Advanced Techniques in Diagnostic Microbiology

18. Real-Time PCR Based on FRET 297
Because the hybrid hairpin configuration is very thermostable, molecular beacons
have a high specificity to hybridize to a target, which are used to distinguish
single nucleotide differences. Therefore, molecular beacons are suitable for mutation
analysis and single nucleotide polymorphism detection when specific mutations
are known (McKilli, 2000; Szuhai, 2001; Abravaya, 2003; Bustamante,
2004; Petersen, 2004; Vet, 2005).
All real-time PCR chemistries allow detection of multiple DNA species (multiplexing)
by designing each probe/beacon with a spectrally unique fluor/quench
pair. All of the above can be used in conjunction to melting curve analysis or when
SYBR Green is used only. By multiplexing, the target(s) and endogenous control
can be amplified in a single tube. (Bernard, 1998; Lee, 1999; Vet, 1999; Elnifro,
2000; Read, 2001; Grace, 2003; Rickert, 2004)
Real-Time PCR in Infectious Disease Diagnosis
Molecular diagnostic tools and detection methods such as nucleic acid amplification
are being used increasingly in the clinical microbiology laboratory to enhance
the diagnosis of microbial pathogens (Lanciotti, 2001; Mackay, 2004). Nucleic
acid–based technology is also used to assess drug resistance and epidemiological
surveillance (Piatek, 1998; Makinen, 2001; Huletsky, 2004; Sloan, 2004). The
principle of the real-time PCR is primarily used to detect and amplify a unique
gene or a signature sequence of the microorganism. Quantitative measurements of
viral load can also be made simple. Sensitive detection and accurate identification
can speed up reporting of microbial pathogens without reliance on their phenotypic
characteristics or viability after antibiotic treatment.
The application of real-time PCR in infectious diseases enables the diagnosis of
microbial pathogens both with accuracy and expediency. The clinical significance
of using molecular diagnosis of infectious agents can be characterized by the following
aspects. (1) Pathogens that show fastidious slow growth or inability to grow
in vitro: Mycobacterium, Legionella, Bartonella, Leptospira, Borrelia, Bordetella,
Mycoplasma, and Tropheryma whippelii may require days or weeks of incubation
under specific conditions; (2) obligatory intracellular organisms (Chlamydia,
Rickettsia, Coxiella, Ehrlichia, DNA and RNA viruses); (3) prior antibiotic use;
(4) biochemically inert for phenotypic characterization; (5) additional waiting time
for drug-resistance determination, (6) diagnostic speed from bench to bedside.
Qualitative real-time amplification has outpaced conventional culture methods
in detection of a long list of specific pathogens that are difficult to cultivate:
Bartonella henselae, Bordetella pertussis, Borrelia burgdorferi, Coxiella
burnetii, Ehrlichia spp., Legionella spp., Mycoplasma pneumoniae, Chlamydia
trachomatis, Rickettsia, Toxoplasma gondii, Microsporidium, Cryptosporidium,
Tropheryma whippelii, Mycobacterium tuberculosis and its drug-resistant determinants
(Franzen, 1999; Pretorius, 2000; Hammerschlag, 2001; Bell, 2002;
Fournier, 2002; Gerard, 2002; Kovacova, 2002; Exner, 2003; Templeton, 2003;
Wang, 2003; Fenollar, 2004; Koenig, 2004; Simon, 2004; Wada, 2004; Khanna,