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

158 Berri et al.
Fig. 1. Trans-PCR. Determination of the detection limit using a dilution series of
C. burnetti cells (10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , and 10 1 cells per reaction, lanes 2–8;
lane 1, 100-bp DNA ladder; lane 9, negative control (reagents without DNA).
coli, Klebsiella pneumoniae, Listeria monocytogenes, Salmonella abortus ovis,
Salmonella typhimurium, Staphylococcus aureus, Staphylococcus chromogenese,
Staphylococcus hominis, Streptococcus dysgalactiae, and Streptococcus
agalactiae.
To verify the identity of the PCR amplicon obtained from milk and fecal
material of naturally infected animals, PCR products from these samples and the
reference strain can subsequently be subjected to TaqI and Alu restriction endonuclease
digestion. TaqI digestion will result in the generation of 446-, 201-, and
10-bp fragments, whereas cleavage with AluI will yield bands at 316-, 191-, and
179-bp (9). The assay’s specificity can be further evaluated by sequence analysis
of the Trans-PCR product. In our laboratory, PCR products were cloned and
sequenced using pCR2.1 primers, and in all cases, the sequence of the amplicon
was identical to the sequence in the European Molecular Biology Laboratory
(EMBL)/GenBank ® database (Accession no. M80806) (9).
3.4.4. Inhibitory Effect of Fecal Material
No C. burnetii DNA was amplified from the undiluted DNA solution purified
from artificially contaminated fecal samples. Extraction using the QIAamp
Tissue Kit procedure did not eliminate PCR inhibitors completely, suggesting