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

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to partially compensate this drop in efficiency by prolonging the extension
step in the later cycles or increasing the enzyme concentration of the reaction.
The latter, however, has to be optimized empirically as excessive
enzyme often stimulates the co-synthesis of nonspecific products.
Although thermal stability of commercially available DNA polymerases has
been steadily improved over the last decade (see Chapter 2) there is a measurable
decrease of its activity in the late cycles. This is another reason not to
recommend high numbers of cycles in PCR detection assays, indeed, 30–35
cycles proved sufficient in most applications.
4. Methodologies to Improve the Performance
of Amplification Assays
When using a PCR assay for clinical samples containing only a few cells
of the pathogen, one has to be aware of the special kinetic conditions prevailing
in such a reaction mixture. At the start of the reaction as few as 1 to 100
copies of the target sequence may be present, and the crucial primer annealing
step is particularly difficult to control. This may require additional measures
to insure that primer oligonucleotides specifically bind to as many
targets as possible and, at the same time, avoid nonspecific priming of DNA
synthesis.
4.1. Hot-Start PCR
The basic idea of hot-start PCR is to reduce nonspecific amplification in the
initial phase by releasing active enzyme only immediately before the first
primer binding step (97). This approach is designed to prevent primer–dimer
formation, mispriming, and spontaneous initiation of DNA strand synthesis,
most of which occur already at room temperature between the operations of
mixing reagents and actually starting the PCR run (98,99). The most common
variant of hot-start PCR involves thermostable DNA polymerases (see Chapter
2) that are supplied in an inactive form and require a 10-min heating at 94°–
96°C for activation (100, 101). Many protocols of “conventional” amplification
assays can be improved in terms of specificity by adaptation to the hot-start
procedure and its special reagents.
Another approach involves so-called loop primers that carry additional 5'
tails causing the oligonucleotides to self-anneal or oligomerize at ambient
temperature (102). When the reaction mixture is heated, the primers are linearized
only at elevated temperatures, thus initiating a hot start. To facilitate
specificity of amplification, the protocol begins with six touch-up cycles,
where Tann is gradually increased from 60° to 72°C. Hot-start conditions were
also shown to be created through the addition of short double-stranded DNA
fragments (103).