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

Clostridium PCR 143
3.2.3. DNA Extraction from Biological Samples (see Note 1)
3.2.3.1. INSTAGEN METHOD
Use the same protocol as for DNA extraction from broth culture. Liquid
samples can be processed as broth culture, and solid samples are homogenized
in phosphate buffered saline (PBS) 1:10 (w/v).
3.2.3.2. QIAGEN METHOD
For those samples containing PCR inhibitors, a more reliable method of
DNA extraction is required. The QIAamp DNA Stool Mini Kit has proved
suitable for a variety of sample matrixes.
1. Pipet 200 mL of liquid sample or weigh 180–220 mg of solid sample in a 2-mL
microcentrifuge tube.
2. Add 1.2 mL of ASL buffer (provided in the kit), vortex mix for 1 min or until the
sample is thoroughly homogenized.
3. Incubate at 70°C for 5 min.
4. Vortex mix for 15 s and centrifuge (18000g) for 1 min.
5. Pipet 1.2 mL of the supernatant in another 2-mL microtube and discard the pellet.
6. Add 1 inhibiEx tablet (provided in the kit) and vortex mix until the tablet is completely
suspended. Incubate 1 min at room temperature.
7. Centrifuge at high speed for 3 min.
8. Pipet the supernatant in a 1.5 mL microtube and discard the pellet. Centrifuge at
high speed for 3 min.
9. Pipet 15 µL of proteinase K solution (provided in the kit) into a fresh 1.5-mL
microtube.
10. Pipet 200 µL of supernatant from step 8 into the microtube containing proteinase K.
11. Add 200 µL of buffer AL and vortex mix for 15 s.
12. Incubate at 70°C for 10 min.
13. Add 100 µL of ethanol and vortex mix to homogenize.
14. Apply the complete lysate from step 13 in a QIAamp spin column sitting in a
2-mL microtube, and centrifuge for 1 min. Place the QIAamp spin column in a
new 2-mL microtube and discard the filtrate.
15. Add 500 µL of buffer AW1 into the column, centrifuge for 1 min. Place the
QIAamp spin column in a new 2-mL microtube and discard the filtrate.
16. Add 500 µL of buffer AW2, centrifuge for 2 min and discard the filtrate.
17. Transfer the QIAamp column into a new 1.5-mL microtube, and add 200 µL of
buffer AE onto the QIAamp membrane. Incubate for 1 min and centrifuge for
1 min. Use 5 µL for PCR. Add bovine serum albumin (BSA) to a final concentration
of 0.1 µg/µL in the PCR mixture. The DNA extract can be stored at –20°C
for later use.
3.3. Amplification by PCR (General Protocol)
3.3.1. Preparation of the Master Mixture
Prepare a master mix for all amplification reaction of the series. It should
contain the following concentrations of reagents in each 50-µL reaction: