The performance of strain typing technology, including PFGE, is measured by the following criteria (Struelens, 1998; Pfaller et al., 2001):
PFGE has high discriminatory power and reproducibility. This performance feature is based on direct analysis of greater than 90% bacterial chromosomal polymorphism (Goering, 2000). It has significant advantages compared with other nonamplification methods, which include plasmid DNA analysis and restriction endonuclease analysis of chromosomal DNA (REA). First, PFGE digests large DNA chromosomal fragments with infrequent-cutting restriction endonucleases, yielding well-separated bands that are easy to read. Second, because conventional electrophoresis is limited to the separation of relatively small (<50 kb) DNA fragments, the chromosomal DNA must be digested with frequent-cutting restriction endonucleases, thus generating hundreds of uninterpretable bands.
In recent years, a number of PCR amplification-based methods have been developed for genotyping microbial pathogens, such as arbitrarily primed polymerase chain reaction (AP-PCR), random amplified polymorphic DNA (RAPD), multi-locus primed PCR or repetitive chromosomal elements PCR (rep-PCR), and amplified restriction fragment length polymorphism (AFLP). Amplification-based technologies are less discriminatory but have the advantages of being less costly and labor intensive, taking approximately 2 days to obtain results, as compared with 4 to 5 days for PFGE (Wu and Della-Latta, 2002). In addition, DNA fragments smaller than 50 kb cannot be reliably separated by PFGE, because the system is not able to switch the field orientation quickly enough to separate these smaller molecules. Certain organisms such as Clostridium difficile and Aspergillus spp., which are difficult to type by PFGE because they are either uncultivable or their DNA cannot be isolated intact, can be analyzed using PCR-based typing methods. The typeability of PFGE may not be excellent for some bacterial species, such as Acinetobacter spp. because of DNA degradation challenges (Silbert et al., 2003). The comparison of the procedural features of PFGE and amplification-based typing methods are summarized in Table 9.2.
Many different PFGE protocols have been developed, and this has led to some variability in assay design and reproducibility among laboratories. It is important to
Table 9.2. Comparison of the procedural features of pulsed-field gel electrophoresis and PCR-based typing methods.
Procedural Nonamplification typing Amplification typing characteristics PFGE (RAPD, rep-PCR, MLST)
Genomic region Entire chromosome Selected region on the chromosome
Sample preparation Intact cells embedded in agarose DNA extraction
Fragment generation Restriction endonuclease digestion DNA polymerase amplification
Electrophoresis Pulsed field Single homogeneous
Fragment size 50-2000 kb < 10 kb
Time to results 3-4 days 2-3 days establish standardized PFGE protocols, particularly with critical elements such as the DNA concentration, the effectiveness of restriction enzyme digestion, and the electrophoresis conditions including agarose gel volume and concentration, buffer volume, and ionic strength. The running conditions including voltage, switching times, reorientation angle, and total run times of electrophoresis are other variables to consider (Chung et al., 2000; Murchan et al., 2003). To insure good-quality gels and consistent reproducibility, a quality control strain should be included with each gel run for comparison. Using a standardized approach, and computer-assisted programs that demonstrate enhanced capability of comparing DNA fragment patterns present on multiple gels, investigators can create a searchable database of PFGE fragment patterns for interlaboratory comparison and facilitate cluster analyses.
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