PCR Detection of Microbial Pathogens PCR Detection of Microbial ...

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almost all C. perfringens strains, it represents a marker of C. perfringens species,
and the enterotoxin gene, which is present only in some strains responsible
for food intoxication, is a suitable target for the identification of
enterotoxigenic strains (20). In some circumstances, a quantitative result is
required (see Note 2).
The standard protocol of C. perfringens typing includes: DNA extraction
from broth culture (see Subheading 3.2.1.) or colonies (see Subheading
3.2.2.). PCR amplification (see Subheading 3.3.) using primers specific for
each toxin gene (Table 3).
3.7. Differentiation of C. chauvoei and C. septicum
C. chauvoei and C. septicum are two closely related pathogenic bacteria.
The gene of the α-toxin, which is the main toxin produced by C. septicum, has
been characterized and can be used as target for PCR detection (see Table 4).
However, no toxin gene of C. chauvoei has been cloned or sequenced yet.
The method proposed here targets the ribosomal RNA gene region (29,30).
Primers CC16S-L/CC16S-R (Table 4) give rise to an amplification product
of 960 bp for C. chauvoei and, to a lesser extent, for C. septicum. The distinction
between C. chauvoei and C. septicum can be made by analysis of the
restriction digestion pattern of PCR products using HincII/SspI enzymes and
2%-agarose gel electrophoresis. Digestion by HincII/SspI will produce 118,
182, 186, and 474-bp fragments for C. chauvoei, and 182, 304, and 474-bp
fragments for C. septicum.
3.8. Identification of C. tetani
Samples (suppuration, necrotic tissue) from the inoculation wound are processed
for enrichment culture as described in Subheading 3.1.3., and subsequent
PCR is conducted with specific primers (P476–P477) for the tetanus
toxin gene (Table 4).
3.9. Strategy for Identification
of Other Toxigenic Clostridium Species
Detection of other toxigenic Clostridium species from intestinal content or
organ samples is based on probes specific for toxin genes using the general
protocols of enrichment culture (see Subheading 3.1.), PCR (see Subheading
3.3.), and hybridization (see Subheading 3.10.2.). The probes for Clostridium
toxin genes are listed in Table 4.
Some Clostridium strains can easily lose their toxin gene after subculture.
This is the case with C. sordellii, which often becomes nontoxic after subculture.
Specific probes derived from the sialidase gene, a stable gene in all C.
sordellii strains, are used for detection of this bacterium, and toxin gene probes
permit the identification of toxigenic strains. For the detection of C. sordellii