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

242 Hotzel et al.
4. Notes
1. The use of DNA preparation kits can be recommended for samples of organ tissue,
broth culture, and with some qualification, also for semen and feces. In the
latter instances, the kit should be tested with a series of spiked samples containing
defined numbers of mycoplasma cells in order to examine its suitability.
Most commercial kits are easy to work with. They contain a special buffer
reagent for lysis of the bacterial and tissue cells, the effectiveness of which is
decisive for the kit’s performance. An optional RNase digestion is intended to
remove cellular RNA. The lysate is then centrifuged through a mini-column,
where the released DNA is selectively bound to a solid phase (modified silica,
hydroxyl apatite, or filter membrane). After washing, the DNA can be eluted
with an elution buffer or water. DNA prepared in this manner is usually of high
purity and largely free of PCR inhibitors.
It should be noted, however, that the yield of extracted DNA is limited by the
binding capacity of the mini-column. If maximum recovery of mycoplasmal DNA
is important, e.g., in quantitative assays or for preparation of reference DNA, an
alternative extraction protocol should be followed.
2. Extraction of bacterial DNA from milk samples seems to be particularly difficult
in the case of mycoplasmas. This may be due to adhesive properties and/or the
small size of mycoplasma cells. In a previous study, several extraction procedures
were compared, which included cold and hot phenol extraction, Tween 20 treatment,
protease digestion, commercial DNA extraction kits, and, finally, a combination
of enzymatic treatment and selective membrane binding of DNA (17).
Using the latter, 50–500 cfu of M. bovis/mL milk were detectable. The present
method of pre-PCR enrichment by antigen capture allowed an improvement of the
detection limit up to 20 cfu/mL milk when amplification products were visualized
on agarose gels, and 2 cfu/mL when visualized on Southern blots (24).
3. Several sets of primers have been designed that detect all members of the
M. mycoides cluster, i.e., M. mycoides subsp. mycoides SC, M. mycoides subsp.
mycoides LC, M. mycoides subsp. capri, M. capricolum subsp. capricolum,
M. capricolum subsp. capripneumoniae, Mycoplasma bovine group 7. Those
described by Bashiruddin et al. (25), Dedieu et al. (26), Hotzel et al. (23), and
Rodriguez et al. (27) are derived from the same set of sequence data from the
CAP-21 genomic region, and one or more of them could be used additionally for
the confirmation of results of further differentiation.
4. The target region of the primers PpMB920-1/2 and PpSM5-1/2, used for the
nested PCR system, is the operon of oligopeptide permease (opp) genes encoding
ATP-binding proteins, which are members of the so-called ABC-transporter family.
The oppD and oppF genes of M. bovis were identified on the basis of
sequence homology to the analogous genes of M. hominis.
5. M. bovis-specific primer systems MBOUVRC2-L/R and PpMB920-1/2 were
tested together with other assays in a ring trial in COST Action 826. Both produced
identical results with field isolates from different origins and surpassed
other PCR test systems. All field isolates of M. bovis tested so far were found to