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transcription elongation factor 1 (Bissett et al., 2003), mating-type genes (Dyer
et al., 2001; Foster et al., 2002) and mitochondrial small subunit rDNA (Smith et al.,
1996; Sirven et al., 2002; Martin et al., 2004) have also been deployed for detection
and differentiation of strains and or species.
In bacteria, genus-specific rDNA sequences have been well documented by
Deparasis and Roth (1990) and primers have been developed for pseudomonads,
xanthomonads and most notably for phytoplasmas (Louws et al., 1999). Specific
primer pairs were designed for the detection of Pseudomonas (Widmer et al., 1998;
Alvarez, 2004), Xanthomonas (Maes, 1993) and Ralstonia solanacearum (Seal
et al., 1993) based on sequence variations present in 16S and 23S rDNA regions.
Genes associated with pathogenicity (Prosen et al., 1993; Manulis et al., 2002) and
those present on plasmid DNA (Bereswill et al., 1992; Audy et al., 1994) have also
been used to design specific PCR assays.
The introduction of real-time PCR in the last few years has improved and
simplified methods for PCR-based quantification. Real-time PCR has many
advantages over conventional PCR: it is faster and higher throughput is possible,
post-reaction processing is not required as the amplified products are detected by a
built-in fluorimeter thereby eliminating the risk of contamination, and it is more
specific than conventional PCR if a specific probe is used in addition to the two
specific primers. Real-time PCR involves the detection and measurement of
amplification products at each cycle of amplification. Over last few years many
systems have been developed for performing amplification and detection of nucleic
acids in a single tube. These can be classified into non-specific detection systems
and specific detection systems.
The standard method for non-specific detection is using a DNA binding dye that
fluoresces after binding. The most commonly used dye is SYBR® green I (Morrison
et al., 1998); this is excited at 497 nm and emits at 520 nm. When in solution, the
dye exhibits no fluorescence. However, as PCR products accumulate during the
amplification process, increasing amounts of dye bind to the double stranded DNA.
So in each PCR cycle, the dye fluoresces during the elongation step as it binds to the
DNA and then during the denaturation step, it falls off leading to a decrease in
fluorescence. In this way, when monitored in real time at the end of each cycle,
measurement of SYBR green-borne fluorescence can indicate an accumulation of
the PCR product as the reaction proceeds. Use of SYBR green eliminates the need
for target-specific fluorescent probes as specificity is entirely determined by the
primers. SYBR green will bind to any double stranded DNA present in the reaction
including primer dimmers; this makes it very important for the primers to be very
specific and to generate a single product (Ririe et al., 1997). There are many other
detection methods available that will detect only a specific fragment. These methods
include TaqMan® probes, double dye oligonucleotide probes, molecular beacon
probes, Scorpions primers, hybridization probes, TaqMan® MGB® probes, MGB
Eclipse probes and many others. As the use of qPCR has grown since its development
in 1996, better and more advanced instruments capable of more accurate quantification
and handling more samples in a given time have been developed. Some of the more
popular machines are ABI PRISM ® 7000/7700, ABI PRISM ® 7900HT, Roche
LightCycler ® and Bio-rad iCycler iQ ® .
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