John M. S. Bartlett.pdf - Bio-Nica.info

Nested RT-PCR in a Single Closed Tube 157
Fig. 3. Analysis of RT-nested-PCR products by agarose gel electrophoresis. (A) Detection
of PPV targets from infected plant material. Lane 1: 100 bp molecular weight markers (Gibco
BRL). Lanes 2 through 5: amplification products (243 bp) obtained from PPV-infected GF305
peach seedlings. Lanes 6 and 7: samples from healthy GF305 peach seedlings. (B) Detection of
CTV targets from infected aphid vectors. Lane 1: pUC19DNA/MspI(HpaII) molecular weight
markers 23 (MBI Fermentas). Lanes 2 through 6: amplification products (131 bp) obtained from
individual Aphis gossypii fed on CTV-infected Washington Navel sweet orange. Lane 7: Sample
from Aphis gossypii fed on healthy Washington Navel sweet orange.
paper; ref. 10) for 3 h at 37°C or overnight at 4°C in an uncoated Eppendorf tube. Before
RT-PCR, the tube is washed twice with 150 µL of phosphate-buffered saline–0.05% Tween
20 (washing buffer). However, it may be useful to assay the ability of different plastic tubes
to trap RNA targets in order to select the most appropriate for RT-PCR.
3. There are several commercially available thin-walled Eppendorf tubes for PCR. The
protocol describes the use of 0.5-mL tubes because they are more easily compartmentalized
with the end of a pipet tip (see Fig. 2).
4. The reaction must be performed in a thermal cycler with a heated lid to prevent the
requirement for oil-overlay during the cycling events. The selected thermal cycler must
allow the maintenance of cocktail B (see Fig. 2) in the cone during the cycling. If
evaporation occurs close the tip device as in Note 3 and/or adjust the lid temperature to 70
to 80°C during the first reaction (RT-PCR).
5. Primer design is critical to the success in amplifying RNA targets by conventional
nested RT-PCR but it is even more critical when a multiplex nested strategy is used. The
recommended primer design (see Subheading 3.1.) allows an optimal design of primers.
The size of the amplified product should be small to ensure a good efficiency of the
reaction and high sensitivity (26,27). Primers with a broad range of specificity must be
designed from highly conserved genome sequences. Degenerate primers must be used for a
universal detection of RNA targets belonging to a group. For characterization or typing of
RNA targets, primers must be designed from discriminating or specific regions.
6. The goal of the first round of PCR after RT is to obtain a yield of the amplification product
just enough to ensure adequate target for the second (nested or heminested) amplification.
For this reason, the number of cycles can be as low as 20 to 25.
7. The goal of the second round of PCR (nested or heminested) is to achieve a high sensitivity
and simultaneous specificity (if the method has been designed for typing RNA targets).
The optimum number of cycles ranges from 35 to 40.