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

Clostridium PCR 147
from a sample, we recommend a PCR with primers P708–P709 (sialidase gene)
to identify the species C. sordellii, and another amplification using primers
P535–P536 (lethal toxin gene) (Table 4) to identify the toxigenic strains.
C. difficile is responsible for pseudomembranous colitis and postantibiotherapy
diarrhea, which are the most prominent nosocomial affections in hospitalized
patients. This pathogen is also involved in some digestive diseases in
animals. C. difficile produces two main toxins called ToxA and ToxB, but some
strains only synthesize ToxA or ToxB, and others produce another toxin
(C. difficile transferase or CDT) which probably represents an additional virulence
factor. Specific ToxA, ToxB, and CDT gene probes are listed in Table 4.
3.10. Detection of PCR Products
3.10.1. Agarose Gel Electrophoresis
Agarose gel electrophoresis is the method of choice for checking the size
and purity of a PCR product.
1. To prepare a 2% agarose minigel, place 1 g of DNA-grade agarose in a 100-mL
flask or bottle.
2. Add 50 mL of 1X TAE buffer (49 mL distilled water and 1 mL of 50X TAE buffer).
3. Heat the mixture in boiling water or in a microwave for 2– 5 min for the agarose
to dissolve completely.
4. Cool the solution on the bench top for few min.
5. Add 10 mL of 10 mg/mL ethidium bromide stock solution.
6. Pour onto a gel casting stand with well comb(s) in place, and allow about 20 min
for the gel to set. Remove the comb(s) and transfer the solid gel to a gel tank and
add enough 1X TAE buffer (usually 500 mL) to cover the gel by at least several
millimeters.
7. Mix 10 µL of PCR product and 2 µL of gel-loading dye, and load the samples
into the wells. A sample DNA ladder is loaded in each agarose gel.
8. Run the gel at constant voltage of 100–120 V for approx 1 h until the dye front is
one-half to two-thirds of the way down the gel.
9. View the DNA by placing the stained gel on a UV lightbox.
10. (Optional) Photograph the gel on a UV illuminator using a Polaroid camera.
3.10.2. Hybridization
When many samples have to be routinely analyzed, agarose gel electrophoresis
is not an appropriate method. This is the case for C. botulinum A, B,
and E examination of food samples. We have proposed a method that includes
transfer of PCR products onto nitrocellulose membrane and hybridization with
internal probes specific of each toxin type A, B, and E, respectively (31). A
preferred method is sandwich hybridization which can be automated (32).
The following protocol is from Fach et al. (manuscript submitted):
1. PCR amplification with BotU and BotR (see Subheading 3.3.).