Advanced Techniques in Diagnostic Microbiology

450 S. R. Frye and M. Healy
TABLE 26.2. Organism-specific applications of each technology.
Filamentous
Protocol consistency
Technique Bacteria a Mycobacterium a fungi Yeast a between organisms
PFGE Yes Limited h No Yes Low, RE and
electrophoresis
parameters change
with each organism b
Sequencing Yes Yes Yes Yes Moderate,
species-specific
gene-related assays
required for
strain-typing c
RAPD Yes Yes Yes Yes Low, very sensitive to
primer and annealing
temperature changes c
RFLP Yes Yes Yes Yes Moderate, gene-specific
primers change with
each organism c
AFLP Yes Yes Yes Yes Moderate, nonspecific
primer kits available d
Ribotyping Yes Yes No No Moderate, nonspecific
restriction enzyme kits
available e
MLST Yes No Limited i Yes Low, multiple primers
change with each
organism f
Rep-PCR Yes Yes Yes Yes High, quality-controlled
genus-specific kits
available g
PFGE, pulsed-field gel electrophoresis; RAPD, randomly amplified polymorphic DNA; RFLP, restriction
fragment length polymorphism; AFLP, amplified fragment length polymorphism; MLST,
multilocus sequence typing.
a Data taken from Table 4, Pfaller (2001) Emerg Infect Dis, 7(2):312–318.
b http://www.cdc.gov/pulsenet/protocols.htm.
c Olive and Bean (1999) J Clin Micribiol, 37(6):1661–1669.
d http://www.appliedbiosystems.com.
e http://www.qualicon.com/riboprinter.html.
f Trinidade, et al. (2003) Braz J Infect Dis, 7(1):32–43.
g http://www.bacterialbarcodes.com.
h Zhang, J Clin Microbial, 2004; 42(12):5582–5587.
i Taylor, Curr Opin Microbiol, 2003; 6:351–356.
Reproducibility of a typing method is critical for longitudinal studies, including
tracking and trending, for comparing archived fingerprint patterns, and for
validation of the system. The data presented in several studies (Versalovic et al.,
1992; Kang and Dunne, 2003) indicate that rep-PCR fingerprints are stable over
multiple generations of growth, reproducible within a plate of isolated colonies
within a strain, and distinct between strains. Additionally, automated rep-PCR has
shown high interlaboratory reproducibility (Healy et al., 2005; Shutt et al., 2005).