detect bacterial isolates (Table 9.1). Microorganisms contain a number of wellconserved genes, such as the ribosomal 16S gene and the heat-shock protein/
chaperonin Hsp60/65 (or GRAEL), which are excellent targets for PCR. Analysis
of the16S ribosomal RNA gene in bacteria in PCR and subsequent sequencing is
particularly informative, as there are well-conserved sequences that can be used as
binding sites for universal PCR primers adjacent to variable sequences and then a
database of known sequences can be compared and analyzed. Other forms of PCR
are real-time PCR, nested PCR, reverse-transcription PCR, and many more. Polymerase chain reaction assays have been routinely used for rapid detection, identification, and differentiation of pathogens. They have been used in areas such as DNA
cloning, diagnosis of hereditary and infectious diseases, identification of genetic
fingerprints, and detection and diagnosis of infectious diseases. Polymerase chain
reaction technique plays an important role in the identification of typical bacterial
strains that exist in viable but nonculturable coccoid forms (e.g., Campylobacter
spp.), which are often missed by the conventional method (Magistrado et al. 2001).
The use of PCR also avoids situations where phenotypic characteristics are ambiguous and wrongly interpreted, for instance, the occurrence of hippurate negative
C. jejuni strains (Adzitey and Corry 2011). However, some PCRs may not be
suitable for processed and certain foods because amplification can be obtained
from DNA originating from both viable and nonviable cells (Sails et al. 1998;
Wang et al. 2000). The technique can be expensive and its sensitivity and performance can be inhibited by components of enrichment broth and DNA extraction
solution, concentration of the PCR mixtures (primers, DNA templates, dNTPs, and
Mg
2+ ), and temperature and cycling conditions (Rossen et al. 1992; Wilson 1997;
Wassenaar and Newell 2000). Table 9.1 shows commonly available molecular
techniques that have been applied to identify bacteria isolated from environmental
samples, while Table 9.2 summarizes the advantages and disadvantages of some
commonly available molecular techniques for identifying pathogens. After popularization of polymerase chain reaction (PCR), nucleic acid-based assays for the
detection and identification of environmental pathogens have been successfully
developed. There are several DNA-based assay formats here, but only nucleic acid
amplification techniques have been developed commercially to detect pathogens.
PCRs, involving amplification step, are becoming more popular due to their higher
sensitivity and fast identification of the pathogens and their toxins. Naravaneni and
Jamil (2005) had standardized PCR-based technique for detection of Salmonella and
Escherichia coli. They designed specific genes for examples for Salmonella used
fimA, pathogenic E. coli used afa gene primers for amplification. Adleyb et al.’s
(2009) studies have established that BCFomp1/BCRomp1, the DNA sequences, can
be used for the specific detection of the B. cereus group spp. Analysis of these
primers using standard PCR analysis showed that the minimum level of detection
was 10
3 CFU/ml and the lowest number of bacterial cell per reaction tube amplified
was 5 CFU with initial need of DNA found to be 1 pg. Malorny et al. (2004)
developed robust real-time PCR for the specific detection of Salmonella. The assay
used specifically designed primers and a probe targeted within the ttrRSBCA locus,
which is located near the Salmonella pathogenicity island 2 at centisome 30.5. The
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S. Purwar and S. Srivastava
chaperonin Hsp60/65 (or GRAEL), which are excellent targets for PCR. Analysis
of the16S ribosomal RNA gene in bacteria in PCR and subsequent sequencing is
particularly informative, as there are well-conserved sequences that can be used as
binding sites for universal PCR primers adjacent to variable sequences and then a
database of known sequences can be compared and analyzed. Other forms of PCR
are real-time PCR, nested PCR, reverse-transcription PCR, and many more. Polymerase chain reaction assays have been routinely used for rapid detection, identification, and differentiation of pathogens. They have been used in areas such as DNA
cloning, diagnosis of hereditary and infectious diseases, identification of genetic
fingerprints, and detection and diagnosis of infectious diseases. Polymerase chain
reaction technique plays an important role in the identification of typical bacterial
strains that exist in viable but nonculturable coccoid forms (e.g., Campylobacter
spp.), which are often missed by the conventional method (Magistrado et al. 2001).
The use of PCR also avoids situations where phenotypic characteristics are ambiguous and wrongly interpreted, for instance, the occurrence of hippurate negative
C. jejuni strains (Adzitey and Corry 2011). However, some PCRs may not be
suitable for processed and certain foods because amplification can be obtained
from DNA originating from both viable and nonviable cells (Sails et al. 1998;
Wang et al. 2000). The technique can be expensive and its sensitivity and performance can be inhibited by components of enrichment broth and DNA extraction
solution, concentration of the PCR mixtures (primers, DNA templates, dNTPs, and
Mg
2+ ), and temperature and cycling conditions (Rossen et al. 1992; Wilson 1997;
Wassenaar and Newell 2000). Table 9.1 shows commonly available molecular
techniques that have been applied to identify bacteria isolated from environmental
samples, while Table 9.2 summarizes the advantages and disadvantages of some
commonly available molecular techniques for identifying pathogens. After popularization of polymerase chain reaction (PCR), nucleic acid-based assays for the
detection and identification of environmental pathogens have been successfully
developed. There are several DNA-based assay formats here, but only nucleic acid
amplification techniques have been developed commercially to detect pathogens.
PCRs, involving amplification step, are becoming more popular due to their higher
sensitivity and fast identification of the pathogens and their toxins. Naravaneni and
Jamil (2005) had standardized PCR-based technique for detection of Salmonella and
Escherichia coli. They designed specific genes for examples for Salmonella used
fimA, pathogenic E. coli used afa gene primers for amplification. Adleyb et al.’s
(2009) studies have established that BCFomp1/BCRomp1, the DNA sequences, can
be used for the specific detection of the B. cereus group spp. Analysis of these
primers using standard PCR analysis showed that the minimum level of detection
was 10
3 CFU/ml and the lowest number of bacterial cell per reaction tube amplified
was 5 CFU with initial need of DNA found to be 1 pg. Malorny et al. (2004)
developed robust real-time PCR for the specific detection of Salmonella. The assay
used specifically designed primers and a probe targeted within the ttrRSBCA locus,
which is located near the Salmonella pathogenicity island 2 at centisome 30.5. The
190
S. Purwar and S. Srivastava