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

10 Sachse
proteins (66–68), invasion factors (69,70) and various virulence factors (71–73)
being used in PCR assays.
Apart from the potential to fine-tune specificity of detection as mentioned
above, the most evident advantage from the utilization of protein gene-based
PCR assays is the concomitant information provided on toxins, surface antigens,
or other virulence markers, as these factors are supposed to be directly
involved in pathogenesis. In this respect, such tests deliver more evidence on a
given microorganism than just confirming its presence in a sample.
2.4. Repetitive Elements
Some microorganisms possess repetitive sequences or insertion elements.
Since these segments are present in multiple copies the idea of targeting them
appears straightforward. Indeed, this is a favorable prerequisite for the
development of highly sensitive detection methods. In the literature, amplification
assays based on repetitive elements were reported for Coxiella burnetii
(74,75), Mycobacterium bovis (76), the Mycobacterium tuberculosis complex
(77–79) Leptospira interrogans (80), and trichinellae (81). In combination
with sequence-specific DNA capture prior to amplification, a detection
limit of one mycobacterial genome was attained (79).
3. Efficiency of the Amplification Reaction
3.1. Early, Middle, and Late Cycles
DNA amplification by PCR is based on a cyclical enzymatic reaction,
where the products (amplicons) of the previous cycle are used as substrate
for the subsequent cycle. Thus, in theory, the number of target molecules is
expected to increase exponentially, i.e. double, after each cycle. As the efficiency
of the reaction is not 100% in practice, the real amplification curves
are known to deviate from the exponential shape (82–85). The course of DNA
amplicon production during 30 cycles in an ideal and a real PCR is illustrated
in Fig. 2. The extent of deviation from the theoretical product yield is determined
by the efficiency of amplification, which can be approx assessed by
Equation 1 (86):
Y (1 ε ) n [Eq.1]
where Y is the amplification yield (expressed as quotient of the number of molecules
of PCR product and the initial number of target molecules), n is the
number of cycles, and ε is the mean efficiency of all cycles with 0 ≤ ε ≤ 1.
The reaction efficiency may, in principle, assume a different value in each
cycle. The parameters affecting ε include the concentration of DNA polymerase,
dNTPs, MgCl 2, DNA template, primers, temperatures of denaturation,