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

PCR Technology 71
4.1. Primer Design
A critical aspect of any PCR assay is the design and selection of optimal
primers. Poorly designed primers are among the main causes for nonspecific
amplification of nontarget DNA or complete failure of amplification of target
DNA. Commercially available primer design software, e.g., OLIGO (51), can
facilitate a systematic search for suitable primers.
An important feature of an efficient primer is its complete match with the
target DNA. When primers have to be designed on the basis of a single or a few
available sequences of the target organism, it may turn out later that they do
not match completely to all strains because of sequence variation. In these circumstances,
the primers must match the target sequence at least at the 3' end,
covering 5 to 6 nucleotides towards the 3' end. A few mismatches of the primer
at the 5' end can be tolerated. The amount of tolerable mismatch is dependent
on the nucleobases facing each other. While GC and AT pairing are perfect,
and AC pairs are acceptable, GT, CT and GA pairs are unacceptable because of
weak hybridization or steric hindrance. The consequences of poor primer
annealing due to mismatches can be false negative results of the PCR assay.
Another important factor impairing the performance of an amplification
assay is the formation of primer–dimers as a result of complementary base
pairing between primer molecules. Complementary stretches of 2 to 3 bases
in the two primers are sufficient to cause this phenomenon. Moreover, primers
forming hairpin structures by internal hybridization will reduce the efficiency
of amplification. In both cases, high annealing temperatures are
necessary to prevent the unwanted hybridization, or new primers should be
designed to insure optimal amplification.
Following the design of specific primers, it is necessary to check their
selectivity in diagnostic PCR (see Chapter 3). There should be a positive
PCR signal for all members of the target taxon and no amplification of DNA
from organisms outside the taxon. Specificity in PCR is dependent on the
annealing temperature. For specific detection with a completely matching
primer set, it is recommended to use the highest possible annealing temperature
to insure high stringency (45,49,52). Optimal annealing temperatures
are typically at 55–60°C. In contrast, primers not matching 100 % will
anneal only at lower temperatures, which leads to lower sensitivity and specificity.
This can be compensated by nested amplification (53,54) (see Chapters
1 and 3).
4.2. Diagnostic Specificity
When developing a technique for detection and identification of organisms
in a complex environment, one of the most central aspects to be considered is its
diagnostic specificity (see Chapters 1 and 3). In most cases, the criteria of