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

PCR for Veterinary Mycobacteria 203
with identification, as some species are difficult to differentiate from closely
related mycobacterial species or from species in genera such as Corynebacterium,
Nocardia, and Rhodococcus .
Consequently in recent years, there has been considerable and justifiable
interest in applying the modern technologies of molecular biology to detection,
speciation, and even to the epidemiology of diseases caused by mycobacteria.
Such technologies have potential to provide improvements in these areas, not
least possibly obviating the process of time-consuming culture. However, there
has been a tendency to underestimate the difficulties inherent in introducing
and applying these technologies to such recalcitrant pathogens as mycobacteria.
Their intracellular nature, the intractability of the mycobacterial cell wall
and the potential presence of polymerase chain reaction (PCR) inhibitors in
clinical specimens are limitations on PCR effectiveness. The paucibacillary
nature of specimens in some scenarios can be particularly problematic. Additionally,
the standard configuration of PCR does not currently distinguish
between live and dead mycobacteria, a factor of importance, e.g., when considering
its use for detecting the survival of M. avium subsp paratuberculosis
post-pasteurization (10). However, if molecular diagnostic methods are to be
considered as acceptable replacements for traditional procedures, there must
be sustained improvement in sensitivity, specificity, reproducibility, and convenience,
together with possibly a capability for increased throughput capacity.
Reduced costs and availability of appropriate resources and trained staff
are also important factors for their successful adoption by routine laboratories.
The more conventional technologies may be more appropriate in some countries
and particularly so for some pathogens. For mycobacteria, in both developed
and developing countries, culture is still considered to be the gold standard
(11), despite lacking specificity and being time-consuming.
There are several published protocols for PCR detection of clinically important
mycobacteria. Many have focused on mycobacterial pathogens of humans
(12). However, among these, there appears to be no consensus regarding
extraction or amplification methodologies. There are also difficulties in technology
transfer and reproducibility between reputable laboratories in
multicenter studies (13). Potential problems with false negative reactions, possibly
as a result of inhibitors, and also with false positive reactions, possibly
due to contamination have been recorded. Stringent design and use of PCR
facilities and inclusion of relevant controls should reduce and allow monitoring
of such occurrences.
In veterinary use, the primary focus has been mainly on in-house amplification-based
detection of M. bovis (14–17). Other applications of nucleic acidbased
diagnostics in veterinary mycobacteriology laboratories have tended
to be the evaluation of more robust and sometimes prohibitively expensive