sequences and generates a set of finger printing patterns of different sizes specific to
each strain (Farber 1996; Trindade et al. 2003). The advantages of RAPD are that it
is relatively cheap, rapid, readily available, and easy to perform (Wassenaar and
Newell 2000; Shi et al. 2010; Rezk et al. 2012). In RAPD, the efficiency of
amplification, annealing, and the length of the product varies with the primed
sites, giving rise to both weak and strong amplicons, which makes interpretation
of the results difficult. In addition, RAPD has low reproducibility, average discriminatory power, and approximately 80% typeability (Wassenaar and Newell 2000).
The use of two or more primers improves the discriminatory power of RAPD
(Trindade et al. 2003).
9.2.1.6 Deoxyribonucleic Acid (DNA) Sequencing Techniques
Deoxyribonucleic acid (DNA) sequencing techniques involve technologies used to
determine the order of the nucleotide bases (namely adenine, cytosine, guanine, and
thymine) in a DNA molecule. In recent times, DNA sequencing is widely and
routinely used in the identification, typing, characterization, and/or taxonomic classification of unknown or novel pathogens isolates by many researchers. DNA
sequencing has always been preceded by PCR to amplify the target genes. 16S
rRNA is a common gene that is amplified for sequencing and subsequently for the
identification, typing, and/or taxonomic classification of the pathogen in question.
Sequencing has high discriminatory power, 100% typeability, and good reproducibility (Newell et al. 2000; Wassenaar and Newell 2000). The disadvantage is that it
requires 2–3 days to complete a test, has limited availability, and costs are higher
than other typing methods (Newell et al. 2000; Wassenaar and Newell 2000).
Other typing methods are enterobacterial repetitive intergenic consensus (ERIC),
repetitive extragenic palindromic (REP), ribotyping, amplified fragment length
polymorphism (AFLP), and restriction fragment length polymorphism (RFLP).
Enterobacterial repetitive intergenic consensus (ERIC) PCR uses primers specific
for enterobacterial repetitive intergenic consensus sequences. These primers can be
used under high stringency conditions to match the target DNA to produce DNA
finger printing that are different in sizes (Wassenaar and Newell 2000; Trindade et al.
2003). Enterobacterial repetitive intergenic consensus (ERIC) PCR is quick, easy to
perform, and cost effective. Nonetheless, reproducibility is low compared to pulsed
field gel electrophoresis. Repetitive extragenic palindromic sequences (REP) also
depend on repetitive DNA elements present in pathogens (Trindade et al. 2003). In
repetitive extragenic palindromic sequences, repetitive DNA elements present
within bacterial genome are amplified to produce finger printing of different sizes
specific to each strain (Versalovic et al. 1991). Trindade et al. (2003) reported that
REP is cheaper, easy to perform, and applicable to small or large number of isolates,
and the results have a good correlation with those obtained by PFGE but have lower
discriminatory power. Ribotyping is a molecular technique that uses unique DNA
sequences to differentiate strains of bacteria. In ribotyping, first isolation of genomic
DNA then digestion of isolated DNA with selected restriction endonuclease at
specific sites and generates pieces of DNA of different lengths, then go for separation
of pieces of DNA by gel electrophoresis and at last identified bands of DNA using
9 Development of Modern Tools for Environmental Monitoring of Pathogens and. . .
193
each strain (Farber 1996; Trindade et al. 2003). The advantages of RAPD are that it
is relatively cheap, rapid, readily available, and easy to perform (Wassenaar and
Newell 2000; Shi et al. 2010; Rezk et al. 2012). In RAPD, the efficiency of
amplification, annealing, and the length of the product varies with the primed
sites, giving rise to both weak and strong amplicons, which makes interpretation
of the results difficult. In addition, RAPD has low reproducibility, average discriminatory power, and approximately 80% typeability (Wassenaar and Newell 2000).
The use of two or more primers improves the discriminatory power of RAPD
(Trindade et al. 2003).
9.2.1.6 Deoxyribonucleic Acid (DNA) Sequencing Techniques
Deoxyribonucleic acid (DNA) sequencing techniques involve technologies used to
determine the order of the nucleotide bases (namely adenine, cytosine, guanine, and
thymine) in a DNA molecule. In recent times, DNA sequencing is widely and
routinely used in the identification, typing, characterization, and/or taxonomic classification of unknown or novel pathogens isolates by many researchers. DNA
sequencing has always been preceded by PCR to amplify the target genes. 16S
rRNA is a common gene that is amplified for sequencing and subsequently for the
identification, typing, and/or taxonomic classification of the pathogen in question.
Sequencing has high discriminatory power, 100% typeability, and good reproducibility (Newell et al. 2000; Wassenaar and Newell 2000). The disadvantage is that it
requires 2–3 days to complete a test, has limited availability, and costs are higher
than other typing methods (Newell et al. 2000; Wassenaar and Newell 2000).
Other typing methods are enterobacterial repetitive intergenic consensus (ERIC),
repetitive extragenic palindromic (REP), ribotyping, amplified fragment length
polymorphism (AFLP), and restriction fragment length polymorphism (RFLP).
Enterobacterial repetitive intergenic consensus (ERIC) PCR uses primers specific
for enterobacterial repetitive intergenic consensus sequences. These primers can be
used under high stringency conditions to match the target DNA to produce DNA
finger printing that are different in sizes (Wassenaar and Newell 2000; Trindade et al.
2003). Enterobacterial repetitive intergenic consensus (ERIC) PCR is quick, easy to
perform, and cost effective. Nonetheless, reproducibility is low compared to pulsed
field gel electrophoresis. Repetitive extragenic palindromic sequences (REP) also
depend on repetitive DNA elements present in pathogens (Trindade et al. 2003). In
repetitive extragenic palindromic sequences, repetitive DNA elements present
within bacterial genome are amplified to produce finger printing of different sizes
specific to each strain (Versalovic et al. 1991). Trindade et al. (2003) reported that
REP is cheaper, easy to perform, and applicable to small or large number of isolates,
and the results have a good correlation with those obtained by PFGE but have lower
discriminatory power. Ribotyping is a molecular technique that uses unique DNA
sequences to differentiate strains of bacteria. In ribotyping, first isolation of genomic
DNA then digestion of isolated DNA with selected restriction endonuclease at
specific sites and generates pieces of DNA of different lengths, then go for separation
of pieces of DNA by gel electrophoresis and at last identified bands of DNA using
9 Development of Modern Tools for Environmental Monitoring of Pathogens and. . .
193