30 cycles in only 6 min. The requirement of the
initial DNA and RNA input molecules was used during these studies in the range of
2.5 Â 10
6
À 1.6 Â 10
8 , respectively.
Emerging molecular techniques, such as pulsed field gel electrophoresis (PFGE),
denaturing gradient gel electrophoresis (DGGE), multilocus sequence typing
(MLST), random amplified polymorphism deoxyribonucleic acid (RAPD), plasmid
profile analysis, and deoxyribonucleic acid (DNA) sequencing are among most often
used typing techniques and have been applied to pathogens isolated from environmental samples (Table 9.2). Others such as repetitive extragenic palindromic (REP),
enterobacterial repetitive intergenic consensus (ERIC), ribotyping, amplified fragment length polymorphism (AFLP), and restriction fragment length polymorphism
(RFLP) and so on are yet to be reported in terms of their application. Table 9.1
summarizes the advantages and disadvantages of some commonly available molecular techniques for typing or characterizing pathogens.
9.2.1.2 Denaturing Gradient Gel Electrophoresis (DGGE)
Denaturing gradient gel electrophoresis (DGGE) is often used to examine microbial
diversity of environmental samples and to monitor changes in microbial
communities. The number, exact position, and intensity of bands in a gel track in
DGGE gel numerically approximate the number and relative abundance of dominant
ribotypes in the sample. This approach allows comparison of different microbial
communities. Banding patterns of highly diverse microbial communities, present in
soils, activated sludge and sediment, are usually very complex when bacterial primer
is used. Furthermore, only the major populations of the analyzed community are
represented on these DGGE patterns and thus are relatively less abundant but
possibly very important species that cannot be detected by this molecular method.
The DGGE gel provides a valuable tool for monitoring the structure and dynamics of
microbial populations over time or under the influence of environmental changes.
This approach has already been used in a few studies, which investigated specific
microbial groups such as methanotrophic members of the Proteobacteria,
actinomycetes, ammonia-oxidizing bacteria, Archaea, and fungi. Lee et al. (2009)
9 Development of Modern Tools for Environmental Monitoring of Pathogens and. . .
191
10
3 CFU/ml was used as a template in the PCR (5 CFU per reaction) and 100%
when a suspension of 10
4 CFU/ml was used. Sharma (2006) developed a method for
detection of mRNA encoded by rfbE and eae genes of enterohemorrhagic
Escherichia coli (EHEC) O157:H7. A 129-bp and a 106-bp sequence specific to
rfbE and eae, respectively, were targeted for real-time detection. This method may
contribute to meet the enhancing demand for quality assurance laboratories as
standard diagnostic methods. Obeid et al. (2003) characterized and developed
reusable glass chip-based microfibricated monolithic microdevices using reverse
transcription (RT) and functional integration of PCR in a continuous flow mode.
This allows the selection of the number of chip amplification cycles. Samples and
reagents for PCR were pumped continuously through appropriate entry holes. After
cycles 20, 25, 30, 35, and 40, products were collected from outlet channels. Products
were collected in 0.2 ml tubes and analyzed by agarose gel electrophoresis and
ethidium bromide staining after 30 cycles in only 6 min. The requirement of the
initial DNA and RNA input molecules was used during these studies in the range of
2.5 Â 10
6
À 1.6 Â 10
8 , respectively.
Emerging molecular techniques, such as pulsed field gel electrophoresis (PFGE),
denaturing gradient gel electrophoresis (DGGE), multilocus sequence typing
(MLST), random amplified polymorphism deoxyribonucleic acid (RAPD), plasmid
profile analysis, and deoxyribonucleic acid (DNA) sequencing are among most often
used typing techniques and have been applied to pathogens isolated from environmental samples (Table 9.2). Others such as repetitive extragenic palindromic (REP),
enterobacterial repetitive intergenic consensus (ERIC), ribotyping, amplified fragment length polymorphism (AFLP), and restriction fragment length polymorphism
(RFLP) and so on are yet to be reported in terms of their application. Table 9.1
summarizes the advantages and disadvantages of some commonly available molecular techniques for typing or characterizing pathogens.
9.2.1.2 Denaturing Gradient Gel Electrophoresis (DGGE)
Denaturing gradient gel electrophoresis (DGGE) is often used to examine microbial
diversity of environmental samples and to monitor changes in microbial
communities. The number, exact position, and intensity of bands in a gel track in
DGGE gel numerically approximate the number and relative abundance of dominant
ribotypes in the sample. This approach allows comparison of different microbial
communities. Banding patterns of highly diverse microbial communities, present in
soils, activated sludge and sediment, are usually very complex when bacterial primer
is used. Furthermore, only the major populations of the analyzed community are
represented on these DGGE patterns and thus are relatively less abundant but
possibly very important species that cannot be detected by this molecular method.
The DGGE gel provides a valuable tool for monitoring the structure and dynamics of
microbial populations over time or under the influence of environmental changes.
This approach has already been used in a few studies, which investigated specific
microbial groups such as methanotrophic members of the Proteobacteria,
actinomycetes, ammonia-oxidizing bacteria, Archaea, and fungi. Lee et al. (2009)
9 Development of Modern Tools for Environmental Monitoring of Pathogens and. . .
191