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

306 Pozio and La Rosa
Table 3
PCR-RFLP Amplicon Sizes
of Principal Bands of Trichinella britovi Genotypes
Restriction enzymes
Trichinella genotype Dde I Ssp I
T. britovi 700, 680, 560, 520 2100
T8 700, 650, 560, 330 2300, 2100
T9 700, 650, 560, 520 1500, 1200, 650
3.4. 43 kDa (E/S) PCR-RFLP Digestion Protocols
1. Thaw a sample of crude DNA extraction on ice (at this point, each tube should
contain 10 µL of the larva preparation).
2. To set up PCR, add sequentially 5 µL 10X PCR buffer, 4 µL dNTPs, 2 µL set of
primers, 0.2 µL Taq DNA polymerase, 10 µL of crude DNA extraction, and H 2O
up to 50 µL in a 0.2-mL thin-walled tube.
3. Place tubes on ice.
4. PCR cycle: pre-amplification cycle at 94°C for 5 min, followed by 30 cycles at
98°C for 20 s, and 60°C for 15 min, followed by an extension cycle at 72°C for
4 min, and then place on ice.
5. Hot start at 94°C: wait until the thermal cycler reaches 94°C and then place the
tubes on the hot plate.
6. Electrophoresis: use 10 µL of the amplification reaction. Select samples showing
good single-band amplification for restriction analysis.
7. Restriction analysis: transfer 20 µL of the amplification reaction into a 1.5-mL
conical tube, add 5 µL of the respective 10X restriction buffer, 10 U of the
selected enzyme (see Note 12), and H 2O up to 50 µL.
8. Incubate at 37°C for 2 h.
9. Transfer on ice and stop the reaction with 5 µL of 0.5 M EDTA.
10. Electrophoresis: load all of the reaction onto the agarose gel (see Table 3 and
Fig. 2).
3.5. Electrophoresis Conditions
1. Standard agarose gel: follow standard procedures to prepare 1–1.5% agarose gel
in TBE or TAE buffer and run at 10 V/cm.
2. High-resolution agarose gel: to have an adequate resolution of T. pseudospiralis
isolates, run the amplification products on 3% metaphor agarose gel at 10 V/cm.
4. Notes
1. In the original description of multiplex-PCR (7), a nested-PCR was used. However,
this is not necessary with the present protocol.