Southern blot hybridization with specific probe of rRNA genes (Shi et al. 2010).
Ribotyping has 100% typeability and good reproducibility, but it is a complex
method, is sensitive to genetic instability, and requires 3–4 days to complete a test
(Wassenaar and Newell 2000). Ribotyping has higher discriminatory power at the
species and subspecies level compared to the strain level (Denes et al. 1997; Shi et al.
2010). Amplified fragment length polymorphism (AFLP) involves the use of two
restriction enzymes to digest total genome DNA, one with an average cutting
frequency (4-bp recognition site) and the other with a higher cutting frequency
(6-bp recognition site) followed by linking of adapters to the sticky ends of the
restriction fragments and amplification of a subset of selected restriction fragments
(Wassenaar and Newell 2000; Shi et al. 2010). The primers used for amplification
are radioactive or fluorescent labeled, and denaturing polyacrylamide gel analysis is
used to determine the presence or absence of DNA fragments to identify
polymorphisms (Blears et al. 1998; Wassenaar and Newell 2000). Amplified restriction length polymorphism has good discriminatory power, good reproducibility, and
100% typeability, needs no prior sequence information for amplification, and is
insensitive to genetic instability, but AFLP is a complex method, requires 3–4 days
to complete a test, and requires major capital investment (Wassenaar and Newell
2000; Meudt and Clarke 2007). Restriction fragment length polymorphism (RFLP)
involves the use of restriction enzyme to digest DNA and to separate the resulting
restriction fragments according to their length on agarose gel electrophoresis.
Restriction fragments are then transferred into a membrane through Southern blot
procedure and hybridized to a membrane bound labeled DNA probe (Babalola 2003;
Foley et al. 2009). This method utilizes the variations in homologous DNA
sequences to characterize bacteria. This technique is inexpensive, is very sensitive
for strain identification or differentiation, and has widespread application, although it
has become obsolete in the present time due to the emergence of relatively inexpensive sequencing technologies (Mohran et al. 1996; Babalola 2003). The technology
is also slow and difficult and could take up to a month to complete (Mohran et al.
1996; Nachamkin et al. 1996).
9.2.2 Enzyme-Linked Immunsorbent Assay (ELISA)
Many immunological techniques provide quantitative assessment of the concentration of analytes in pure solutions or complex mixtures. In this area have great
potential due to its sensitive and specificity towards diverse range of chemical and
biological molecules and the immunoassays can also be used to provide real-time
information.
The enzyme-linked immunosorbent assay (ELISA) is a test that identifies the
substrate by the interaction of antibodies with antigen. An antibody is “Y”-shaped
immunoglobin (Ig) that is made up of two heavy chains (H) and two light chains (L).
Each of the chain has a constant and a variable part. The variable part is specific to
the antigen that binds with corresponding antigen that is highly specific and selective
(Conroy et al. 2009; Donahue and Albitar 2010). An ELISA required at least one
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S. Purwar and S. Srivastava
Ribotyping has 100% typeability and good reproducibility, but it is a complex
method, is sensitive to genetic instability, and requires 3–4 days to complete a test
(Wassenaar and Newell 2000). Ribotyping has higher discriminatory power at the
species and subspecies level compared to the strain level (Denes et al. 1997; Shi et al.
2010). Amplified fragment length polymorphism (AFLP) involves the use of two
restriction enzymes to digest total genome DNA, one with an average cutting
frequency (4-bp recognition site) and the other with a higher cutting frequency
(6-bp recognition site) followed by linking of adapters to the sticky ends of the
restriction fragments and amplification of a subset of selected restriction fragments
(Wassenaar and Newell 2000; Shi et al. 2010). The primers used for amplification
are radioactive or fluorescent labeled, and denaturing polyacrylamide gel analysis is
used to determine the presence or absence of DNA fragments to identify
polymorphisms (Blears et al. 1998; Wassenaar and Newell 2000). Amplified restriction length polymorphism has good discriminatory power, good reproducibility, and
100% typeability, needs no prior sequence information for amplification, and is
insensitive to genetic instability, but AFLP is a complex method, requires 3–4 days
to complete a test, and requires major capital investment (Wassenaar and Newell
2000; Meudt and Clarke 2007). Restriction fragment length polymorphism (RFLP)
involves the use of restriction enzyme to digest DNA and to separate the resulting
restriction fragments according to their length on agarose gel electrophoresis.
Restriction fragments are then transferred into a membrane through Southern blot
procedure and hybridized to a membrane bound labeled DNA probe (Babalola 2003;
Foley et al. 2009). This method utilizes the variations in homologous DNA
sequences to characterize bacteria. This technique is inexpensive, is very sensitive
for strain identification or differentiation, and has widespread application, although it
has become obsolete in the present time due to the emergence of relatively inexpensive sequencing technologies (Mohran et al. 1996; Babalola 2003). The technology
is also slow and difficult and could take up to a month to complete (Mohran et al.
1996; Nachamkin et al. 1996).
9.2.2 Enzyme-Linked Immunsorbent Assay (ELISA)
Many immunological techniques provide quantitative assessment of the concentration of analytes in pure solutions or complex mixtures. In this area have great
potential due to its sensitive and specificity towards diverse range of chemical and
biological molecules and the immunoassays can also be used to provide real-time
information.
The enzyme-linked immunosorbent assay (ELISA) is a test that identifies the
substrate by the interaction of antibodies with antigen. An antibody is “Y”-shaped
immunoglobin (Ig) that is made up of two heavy chains (H) and two light chains (L).
Each of the chain has a constant and a variable part. The variable part is specific to
the antigen that binds with corresponding antigen that is highly specific and selective
(Conroy et al. 2009; Donahue and Albitar 2010). An ELISA required at least one
194
S. Purwar and S. Srivastava