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

Coxiella PCR 157
genome (12): Trans-1 (5'-TAT GTA TCC ACC GTA GCC AGT C-3') and
Trans-2 (5'-CCC AAC AAC ACC TCC TTA TTC-3'). The expected product
of amplification of the target sequence will be 687 bp in length.
3.4.1. PCR Procedure
1. Perform the assay in 25-µL reactions, each of which should contain: 2 µΜ of
each primer, 200 µM of each deoxynucleoside triphosphate, 3 mM MgCl2, 0.5 U
of Taq DNA polymerase, 2.5 µL of DNA extract prepared as described under
Subheading 3.3.
2. Perform the Trans-PCRs according to a modified touchdown protocol (9):
5 cycles consisting of denaturation at 94°C for 30 s, annealing at 66–61°C (the
temperature was decreased 1°C between consecutive cycles) for 1 min and
extension at 72°C for 1 min, and then 40 cycles of denaturation at 94°C for
30 s, annealing at 61°C for 30 s, and extension at 72 °C for 1 min.
3. Load 10 µL of PCR product onto 1.5% agarose gel and run electrophoresis.
4. Stain the gel with ethidium bromide and visualize under UV transilluminator.
3.4.2. Evaluation of the Sensitivity of the PCR Assay (see Note 4)
In order to evaluate the sensitivity of PCR detection, three different types of
samples are used. Each sample should be tested in triplicate.
3.4.2.1. CELL SUSPENSION OF C. BURNETII
1. Prepare serial dilutions in PBS of one set of C. burnetii suspension containing
between 10 8 and 10 1 Coxiella cells/mL.
2. Boil each dilution for 10 min or treat with proteinase K as described in Subheading
3.3.2. step 2.
3. Use 2.5 µL in the PCR assay (an example is shown in Fig. 1).
3.4.2.2. SPIKED MILK SAMPLES
1. Add 50 µL of C. burnetii Nine Mile strain (serial dilutions containing between
10 8 and 10 1 Coxiella cells/mL) to 1 mL of Coxiella-free milk (9). Commercial
pasteurized milk, bovine, caprine, and ovine raw milk may be used.
2. Treat 1 mL of each dilution as described in Subheading 3.3.3.
3.4.2.3. SPIKED FECAL SAMPLES
1. Mix 50 µL of serially diluted Coxiella suspension (10 8 to 10 1 C. burnetii cells/
mL) with 20 mg of Coxiella-free feces.
2. Extract the DNA as described in Subheading 3.3.4.
3.4.3. Evaluation of Specificity
Specificity can be examined by subjecting genomic DNA (106 template copies)
of the following bacteria to Trans-PCR : Chlamydophila psittaci,
Chlamydophila pecorum, Brucella melitensis, Brucella abortus, Escherichia