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

PCR for Veterinary Mycobacteria 205
Table 2
Detection of Mycobacteria in Different Hosts and Tissues
by DNA Sequence-Capture PCR
Species Specimen site Specimen type
Badger Kidney Decontaminated homogenate
Boar Lung Formalin-fixed paraffin-embedded
Bovine Lymph node Fresh
Companion animals Muscle Frozen
Deer Nasal Mucus Lypholized
Ferret
Sequence capture procedure has enabled detection of mycobacteria in a wide range of host
species and specimen types in this laboratory (as above). We intend to adapt specimen pretreatment
further to apply sequence capture to other specimen types including milk. feces, blood, and urine.
tissues. Several mycobacterial DNA extraction methodologies for PCR analyses
of veterinary specimens have been used previously, both by this laboratory
(26) and by others (14–16, 27), but PCR assays have been shown not to be as
sensitive as culture methods. Sequence capture PCR was subsequently reported
to be successful in the detection of M. tuberculosis in paucibacillary specimens
from patients with tuberculous pleurisy (28,29). This procedure involved
enrichment of target DNA by hybridization to biotinylated oligonucleotides
and subsequent capture of hybridized DNA onto streptavidin-coated magnetic
beads (see Fig. 1). Magnetic bead attractors enabled potential inhibitors to be
removed easily by washing the beads. The method was advantageous from a
health and safety perspective in not requiring solvent extraction.
We have now adapted this approach for detection of M. bovis in animal
specimens. For PCR-based confirmation from culture, DNA was released simply
by heat treatment and a modified sequence capture protocol (see Fig. 1)
was used to concentrate the target DNA. Pretreatment involving mechanical
and enzymatic disruption (28) has been modified for different specimen types
(see Fig. 2). This approach has flexibility with regard to specimen type and
mycobacterial pathogen. In addition, the method was robust in allowing successful
adoption and adaptation by our laboratory without recourse to the
developers of the methodology.
The basic sequence capture and PCR protocols, introducing different capture
oligonucleotides and PCR primer sets, have been modified to facilitate
a range of different PCR amplifications. We have used this procedure in a
number of ways: for definitive diagnosis and simultaneous strain typing of
M. tuberculosis complex from radiometric cultures at early growth stages (30);
and directly in decontaminated tissue homogenates and in lesioned tissue from